tretinoin and Neuroblastoma

Neuroblastoma is a rare malignant disease and mainly affects infants and very young children. The tumours mainly develop in the adrenal medullary tissue, with an abdominal mass as the most common presentation. About 50% of patients have metastatic disease at diagnosis. The high-risk group is characterised by metastasis and other features that increase the risk of an adverse outcome. High-risk patients have a five-year event-free survival of less than 50%. Retinoic acid has been shown to inhibit growth of human neuroblastoma cells and has been considered as a potential candidate for improving the outcome of patients with high-risk neuroblastoma. This review is an update of a previously published Cochrane Review.. To evaluate the efficacy and safety of additional retinoic acid as part of a postconsolidation therapy after high-dose chemotherapy (HDCT) followed by autologous haematopoietic stem cell transplantation (HSCT), compared to placebo retinoic acid or to no additional retinoic acid in people with high-risk neuroblastoma (as defined by the International Neuroblastoma Risk Group (INRG) classification system).. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library (2016, Issue 11), MEDLINE in PubMed (1946 to 24 November 2016), and Embase in Ovid (1947 to 24 November 2016). Further searches included trial registries (on 22 December 2016), conference proceedings (on 23 March 2017) and reference lists of recent reviews and relevant studies. We did not apply limits by publication year or languages.. Randomised controlled trials (RCTs) evaluating additional retinoic acid after HDCT followed by HSCT for people with high-risk neuroblastoma compared to placebo retinoic acid or to no additional retinoic acid. Primary outcomes were overall survival and treatment-related mortality. Secondary outcomes were progression-free survival, event-free survival, early toxicity, late toxicity, and health-related quality of life.. We used standard methodological procedures expected by Cochrane.. The update search did not identify any additional studies. We identified one RCT that included people with high-risk neuroblastoma who received HDCT followed by autologous HSCT (N = 98) after a first random allocation and who received retinoic acid (13-cis-retinoic acid; N = 50) or no further therapy (N = 48) after a second random allocation. These 98 participants had no progressive disease after HDCT followed by autologous HSCT. There was no clear evidence of difference between the treatment groups either in overall survival (hazard ratio (HR) 0.87, 95% confidence interval (CI) 0.46 to 1.63; one trial; P = 0.66) or in event-free survival (HR 0.86, 95% CI 0.50 to 1.49; one trial; P = 0.59). We calculated the HR values using the complete follow-up period of the trial. The study also reported overall survival estimates at a fixed point in time. At the time point of five years, the survival estimate was reported to be 59% for the retinoic acid group and 41% for the no-further-therapy group (P value not reported). We did not identify results for treatment-related mortality, progression-free survival, early or late toxicity, or health-related quality of life. We could not rule out the possible presence of selection bias, performance bias, attrition bias, and other bias. We judged the evidence to be of low quality for overall survival and event-free survival, downgraded because of study limitations and imprecision.. We identified one RCT that evaluated additional retinoic acid as part of a postconsolidation therapy after HDCT followed by autologous HSCT versus no further therapy in people with high-risk neuroblastoma. There was no clear evidence of a difference in overall survival and event-free survival between the treatment alternatives. This could be the result of low power. Information on other outcomes was not available. This trial was performed in the 1990s, since when many changes in treatment and risk classification have occurred. Based on the currently available evidence, we are therefore uncertain about the effects of retinoic acid in people with high-risk neuroblastoma. More research is needed for a definitive conclusion.

Topics: Adrenal Gland Neoplasms; Antineoplastic Agents; Bone Marrow Transplantation; Consolidation Chemotherapy; Disease-Free Survival; Hematopoietic Stem Cell Transplantation; Humans; Infant; Neuroblastoma; Randomized Controlled Trials as Topic; Selection Bias; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

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Neuroblastoma is a rare malignant disease and patients with high-risk neuroblastoma have a poor prognosis. Retinoic acid has been shown to inhibit growth of human neuroblastoma cells and has been considered as a potential candidate for improving the outcome.. The objective was to evaluate effects of retinoic acid after consolidation with high-dose chemotherapy and bone marrow transplantation as compared to placebo or no further treatment in patients with high-risk neuroblastoma. We searched the databases CENTRAL, MEDLINE, and EMBASE from inception to 01 October 2014 and included randomized controlled trials.. We identified one relevant randomized controlled trial with 50 participants receiving retinoic acid and 48 participants receiving no further therapy. There was no statistically significant difference between the treatment groups in overall survival (hazard ratio 0.87, 95% confidence interval 0.46-1.63, P=0.66) and event-free survival (hazard ratio 0.86, 95% confidence interval 0.50-1.49, P=0.59). We did not identify results for other outcomes, including toxicity.. The difference in overall and event-free survival between treatment alternatives was not statistically significantly different. Based on the currently available evidence, we are uncertain about the effects of retinoic acid after bone marrow transplantation in patients with high-risk neuroblastoma.

Topics: Bone Marrow Transplantation; Chemotherapy, Adjuvant; Child; Child, Preschool; Combined Modality Therapy; Disease-Free Survival; Humans; Infant; Infant, Newborn; Neoplasm Staging; Neuroblastoma; Prognosis; Randomized Controlled Trials as Topic; Risk Assessment; Tretinoin

Neuroblastoma is a rare malignant disease and mainly affects infants and very young children. The tumors mainly develop in the adrenal medullary tissue and an abdominal mass is the most common presentation. About 50% of patients have metastatic disease at diagnosis. The high-risk group is characterized by metastasis and other characteristics that increase the risk for an adverse outcome. High-risk patients have a five-year event-free survival of less than 50%. Retinoic acid has been shown to inhibit growth of human neuroblastoma cells and has been considered as a potential candidate for improving the outcome of patients with high-risk neuroblastoma.. To evaluate efficacy and adverse events of retinoic acid after consolidation with high-dose chemotherapy followed by bone marrow transplantation as compared to placebo or no therapy in patients with high-risk neuroblastoma (as defined by the International Neuroblastoma Risk Group (INRG) classification system). Our outcomes of interest were overall survival and treatment-related mortality as primary outcomes; and progression- and event-free survival, early and late toxicity, and health-related quality of life as secondary outcomes.. We searched the electronic databases CENTRAL (2014, Issue 8) on The Cochrane Library, MEDLINE (1946 to October 2014), and EMBASE (1947 to October 2014). Further searches included trial registries, conference proceedings, and reference lists of recent reviews and relevant articles. We did not apply limits on publication year or languages.. Randomized controlled trials (RCTs) evaluating retinoic acid post consolidation therapy for high-risk neuroblastoma patients treated with autologous hematopoietic stem cell transplantation (HSCT) compared to placebo or no further treatment.. Two review authors performed the study selection, extracted the data on study and patient characteristics and assessed the risk of bias independently. We resolved differences by discussion or by appeal to a third review author. We performed analyses according to the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions. The authors of the included study did not report the results specifically for the treatment groups relevant to this Cochrane Review. Therefore, we deduced the appropriate survival data from the published survival curves and calculated a hazard ratio (HR) based on the deduced data.. We identified one RCT (CCG-3891) that included patients with high-risk neuroblastoma who received high-dose chemotherapy followed by autologous HSCT (N = 98) after a first random allocation and who received retinoic acid (13-cis-retinoic acid; N = 50) or no further therapy (N = 48) after a subsequent second random allocation. These patients had no progressive disease after consolidation therapy. There was no clear evidence of difference between the treatment groups in both overall survival (HR 0.87, 95% CI 0.46 to 1.63; one trial; P = 0.66, low quality of evidence) and event-free survival (HR 0.86, 95% CI 0.50 to 1.49; one trial; P = 0.59, low quality of evidence). We calculated these HR values using the complete follow-up period of the trial. The study also reported five-year overall survival rates: 59% for the retinoic acid group and 41% for the no further therapy group (P value not reported). We did not identify results for treatment-related mortality, progression-free survival, early or late toxicity, or health-related quality of life. Also, we could not rule out the possible presence of selection bias, performance bias, attrition bias, and other bias.. We identified one RCT that evaluated retinoic acid as a consolidation therapy versus no further therapy after high-dose chemotherapy followed by bone-marrow transplantation in patients with high-risk neuroblastoma. The difference in overall survival and event-free survival between both treatment alternatives was not statistically significantly different. This could be the result of low power. Information on other outcomes was not available. This trial was performed in the 1990s, since then many changes in for example treatment and risk classification have occurred. Therefore, based on the currently available evidence, we are uncertain about the effects of retinoic acid in patients with high-risk neuroblastoma. More research is needed for a definitive conclusion.

Topics: Adrenal Gland Neoplasms; Antineoplastic Agents; Bone Marrow Transplantation; Consolidation Chemotherapy; Disease-Free Survival; Hematopoietic Stem Cell Transplantation; Humans; Infant; Neuroblastoma; Randomized Controlled Trials as Topic; Tretinoin

Retinoids are lipophilic compounds derived from vitamin A, which have been extensively studied in cancer prevention and therapy. In pediatric oncology, they are successfully used for the treatment of acute promyelocytic leukemia (APL) and high-risk neuroblastoma (HR-NBL). APL is a subtype of acute myeloid leukemia (AML) clinically characterized by a severe bleeding tendency with a highrisk of fatal hemorrhage. The molecular hallmark of this disease is the presence of the promyelocytic leukemia (PML)-retinoic acid receptor-α (RAR α) gene fusion that plays a critical role in promyelocytic leukemogenesis and represents the target of retinoid therapy. The introduction in the late 1980s of all-trans retinoic acid (ATRA) into the therapy of APL radically changed the management and the outcome of this disease. Presently, the standard front-line therapeutic approach for pediatric APL includes anthracycline-based chemotherapy and ATRA, leading to a complete remission in almost 90% of the patients. Neuroblastoma (NBL) is an aggressive childhood tumor derived from the peripheral neural crest. More than half of patients have a high-risk disease, with a poor outcome despite intensive multimodal treatment. Although the exact mechanism of action remains unclear, the introduction of 13-cis-retinoic acid (13-cis-RA) in the therapy of NBL has improved the prognosis of this disease. Currently, the standard treatment for HR-NBL consists of myeloablative therapy followed by autologous hematopoietic stem cell transplantation (HSCT) and maintenance with 13-cis-RA for the treatment of minimal residual disease, leading to a 3-year disease-free survival rate (DFS) of about 50%. In this paper the authors provide a review of the peer-reviewed literature on the role of retinoids in the treatment of pediatric APL and HR-NBL, summarizing the most relevant clinical trial results of the last decades, analyzing the ongoing trials, and investigating future therapeutic perspectives of children affected by these diseases.

Topics: Antineoplastic Agents; Child; Disease-Free Survival; Humans; Isotretinoin; Leukemia, Promyelocytic, Acute; Neuroblastoma; Tretinoin

Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; DNA Methylation; Humans; MicroRNAs; Neuroblastoma; Tretinoin

Neuroblastoma arises from precursor cells of the sympathetic nervous system and presently accounts for 15% of all childhood cancer deaths. These tumors display remarkable heterogeneity in clinical behavior, ranging from spontaneous regression to rapid progression and resistance to therapy. The clinical behavior of these tumors is associated with many factors, including patient age, histopathology and genetic abnormalities such as MYCN amplification. More recently, the dysregulation of some miRNAs, including the miR-17-5p-92 cluster and miR-34a, has been implicated in the pathobiology of neuroblastoma. MiR-17-5p-92 family members act in an oncogenic manner while miR-34a has tumor suppressor functions. The evidence for the contribution of miRNAs in the aggressive neuroblastoma phenotype is reviewed in this article, along with exciting possibilities for miRNA mediated therapeutics.

Topics: Apoptosis; Gene Expression Profiling; Gene Expression Regulation; Humans; MicroRNAs; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Tretinoin

Retinoic acid isomers have been used with some success as chemotherapeutic agents, most recently with 13-cis retinoic acid showing impressive clinical efficacy in the paediatric malignancy neuroblastoma. The aim of this commentary is to review the evidence that 13-cis retinoic acid is a pro-drug, and consider the implications of retinoid metabolism and isomerisation for the further development of retinoic acid for cancer therapy. The low binding affinity of 13-cis retinoic acid for retinoic acid receptors, low activity in gene expression assays and the accumulation of the all-trans isomer in cells treated with 13-cis retinoic acid, coupled with the more-favourable pharmacokinetic profile of 13-cis retinoic acid compared to other isomers, suggest that intracellular isomerisation to all-trans retinoic acid is the key process underlying the biological activity of 13-cis retinoic acid. Intracellular metabolism of all-trans retinoic acid by a positive auto-regulatory loop may result in clinical resistance to retinoic acid. Agents that block or reduce the metabolism of all-trans retinoic acid are therefore attractive targets for drug development. Devising strategies to deliver 13-cis retinoic acid to tumour cells and facilitate the intracellular isomerisation of 13-cis retinoic acid, while limiting metabolism of all-trans retinoic acid, may have a major impact on the efficacy of 13-cis retinoic acid in paediatric oncology.

Topics: Animals; Antineoplastic Agents; Child; Humans; Isotretinoin; Molecular Structure; Neuroblastoma; Retinol-Binding Proteins; Stereoisomerism; Tretinoin

Neuroblastoma (NB), an embryonal malignancy, poses a major challenge in pediatric oncology for the treatment of disseminated forms. Here, we report the decrease of Wnt-5a gene expression in high-risk NB (HR-NB) as well as in cultured metastatic neuroblasts. Wnt-5a is a member of the Wnt signaling pathway which is mainly associated with patterning decisions in the embryonic nervous system. Moreover, Wnt-5a has been involved in metastatic melanoma progression and invasive ductal breast cancer via adhesion and migration alterations. As retinoic acid (RA) plays a major role in the neural crest induction and differentiation, we showed that RA reverses the aberrant negative regulation of Wnt-5a in metastatic neuroblasts. While beta-catenin expression remained unchanged, PKC-theta, a protein kinase C isoform, was evidenced to increase and parallel Wnt-5a level. For the first time, the involvement of Wnt-5a through the Wnt/calcium signaling is highlighted in the pathogenesis of a pediatric embryonal malignancy, NB.

Topics: Cell Differentiation; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Protein Kinase C; Proto-Oncogene Proteins; Signal Transduction; Tretinoin; Wnt Proteins; Wnt-5a Protein

Numerous reports have indicated that the biological activity of all-trans retinoyl beta-glucuronide (RAG) is similar to that of all-trans-retinoic acid (RA), but without the toxic side effects of RA. In the present series of studies, we report new findings that support the contention that RAG can function as a nontoxic substitute for RA in a variety of clinic settings. One study on the effects of s.c. injected graded doses of RA and RAG (20-480 micromol/kg BW) into pregnant Sprague-Dawley rats showed that any differences between RAG and RA could be observed only at the highest dose levels of 360 and 420 micromol/kg BW, with RAG being much less toxic than RA. Similarly, daily topical application of RAG (0.16-1.6%) and RA (0.1-0.5%) to shaved swine dorsal skin for six mo resulted in redness and scabbing in RA-treated patches, and to a lesser extent in 1.6% RAG-treated, but not in other RAG-treated patches. Histological scores were significantly higher in the dermis and epidermis of RA-treated pigs than in RAG-treated pigs. Studies to document the pharmacokinetics of chronically administered RAG in mice indicated that, unlike RA, sustained blood levels of parent retinoid (RAG) can be achieved during at least 2 mo of daily administration. Another investigation to study the effects of RAG on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5 showed that s.c. injection of RAG (30 micromol/kg BW) reduced tumor formation when the retinoid was first administered 3 d before tumor injection and continued daily for 30 d thereafter. In established tumors, RAG was shown to inhibit progressive tumor growth, the antitumor effects of RAG being comparable with RA. However, with RAG, as opposed to RA, there were no significant adverse physical side effects. Based on the results of these series of studies along with ample published reports over the last 15 y, we conclude that RAG may be a safe and effective alternative to RA and some other retinoids that are presently being utilized in the clinic.

Topics: Animals; Cell Division; Humans; Injections, Subcutaneous; Neoplasm Transplantation; Neuroblastoma; Tretinoin

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Bone Morphogenetic Proteins; Cell Differentiation; Chromosomes, Human, Pair 1; Combined Modality Therapy; Diploidy; DNA-Binding Proteins; Fenretinide; Gangliosides; Humans; Infant; Loss of Heterozygosity; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Sympathetic Nervous System; Transcription Factors; Tretinoin

The high mobility group A (HMGA) proteins are thought to work as ancillary transcription factors and to regulate the expression of a growing number of genes through direct binding to DNA or via protein-protein interactions. Both HMGA1 and HMGA2 are important regulators of basic biological processes, including cell growth, differentiation and transformation. Their qualitatively or quantitatively altered expression has been described in a number of human tumors. We studied and review here their expression in neuroblastic tumors. HMGA2 is expressed only in a subset of ex vivo neuroblastoma (NB) tumors and in the embryonic adrenal gland, but it is undetectable in the adult adrenal gland, suggesting that its anomalous expression might be associated with NB tumorigenesis and/or tumor progression. In vitro, its expression is easily detectable in retinoic acid (RA)-resistant cell lines. The exogenous expression of HMGA2 is sufficient to convert RA-sensitive SY5Y NB cells into RA-resistant cells, thus suggesting that HMGA2 might be a relevant player in determining NB cell responses to endogenous or therapeutically important growth inhibitory substances. In contrast, HMGA1 expression is readily detectable in all NB cell lines and tumors, but its expression is consistently higher in less differentiated NBs compared with ganglioneuromas and ganglioneuroblastomas. Interestingly, RA increases HMGA1 expression in RA-resistant NB cells but inhibits it in cells undergoing RA-induced growth inhibition and neuronal differentiation. Our studies indicate that HMGA molecules might be biologically and pathologically relevant factors in neuroblastic tumor development and progression.

Topics: Amino Acid Sequence; Blotting, Northern; Cell Differentiation; Cell Line, Tumor; Disease Progression; DNA; Gene Deletion; HMGA1a Protein; HMGA1b Protein; HMGA2 Protein; Humans; Models, Biological; Molecular Sequence Data; Neoplasms; Neuroblastoma; Phenotype; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Transcription, Genetic; Tretinoin

Retinoids are derivatives of vitamin A that include all trans-retinoic acid (ATRA), 13-cis-retinoic acid, (13-cis-RA), and fenretinide (4-HPR). High levels of either ATRA or 13-cis-RA can cause arrest of cell growth and morphological differentiation of human neuroblastoma cell lines, and phase I trials showed that higher and more sustained drug levels were obtained with 13-cis-RA relative to ATRA. A phase III randomized trial showed that high-dose, pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improved event-free survival in high-risk neuroblastoma. The cytotoxic retinoid 4-HPR achieved multi-log cell kills in neuroblastoma cell lines resistant to ATRA and 13-cis-RA, and a pediatric phase I trial has shown it to be well tolerated. Cytotoxicity of 4-HPR is mediated at least in part by increasing tumor cell ceramide levels and combining 4-HPR with ceramide modulators increased anti-tumor activity in pre-clinical models. Thus, further clinical trials of 4-HPR in neuroblastoma, and of 4-HPR in combination with ceramide modulators, are warranted.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Humans; Neuroblastoma; Receptors, Retinoic Acid; Tretinoin

Topics: Antineoplastic Agents; Child; Child, Preschool; Down-Regulation; Genes, myc; Humans; Neoplasm Regression, Spontaneous; Nervous System Neoplasms; Neuroblastoma; Tretinoin

In vitro studies that showed RA could cause growth arrest and differentiation of myelogenous leukemia and neuroblastoma led to clinical trials of retinoids in APL and neuroblastoma that increased survival for both of those diseases. In the case of APL, ATRA has been the drug of choice, and preclinical and clinical data support direct combinations of ATRA with cytotoxic chemotherapy. For neuroblastoma, a phase I study defined a dose of 13-cis-RA, which was tolerable in patients after myeloablative therapy, and a phase III trial that showed postconsolidation therapy with 13-cis-RA improved EFS for patients with high-risk neuroblastoma. Preclinical studies in neuroblastoma indicate that ATRA or 13-cis-RA can antagonize cytotoxic chemotherapy and radiation, so use of 13-cis-RA in neuroblastoma is limited to maintenance after completion of cytotoxic chemotherapy and radiation. A limitation on the antitumor benefit of ATRA in APL is the marked decrease in drug levels that occurs during therapy as a result of induction of drug metabolism, resulting in a shorter drug half-life and decreased plasma levels. Although early studies sought to overcome the pharmacologic limitations of ATRA therapy in APL, the demonstration that ATO is active against APL in RA-refractory patients has led to a focus on studies employing ATO. Use of 13-cis-RA in neuroblastoma has avoided the decreased plasma levels seen with ATRA. It is likely that recurrent disease seen during or after 13-cis-RA therapy in neuroblastoma is due to tumor cell resistance to retinoid-mediated differentiation induction. Studies in neuroblastoma cell lines resistant to 13-cis-RA and ATRA have shown that they can be sensitive, and in some cases collaterally hypersensitive, to the cytotoxic retinoid fenretinide. Fenretinide induces tumor cell cytotoxicity rather than differentiation, acts independently from RA receptors, and in initial phase I trials has been well tolerated. Clinical trials of fenretinide, alone and in combination with ceramide modulators, are in development.

Topics: Antineoplastic Agents; Child; Clinical Trials as Topic; Fenretinide; Humans; Isotretinoin; Leukemia, Myeloid, Acute; Neoplasms; Neuroblastoma; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Retinoids; Tretinoin

Retinoids are derivatives of vitamin A that include all- trans-retinoic acid (ATRA), 13-cis-retinoic acid, (13-cis-RA), and fenretinide (4-HPR). High levels of either ATRA or 13-cis-RA can cause arrest of cell growth and morphologic differentiation of human neuroblastoma cell lines. Phase I trials have shown that higher and more sustained drug levels were obtained with 13-cis-RA relative to ATRA. A phase III randomized trial showed that high-dose pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improves event-free survival in high-risk neuroblastoma. Because 4-HPR achieves multi-log cell kills in neuroblastoma cell lines that are resistant to ATRA and 13-cis-RA, a pediatric phase I trial is in progress to determine the maximum tolerated dose of 4-HPR, with a view toward giving 4-HPR after completion of myeloablative therapy and 13-cis-RA.

Topics: Antineoplastic Agents; Child; Drug Administration Schedule; Fenretinide; Humans; Isotretinoin; Neuroblastoma; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma cell lines are established from high-stage, highly malignant tumours. Despite this and the fact that these tumours are arrested at an early, immature stage, many cell lines have the capacity to undergo neuronal differentiation under proper growth conditions. One such cell line is the noradrenergic SH-SY5Y cell line. These cells can be induced to mature by a variety of modalities, resulting in different mature phenotypes. The use of this cell system as a model to study the stem cell character of neuroblastoma is reviewed and discussed. In particular, we focus on growth factor dependencies in the SH-SY5Y system, and compare that to the normal situation, i.e. growth factor control of sympathetic neuronal and neuroendocrine differentiation during human and rat embryogenesis.

Topics: Cell Differentiation; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplastic Stem Cells; Neuroblastoma; Phenotype; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid has considerable potential for the chemoprevention and chemotherapy of cancer. Neuroblastoma cells differentiate in response to retinoic acid in vitro, an observation that has led to clinical trials using either the 13-cis or all-trans isomers of retinoic acid. We review the effects of retinoic acid on neuroblastoma, and the potential involvement of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis retinoic acid is a ligand for RXRs, and we review recent data on the differential effects of 9-cis and all-trans retinoic acid on neuroblastoma differentiation and proliferation in vitro, and possible mechanisms of action via hetero- and homodimers of RARs and RXRs. Although there is uncertainty whether or not 9-cis retinoic acid produces its biological effects primarily via RXR homodimers, in vitro data suggest that this isomer of retinoic acid or stable analogues may have considerable potential for the treatment of resistant, disseminated neuroblastoma.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Phenotype; Tretinoin; Tumor Cells, Cultured

The pediatric peripheral neuroectodermal tumors which include neuroblastoma, peripheral neuroepithelioma and Ewing's sarcoma may correspond to distinct neural crest cell lineages or tumors arrested at different stages of neural crest development. Besides a brief commentary on the salient clinical features of these tumors, this review examines how cell and molecular biological studies have contributed to a re-classification of these tumors. The differentiation of these tumors is reviewed with a particular emphasis on retinoic acid induced differentiation of neuroblastoma as a model to identify genes important in controlling cell growth, suppression of tumorigenicity and induction of differentiation.

Topics: Adolescent; Cell Differentiation; Child; Child, Preschool; Humans; Infant; Infant, Newborn; Nervous System Neoplasms; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Sarcoma, Ewing; Tretinoin

Neuroblastoma is a childhood solid tumor composed of primitive cells derived from precursors of the autonomic nervous system. This neoplasm has the highest rate of spontaneous regression of all cancer types and has been noted to undergo spontaneous and chemically induced differentiation into elements resembling mature nervous tissue. As such, neuroblastoma has been a prime model system for the study of neuronal differentiation and the process of cancer cell maturation. In this paper we review those agents that have been described to induce the differentiation of neuroblastoma, with an emphasis on the effects and possible mechanisms of action of a group of related compounds, the retinoids. With this model system and the availability of subclones that are both responsive and resistant to chemically induced differentiation, fundamental questions regarding the mechanisms and processes underlying cell maturation have become more amenable to in vitro study.

Topics: Butyrates; Butyric Acid; Carrier Proteins; Cell Differentiation; Cell Line; Cyclic AMP; Humans; In Vitro Techniques; Nerve Growth Factors; Neuroblastoma; Neurons; Oncogenes; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Topics: Cell Transformation, Neoplastic; Gene Expression Regulation; Humans; Neuroblastoma; Peripheral Nervous System Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Transcription, Genetic; Tretinoin

Neuroblastoma, the second commonest solid tumor in children, is a neoplasm of the peripheral autonomic nervous system that usually occurs before children are 6 years old. Therapy of localized tumor (clinical stage I and II) and of a special form of metastatic tumor (clinical stage IV-S) is usually successful, but treatment of widespread regional (clinical stage III) or metastatic (clinical stage IV) neuroblastoma is almost uniformly unsuccessful. Unfortunately, two thirds of children have stage III or IV disease at diagnosis. Several clinical trials are in progress. Preclinical investigations with monoclonal antibodies and retinoic acid may lead to new therapies. Monoclonal antibodies that react relatively selectively with neuroblastoma cells may be useful for diagnosis and therapy. Neuroblastoma cells have cytoplasmic retinoic-acid-binding proteins; treatment of cultured cells with retinoic acid induces morphologic maturation and markedly inhibits proliferation.

Topics: Antibodies, Monoclonal; Cell Division; Cell Line; Child; Humans; Neoplasms, Experimental; Neuroblastoma; Prognosis; Tretinoin

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; 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Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA.. Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples.. Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels.. We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Humans; Isotretinoin; Mice, Inbred BALB C; Microsomes; N-Myc Proto-Oncogene Protein; Neurites; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Receptors, Retinoic Acid; RNA, Messenger; Tandem Mass Spectrometry; Tretinoin

The combination of the antiproliferative and differentiation-inducing effects of retinoids together with the antiproliferative, immunostimulatory, and differentiation-potentiating effects of interferon-alpha (IFN-alpha) were the basis for the development of this combination in pediatric patients with refractory neuroblastoma or Wilms tumor.. A phase 2 trial of all-trans-retinoic acid (ATRA), administered orally at a dose of 90 mg/m(2)/day in three divided doses for 3 consecutive days per week, and IFN-alpha2a, administered subcutaneously daily at a dose of 3 x 10(6) U/m(2)/day for 5 consecutive days per week, in 4 week cycles was performed. A two-stage design was used for each disease stratum.. Seventeen patients (16 evaluable) with neuroblastoma, median age 9 years, and 15 patients (14 evaluable) with Wilms tumor, median age 6 years, were enrolled. Overall, the combination was well tolerated, with headache being the most common toxicity observed. There were no complete or partial responses. The median number of cycles administered was 1 (range 1-9). Four patients with neuroblastoma had stable disease for 12 or more weeks.. The combination of ATRA and IFN-alpha2a was inactive in children with relapsed or refractory neuroblastoma and Wilms tumor. The lack of activity with this combination in children with refractory neuroblastoma is similar to the disappointing phase 2 results of single agent 13-cis-retinoic-acid (13cRA) and does not support further development of ATRA for children with relapsed neuroblastoma.

Topics: Adolescent; Child; Child, Preschool; Drug Therapy, Combination; Humans; Infant; Interferon alpha-2; Interferon-alpha; Neuroblastoma; Recombinant Proteins; Remission Induction; Salvage Therapy; Treatment Outcome; Tretinoin; Wilms Tumor

This review will summarize the rationale for pursuing investigations into the use of retinoids for pediatric patients with cancer, describe the phase I results of all-trans-retinoic acid (ATRA) in children, and discuss the results of a series of preclinical and clinical pharmacokinetic studies of ATRA. The prognosis for children with advanced stage neuroblastoma, the most common extracranial solid tumor of childhood, has remained poor despite significant increases in the intensity of multi-modality therapy. Observations that neuroblastoma has the potential in vivo to differentiate into the more mature neuronal phenotype of a ganglioneuroma or to spontaneously regress, combined with the ability of ATRA to induce differentiation of neuroblastoma cell lines in vitro, suggests that neuroblastoma may be a prime candidate for a retinoid-based approach to differentiation therapy. We previously performed a standard pediatric phase I trial of ATRA and defined the maximum tolerated dose (MTD) in children to be 60 mg/m2/day, significantly lower than the MTD in adult patients. Pharmacokinetic results revealed that the plasma half-life of ATRA was short (45 min) relative to 13-cis-RA (12-24 h), and that plasma drug exposure decreased significantly by day 28 of daily drug administration. Preclinical studies using an i.v. formulation of ATRA in a Rhesus monkey pharmacokinetic model then demonstrated that ATRA is eliminated by a capacity-limited (saturable) process. This elimination process was rapidly induced within the first week of daily i.v. ATRA administration, and suggested that an intermittent schedule of drug administration might allow for down-regulation of the elimination process. These pre-clinical studies formed the basis for investigating whether an intermittent schedule of ATRA administration would allow for repeated periods of relatively higher plasma drug concentrations. Preliminary results of two clinical trials using intermittent schedules of administration suggest that this approach may result in significantly higher plasma drug exposure over time. Plans to study the role of intermittent schedules of ATRA administration in pediatric phase II trials in patients with neuroblastoma are underway.

Topics: Adolescent; Antineoplastic Agents; Child; Child, Preschool; Female; Humans; Male; Neoplasms; Neuroblastoma; Tretinoin; Up-Regulation

Reexpressed PAX3 transcription factor is believed to be responsible for the differentiation defects observed in neuroblastoma. Although the importance of PAX3 in neuronal differentiation is documented how it is involved in the defective differentiation remains unexplored particularly with its isoforms. Here, first we have analyzed PAX3 expression, its functional status, and its correlation with the neuronal marker expression in SH-SY5Y and its parental SK-N-SH cells. We have found that SH-SY5Y cells which expressed more PAX3 showed increased expression of neuronal marker genes (TUBB, MAP2, NEFL, NEUROG2, SYP) and reported PAX3 target genes (MET, TGFA, and NCAM1) than the SK-N-SH cells that had low PAX3 level. Retinoic acid treatment is unable to induce neuronal differentiation in cells (SK-N-SH) with low PAX3 level/activity. Moreover, ectopic expression of PAX3 in SK-N-SH cells neither induces neuronal marker genes nor its target genes. PAX3 isoform expression analysis revealed the expression of PAX3b isoform that contains only paired domain in SK-N-SH cells, whereas in SH-SY5Y cells, we could also observe PAX3c isoform that contains all functional domains. Further, PAX3b depletion in SK-N-SH cells is not induced PAX3 target genes, and the cells remain poorly differentiated. Interestingly, ectopic PAX3 expression in PAX3b-depleted SK-N-SH cells enhanced neuronal outgrowth along with neuronal marker gene induction. Collectively, these results showed that the PAX3b isoform may be responsible for the differentiation defect observed in SK-N-SH cells and restoration of functional PAX3 in the absence of PAX3b can induce neurogenesis in these cells.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Neuroblastoma; PAX3 Transcription Factor; Protein Isoforms; Tretinoin

The aim of the study is to check whether dyngo-4a can inhibit neuroblastoma (NB) proliferation and induce NB cell differentiation Background: Dynamin plays a role in regulating neurotransmission, signaling pathways, nutrient uptake, and pathogen infection, enhancing cell proliferation, tumor invasion, and metastasis. Studies have reported that dyngo-4a, a dynamin inhibitor, can be used to identify potential biomarkers and promising novel therapeutic targets for cancer treatment.. To our knowledge, no published reports are showing that dynamin inhibitors can reduce NB cell proliferation and induce differentiation. In this study, we report that dyngo-4a can inhibit NB proliferation and induce NB cell differentiation.. In this study, mouse neuroblastoma (Neuro-2a) cells were cultured in the presence or absence of dyngo-4a or retinoic acid (RA), or in the presence of both dyngo-4a and RA, or in the presence of sequential administration of dyngo-4a and RA to compare the effects on the inhibition of cell proliferation and effects on neuroblastoma cell differentiation induction. The neural cell markers, Nestin and Tuj 1 (Neuron-specific class III beta-tubulin), were used to demonstrate that the differentiated cells have neuronal cell features. The phosphorylation of Protein Kinase B (AKT), extracellular signalregulated kinases1/2 (ERK1/2), and epidermal growth factor receptor (EGFR) were determined to examine the potential mechanisms of induced differentiation.. At a level comparable to RA, dynamin inhibition with dyngo-4a lowers proliferation and causes differentiation of Neuro-2a mouse NB cells

Topics: Animals; Cell Differentiation; Cell Line, Tumor; ErbB Receptors; MAP Kinase Signaling System; Mice; Neuroblastoma; Proto-Oncogene Proteins c-akt; Tretinoin

While the goal of most anticancer treatments is to kill cancer cells, some therapies halt cancer progression by inducing cancer cell differentiation. For example, retinoic acid induces neuroblastoma cell differentiation in vitro and is used as maintenance therapy for children with high-risk neuroblastoma. A new study by Jiang and colleagues has revealed the mitochondrial uncoupler niclosamide ethanolamine (NEN) induces neuroblastoma cell differentiation in vitro and slows neuroblastoma tumor growth in vivo. Mitochondrial uncoupler molecules alter cell metabolism by forcing cells to "burn" more nutrients, resulting in a switch from anabolic to catabolic metabolism. NEN-induced neuroblastoma cell differentiation was associated with disruption of Warburg metabolism, epigenetic remodeling, and downregulation of key oncogenic drivers of neuroblastoma development, including MYCN. NEN is currently used as an antiparasitic worm treatment and is safe to use in children but has poor pharmacokinetic properties. However, derivatives of NEN and structurally distinct uncouplers that have improved pharmacokinetic properties are in development. Results of this study ignite the idea that mitochondrial uncouplers could be used as differentiating agents and expand the pharmacotherapy toolkit to treat cancer, including neuroblastoma. See related article by Jiang et al., p. 181.

Topics: Antineoplastic Agents; Cell Differentiation; Epigenome; Ethanolamine; Humans; Neuroblastoma; Niclosamide; Tretinoin

Neuroblastoma (NB) is a pediatric malignancy that arises in the peripheral nervous system, and the prognosis in the high-risk group remains dismal, despite the breakthroughs in multidisciplinary treatments. The oral treatment with 13-cis-retinoic acid (RA) after high-dose chemotherapy and stem cell transplant has been proven to reduce the incidence of tumor relapse in children with high-risk neuroblastoma. However, many patients still have tumors relapsed following retinoid therapy, highlighting the need for the identification of resistant factors and the development of more effective treatments. Herein, we sought to investigate the potential oncogenic roles of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family in neuroblastoma and explore the correlation between TRAFs and retinoic acid sensitivity. We discovered that all TRAFs were efficiently expressed in neuroblastoma, but TRAF4, in particular, was found to be strongly expressed. The high expression of TRAF4 was associated with a poor prognosis in human neuroblastoma. The inhibition of TRAF4, rather than other TRAFs, improved retinoic acid sensitivity in two human neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells. Further in vitro studies indicated that TRAF4 suppression induced retinoic acid-induced cell apoptosis in neuroblastoma cells, probably by upregulating the expression of Caspase 9 and AP1 while downregulating Bcl-2, Survivin, and IRF-1. Notably, the improved anti-tumor effects from the combination of TRAF4 knockdown and retinoic acid were confirmed in vivo using the SK-N-AS human neuroblastoma xenograft model. In conclusion, the highly expressed TRAF4 might be implicated in developing resistance to retinoic acid treatment in neuroblastoma, and the combination therapy with retinoic acid and TRAF4 inhibition may offer significant therapeutic advantages in the treatment of relapsed neuroblastoma.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Neoplasm Recurrence, Local; Neuroblastoma; TNF Receptor-Associated Factor 4; Tretinoin

Neuroblastoma arises when immature neural precursor cells do not mature into specialized cells. Although retinoic acid (RA), a pro-differentiation agent, improves the survival of low-grade neuroblastoma, resistance to retinoic acid is found in high-grade neuroblastoma patients. Histone deacetylases (HDAC) inhibitors induce differentiation and arrest the growth of cancer cells; however, HDAC inhibitors are FDA-approved mostly for liquid tumors. Therefore, combining histone deacetylase (HDAC) inhibitors and retinoic acid can be explored as a strategy to trigger the differentiation of neuroblastoma cells and to overcome resistance to retinoic acid. Based on this rationale, in this study, we linked evernyl group and menadione-triazole motifs to synthesize evernyl-based menadione-triazole hybrids and asked if the hybrids cooperate with retinoic acid to trigger the differentiation of neuroblastoma cells. To answer this question, we treated neuroblastoma cells using evernyl-based menadione-triazole hybrids (6a-6i) or RA or both and examined the differentiation of neuroblastoma cells. Among the hybrids, we found that compound 6b inhibits class-I HDAC activity, induces differentiation, and RA co-treatments increase 6b-induced differentiation of neuroblastoma cells. In addition, 6b reduces cell proliferation, induces expression of differentiation-specific microRNAs leading to N-Myc downregulation, and RA co-treatments enhance the 6b-induced effects. We observed that 6b and RA trigger a switch from glycolysis to oxidative phosphorylation, maintain mitochondrial polarization, and increase oxygen consumption rate. We conclude that in evernyl-based menadione-triazole hybrid, 6b cooperates with RA to induce differentiation of neuroblastoma cells. Based on our results, we suggest that combining RA and 6b can be pursued as therapy for neuroblastoma. Schematic representation of RA and 6b in inducing differentiation of neuroblastoma cells.

Topics: Cell Differentiation; Cell Line, Tumor; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Neural Stem Cells; Neuroblastoma; Tretinoin; Vitamin K 3

Histone deacetylase (HDAC) inhibitors affect cell homeostasis, gene expression, and cell cycle progression and promote cell terminal differentiation or apoptosis. However, the effect of HDAC inhibition on SH-SY5Y cells, which are neuroblastoma cells capable of differentiating into neurons under specific conditions, such as in the presence of retinoic acid (RA), is unknown. In this study, we hypothesized that HDAC inhibitors induced the neuronal differentiation of SH-SY5Y cells. To test this hypothesis, we used phase contrast microscopy, immunocytochemistry (ICC), qPCR, and western blotting analysis. MS-275 and valproic acid (VPA), two HDAC inhibitors, were selected to evaluate neuronal differentiation. It was confirmed that cells treated with MS-275 or VPA differentiated into mature neurons, which were distinguished by bipolar or multipolar morphologies with elongated branches. In addition, the mRNA expression of neuronal markers (Tuj1 and NEFH) and the oligodendrocyte marker (CNP) was significantly increased with MS-275 or VPA treatment compared to that with RA treatment. In addition, the protein expression of the other neuronal markers, Tuj1 and NeuN, was highly increased with HDAC inhibitor treatments compared to that with RA treatment. Furthermore, we confirmed that noncanonical Wnt signaling was upregulated by HDAC inhibitors via MAPK signaling and the Wnt/JNK pathway. Therefore, both MS-275 and VPA promoted the differentiation of SH-SY5Y cells into mature neurons via the Wnt signaling pathway.

Topics: Cell Differentiation; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Neuroblastoma; Neurons; Tretinoin; Valproic Acid; Wnt Signaling Pathway

Parkinson's disease (PD), the second-most prevalent neurodegenerative disorder, is characterized by the aberrant deposition of α-synuclein (α-Syn) aggregation in neurons. Recent reports have shown that retinoic acid (RA) ameliorates motor deficits. However, the underlying molecular mechanisms remain unclear. In this article, we investigated the effects of RA on cellular and animal models of PD. We found that RA is beneficial for neuronal survival in PD-associated models. In α-Syn preformed fibrils-treated mice, RA administration relieved the formation of intracellular inclusions, dopaminergic neuronal loss, and behavioral deficits. α-Syn preformed fibrils-treated SH-SY5Y cells manifested decreased cell viability, apoptosis, α-Syn aggregation, and autophagy defects. All these negative phenomena were alleviated by RA. More importantly, RA could inhibit the neurotoxicity via inhibiting α-Syn preformed fibrils-induced STAT1-PARP1 signaling, which could also be antagonized by IFN-γ. In conclusion, RA could hinder α-Syn preformed fibrils-induced toxicity by inhibiting STAT1-PARP1 signaling. Thus, we present new insight into RA in PD management.

Topics: alpha-Synuclein; Animals; Humans; Mice; Neuroblastoma; Neurons; Parkinson Disease; Poly (ADP-Ribose) Polymerase-1; STAT1 Transcription Factor; Tretinoin

Retinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photolability where any findings from prolonged time course observations using microscopy may alter outcomes. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening of differentiation events using Electrical cell-substrate impedance sensing (ECIS).. A differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9 day period in order to be more representative of differentiation over a prolonged timeline. Specific features associated with differentiation (growth inhibition, neurite outgrowths) were confirmed by end-point analysis. RA-induced growth inhibition and assumed phenotypic changes (i.e. neurite outgrowth) were identified by the xCELLigence analysis and further confirmed by end-point metabolic and phenotypic assays. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. Based on this it was possible to identify SH-SY5Y phenotypic differentiation with distinct phases observed over 9 days using Electric cell-substrate impedance sensing (ECIS) cell index traces providing a path to application in larger scale neurotrophic factor screening using this scalable technology.

Topics: Cell Differentiation; Cell Line, Tumor; Electric Impedance; Humans; Neuroblastoma; Neurogenesis; Tretinoin; Workflow

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Lipids; Lipoylation; Neuroblastoma; Proteins; Tretinoin

High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation.. Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins.. Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability.. Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.

Topics: Agar; Animals; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Histone Demethylases; Humans; Mice; Mice, Knockout; Mice, Nude; Mice, Transgenic; Neuroblastoma; Tretinoin

Neuroblastoma is the most common extracranial solid tumour in childhood, originated from cells of the neural crest during the development of the Sympathetic Nervous System. Retinoids are vitamin-A derived differentiating agents utilised to avoid disease resurgence in high-risk neuroblastoma treatment. Several studies indicate that hypoxia-a common feature of the tumoural environment-is a key player in cell differentiation and proliferation. Hypoxia leads to the accumulation of the hypoxia-inducible factor-1α (HIF-1α). This work aims to investigate the effects of the selective inhibition of HIF-1α on the differentiation induced by retinoic acid in human neuroblastoma cells from the SH-SY5Y lineage to clarify its role in cell differentiation. Our results indicate that HIF-1α inhibition impairs RA-induced differentiation by reducing neuron-like phenotype and diminished immunolabeling and expression of differentiation markers. HIF1A is involved in Retinoic Acid (RA) induced differentiation in SH-SY5Y neuroblastoma cells. siRNA HIF1A gene silencing leads to a weaker response to RA, demonstrated by changes in the neuro-like phenotype and diminished expression of differentiation markers.

Topics: Cell Differentiation; Cell Line, Tumor; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neurites; Neuroblastoma; Tretinoin

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

Topics: Cell Differentiation; Cell Line, Tumor; Chromatography, Liquid; Energy Metabolism; Humans; Neuroblastoma; Tandem Mass Spectrometry; Tretinoin

Luteolin is one of the most common flavonoids present in edible plants and its potential benefits to the central nervous system include decrease of microglia activation, neuronal damage and high antioxidant properties. The aim of this research was to evaluate the neuroprotective, antioxidant and anti-inflammatory activities of luteolin-7-O-glucoside (Lut7). Undifferentiated and retinoic acid (RA)-differentiated SH-SY5Y cells were pretreated with Lut7 and incubated with 6-hydroxydopamine (6-OHDA). Cytotoxic and neuroprotective effects were determined by MTT assay. Antioxidant capacity was determined by DPPH, FRAP, and ORAC assays. ROS production, mitochondrial membrane potential (ΔΨm), Caspase-3 activity, acetylcholinesterase inhibition (AChEI) and nuclear damage were also determined in SH-SY5Y cells. TNF-α, IL-6 and IL-10 release were evaluated in LPS-induced RAW264.7 cells by ELISA. In undifferentiated SH-SY5Y cells, Lut7 increased cell viability after 24 h, while in RA-differentiated SH-SY5Y cells, Lut7 increased cell viability after 24 and 48 h. Lut7 showed a high antioxidant activity when compared with synthetic antioxidants. In undifferentiated cells, Lut7 prevented mitochondrial membrane depolarization induced by 6-OHDA treatment, decreased Caspase-3 and AChE activity, and inhibited nuclear condensation and fragmentation. In LPS-stimulated RAW264.7 cells, Lut7 treatment reduced TNF-α levels and increased IL-10 levels after 3 and 24 h, respectively. In summary, the results suggest that Lut7 has neuroprotective effects, thus, further studies should be considered to validate its pharmacological potential in more complex models, aiming the treatment of neurodegenerative diseases.

Topics: Acetylcholinesterase; Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Flavones; Glucosides; Humans; Interleukin-10; Lipopolysaccharides; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Oxidopamine; Tretinoin; Tumor Necrosis Factor-alpha

Neuroblastoma (NB) is one of the most common extracranial tumors with limited therapeutic options. Retinoic acid (RA) has been identified to play anticancer role against NB cells by inducing the differentiation and apoptosis of immature neuroblasts. However, silencing HoxC9 promoter by EZH2-induced H3K27me3 hypermethylation can lead to RA resistance. Previous studies have suggested that arsenic trioxide (ATO), an inhibitor of DNA methylation, could downregulate the expression of EZH2 in breast cancer cells.. In our study, we attempted to obtain some insight into the mechanisms of differentiation of RA-resistant NB cells by detecting the expressions of HoxC9 and EZH2 in NB cells treated with ATO, so as to provide a basis for the subsequent treatment of RA-resistant NB by ATO.. Two NB cell lines, SK-N-AS (retinoic acid-resistant neuroblastoma cells) and SK-N-SH (retinoic acid-sensitive neuroblastoma cells), were used in our experiments. Cell proliferation and apoptosis were respectively determined with Cell Counting Kit-8 (CCK-8) assay kit and Annexin V staining. The inverted phase contrast microscope was used to observe cell growth and measure the total length of nerve synapses. We employed label-free quantitative proteomic analysis to profile ATO-dependent changes in the proteome of NB cells. Western blot was used to detect the expressions of HoxC9, HoxD8 and EZH2.. Arsenic trioxide inhibited the cell proliferation and increased apoptosis and total length of synapses in two NB cell lines. The expressions of HoxC9 and HoxD8 were upregulated, while the expression of EZH2 was downregulated in the SK-N-AS cell line. No significant changes in the 3 proteins mentioned above were observed in the SK-N-SH cell line after ATO treatment.. Arsenic trioxide may reactivate the expression of HoxC9 by downregulating EZH2, which leads to restoring RA sensitivity and promoting the differentiation and apoptosis of RA-resistant NB cells.

Topics: Apoptosis; Arsenic Trioxide; Cell Differentiation; Cell Line, Tumor; Homeodomain Proteins; Humans; Neuroblastoma; Proteomics; Tretinoin; Up-Regulation

Neuroblastoma is a deadly childhood cancer, and MYCN-amplified neuroblastoma (MNA-NB) patients have the worst prognoses and are therapy-resistant. While retinoic acid (RA) is beneficial for some neuroblastoma patients, the cause of RA resistance is unknown. Thus, there remains a need for new therapies to treat neuroblastoma. Here we explored the possibility of combining a MYCN-specific antigene oligonucleotide BGA002 and RA as therapeutic approach to restore sensitivity to RA in NB.. By molecular and cellular biology techniques, we assessed the combined effect of the two compounds in NB cell lines and in a xenograft mouse model MNA-NB.. We found that MYCN-specific inhibition by BGA002 in combination with RA (BGA002-RA) act synergistically and overcame resistance in NB cell lines. BGA002-RA also reactivated neuron differentiation (or led to apoptosis) and inhibited invasiveness capacity in MNA-NB. Moreover, we found that neuroblastoma had the highest level of mRNA expression of mTOR pathway genes, and that BGA002 led to mTOR pathway inhibition followed by autophagy reactivation in MNA-NB cells, which was strengthened by BGA002-RA. BGA002-RA in vivo treatment also eliminated tumor vascularization in a MNA-NB mouse model and significantly increased survival.. Taken together, MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, a cancer-specific mTOR pathway inhibition occurs only in MNA-NB, thus avoiding the side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a strategy for overcoming RA resistance in MNA-NB.

Topics: Animals; Apoptosis; Child; Humans; Mice; N-Myc Proto-Oncogene Protein; Neuroblastoma; TOR Serine-Threonine Kinases; Tretinoin

The translation elongation factor-1, alpha-2 (eEF1A2) plays an important role in protein synthesis. Mutations in this gene have been described in individuals with neurodevelopmental disorders. Here, we silenced the expression of eEFA2 in human SH-SY5Y neuroblastoma cells and observed its roles in neuronal proliferation and differentiation upon induction with retinoic acid.. eEF1A2 were silenced using siRNA transfection. Cell proliferation was qualitatively evaluated by Ki-67 immunocytochemistry. Neuronal differentiation was induced with retinoic acid for 3, 5, 7 and 10 days. Neurite length was measured. The expression of microtubule-associated protein 2 (MAP2) was analyzed by western blotting. Tyrosine hydroxylase expression was visualized by immunofluorescence. Cytotoxicity to a neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and western blotting of cleaved caspase-3.. eEF1A2 knockdown suppressed the proliferative activity of undifferentiated SH-SY5Y cells as shown by decreased Ki-67 immunostaining. Upon retinoic acid-induction, differentiated neurons with eEF1A2 knockdown exhibited shorter neurite length than untransfected cells, which was associated with the reduction of tyrosine hydroxylase and suppression of MAP2 at 10 days of differentiation. eEF1A2 knockdown decreased the survival of neurons, which was clearly observed in undifferentiated and short-term differentiated cells. Upon treatment with MPP+, cells with eEF1A2 knockdown showed a further reduction in cell survival and an increase of cleaved caspase-3 protein.. Our results suggest that eEF1A2 may be required for neuronal proliferation and differentiation of SH-SY5Y cells. Increased cell death susceptibility against MPP+ in eEF1A2-knockdown neurons may imply the neuroprotective role of eEF1A2.

Topics: 1-Methyl-4-phenylpyridinium; Caspase 3; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Ki-67 Antigen; Neuroblastoma; Neuronal Outgrowth; Neurons; Peptide Elongation Factor 1; Tretinoin; Tyrosine 3-Monooxygenase

The neuroblastoma cell line SH-SY5Y is commonly employed to study neuronal function and disease. This includes cells grown under standard conditions or differentiated to neuron-like cells by administration of chemical reagents such as retinoic acid (RA) or phorbol-12-myristate-13-acetate (PMA). Even though SH-SY5Y cells are widely explored, a complete description of the resulting proteomes and cellular reorganisation during differentiation is still missing. Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Relative quantification and KEGG pathway analysis of the proteins reveals the presence of early differentiating cells and provides a list of marker proteins for undifferentiated and differentiated cells. For characterisation of neuronal sub-types, we analyse expression of marker genes and find that RA-differentiated cells are acetylcholinergic and cholinergic, while RA/PMA-differentiated cells show high expression of acetylcholinergic and dopaminergic marker genes. In-cell cross-linking further allows capturing protein interactions in different cellular organelles. Specifically, we observe structural reorganisation upon differentiation involving regulating protein factors of the actin cytoskeleton.

Topics: Biomarkers; Cell Differentiation; Cell Line, Tumor; Humans; Neuroblastoma; Proteome; Proteomics; Tretinoin

Besides being anti-diabetic drug, metformin also has anti-proliferation and growth in several tumors; however, details of possible mechanism have not been elucidated. Here, we investigated the effects of metformin in neuroblastoma which has been termed as extra-cranial solid tumor that is due to a differentiation block with more stemness. The results showed that 5 mM metformin inhibited cell cycle progression at G0/G1 phase. Metformin also induced morphological differentiation of neuroblastoma into neuron-like phenotypes by which upregulation of MAP2, β-tubulin III and tyrosine hydroxylase expressions with no significant difference to retinoic acid (RA)-treated cells. We also tested proliferative, growth and self-renewal ability after neuroblastoma being differentiated by metformin for 24 h. The proliferative rate, sizes and numbers of colonies and spheroids were significantly reduced in differentiated neuroblastoma compared to undifferentiated neuroblastoma. A significant increase of ROS and ADP/ATP ratio with decreased mitochondrial membrane potential (MMP) were observed in metformin-treated cells, indicating mitochondrial biogenesis and metabolic change during metformin-mediated differentiation. The further studies exhibited that p-Erk1/2 and Cdk5 levels were reduced in metformin treatment whereas using PD98095 and roscovitine, selectively inhibited Erk1/2 and Cdk5, respectively, significantly increased neurite length and MAP2 expression. In addition, cell proliferation was decreased by cell cycle arrested at G0/G1 phase. Taken together, this study suggests the inhibitory effects of metformin against proliferation and growth of neuroblastoma due to induced morphological differentiation may be through Erk1/2 and Cdk5 pathways. Therefore, metformin might be eventually considered as a differentiation agent for neuroblastoma treatment in term of differentiation therapy.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Metformin; Neuroblastoma; Tretinoin

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state.

Topics: Adrenergic Agents; Cell Differentiation; Cell Line, Tumor; DNA Topoisomerases, Type II; Humans; Neuroblastoma; Phenotype; Poly-ADP-Ribose Binding Proteins; Tretinoin

Abuse of pyrrolidinophenone derivatives (PPs) is known to cause severe damage to the central nervous system due to their high lipophilicity. In this study, we compared sensitivity to toxicity elicited by 4'-iodo-α-pyrrolidinononanophenone (I-α-PNP), one of the most potent cytotoxic derivatives among PPs synthesized previously, between SH-SY5Y cells differentiated by all-trans-retinoic acid (ATRA) and the undifferentiated cells, and found that the differentiated cells are more sensitive to I-α-PNP toxicity than the undifferentiated cells. Treatment with I-α-PNP elicited some apoptotic alterations (Bax expression, loss of mitrochondrial membrane potential, and activation of caspases) in the differentiated cells, whose patterns were similar to those in the undifferentiated cells. I-α-PNP treatment resulted in no significant alteration in Bcl-2 expression in the undifferentiated cells, whereas it considerably downregulated the protein expression in the differentiated cells, suggesting that the high I-α-PNP sensitivity of the differentiated cells is mainly due to downregulation of Bcl-2 expression. I-α-PNP treatment decreased nitric oxide (NO) production and neuronal NOS (nNOS) expression in the differentiated cells, and the patterns of I-α-PNP-evoked alterations in phosphorylation of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression were almost the same as that in nNOS expression. Additionally, the addition of an NO donor restored the I-α-PNP-evoked alterations in expressions of Bcl-2, BDNF, and nNOS in the differentiated cells. These findings suggest that the downregulation of Bcl-2 expression by I-α-PNP in differentiated cells is attributed to the acceleration of two negative feedback loops (nNOS/NO/CREB loop and CREB/BDNF loop) triggered by decreased NO production.

Topics: Apoptosis; bcl-2-Associated X Protein; Brain-Derived Neurotrophic Factor; Caspases; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Humans; Ketones; Neuroblastoma; Nitric Oxide; Pyrrolidines; Tretinoin

Nicotinamide adenine dinucleotide (NAD) metabolism is involved in redox and non-redox reactions that regulate several processes including differentiation of cells of different origins. Here, the role of NAD metabolism in neuronal differentiation, which remains elusive so far, was investigated.. A protein-protein interaction network between neurotrophin signaling and NAD metabolic pathways was built. Expression of NAD biosynthetic enzymes in SH-SY5Y cells during retinoic acid (RA)/brain derived neurotrophic factor (BDNF) differentiation, was evaluated. The effects of NAD biosynthetic enzymes QPRT and NAPRT inhibition in neurite outgrowth, cell viability, NAD availability and histone deacetylase (HDAC) activity, were analyzed in RA- and BDNF-differentiated cells.. Bioinformatics analysis revealed the interaction between NAD biosynthetic enzyme NMNAT1 and NTRK2, a receptor activated by RA/BDNF sequential treatment. Differences were found in the expression of NAD biosynthetic enzymes during neuronal differentiation, namely, increased QPRT gene expression along the course of RA/BDNF treatment and NAPRT protein expression after a 5-day treatment with RA. QPRT inhibition in BDNF-differentiated SH-SY5Y cells resulted in less neuritic length per cell, decreased expression of the neuronal marker β-III Tubulin and also decreased NAD. Taken together, our results show the involvement of NAD metabolism in neuronal differentiation, specifically, the importance of QPRT-mediated NAD biosynthesis in BDNF-associated SH-SY5Y differentiation and suggest additional roles for NAPRT beyond NAD production in RA-differentiated cells.

Topics: Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Humans; NAD; Neuroblastoma; Nicotinamide-Nucleotide Adenylyltransferase; Tretinoin; Tubulin

Neuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR).. Cell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo.. Here, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome.. Together, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

Topics: Cell Differentiation; Cell Line, Tumor; Child; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Prognosis; Receptors, Cytoplasmic and Nuclear; Tretinoin

Neuronal growth and synaptic function are dependent on precise protein production and turnover at the synapse. AMPK-activated protein kinase (AMPK) represents a metabolic node involved in energy sensing and in regulating synaptic protein homeostasis. However, there is ambiguity surrounding the role of AMPK in regulating neuronal growth and health. This study examined the effect of chronic AMPK activation on markers of synaptic function and growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 nM) for 1, 3, or 5 days before AMPK, mTORC1, and markers for synapse function were examined. Cell morphology, neuronal marker content, and location were quantified after 5 days of treatment. AMPK phosphorylation was maintained throughout all 5 days of treatment with A-769662 and resulted in chronic mTORC1 inhibition. Lower total, soma, and neuritic neuronal marker contents were observed following 5 d of AMPK activation. Neurite protein abundance and distribution was lower following 5 days of A-769662 treatment. Our data suggest that chronic AMPK activation impacts synaptic protein content and reduces neurite protein abundance and distribution. These results highlight a distinct role that metabolism plays on markers of synapse health and function.

Topics: AMP-Activated Protein Kinases; Humans; Mechanistic Target of Rapamycin Complex 1; Neuroblastoma; Neurons; Tretinoin

Epilepsy is a chronic brain disease with recurrent seizures. Autophagy plays a crucial role in the progression of epilepsy. This study aimed to explore the function and intrinsic mechanism of the long non-coding RNA (lncRNA) UCA1/miR-132-3p/ATG16L1 axis in epilepsy via regulation of autophagy.. The expression of lncRNA UCA1, miR-132-3p and ATG16L1 was measured in serum from epileptic patients by quantitative RT-PCR. A SH-SY5Y cell model was further constructed using retinoic acid to investigate the UCA1/ miR-132-3p/ATG16L1 axis by quantitative RT-PCR, western blotting, fluorescence in situ hybridisation, RNA immunoprecipitation, chromatin immunoprecipitation, and a dual-luciferase reporter gene assay.. In the serum of epileptic patients, the level of lncRNA UCA1 and ATG16L1 was reduced and miR-132-3p elevated, compared to controls. Similarly, in the SH-SY5Y cell model, the level of lncRNA UCA1 and ATG16L1 was reduced and miR-132-3p elevated in retinoic acid-treated cells; lncRNA UCA1 was mainly located in the cytoplasm. lncRNA UCA1 overexpression was shown to promote autophagic gene expression, which was reversed by miR-132-3p overexpression. Moreover, autophagic gene expression induced by miR-132-3p knockdown was reversed by ATG16L1 knockdown. Based on precipitation assays, lncRNA UCA1 and miR-132-3p were shown to form a complex with the transcription factor, EZH2, and miR-132-3p was shown to interact with ATG16L1 based on a luciferase assay. Finally, lncRNA UCA1 was shown to negatively regulate miR-132-3p expression, and miR-132-3p was shown to negatively regulate ATG16L1.. In this cell model, lncRNA UCA1 promotes autophagic gene expression via epigenetic regulation mediated by ATG16L1 and miR-132-3p.

Topics: Autophagy; Autophagy-Related Proteins; Epigenesis, Genetic; Gene Expression; Humans; MicroRNAs; Neuroblastoma; RNA, Long Noncoding; Transcription Factors; Tretinoin

Among the various methods, Non Thermal Plasma (NTP) has been recently introduced and is being studied to recover the damaged nerve. In the recent years, several studies have suggested that NTP accelerates nerve cell regeneration, but the mechanism remains unknown. This study evaluated the effect of NTP on neuronal proliferation in SH-SY5Y (Human neuroblastoma cells) cells differentiated by retinoic acid (RA) and investigated the mechanism by which NTP promotes cell proliferation. We analyzed the morphology of differentiated SH-SY5Y cells, and performed western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence analysis was performed in an in vivo study by categorizing Wistar A rats into three groups: non-nerve damage (Non-ND), nerve damage (ND), and nerve damage + NTP treatment (ND + NTP). The cell morphology analysis revealed that the number of cells increased and axonal elongation progressed after NTP treatment. In addition, western blots indicated that tau expression increased significantly after NTP treatment. The RT-PCR results revealed that the expression of tau, wnt3a, and β-catenin increased after NTP treatment. The in vivo immunofluorescence assay showed that NTP increased the markers for tau and S100B while regulating the over-expression of MAP2 and GAP43. NTP treatment accelerated cell proliferation and regeneration of damaged neurons in differentiated SH-SY5Y cells. These results establish the fact of NTP as a noninvasive and effective treatment for nerve injury.

Topics: Animals; Axons; beta Catenin; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Neuroblastoma; Neurons; Plasma Gases; Rats; Rats, Wistar; Tretinoin

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins βIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

Topics: Acetylcholinesterase; Cell Differentiation; Cells, Cultured; Cholinergic Agents; Dental Pulp; Homeodomain Proteins; Humans; NAV1.7 Voltage-Gated Sodium Channel; Neuroblastoma; Stem Cells; Transcription Factors; Tretinoin

Neuroblastoma (NB), the most common cancer in the first year of life, presents almost exclusively in the trunk. To understand why an early-onset cancer would have such a specific localization, we xenotransplanted human NB cells into discrete neural crest (NC) streams in zebrafish embryos. Here, we demonstrate that human NB cells remain in an undifferentiated, tumorigenic state when comigrating posteriorly with NC cells but, upon comigration into the head, differentiate into neurons and exhibit decreased survival. Furthermore, we demonstrate that this in vivo differentiation requires retinoic acid and brain-derived neurotrophic factor signaling from the microenvironment, as well as cell-autonomous intersectin-1-dependent phosphoinositide 3-kinase-mediated signaling, likely via Akt kinase activation. Our findings suggest a microenvironment-driven explanation for NB's trunk-biased localization and highlight the potential for induced differentiation to promote NB resolution in vivo.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cell Movement; Female; Humans; Male; Mice; Neural Crest; Neuroblastoma; Neurons; Signal Transduction; Transplantation, Heterologous; Tretinoin; Tumor Microenvironment; Zebrafish

The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2'-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination.. Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment.. Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes.. Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neuroblastoma; Retinoids; Signal Transduction; Tretinoin

Neuroblastoma is the second most common pediatric cancer involving the peripheral nervous system in which stage IVS metastatic tumors regress due to spontaneous differentiation. 13-cis retinoic acid (13-cis RA) is currently used in the clinic for its differentiation effects and although it improves outcomes, relapse is seen in half of high-risk patients. Combinatorial therapies have been shown to be more effective in oncotherapy and since cathepsin inhibition reduces tumor growth, we explored the potential of coupling 13-cis RA with a cathepsin inhibitor (K777) to enhance therapeutic efficacy against neuroblastoma. Shotgun proteomics was used to identify proteins affected by K777 and dual (13-cis RA/K777) treatment in neuroblastoma SK-N-SH cells. Cathepsin inhibition was more effective in increasing proteins involved in neuronal differentiation and neurite outgrowth than 13-cis RA alone, but the combination of both treatments enhanced the neuronal differentiation effect. SIGNIFICANCE: As neuroblastoma can spontaneously differentiate, determining which proteins are involved in differentiation can guide development of more accurate diagnostic markers and more effective treatments. In this study, we established a differentiation proteomic map of SK-N-SH cells treated with a cathepsin inhibitor (K777) and K777/13-cis RA (dual). Bioinformatic analysis revealed these treatments enhanced neuronal differentiation and axonogenesis pathways. The most affected proteins in these pathways may become valuable biomarkers of efficacy of drugs designed to enhance differentiation of neuroblastoma [1].

Topics: Cathepsins; Cell Differentiation; Child; Humans; Isotretinoin; Neuroblastoma; Proteomics; Tretinoin

Since most models used to study neuronal dysfunction display disadvantages and ethical concerns, a fast and reproducible in vitro model to study mitochondria-related neurodegeneration is required. Here, we optimized and characterized a 3-day retinoic acid-based protocol to differentiate the SH-SY5Y cell line into a neuronal-like phenotype and investigated alterations in mitochondrial physiology and distribution. Differentiation was associated with p21-linked cell cycle arrest and an increase in cell mass and area, possibly associated with the development of neurite-like extensions. Notably, increased expression of mature neuronal markers (neuronal-specific nuclear protein, microtubule-associated protein 2, βIII tubulin and enolase 2) was observed in differentiated cells. Moreover, increased mitochondrial content and maximal area per cell suggests mitochondrial remodeling. To demonstrate that this model is appropriate to study mitochondrial dysfunction, cells were treated for 6 h with mitochondrial toxicants (rotenone, antimycin A, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 6-hydroxydopamine (6-OHDA)). Differentiated cells were more susceptible to increasing concentrations of FCCP, antimycin A, and rotenone, while 6-OHDA showed a distinct dose-dependent neurotoxicity pattern. Even though differentiated cells did not exhibit a fully mature/differentiated neuronal phenotype, the protocol developed can be used to study neurotoxicity processes, mitochondrial dynamics, and bioenergetic impairment, representing an alternative to study mitochondrial impairment-related pathologies in vitro.

Topics: Cell Differentiation; Cell Line, Tumor; Colorimetry; Humans; Microscopy; Mitochondria; Neuroblastoma; Neurons; Neurotoxicity Syndromes; Rhodamines; Tretinoin

The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation.. Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed.. The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells.. The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function.

Topics: Binding Sites; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Nerve Tissue Proteins; Neuroblastoma; Promoter Regions, Genetic; Transcription Factors; Transcription Initiation Site; Tretinoin; Vasoactive Intestinal Peptide

Neuroblastoma (NB) is the most common malignant extra cranial solid tumors in children. It has been well established that retinoic acid (RA) inhibits proliferation of neuroblastoma (NB) by blocking cells at G1 phase of the cell cycle. Clinically, RA has been successfully used to treat NB patients. However, the precise mechanism underlying the potent action of RA-treated NB is not fully explored. In this work, we carried out a gene expression profiling by RNA sequencing on all-trans retinoic acid (ATRA)-treated NB cells. Cancer-related pathway enrichment and subsequent protein-protein interaction (PPI) network analysis identified fibronectin 1 (FN1) as one of the central molecules in the network, which was significantly upregulated during ATRA treatment. In addition, we found that although downregulation of FN1 had no significant effects on either cell proliferation or cell cycle distributions in the presence or absence of ATRA, it increased cell migration and invasion in NB cells and partially blocked ATRA-induced inhibition of cell migration and invasion in SY5Y NB cells. Consistent with this finding, FN1 expression levels in NB patients positively correlate with their overall survivals. Taken together, our data suggest that FN1 is a potential target for effective ATRA treatment on NB patients, likely by facilitating ATRA-induced inhibition of cell migration and invasion.

Topics: Cell Line, Tumor; Cell Movement; Fibronectins; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Neuroblastoma; Tretinoin

The use of primary mammalian neurons derived from embryonic central nervous system tissue is limited by the fact that once terminally differentiated into mature neurons, the cells can no longer be propagated. Transformed neuronal-like cell lines can be used in vitro to overcome this limitation. However, several caveats exist when utilizing cells derived from malignant tumors. In this context, the popular SH-SY5Y neuroblastoma cell line and its use in in vitro systems is described. Originally derived from a metastatic bone tumor biopsy, SH-SY5Y (ATCC

Topics: Biomarkers; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cryopreservation; Humans; Neurobiology; Neuroblastoma; Neurogenesis; Neurons; Phenotype; Tretinoin

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Topics: ADP Ribose Transferases; Adrenal Gland Neoplasms; Animals; Botulinum Toxins; Cell Culture Techniques; Humans; Neuroblastoma; Neurogenesis; Neuronal Outgrowth; Neurons; PC12 Cells; Phenotype; Pheochromocytoma; Rats; Teratocarcinoma; Transfection; Tretinoin

Neuroblastoma cell line SH-SY5Y, due to its capacity to differentiate into neurons, easy handling, and low cost, is a common experimental model to study molecular events leading to Alzheimer's disease (AD). However, it is prevalently used in its undifferentiated state, which does not resemble neurons affected by the disease. Here, we show that the expression and localization of amyloid-β protein precursor (AβPP), one of the key molecules involved in AD pathogenesis, is dramatically altered in SH-SY5Y cells fully differentiated by combined treatment with retinoic acid and BDNF. We show that insufficient differentiation of SH-SY5Y cells results in AβPP mislocalization.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Humans; Intravital Microscopy; Models, Biological; Neuroblastoma; Neurons; Oxidative Stress; Proteolysis; Tretinoin

High-risk neuroblastoma remains the most difficult pediatric solid tumor to treat and is associated with chemotherapy and radiation resistance that may be secondary to epigenetic modifications. We have previously found that α-N-catenin, a cell-adhesion protein encoded by the gene CTNNA2, plays a tumor suppressor role in neuroblastoma by inhibiting the NF-κB signaling pathway. A subset of neuroblastoma tumors that lack α-N-catenin are resistant to all-trans retinoic acid. However, the mechanism of CTNNA2 silencing in neuroblastoma remains unknown. Herein, we sought to determine the mechanism of α-N-catenin silencing in neuroblastoma.. Two human neuroblastoma cell lines, SK-N-AS and BE(2)-C, were stably transfected with a plasmid expressing CTNNA2. Both cell lines were treated with the histone deacetylase inhibitor Trichostatin A alone and in combination with retinoic acid. Cell survival and colony formation were measured. Cellular differentiation and expression of cell survival signaling pathways were analyzed. Immunoblotting and reverse transcription quantitative polymerase chain reaction were used to examine protein and messenger RNA expression.. Retinoic acid treatment induced cellular differentiation and inhibited cellular proliferation in BE(2)-C cells but did not induce differentiation in SK-N-AS cells. Re-expression of α-N-catenin enhanced the sensitivity to retinoic acid-induced cell growth arrest and downregulated key cell survival pathways in both cell lines. Trichostatin A treatment induced CTNNA2 expression in SK-N-AS cells, and combination treatment with Trichostatin A induced retinoic acid sensitivity in retinoic acid-resistant cells.. Re-expression of α-N-catenin in retinoic acid-resistant cells induced sensitivity to retinoic acid treatment and is controlled epigenetically via histone deacetylase. α-N-catenin is a potential biomarker for retinoic acid sensitivity and combination treatment with Trichostatin A and retinoic acid may improve survival among children with high-risk, retinoic acid-resistant neuroblastoma.

Topics: alpha Catenin; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neuroblastoma; Tretinoin; Tumor Suppressor Proteins

Studying human ageing is of increasing importance due to the worldwide ageing population. However, it faces the challenge of lengthy experiments to produce an ageing phenotype. Often, to recreate the hallmarks of ageing requires complex empirical conditions that can confound data interpretation. Indeed, many studies use whole organisms with relatively short life spans, which may have little, or limited, relevance to human ageing. There has been extensive use of cell lines to study ageing in human somatic cells, but the modelling of human neuronal ageing is somewhat more complex in vitro.. We cultured the well-characterised SH-SY5Y human neural cell line to produce high purity cultures of cells differentiated to express a neuronal phenotype, and designed a protocol to maintain these cells in culture until they accumulated biomarkers of cellular ageing.. Our data validate a novel and simple technique for the efficient differentiation and long-term maintenance of SH-SY5Y cells, expressing markers of neuronal differentiation and demonstrating electrical activity in culture. Over time in vitro, these cells progressively accumulate markers of ageing such as enhanced production of reactive oxygen species and accumulation of oxidative damage.. In comparison to existing techniques to model neuronal ageing our method is cost effective, requiring no specialist equipment or growth factors.. We demonstrate that SH-SY5Y cells, grown under these culture conditions, represent a simple model of neuronal ageing that is amenable to cell biological, biochemical and electrophysiological investigation.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Nerve Growth Factors; Neuroblastoma; Neurons; Tretinoin

Neuroblastoma is the third most common pediatric cancer composed of malignant immature cells that are usually treated pharmacologically by all trans-retinoic acid (ATRA) but sometimes, they can spontaneously differentiate into benign forms. In that context, biomimetic cell culture models are warranted tools as they can recapitulate many of the biochemical and biophysical cues of normal or pathological microenvironments. Inspired by that challenge, we developed a neuroblastoma culture system based on biomimetic LbL films of physiological biochemical composition and mechanical properties. For that, we used chondroitin sulfate A (CSA) and poly-L-lysine (PLL) that were assembled and mechanically tuned by crosslinking with genipin (GnP), a natural biocompatible crosslinker, in a relevant range of stiffness (30-160 kPa). We then assessed the adhesion, survival, motility, and differentiation of LAN-1 neuroblastoma cells. Remarkably, increasing the stiffness of the LbL films induced neuritogenesis that was strengthened by the combination with ATRA. These results highlight the crucial role of the mechanical cues of the neuroblastoma microenvironment since it can dramatically modulate the effect of pharmacologic drugs. In conclusion, our biomimetic platform offers a promising tool to help fundamental understanding and pharmacological screening of neuroblastoma differentiation and may assist the design of translational biomaterials to support neuronal regeneration. STATEMENT OF SIGNIFICANCE: Neuroblastoma is one of the most common pediatric tumor commonly treated by the administration of all-trans-retinoic acid (ATRA). Unfortunately, advanced neuroblastoma often develop ATRA resistance. Accordingly, in the field of pharmacological investigations on neuroblastoma, there is a tremendous need of physiologically relevant cell culture systems that can mimic normal or pathological extracellular matrices. In that context, we developed a promising matrix-like cell culture model that provides new insights on the crucial role of mechanical properties of the microenvironment upon the success of ATRA treatment on the neuroblastoma maturation. We were able to control adhesion, survival, motility, and differentiation of neuroblastoma cells. More broadly, we believe that our system will help the design of in vitro pharmacological screening strategy.

Topics: Biomimetics; Cell Differentiation; Cell Line, Tumor; Extracellular Matrix; Humans; Neuroblastoma; Tretinoin; Tumor Microenvironment

Neuroblastoma is the most common extracranial solid tumor in children and originates from poorly differentiated neural crest progenitors. High-risk neuroblastoma patients frequently present with metastatic disease at diagnosis. Despite intensive treatment, patients often develop refractory disease characterized by poorly differentiated, therapy resistant cells. Although adjuvant therapy using retinoic acid (RA)-induced differentiation may increase event-free survival, in the majority of cases response to RA-therapy is inadequate. Consequently, current research aims to identify novel therapeutic targets that enhance the sensitivity to RA and induce neuroblastoma cell differentiation. The similarities between neural crest development and neuroblastoma progression provide an appealing starting point. During neural crest development the EMT-transcription factor SNAI2 plays an important role in neural crest specification as well as neural crest cell migration and survival. Here, we report that CRISPR/Cas9 mediated deletion as well as shRNA mediated knockdown of the EMT-transcription factor SNAI2 promotes cellular differentiation in a variety of neuroblastoma models. By comparing mRNA expression data from independent patient cohorts, we show that a SNAI2 activity-based gene expression signature significantly correlates with event-free survival. Loss of SNAI2 function reduces self-renewal, 3D invasion as well as metastatic spread in vivo, while strongly sensitizing neuroblastoma cells to RA-induced growth inhibition. Together, our data demonstrate that SNAI2 maintains progenitor-like features in neuroblastoma cells while interfering with RA-induced growth inhibition. We propose that targeting gene regulatory circuits, such as those controlling SNAI2 function, may allow reversion of RA-therapy resistant neuroblastoma cells to a more differentiated and therapy responsive phenotype.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Movement; Clustered Regularly Interspaced Short Palindromic Repeats; Female; Humans; Mice; Neural Crest; Neural Stem Cells; Neuroblastoma; RNA, Small Interfering; Snail Family Transcription Factors; Transcription, Genetic; Tretinoin

Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties.

Topics: Azoles; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Liposomes; Neuroblastoma; Proto-Oncogene Proteins c-akt; Retinoic Acid 4-Hydroxylase; Signal Transduction; Tretinoin

The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR.. By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets.. We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2.. Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

Topics: 3' Untranslated Regions; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Progression; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; N-Myc Proto-Oncogene Protein; Neuroblastoma; Promoter Regions, Genetic; RNA-Binding Proteins; Survival Analysis; Transcription Factors; Tretinoin

Retinoic acid- (RA-) triggered neuroblastoma cell lines are widely used cell modules of neuronal differentiation in neurodegenerative disease studies, but the gene regulatory mechanism underlying differentiation is unclear now. In this study, system biological analysis was performed on public microarray data from three neuroblastoma cell lines (SK-N-SH, SH-SY5Y-A, and SH-SY5Y-E) to explore the potential molecular processes of all-trans retinoic acid- (ATRA-) triggered differentiation. RT-qPCR, functional genomics analysis, western blotting, chromatin immunoprecipitation (ChIP), and homologous sequence analysis were further performed to validate the gene regulation processes and identify the RA response element in a specific gene. The potential disturbed biological pathways (111 functional GO terms in 14 interactive functional groups) and gene regulatory network (10 regulators and 71 regulated genes) in neuroblastoma differentiation were obtained. 15 of the 71 regulated genes are neuronal projection-related. Among them,

Topics: Binding Sites; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation; Gene Regulatory Networks; Humans; Membrane Glycoproteins; Neurites; Neuroblastoma; Receptor, trkB; Receptors, Retinoic Acid; Transcriptome; Tretinoin

Retinoic acid (RA) is a differentiating agent utilized as maintenance therapy for high-risk neuroblastoma (NB), but associated toxicities limit its use. We have previously shown that a non-toxic, novel rexinoid, 9-cis-UAB30 (UAB30), decreased NB cell proliferation and in vivo tumor growth. A second generation, mono-methylated compound, 6-Methyl-UAB30 (6-Me), has been recently designed having greater potency compared with UAB30. In the current study, we hypothesized that 6-Me would inhibit NB cell proliferation and survival and induce differentiation and cell-cycle arrest.. Proliferation and viability were measured in four human NB cell lines following treatment with UAB30 or 6-Me. Cell-cycle was analyzed and tumor cell stemness was evaluated with extreme limiting dilution assays and immunoblotting for expression of stem cell markers. A xenograft murine model was utilized to study the effects of 6-Me in vivo.. Treatment with 6-Me led to decreased proliferation and viability, induced cell cycle arrest, and increased neurite outgrowth, indicating differentiation of surviving cells. Furthermore, treatment with 6-Me decreased tumorsphere formation and expression of stem cell markers. Finally, inhibition of tumor growth and increased animal survival was observed in vivo following treatment with 6-Me.. These results indicate a potential therapeutic role for this novel rexinoid in neuroblastoma treatment.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Fatty Acids, Unsaturated; Humans; Mice; Naphthalenes; Neuroblastoma; Tretinoin

Neuroblastoma (NB) is the most common solid tumor in childhood. Twenty percent of patients display

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Neurogenesis; RNA Interference; RNA, Small Interfering; Tretinoin

Metastatic neuroblastoma (NB) is an aggressive malignancy with a poor prognosis. Many patients present with relapsed high-risk NB after undergoing first-line treatment, and there is no standard therapy available in this setting.. The present study aimed to present the cases of 2 patients with recurrent high-risk NB.. Two children with International Neuroblastoma Stage System stage 4 high-risk NB chemotherapy. The disease recurrent after finishing the treatment.. Both patients (34 months old and 41 months old) experienced recurrence, received second-line treatment, and then received maintenance treatment using apatinib plus retinoic acid. The apatinib (10 mg/kg per day) and retinoic acid (160 mg/m per day) were administered on alternating 2-week cycles, which was continued for 1 year.. The 2 patients had achieved complete response by the 1-year follow-up after starting apatinib plus retinoic acid, and did not experience any adverse drug reactions.. The outcomes from these cases suggest that apatinib plus isotretinoin might be an option for maintenance therapy in patients with recurrent high-risk NB.

Topics: Abdominal Pain; Antineoplastic Agents; Child, Preschool; Drug Therapy; Humans; Male; Neoplasm Metastasis; Neuroblastoma; Pyridines; Recurrence; Tretinoin

The human neuroblastoma cell line, SH-SY5Y, has been widely used in neuroscience research, especially in studies related to Parkinson's disease. However, differences between clones have been demonstrated, highlighting the importance to characterize the properties of this cell line carefully.. The aim of this study was to characterize the phenotype of undifferentiated and differentiated SH-SY5Y cells using various differentiation protocols.. A morphological and quantitative analysis of markers related to dopaminergic and cholinergic neurons, but also other phenotypes, was performed.. Differentiated cells showed the typical neuronal morphology. Undifferentiated cells expressed low levels of Tyrosine Hydroxylase (TH) and higher levels of the high-affinity Choline Transporter (CHT1). Staurosporine (ST)-differentiation resulted in the highest number of THimmunoreactive cells, followed by phorbol ester Phorbol-12-Myristate-13-Acetate (PMA), whereas differentiation with Brain-Derived Neurotrophic Factor (BDNF) did not increase TH-immunoreactive cells. TH, dopamine β-hydroxylase and vesicular monoamine transporter-2 were also significantly upregulated in ST-differentiated cells compared to both undifferentiated and Retinoic Acid (RA)- differentiated cells. RA induced the highest number of CHT1-immunoreactive cells while ST- and BDNF-differentiation reduced CHT1-immunoreactive cells, indicating a decrease in the cholinergic phenotype. The presynaptic neuronal protein, α-synuclein, was significantly upregulated in RA- and ST-treated cells compared to undifferentiated cells. Ascorbic acid increased the number of CHT1-immunoreactive cells in all differentiation procedures and ST-differentiated TH-positive cells significantly.. Our findings indicate that a quantitative characterization of the phenotype is crucial when using SH-SY5Y cells to study the pathogenesis or evaluate compounds for treatment of neurodegenerative diseases.

Topics: Biomarkers; Cell Differentiation; Cell Line, Tumor; Cholinergic Neurons; Dopamine; Dopaminergic Neurons; Humans; Neuroblastoma; Parkinson Disease; Phenotype; Signal Transduction; Tretinoin; Tyrosine 3-Monooxygenase; Up-Regulation

Neuroblastoma is the most common inheritable, solid neoplasm in children found under the age of 7 and accounts for approximately 7% of childhood cancers. A common treatment that has been prescribed for over a decade is retinoid therapy [using all‑trans retinoic acid (RA)]. Treatment with this differentiating agent has been revealed to progress the cells from their stem‑cell state to a mature neuronal state gaining classical neuronal characteristics, including the suppression of proliferation. However, the molecular mechanism underlying the action of RA treatment remains to be elucidated. In the present study, a novel mechanism of RA‑induced differentiation via regulation of receptor tyrosine kinase‑like orphan receptor 1 (ROR1) is reported. ROR1 is overexpressed in neuroblastoma but significantly downregulated in mature differentiated neurons. Hence, it was hypothesized that RA may modulate ROR1 leading to differentiation and termination of cancerous properties. Immunoblotting revealed that following RA treatment, ROR1 levels initially increased then sharply decreased by 96 h. This was paired with synaptophysin, a mature neuron marker, sharply increasing concurrently, providing evidence of differentiation by 96 h. Investigation of the ROR1 pathway confirmed ROR1‑dependent downstream activation of the PI3K/AKT signaling axis, a growth pathway previously demonstrated to promote differentiation. Chromatin immunoprecipitation revealed an increase in RAR binding to the promoters of ROR1 and its endogenous ligand, Wnt5a. This research provided compelling evidence that RA is able to modulate the expression of ROR1 and Wnt5a to promote differentiation through the expression of synaptophysin. This data combined with the overarching data from the scientific community regarding proliferation and other proliferative factors in early‑stage neurons provides a more in‑depth model of the process of differentiation in neurons.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Proliferation; Child; Datasets as Topic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neuroblastoma; Neurons; Promoter Regions, Genetic; Receptor Tyrosine Kinase-like Orphan Receptors; Receptors, Retinoic Acid; Response Elements; Signal Transduction; Synaptophysin; Tretinoin; Wnt-5a Protein

Neuroblastoma is the most common extracranial solid tumor in childhood. The different treatments available for neuroblastoma are challenged by high rates of resistance, recurrence, and progression, most notably in advanced cases and highly malignant tumors. Therefore, the development of more targeted therapies, which are biocompatible and without undesired side effects, is highly desirable. The mechanisms of actions of platinum nanoparticles (PtNPs) and retinoic acid (RA) in neuroblastoma have remained unclear. In this study, the anticancer effects of PtNPs and RA on neuroblastoma were assessed. We demonstrated that treatment of SH-SY5Y cells with the combination of PtNPs and RA resulted in improved anticancer effects. The anticancer effects of the two compounds were mediated by cytotoxicity, oxidative stress (OS), mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis-associated networks. Cytotoxicity was confirmed by leakage of lactate dehydrogenase (LDH) and intracellular protease, and oxidative stress increased the level of reactive oxygen species (ROS), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and nitric oxide (NO), and protein carbonyl content (PCC). The combination of PtNPs and RA caused mitochondrial dysfunction by decreasing the mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, number of mitochondria, and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Endoplasmic reticulum-mediated stress and apoptosis were confirmed by upregulation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), p53, Bax, and caspase-3 and down regulation of B-cell lymphoma 2 (BCl-2). PtNPs and RA induced apoptosis, and oxidative DNA damage was evident by the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG). Finally, PtNPs and RA increased the differentiation and expression of differentiation markers. Differentiated SH-SY5Y cells pre-treated with PtNPs or RA or the combination of both were more sensitive to the cytotoxic effect of cisplatin than undifferentiated cells. To our knowledge, this is the first study to demonstrate the effect of the combination of PtNPs and RA in neuroblastoma cells. PtNPs may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. The results of this study p

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; beta Carotene; Cell Differentiation; Cell Division; Cell Line, Tumor; Cisplatin; Crystallography, X-Ray; Drug Screening Assays, Antitumor; Drug Synergism; Endoplasmic Reticulum Stress; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Metal Nanoparticles; Neoplasm Proteins; Neuroblastoma; Oxidative Stress; Peptide Hydrolases; Platinum; Tretinoin

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature" that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, highlighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas.

Topics: Adrenergic Agents; Anaplastic Lymphoma Kinase; Animals; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromaffin Cells; Chromosome Deletion; Chromosomes, Human, Pair 11; Female; Gene Expression Regulation, Neoplastic; Guanylate Kinases; Humans; MAP Kinase Signaling System; Mice, Inbred BALB C; Nerve Growth Factor; Neuroblastoma; Neurons; Phenotype; Prognosis; Schwann Cells; Sp1 Transcription Factor; Transcription, Genetic; Treatment Outcome; Tretinoin; Tumor Suppressor Proteins; Up-Regulation

Acrylamide (ACR) is a known neurotoxicant which crosses the blood-brain barrier, passes the placenta and has been detected in breast milk. Hence, early-life exposure to ACR could lead to developmental neurotoxicity. The aim of this study was to elucidate if non-cytotoxic concentrations of ACR alter neuronal differentiation by studying gene expression of markers significant for neurodevelopment in the human neuroblastoma SH-SY5Y cell model. Firstly, by using RNASeq we identified two relevant pathways that are activated during 9 days of retinoic acid (RA) induced differentiation i.e. RA receptor (RAR) activation and the cAMP response element-binding protein (CREB) signalling pathways. Next, by qPCR we showed that 1 and 70 µM ACR after 9 days exposure alter the expression of 13 out of 36 genes in the RAR activation pathway and 18 out of 47 in the CREB signalling pathway. Furthermore, the expression of established neuronal markers i.e. BDNF, STXBP2, STX3, TGFB1 and CHAT were down-regulated. Decreased protein expression of BDNF and altered ratio of phosphorylated CREB to total CREB were confirmed by western blot. Our results reveal that micromolar concentrations of ACR sustain proliferation, decrease neurite outgrowth and interfere with signalling pathways involved in neuronal differentiation in the SH-SY5Y cell model.

Topics: Acrylamide; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Humans; Neuroblastoma; Neuronal Outgrowth; Neurons; Signal Transduction; Tretinoin

Neuroblastoma (NB) is the most common solid childhood tumor, and all-trans retinoic acid (ATRA) is used as a treatment to decrease minimal residual disease. Molecular iodine (I2) induces differentiation and/or apoptosis in several neoplastic cells through activation of PPARγ nuclear receptors. Here, we analyzed whether the coadministration of I2 and ATRA increases the efficacy of NB treatment. ATRA-sensitive (SH-SY5Y), partially-sensitive (SK-N-BE(2)), and non-sensitive (SK-N-AS) NB cells were used to analyze the effect of I2 and ATRA in vitro and in xenografts (Foxn1 nu/nu mice), exploring actions on cellular viability, differentiation, and molecular responses. In the SH-SY5Y cells, 200 μM I2 caused a 100-fold (0.01 µM) reduction in the antiproliferative dose of ATRA and promoted neurite extension and neural marker expression (tyrosine hydroxylase (TH) and tyrosine kinase receptor alpha (Trk-A)). In SK-N-AS, the I2 supplement sensitized these cells to 0.1 μM ATRA, increasing the ATRA-receptor (RARα) and PPARγ expression, and decreasing the Survivin expression. The I2 supplement increased the mitochondrial membrane potential in SK-N-AS suggesting the participation of mitochondrial-mediated mechanisms involved in the sensibilization to ATRA. In vivo, oral I2 supplementation (0.025%) synergized the antitumor effect of ATRA (1.5 mg/kg BW) and prevented side effects (body weight loss and diarrhea episodes). The immunohistochemical analysis showed that I2 supplementation decreased the intratumoral vasculature (CD34). We suggest that the I2 + ATRA combination should be studied in preclinical and clinical trials to evaluate its potential adjuvant effect in addition to conventional treatments.

Topics: Animals; Antineoplastic Agents; Humans; Iodine; Mice; Neuroblastoma; Tretinoin

Accumulation of toxic strands of amyloid beta (AB), which cause neurofibrillary tangles and, ultimately, cell death, is suspected to be the main culprit behind clinical symptoms of Alzheimer's disease. Although the mechanism of cell death due to AB accumulation is well known, the intermediate phase between the start of accumulation and cell death is less known and investigated, partially due to technical challenges in identifying partially affected cells.. First, we aimed to establish an in vitro model that would show resilience against AB toxicity. Then we used morphological, molecular and electrophysiological assays to investigate how the characteristics of the surviving cells changed after AB toxicity.. To investigate this phase, we used differentiation of SH-SY5Y neuroblastoma stem cells by Retinoic Acid (RA) and Brain Derived Neurotrophic Factor (BDNF) to establish an in vitro model which would be able to demonstrate various levels of resistance to AB toxicity. We utilized fluorescent microscopy and whole cell patch clamp recordings to investigate behavior of the model.. We observed significantly higher morphological resilience against AB toxicity in cells which were differentiated by both Retinoic Acid and Brain Derived Neurotrophic Factor compared to Retinoic Acid only. However, the electrophysiological properties of the Retinoic Acid + Brain-Derived Neurotrophic Factor differentiated cells were significantly altered after AB treatment.. We established a transient survival model for AB toxicity and observed the effects of AB on transmembrane currents of differentiated neurons.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Brain-Derived Neurotrophic Factor; Cell Death; Cell Differentiation; Cell Line; Cell Survival; Humans; In Vitro Techniques; Neuroblastoma; Neurofibrillary Tangles; Tretinoin

3,4-Methylenedioxypyrovalerone (MDPV), one of the most commonly abused synthetic cathinones, has caused several intoxications and deaths despite its short presence on the market. Apart from its effects on the monoamine systems in the brain, recent in vitro investigations have revealed cytotoxicity. In this study, the effects of increasing concentrations (10-1000 μM) of 3,4-Catechol-PV, one of major MDPV metabolites, on cell viability, morphology, and apoptosis have been evaluated after acute exposure (24-48 h) in human neuroblastoma SH-SY5Y cells-undifferentiated and differentiated to a more mature neuronal-like phenotype. Results indicated the following: (i) Cell viability: concentration-dependent decrease (15-55%) in differentiated SH-SY5Y after 24 h, with no exacerbation after 48 h (LC

Topics: Antineoplastic Agents; Benzodioxoles; Caspase 3; Cell Differentiation; Cell Line, Tumor; Cell Size; Dopamine; Dopamine beta-Hydroxylase; Dopamine Uptake Inhibitors; Dopaminergic Neurons; Dose-Response Relationship, Drug; Guaiacol; Humans; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Pyrrolidines; Statistics, Nonparametric; Synthetic Cathinone; Time Factors; Tretinoin; Tyrosine 3-Monooxygenase

Of the mammalian topoisomerase (Topo)-2 isozymes (α and β), Topo-2β protein has been reported to regulate neuronal development and differentiation. However, the status of Topo-2β in all-trans retinoic acid (ATRA)-treated human neuroblastoma (SK-N-SH) cells is not understood. More information about the effects of ATRA on SK-N-SH cells is needed to reveal the role of ATRA in the regulation of Topo-2β levels and spontaneous regression of SK-N-SH cells to predict the clinical activity. This study was proposed to investigate the status and role of Topo-2β protein in ATRA-induced survival and neuronal differentiation of SK-N-SH cells. Microscopic, sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitations and Western blot analysis were used to study and compare Topo-2β protein among 10 µM ATRA-treated SK-N-SH cells and controls at different time points. The level of Topo-2β protein increased in the initial days of treatment but markedly decreased upon induction of differentiation by ATRA in later stages. Upon ATRA treatment, SK-N-SH cells stretched, exhibited neurite extensions, and acquired a neuronal phenotype. Both treated and untreated SK-N-SH cells were able to migrate, occupy the scratched area, and completely recolonized 24 hours later. These results suggest an indirect role of Topo-2β protein in regulation of genes involved in cell migration and differentiation of ATRA-treated SK-N-SH cells. This study suggests that Topo-2β may be part of activation/repression of protein complexes activated by epigenetic modifying agents, differentiating signals, and inducible locus. However, detailed studies are needed to explore the ATRA-downstream genes leading to Topo-2β regulation and regulatory proteins of neuronal differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA Topoisomerases, Type II; Humans; Neuroblastoma; Neuronal Outgrowth; Poly-ADP-Ribose Binding Proteins; Tretinoin

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Humans; Immunoprecipitation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Plasmids; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Tretinoin; Tumor Suppressor Protein p53

Retinoic acid (RA) promotes differentiation in multiple neurogenic cell types by promoting gene reprogramming through retinoid receptors and also by inducing cytosolic signaling events. The nuclear RXR receptors are one of the main mediators of RA cellular effects, classically by joining the direct receptors of RA, the nuclear RAR receptors, in RAR/RXR dimers which act as transcription factors. Distinct RXR genes lead to RXRα, RXRβ and RXRγ subtypes, but their specific roles in neuronal differentiation remain unclear. We firstly investigated both RXRs and RARs expression profiles during RA-mediated neuronal differentiation of human neuroblastoma cell line SH-SY5Y, and found varying levels of retinoid receptors transcript and protein contents along the process. In order to understand the roles of the expression of distinct RXR subtypes to RA signal transduction, we performed siRNA-mediated silencing of RXRα and RXRβ during the first stages of SH-SY5Y differentiation. Our results showed that RXRα is required for RA-induced neuronal differentiation of SH-SY5Y cells, since its silencing compromised cell cycle arrest and prevented the upregulation of neuronal markers and the adoption of neuronal morphology. Besides, silencing of RXRα affected the phosphorylation of ERK1/2. By contrast, silencing of RXRβ improved neurite extension and led to increased expression of tau and synaptophysin, suggesting that RXRβ may negatively regulate neuronal parameters related to neurite outgrowth and function. Our results indicate distinct functions for RXR subtypes during RA-dependent neuronal differentiation and reveal new perspectives for studying such receptors as clinical targets in therapies aiming at restoring neuronal function.

Topics: Animals; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Dopaminergic Neurons; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neurites; Neuroblastoma; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptor alpha; Retinoid X Receptor beta; Retinoid X Receptors; Signal Transduction; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Choline Kinase; Epigenesis, Genetic; F-Box Proteins; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Jumonji Domain-Containing Histone Demethylases; Mice; Neural Stem Cells; Neuroblastoma; Prognosis; Promoter Regions, Genetic; RNA, Small Interfering; Tretinoin; Up-Regulation

Topics: Bismuth; Cell Differentiation; Cell Line, Tumor; Drug Carriers; Endocytosis; Humans; Metal Nanoparticles; MicroRNAs; Microscopy, Confocal; Neuroblastoma; Oligonucleotides; Organoselenium Compounds; Povidone; Selenium Compounds; Silver; Spectrum Analysis, Raman; Tretinoin

Cholinergic transmission is critical to high-order brain functions such as memory, learning, and attention. Alzheimer's disease (AD) is characterized by cognitive decline associated with a specific degeneration of cholinergic neurons. No effective treatment to prevent or reverse the symptoms is known. Part of this might be due to the lack of in vitro models that effectively mimic the relevant features of AD. Here, we describe the characterization of an AD in vitro model using the SH-SY5Y cell line. Exponentially growing cells were maintained in DMEM/F12 medium and differentiation was triggered by the combination of retinoic acid (RA) and BDNF. Both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzymatic activities and immunocontent were determined. For mimicking tau and amyloid-β pathology, RA + BDNF-differentiated cells were challenged with okadaic acid (OA) or soluble oligomers of amyloid-β (AβOs) and neurotoxicity was evaluated. RA + BDNF-induced differentiation resulted in remarkable neuronal morphology alterations characterized by increased neurite density. Enhanced expression and enzymatic activities of cholinergic markers were observed compared to RA-differentiation only. Combination of sublethal doses of AβOs and OA resulted in decreased neurite densities, an in vitro marker of synaptopathy. Challenging RA + BDNF-differentiated SH-SY5Y cells with the combination of sublethal doses of OA and AβO, without causing considerable decrease of cell viability, provides an in vitro model which mimics the early-stage pathophysiology of cholinergic neurons affected by AD.

Topics: Alzheimer Disease; Biomarkers; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cholinergic Neurons; Gene Expression Regulation; Humans; Models, Biological; Neurites; Neuroblastoma; Signal Transduction; Synapses; Tretinoin

Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.

Topics: Adolescent; Antineoplastic Agents; Bexarotene; Biomarkers, Pharmacological; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; DEAD-box RNA Helicases; Drug Resistance, Neoplasm; Female; Fenretinide; HMGA1a Protein; HMGA2 Protein; Homeodomain Proteins; Humans; Infant; Infant, Newborn; Isotretinoin; Male; Nervous System Neoplasms; Neuroblastoma; Paraffin Embedding; Pre-B-Cell Leukemia Transcription Factor 1; Tissue Fixation; Tretinoin; Young Adult

Despite intensive chemotherapy, the survival rates for high-risk neuroblastoma, most of which have MYCN amplification, remain low. Overexpression of N-myc oncoprotein promotes expression of cancer-associated properties. We recently found that combination of all-trans retinoic acid (ATRA) with the β1-integrin-activating peptide TNIIIA2 attenuated cancer-associated properties of neuroblastoma cells through N-Myc degradation. However, ATRA has serious side-effects and there are concerns about late adverse effects. The aim of this study was to examine the effects of the combination of acyclic retinoid (ACR) with TNIIIA2 on neuroblastoma.. The effects of ACR and TNIIIA2 were examined by neuroblastoma cell proliferation and survival assays as well as by using a neuroblastoma xenograft model. The levels of N-Myc and cancer-associated malignant properties were assayed by western blot and colony formation assay, respectively.. Combining ACR, which is clinically safe, with TNIIIA2 induced proteasomal degradation of N-Myc and reduction of neuroblastoma cell malignant properties. An in vivo experiment showed therapeutic potential.. ACR-TNIIIA2 combination treatment may be efficacious and clinical safe chemotherapy for high-risk neuroblastoma.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Mice, Inbred BALB C; Mice, Nude; N-Myc Proto-Oncogene Protein; Neuroblastoma; Peptides; Phenotype; Tenascin; Tretinoin; Tumor Burden

Aggressive, high-risk neuroblastoma (NB) exhibits an immature differentiation state, profound epigenetic dysregulation and high telomerase activity. It has been suggested that aggressive NB may be treatable by inducing differentiation whereas therapeutic targeting of telomerase is under investigation for multiple cancer types. While epigenetic regulation of the telomerase reverse transcriptase (TERT) promoter has been described in high-risk NB, the exact molecular mechanisms are still not completely understood. Here we used quantitative real-time polymerase chain reaction (PCR), chromatin immunoprecipitation qPCR, quantitative telomeric repeat amplification protocol, and immunoblot techniques to investigate epigenetic regulation of TERT in wild-type and genetically modified NB cell lines. We demonstrated that TERT expression is reduced during 13-cis retinoic acid-induced NB differentiation and that this inversely correlated with increased expression of AT-rich interaction domain 1A (ARID1A), a subunit of the SWItch/sucrose nonfermentable chromatin remodeling complex. We showed that ARID1A directly caused suppression of TERT and was reliant on DNA binding and co-occupancy of the TERT promoter by the SIN3 transcription regulator family member A (SIN3A) repressor complex allowing NB differentiation to proceed. Finally, using data from NB patient cohorts, we reported a significant correlation between low ARID1A expression, elevated expression of TERT, and poorly differentiated, high-risk NB. These results provide insights into a key epigenetic pathway responsible for modulating TERT-driven NB progression, which could represent a target for therapeutic intervention.

Topics: Cell Differentiation; Cell Line, Tumor; Child, Preschool; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Infant, Newborn; Male; Neuroblastoma; Repressor Proteins; Sin3 Histone Deacetylase and Corepressor Complex; Telomerase; Transcription Factors; Transcription, Genetic; Tretinoin

The function, regulation and cellular distribution of GABA

Topics: Adolescent; Adult; Age Factors; Amyloid beta-Peptides; Animals; Animals, Newborn; Case-Control Studies; Doublecortin Domain Proteins; Down Syndrome; Embryo, Mammalian; Female; Fetus; Gene Expression Regulation, Developmental; Gestational Age; Glutamate Decarboxylase; Hippocampus; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Middle Aged; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neuropeptides; Peptide Fragments; POU Domain Factors; Protein Subunits; Receptors, GABA-A; Tretinoin; Vesicular Inhibitory Amino Acid Transport Proteins; Young Adult

Although anti-disialoganglioside (GD2) antibodies are successfully used for neuroblastoma therapy, a third of patients with neuroblastoma experience treatment failure or serious toxicity. Various strategies have been employed in the clinic to improve antibody-dependent cell-mediated cytotoxicity (ADCC), such as the addition of interleukin (IL)-2 to enhance natural killer (NK) cell function, adoptive transfer of allogeneic NK cells to exploit immune surveillance, and retinoid-induced differentiation therapy. Nevertheless, these mechanisms are not fully understood. We developed a quantitative assay to test ADCC induced by the anti-GD2 antibody Hu14.18K322A in nine neuroblastoma cell lines and dissociated cells from orthotopic patient-derived xenografts (O-PDXs) in culture. IL-2 improved ADCC against neuroblastoma cells, and differentiation with all-trans retinoic acid stabilized GD2 expression on tumor cells and enhanced ADCC as well. Degranulation was highest in licensed NK cells that expressed CD158b (P < 0.001) and harbored a killer-cell immunoglobulin-like receptor (KIR) mismatch against the tumor-specific human leukocyte antigen (HLA; P = 0.016). In conclusion, IL-2 is an important component of immunotherapy because it can improve the cytolytic function of NK cells against neuroblastoma cells and could lower the antibody dose required for efficacy, thereby reducing toxicity. The effect of IL-2 may vary among individuals and a biomarker would be useful to predict ADCC following IL-2 activation. Sub-populations of NK cells may have different levels of activity dependent on their licensing status, KIR expression, and HLA-KIR interaction. Better understanding of HLA-KIR interactions and the molecular changes following retinoid-induced differentiation is necessary to delineate their role in ADCC.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Gangliosides; Humans; Interleukin-2; Killer Cells, Natural; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Sirtuins (SIRTs) play crucial roles in various signaling pathways that modulate differentiation and proliferation. We sought to elucidate the role of SIRTs in differentiation and proliferation of human neuroblastoma (NB).. NB cells were treated with nicotinamide (NAM), a non-specific SIRT inhibitor, SIRT-targeted short hairpin RNAs, and retinoic acid to assess cell growth and differentiation.. SIRTs are involved in proliferation and differentiation using NAM in BE(2)-C cells. Specifically, SIRT6 knockdown in BE(2)-C cells reduced cell proliferation, induced neurite extension, corresponding with induction of p21. SIRTs have important oncogenic properties in NB beyond its established functions in aging and genome stability. SIRT6 may represent a novel target for developing future therapeutics for the treatment of aggressive NBs.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Gene Knockdown Techniques; Humans; Neuroblastoma; Niacinamide; RNA, Small Interfering; Sirtuins; Tretinoin

Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of

Topics: Alternative Splicing; Animals; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Exons; Gene Knockdown Techniques; Gene Silencing; Homeodomain Proteins; Humans; Male; Mice; Mice, Nude; Neuroblastoma; Prognosis; Protein Isoforms; RNA, Messenger; Transcription Factors; Tretinoin

We have previously demonstrated the potential of biologically synthesized silver nanoparticles (AgNP) in the induction of neuronal differentiation of human neuroblastoma, SH-SY5Y cells; we aimed herein to unveil its molecular mechanism in comparison to the well-known neuronal differentiation-inducing agent, all-trans-retinoic acid (RA). AgNP-treated SH-SY5Y cells showed significantly higher reactive oxygen species (ROS) generation, stronger mitochondrial membrane depolarization, lower dual-specificity phosphatase expression, higher extracellular-signal-regulated kinase (ERK) phosphorylation, lower AKT phosphorylation, and lower expression of the genes encoding the antioxidant enzymes than RA-treated cells. Notably, pretreatment with

Topics: Acetylcysteine; Cell Line, Tumor; Humans; Membrane Potential, Mitochondrial; Metal Nanoparticles; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Neurogenesis; Neurons; Reactive Oxygen Species; Silver; Tretinoin

High-risk neuroblastoma is often distinguished by amplification of

Topics: Animals; Axons; Benzazepines; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Demethylation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Histone Demethylases; Histones; Humans; Lysine; Mice, Nude; Neuroblastoma; Pyrimidines; Risk Factors; Sulfonamides; Tretinoin

The chemokine CCL5 prevents neuronal cell death mediated both by amyloid β, as well as the human immunodeficiency virus viral proteins gp120 and Tat. Because CCL5 binds to CCR5, CCR3 and/or CCR1 receptors, it remains unclear which of these receptors plays a role in neuroprotection. Indeed, CCL5 also has neuroprotective activity in cells lacking these receptors. CCL5 may bind to a G-protein-coupled receptor 75 (GPR75), which encodes for a 540 amino-acid orphan receptor of the Gqα family. In this study, we have used SH-SY5Y human neuroblastoma cells to characterize whether CCL5 could activate a Gq signaling through GPR75. Both qPCR and flow cytometry show that these cells express GPR75 but do not express CCR5, CCR3 or CCR1 receptors. SY-SY5Y cells were then used to examine CCL5-mediated signaling. We report that CCL5 promotes a time- and concentration-dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3β, and extracellular signal-regulated kinase (ERK) 1/2. Specific antagonists of CCR5, CCR3, and CCR1 did not prevent CCL5 from increasing phosphorylated AKT or ERK. Moreover, CCL5 promotes a time-dependent internalization of GPR75. Lastly, knocking down GPR75 expression by a CRISPR-Cas9 approach inhibited the ability of CCL5 to activate pERK in SH-SY5Y cells. Therefore, we propose that GPR75 is a novel receptor for CCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small molecule GPR75 agonists that mimic CCL5. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Cerebral Cortex; Chemokine CCL5; CRISPR-Associated Protein 9; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Humans; Mutagenesis; Neuroblastoma; Neurons; Pertussis Toxin; Protein Transport; Rats; Receptors, G-Protein-Coupled; Signal Transduction; T-Lymphocytes; Tretinoin

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Checkpoints; Cell Death; Cell Differentiation; Cell Line, Tumor; DNA Damage; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Neuroblastoma; Repressor Proteins; Tretinoin; Tumor Cells, Cultured

Neuroblastoma (NB) originates from immature neuronal cells and currently has a poor clinical outcome. NB cells possess cancer stem cells (CSCs) characteristics that facilitate the initiation of a tumor, as well as its metastasis. Human bitter taste receptors, referred to as TAS2Rs, are one of five types of basic taste receptors and they belong to a family of G-protein coupled receptors. The recent finding that taste receptors are expressed in non-gustatory tissues suggest that they mediate additional functions distinct from taste perception. While it is generally admitted that the recognition of bitter tastes may be associated with a self-defense system to prevent the ingestion of poisonous food compounds, this recognition may also serve as a disease-related function in the human body. In particular, the anti-cancer stemness and invasion effects of TAS2Rs on NB cells remain poorly understood. In the present study, endogenous expression of TAS2R8 and TAS2R10 in SK-N-BE(2)C and SH-SY5Y cells was examined. In addition, higher levels of TAS2R8 and TAS2R10 expression were investigated in more differentiated SY5Y cells. Both TAS2Rs were up-regulated following the induction of neuronal cell differentiation by retinoic acid. In addition, ectopic transfection of the two TAS2Rs induced neurite elongation in the BE(2)C cells, and down-regulated CSCs markers (including DLK1, CD133, Notch1, and Sox2), and suppressed self-renewal characteristics. In particular, TAS2RS inhibited tumorigenicity. Furthermore, when TAS2Rs was over-expressed, cell migration, cell invasion, and matrix metalloproteinases activity were inhibited. Expression levels of hypoxia-inducible factor-1α, a well-known regulator of tumor metastasis, as well as its downstream targets, vascular endothelial growth factor and glucose transporter-1, were also suppressed by TAS2Rs. Taken together, these novel findings suggest that TAS2Rs targets CSCs by suppressing cancer stemness characteristics and NB cell invasion, thereby highlighting the chemotherapeutic potential of bitter taste receptors.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplastic Stem Cells; Neuroblastoma; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Taste Perception; Tretinoin; Vascular Endothelial Growth Factor A

The incidence of brain degenerative disorders like Alzheimer's disease (AD) will increase as the world population ages. While there is presently no known cure for AD and current treatments having only a transient effect, an increasing number of publications indicate that growth factors (GF) may be used to treat AD. GFs like the bone morphogenetic proteins (BMPs), especially BMP-9, affect many aspects of AD. However, BMP-9 is a big protein that cannot readily cross the blood-brain barrier. We have therefore studied the effects of two small peptides derived from BMP-9 (pBMP-9 and SpBMP-9). We investigated their capacity to differentiate SH-SY5Y human neuroblastoma cells into neurons with or without retinoic acid (RA). Both peptides induced Smad 1/5 phosphorylation and their nuclear translocation. They increased the number and length of neurites and the expression of neuronal markers MAP-2, NeuN and NSE better than did BMP-9. They also promoted differentiation to the cholinergic phenotype more actively than BMP-9, SpBMP-9 being the most effective as shown by increases in intracellular acetylcholine, ChAT and VAchT. Finally, both peptides activated the PI3K/Akt pathway and inhibited GSK3beta, a current AD therapeutic target. BMP-9-derived peptides, especially SpBMP-9, with or without RA, are promising molecules that warrant further investigation.

Topics: Alzheimer Disease; Binding Sites; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Growth Differentiation Factor 2; Growth Differentiation Factors; Humans; Models, Biological; Neuroblastoma; Neurons; Peptides; Signal Transduction; Tretinoin

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as

Topics: Animals; Azacitidine; Basic Helix-Loop-Helix Transcription Factors; Cell Proliferation; Chemotherapy, Adjuvant; Decitabine; Female; Genetic Therapy; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Neuroblastoma; Tretinoin

The presence of fibrillary lesions, which are mainly composed of the microtubule associated protein tau (MAPT) in neurons, has gained immense recognition due to their presence in numerous neurodegenerative diseases, including Alzheimer's disease (AD). Dysregulation of tau is related with its altered site-specific phosphorylation which is followed by tau polymerization, neuronal dysfunction and death. Previous reports by us suggest that molecular alterations favor abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates and rodents with past exposure to lead (Pb). Here we report the involvement of Pb-induced alterations in tau and hyperphosphorylation of tau in differentiated Human Neuroblastoma SH-SY5Y cells exposed to a series of Pb concentrations (5-100μM) for 48h. These cells were analyzed for the protein expression of total tau, site-specific tau hyperphosphorylation, cyclin dependent kinase 5 (CDK5) and p35/p25 at selected time points (24-144h), after Pb exposure had ceased. Western blot analysis revealed aberrant tau levels as well as site-specific tau hyperphosphorylation accompanied by elevated CDK5 levels and altered protein ratio of p35/p25 particularly at 72 and 144h. These changes provide additional evidence that neurodegenerative events are subject to environmental influences.

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Densitometry; Dose-Response Relationship, Drug; Humans; Lead; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphorylation; Serine; tau Proteins; Threonine; Time Factors; Tretinoin

Neuroblastoma cells are neural crest derivatives that can differentiate into neuron-like cells in response to exogenous agents, and are known to be particularly sensitive to retinoic acid. The spectrum of neuroblastoma responses, ranging from proliferation, migration, differentiation, or apoptosis, is difficult to predict due to the heterogeneity of these tumors and to the broad effective range of retinoic acid. Our study focused on the effects of nanomolar concentrations of retinoic acid on neuroblastoma differentiation in two cell lines cells: SK-N-SH (HTB-11) and IMR-32. Each cell line was treated with retinoic acid from 1 to 100 nM for up to 6 d. Morphological changes were quantified; immunocytochemistry was used to observe changes in neuronal protein expression and localization, while live-cell calcium imaging utilizing pharmacological agents was conducted to identify neuron-like activity. Retinoic acid-treated HTB-11 but not IMR-32 cells developed specific neuronal phenotypes: acquisition of long neurite-like processes, expression of neurofilament-200, increased responsiveness to acetylcholine, and decreased responsiveness to nicotine and epinephrine. In addition, nanomolar levels of retinoic acid elicited increased nuclear trafficking of the CRABP2, which is traditionally associated with gene expression of cellular pathways related to neuronal differentiation. Collectively, these results show that nanomolar concentrations of retinoic acid are capable of inducing both structural and functional neuron-like features in HTB-11 cells using CRABP2, suggesting differentiation in neuroblastoma cells into neuronal phenotypes. These have important implications for both chemotherapeutic design and for the use of neuroblastomas as in vitro models for neuron differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Humans; Intermediate Filaments; Neurites; Neuroblastoma; Neurogenesis; Neurons; Phenotype; Receptors, Retinoic Acid; Tretinoin; Up-Regulation

SH-SY5Y neuroblastoma cells are frequently used for different neuronal cell culture models. As there is no "gold-standard", miscellaneous protocols exist to differentiate these cells into a neuronal cell type. Here, the aim was to find a differentiation condition making cells suitable for investigation of influenceability of synapses by environmental conditions in pharmacologic experiments. For this purpose, effects on synapse molecules should be somehow rateable and cells should be usable for functional analysis like calcium imaging. A system like this is desirable for example in basic research concerning schizophrenia, depression, autism or neurodegeneration as synaptic plasticity and neuronal maturation are known to have a significant impact in these diseases. Cells grown on laminin-coated glass cover slips and treated with 50 µM retinoic acid (RA) turned out to show most convincing morphological signs of neuronal differentiation and attached strongly to the ground, thereby also fulfilling preconditions for functional analysis. Systematic characterisation of this differentiation condition in comparison to non-treated controls revealed lower methylation rates and higher expression of most candidate molecules relevant for formation, preservation and function of synapses as well as differential function. In conclusion, this combination of differentiation strategy and markers seems to be a suitable system to estimate synapse modifications in basic research as it could help to identify possible dedifferentiating effects. To our knowledge, differentiation of SH-SY5Y has not been described as systematic before regarding comprehensiveness of the set of investigated synapse molecules and coverage of applied methods spanning from epigenetics to protein function. Furthermore, this is the first time that SH-SY5Y cells were differentiated on glass cover slips to an extent making them suitable for investigation of synapse molecules as part of stable intercellular connections in downstream functional analyses.

Topics: Antineoplastic Agents; Brain-Derived Neurotrophic Factor; Calcium; Cell Count; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Dose-Response Relationship, Drug; Epigenesis, Genetic; Glutamic Acid; Humans; Nerve Tissue Proteins; Neurites; Neuroblastoma; Phorbol Esters; Potassium Chloride; Synapses; tau Proteins; Time Factors; Tretinoin

The mouse neuroblastoma N18TG2 clone is unable to differentiate and is defective for the enzymes of the biosynthesis of neurotransmitters. The forced expression of choline acetyltransferase (ChAT) in these cells results in the synthesis and release of acetylcholine (Ach) and hence in the expression of neurospecific features and markers. To understand how the expression of ChAT triggered neuronal differentiation, we studied the differences in genome-wide transcription profiles between the N18TG2 parental cells and its ChAT-expressing 2/4 derived clone. The engagement of the 2/4 cells in the neuronal developmental program was confirmed by the increase of the expression level of several differentiation-related genes and by the reduction of the amount of transcripts of cell cycle genes. At the same time, we observed a massive reorganization of cytoskeletal proteins in terms of gene expression, with the accumulation of the nucleoskeletal lamina component Lamin A/C in differentiating cells. The increase of the Lmna transcripts induced by ChAT expression in 2/4 cells was mimicked treating the parental N18TG2 cells with the acetylcholine receptor agonist carbachol, thus demonstrating the direct role played by this receptor in neuron nuclei maturation. Conversely, a treatment of 2/4 cells with the muscarinic receptor antagonist atropine resulted in the reduction of the amount of Lmna RNA. Finally, the hypothesis that Lmna gene product might play a crucial role in the ChAT-dependent molecular differentiation cascade was strongly supported by Lmna knockdown in 2/4 cells leading to the downregulation of genes involved in differentiation and cytoskeleton formation and to the upregulation of genes known to regulate self-renewal and stemness.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Choline O-Acetyltransferase; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Ontology; Lamin Type A; Mice; Neuroblastoma; Neurons; Protein Interaction Mapping; Receptors, Cholinergic; Receptors, Muscarinic; Transcription, Genetic; Tretinoin; Up-Regulation

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

Topics: Cell Differentiation; Cell Line, Tumor; Gene Expression Profiling; Humans; Membrane Proteins; Neuroblastoma; Neurons; Tretinoin

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.

Topics: Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; DEAD-box RNA Helicases; Down-Regulation; Gene Expression; Humans; MicroRNAs; Neuroblastoma; Neurogenesis; Neurons; Ribonuclease III; Tretinoin

A simple and accurate high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) and N-(4-methoxyphenyl)retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5μm, 50×2.1mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH* 2.4) at a flow rate of 0.5mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2-50ng/mL with a lower limit of quantification of 0.2ng/mL. The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than 90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of the pharmacokinetic study for patients treated with 4-HPR in a clinical trial.

Topics: Acetonitriles; Algorithms; Chromatography, Liquid; Clinical Trials, Phase I as Topic; Fenretinide; Humans; Hydrogen-Ion Concentration; Ions; Limit of Detection; Mass Spectrometry; Neuroblastoma; Quality Control; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Temperature; Tretinoin; Water

Neuroblastoma (NB) is the most common extracranial solid childhood tumor accounting for around 15% of pediatric cancer deaths and most probably originates from a failure in the development of embryonic neural crest cells. Retinoids can inhibit the proliferation and stimulate differentiation of NB cells. In addition, epigenetic events involving changes in chromatin structure and DNA methylation can mediate the effects of retinoids; hence, the scope of this study is to investigate the use of retinoids and epigenetic drugs in NB cell lines. Here, we demonstrate that the combination of retinoid all trans-retinoic acid (ATRA) with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase is more effective in impairing the proliferation of human SH-SY5Y and SK-N-BE(2) NB cells than any drug given alone. Treatments also induced differential changes on the messenger RNA (mRNA) expression of retinoid receptor subtypes and reduced the protein content of c-Myc, the neuronal markers NeuN and β-3 tubulin, and the oncoprotein Bmi1. These results suggest that the combination of retinoids with epigenetic modulators is more effective in reducing NB growth than treatment with single drugs.

Topics: Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Humans; Neuroblastoma; Receptors, Retinoic Acid; Steroids; Tretinoin

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Mice, Nude; Neuroblastoma; PPAR delta; PPAR-beta; PTEN Phosphohydrolase; Retinoic Acid Receptor alpha; SOXB1 Transcription Factors; Tretinoin; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Neurological disorders are characterized by neurodegeneration and/or loss of neuronal function, which cannot be adequately repaired by the host. Therefore, there is need for novel treatment options such as cell-based therapies that aim to salvage or reconstitute the lost tissue or that stimulate host repair. The present study aimed to evaluate the paracrine effects of human dental pulp stem cells (hDPSCs) on the migration and neural maturation of human SH-SY5Y neuroblastoma cells. The hDPSC secretome had a significant chemoattractive effect on SH-SY5Y cells as shown by a transwell assay. To evaluate neural maturation, SH-SY5Y cells were first induced toward neuronal cells, after which they were exposed to the hDPSC secretome. In addition, SH-SY5Y cells subjected to the hDPSC secretome showed increased neuritogenesis compared with nonexposed cells. Maturated cells were shown to increase immune reactivity for neuronal markers compared with controls. Ultrastructurally, retinoic acid (RA) signaling and subsequent exposure to the hDPSC secretome induced a gradual rise in metabolic activity and neuronal features such as multivesicular bodies and cytoskeletal elements associated with cellular communication. In addition, electrophysiological recordings of differentiating cells demonstrated a transition toward a neuronal electrophysiological profile based on the maximum tetrodotoxin (TTX)-sensitive, Na

Topics: Adolescent; Cell Communication; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Cellular Microenvironment; Culture Media, Conditioned; Dental Pulp; Humans; In Vitro Techniques; Neuroblastoma; Neurogenesis; Paracrine Communication; Tretinoin; Young Adult

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.

Topics: Animals; Animals, Newborn; Axons; Cells, Cultured; Electron Transport Complex IV; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neuroblastoma; Neurons; Rats; Rats, Sprague-Dawley; RNA-Binding Protein FUS; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Superior Cervical Ganglion; Transfection; Tretinoin; Y-Box-Binding Protein 1

Mitochondrial ferritin (FtMt) is a type of ferritin that sequesters iron. Previous studies have shown that FtMt is expressed by dopaminergic neurons in the substantia nigra and that it may be involved in the pathology of Parkinson's disease. However, the functional roles of FtMt in dopaminergic neurons remain unclear. In this study, we investigated the function of FtMt in α-synuclein regulation and its antioxidant roles in dopaminergic cells using human dopaminergic neuroblastoma cells, SH-SY5Y. In physiological conditions, FtMt knockdown increased α-synuclein expression at the protein level but not at the mRNA level. By contrast, FtMt overexpression reduced α-synuclein expression at the protein level but not at the mRNA level. FtMt enhanced the iron levels in mitochondria but decreased the iron levels in the intracellular labile iron pool. We found that FeCl

Topics: alpha-Synuclein; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Deferoxamine; Down-Regulation; Ferritins; Ferrous Compounds; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Iron; L-Lactate Dehydrogenase; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Oxidative Stress; Siderophores; Tretinoin; Tyrosine 3-Monooxygenase

Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance.. We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets.. We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-β) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-β. Co-targeting of the retinoic acid and TGF-β pathways, through RA and kartogenin (KGN; a TGF-β signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells.. Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-β signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.

Topics: Anilides; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Phthalic Acids; Precision Medicine; Retinoids; Signal Transduction; Transforming Growth Factor beta; Tretinoin

Oxidative stress is an upsurge in reactive oxygen/nitrogen species (ROS/RNS), which aggravates damage to cellular components viz. lipids, proteins, and nucleic acids resulting in impaired cellular functions and neurological pathologies including Alzheimer's disease (AD). In the present study, we have examined amyloid-β (Aβ)-induced oxidative stress responses, a major cause for AD, in the undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Aβ1-42-induced oxidative damage was evaluated on lipids by lipid peroxidation; proteins by protein carbonyls; antioxidant status by SOD and GSH enzyme activities; and DNA and RNA damage levels by evaluating the number of AP sites and 8-OHG base damages produced. In addition, the neuro-protective role of the phytochemical ginkgolide B (GB) in countering Aβ1-42-induced oxidative stress was assessed. We report that the differentiated cells are highly vulnerable to Aβ1-42-induced oxidative stress events as exerted by the deposition of Aβ in AD. Results of the current study suggest that the pre-treatment of GB, followed by Aβ1-42 treatment for 24 h, displayed neuro-protective potential, which countered Aβ1-42-induced oxidative stress responses in both undifferentiated and differentiated SH-SY5Y neuronal cells by: 1) hampering production of ROS and RNS; 2) reducing lipid peroxidation; 3) decreasing protein carbonyl content; 4) restoring antioxidant activities of SOD and GSH enzymes; and 5) maintaining genome integrity by reducing the oxidative DNA and RNA base damages. In conclusion, Aβ1-42 induces oxidative damage to the cellular biomolecules, which are associated with AD pathology, and are protected by the pre-treatment of GB against Aβ-toxicity. Taken together, this study advocates for phytochemical-based therapeutic interventions against AD.

Topics: Amyloid beta-Peptides; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Ginkgolides; Glutathione; Humans; Hydrogen Peroxide; Lactones; Lipid Peroxidation; Neuroblastoma; Neurons; Nitric Oxide; Oxidative Stress; Peptide Fragments; Phytochemicals; Reactive Nitrogen Species; Reactive Oxygen Species; Superoxide Dismutase; Tretinoin

Minocycline is a tetracycline antibiotic increasingly recognized in psychiatry for its pleiotropic anti-inflammatory and neuroprotective potential. While underlying mechanisms are still incompletely understood, several lines of evidence suggest a relevant functional overlap with retinoic acid (RA), a highly potent small molecule exhibiting a great variety of anti-inflammatory and neuroprotective properties in the adult central nervous system (CNS). RA homeostasis in the adult CNS is tightly controlled through local RA synthesis and cytochrome P450 (CYP450)-mediated inactivation of RA. Here, we hypothesized that minocycline may directly affect RA homeostasis in the CNS via altering local RA degradation.. We used in vitro RA metabolism assays with metabolically competent synaptosomal preparations from murine brain and human SH-SY5Y neuronal cells as well as viable human SH-SY5Y neuroblastoma cell cultures.. We revealed that minocycline potently blocks RA degradation as measured by reversed-phase high-performance liquid chromatography and in a viable RA reporter cell line, even at low micromolar levels of minocycline.. Our findings provide evidence for enhanced RA signalling to be involved in minocycline's pleiotropic mode of action in the CNS. This novel mode of action of minocycline may help in developing more specific and effective strategies in the treatment of neuroinflammatory or neurodegenerative disorders.

Topics: Animals; Brain; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Humans; Male; Minocycline; Neuroblastoma; Neurons; Rats; Rats, Wistar; Regression Analysis; Tretinoin

It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions.

Topics: Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Humans; Neural Stem Cells; Neuroblastoma; Neurons; Tretinoin

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K(+) depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells' population growth by inducing maturation and differentiation.

Topics: Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholesterol; Estradiol; Humans; Intermediate Filaments; Neuroblastoma; Neurons; Synaptic Vesicles; Tretinoin

Topics: Acetylcysteine; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Humans; Models, Biological; Neuroblastoma; NF-E2-Related Factor 2; Phosphorylation; Proto-Oncogene Proteins c-akt; Tretinoin

Retinoic acid induces differentiation in various types of cells including skeletal myoblasts and neuroblasts and maintains differentiation of epithelial cells. The present study demonstrates synthesis and screening of a library of retinoic acid-triazolyl derivatives for their differentiation potential on neuroblastoma cells. Click chemistry approach using copper(I)-catalyzed azide-alkyne cycloaddition was adopted for the preparation of these derivatives. The neurite outgrowth promoting potential of retinoic acid-triazolyl derivatives was studied on neuroblastoma cells. Morphological examination revealed that compounds 8a, 8e, 8f, and 8k, among the various derivatives screened, exhibited promising neurite-outgrowth inducing activity at a concentration of 10 μM compared to undifferentiated and retinoic acid treated cells. Further on, to confirm this differentiation potential of these compounds, neuroblastoma cells were probed for expression of neuronal markers such as NF-H and NeuN. The results revealed a marked increase in the NF-H and NeuN protein expression when treated with 8a, 8e, 8f, and 8k compared to undifferentiated and retinoic acid treated cells. Thus, these compounds could act as potential leads in inducing neuronal differentiation for future studies.

Topics: Animals; Azides; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Mass Spectrometry; Mice; Neuroblastoma; Phosphopyruvate Hydratase; Tretinoin; Triazoles

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

Topics: Adolescent; Adrenergic Neurons; Adult; Azacitidine; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Child; Child, Preschool; CpG Islands; Decitabine; DNA Methylation; Dopamine beta-Hydroxylase; Down-Regulation; Gene Knockdown Techniques; Humans; Infant; Infant, Newborn; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Prognosis; Promoter Regions, Genetic; Repressor Proteins; RNA, Small Interfering; Transcription Factor AP-2; Tretinoin; Tyrosine 3-Monooxygenase; Up-Regulation; Young Adult

Neuroblastoma is the most common extra-cranial solid tumor in childhood; and patients in stage IV of the disease have a high propensity for tumor recurrence. Retinoid therapy has been utilized as a means to induce differentiation of tumor cells and to inhibit relapse. In this study, the expression of a common neuronal differentiation marker [neurofilament M (NF-M)] in human SK-N-SH neuroblastoma cells treated with 10μM all-trans retinoic acid (ATRA) showed significantly increased expression in accordance with reduced cell number. This was accompanied by an increase in MitoSOX and DCFH2 oxidation that could be indicative of increased steady-state levels of reactive oxygen species (ROS) such as O2(•-) and H2O2, which correlated with increased levels of MnSOD activity and immuno-reactive protein. Furthermore PEG-catalase inhibited the DCFH2 oxidation signal to a greater extent in the ATRA-treated cells (relative to controls) at 96h indicating that as the cells became more differentiated, steady-state levels of H2O2 increased in the absence of increases in peroxide-scavenging antioxidants (i.e., glutathione, glutathione peroxidase, and catalase). In addition, ATRA-induced stimulation of NF-M at 48 and 72h was enhanced by decreasing SOD activity using siRNA directed at MnSOD. Finally, treatment with ATRA for 96h in the presence of MnSOD siRNA or PEG-catalase inhibited ATRA induced increases in NF-M expression. These results provide strong support for the hypothesis that changes in steady-state levels of O2(•-) and H2O2 significantly contribute to the process of ATRA-induced differentiation in neuroblastoma, and suggest that retinoid therapy for neuroblastoma could potentially be enhanced by redox-based manipulations of superoxide metabolism to improve patient outcome.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Fluoresceins; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Neuroblastoma; Neurofilament Proteins; Oxidation-Reduction; SOX Transcription Factors; Tretinoin

Neuroblastoma cell lines can differentiate upon treatment with retinoic acid (RA), a finding that provided the basis for the clinical use of RA to treat neuroblastoma. However, resistance to RA is often observed, which limits its clinical utility. Using a gain-of-function genetic screen, we identified an unexpected link between RA signaling and mastermind-like 3 (MAML3), a known transcriptional coactivator for NOTCH. Our findings indicate that MAML3 expression leads to the loss of activation of a subset of RA target genes, which hampers RA-induced differentiation and promotes resistance to RA. The regulatory DNA elements of this subset of RA target genes show overlap in binding of MAML3 and the RA receptor, suggesting a direct role for MAML3 in the regulation of these genes. In addition, MAML3 has RA-independent functions, including the activation of IGF1R and downstream AKT signaling via upregulation of IGF2, resulting in increased proliferation. These results demonstrate an important mechanistic role for MAML3 in proliferation and RA-mediated differentiation.. MAML3 coordinates transcription regulation with receptor tyrosine kinase pathway activation, shedding new light on why this gene is mutated in multiple cancers. Mol Cancer Res; 14(5); 411-22. ©2016 AACR.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Nuclear Proteins; Signal Transduction; Trans-Activators; Transcription Factors; Tretinoin

PHOX2B and its paralogue gene PHOX2A are two homeodomain proteins in the network regulating the development of autonomic ganglia that have been associated with the pathogenesis of neuroblastoma (NB), because of their over-expression in different NB cell lines and tumour samples. We used the SK-N-BE(2)C cell line to show that all-trans retinoic acid (ATRA), a drug that is widely used to inhibit growth and induce differentiation in NBs, regulates both PHOX2A and PHOX2B expression, albeit by means of different mechanisms: it up-regulates PHOX2A and down-regulates PHOX2B. Both mechanisms act at transcriptional level, but prolonged ATRA treatment selectively degrades the PHOX2A protein, whereas the corresponding mRNA remains up-regulated. Further, we show that PHOX2A is capable of modulating PHOX2B expression, but this mechanism is not involved in the PHOX2B down-regulation induced by retinoic acid. Our findings demonstrate that PHOX2A expression is finely controlled during retinoic acid differentiation and this, together with PHOX2B down-regulation, reinforces the idea that they may be useful biomarkers for NB staging, prognosis and treatment decision making.

Topics: Base Sequence; Cell Differentiation; Cell Line, Tumor; Enzyme Induction; Enzyme Repression; Homeodomain Proteins; Humans; Neuroblastoma; Proteasome Endopeptidase Complex; Proteolysis; Response Elements; Transcription Factors; Transcription, Genetic; Tretinoin

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 μM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

Topics: Amino Acid Transport System y+; Antioxidant Response Elements; Bortezomib; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutamate-Cysteine Ligase; Glutathione; Heme Oxygenase-1; Humans; Neuroblastoma; NF-E2-Related Factor 2; Proteasome Endopeptidase Complex; Protein Subunits; RNA, Messenger; Transcription, Genetic; Tretinoin; Up-Regulation

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells.

Topics: Apoptosis; Botulinum Toxins; Cell Differentiation; Cell Line, Tumor; Genetic Therapy; Humans; Neuroblastoma; Phenotype; Protein Synthesis Inhibitors; Qb-SNARE Proteins; Qc-SNARE Proteins; Signal Transduction; Synaptosomal-Associated Protein 25; Syntaxin 1; Transduction, Genetic; Tretinoin

Retinoic acid, the bioactive metabolite of beta-carotene or vitamin A, plays a pleiotropic, multifunctional role in vertebrate development. Studies in rodents revealed that a diet deficient in vitamin A results in a complex neonatal syndrome (the VAD syndrome), manifested in many organs. In humans, the function of retinoic acid (RA) extends into adulthood, where it has important roles in fertility, vision, and suppression of neoplastic growth. In recent years, it has also been suggested that retinoic acid might potentially act as a therapeutically relevant drug in attenuating or even preventing neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report that VAD leads to an increase in A-beta peptide levels while only minor effects were observed on expression levels of the amyloid precursor protein (APP) processing proteinases in wild type mice. In line with these findings, rescue of hypovitaminosis reduced A-beta amount to baseline and induced sApp-alpha secretion in combination with an increase of alpha-secretase Adam10. By comparing retinoic acid treatment starting from a full nutrition status and a "VAD" situation in human neuroblastoma cells, we show that while intensities of differential gene expression were higher in replenished cells, a large overlap in AD-related, regulated genes was observed. Our data suggest that hypovitaminosis A can contribute to onset or progression of AD by increasing synthesis of A-beta peptides and that several AD-related genes such as ADAM10 or BDNF are regulated by retinoic acid. We suggest that dietary supplementation with retinoic acid derivatives is likely to have a beneficial effect on AD-pathology in individuals with hypovitaminosis and patients with normal vitamin A status.

Topics: Acitretin; ADAM10 Protein; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Female; Gene Regulatory Networks; Humans; Keratolytic Agents; Mice; Neuroblastoma; Neurons; Peptide Fragments; Presenilin-2; Rats, Wistar; Tretinoin; Vitamin A Deficiency

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

Topics: Animals; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; MAP Kinase Signaling System; Mice; Neuroblastoma; Prognosis; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Stress, Physiological; Transcriptional Activation; Tretinoin

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

Topics: Antigens, Differentiation; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Humans; Neurites; Neuroblastoma; Stromal Cells; Tretinoin

Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.

Topics: Antineoplastic Agents; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Proteomics; Pyrimidinones; RNA Interference; Survival Analysis; Tretinoin; Wnt Proteins; Wnt Signaling Pathway

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2

Topics: Animals; Cell Line, Tumor; Child; Combined Modality Therapy; Gangliosides; Humans; Immunotherapy, Adoptive; Mice, Inbred NOD; Myeloid-Derived Suppressor Cells; Neuroblastoma; Receptors, Antigen, T-Cell; Sarcoma; T-Lymphocytes; Treatment Outcome; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

In the present study, the changes in the cell viability at different concentrations of hydrogen peroxide (H2O2) for 3 h used to establish a model of oxidative stress. Further assays with 200 μM H2O2 induces significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), reactive oxygen species (ROS) and calcium ion (Ca(2+)) in neuronal cells, but insulin can effectively diminish the oxidative damages. Moreover, cells treated with insulin increased the H2O2-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of Akt, Bcl-2, Bax, IRβ, IGF-1Rβ, IRS-1 and IRS-2 showed that insulin treatment had a protective effect on H2O2-induced oxidative stress in RA-differentiated SH-SY5Y neuroblastoma cells.

Topics: Apoptosis; Calcium; Cell Differentiation; Cell Line, Tumor; Cell Survival; Humans; Hydrogen Peroxide; Insulin; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Tretinoin

CYLD is a deubiquitinating (DUB) enzyme that has a pivotal role in modulating nuclear factor kappa B (NF-κB) signaling pathways by removing the lysine 63- and linear-linked ubiquitin chain from substrates such as tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Loss of CYLD activity is associated with tumorigenicity, and levels of CYLD are lost or downregulated in different types of human tumors. In the present study, we found that high CYLD expression was associated with better overall survival and relapse-free neuroblastoma patient outcome, as well as inversely correlated with the stage of neuroblastoma. Retinoic acid-mediated differentiation of neuroblastoma restored CYLD expression and promoted SUMOylation of CYLD. This posttranslational modification inhibited deubiquitinase activity of CYLD against TRAF2 and TRAF6 and facilitated NF-κB signaling. Overexpression of non-SUMOylatable mutant CYLD in neuroblastoma cells reduced retinoic acid-induced NF-κB activation and differentiation of cells, but instead promoted cell death.

Topics: Antineoplastic Agents; Cell Line, Tumor; Deubiquitinating Enzyme CYLD; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neuroblastoma; NF-kappa B; Signal Transduction; Sumoylation; TNF Receptor-Associated Factor 2; TNF Receptor-Associated Factor 6; Tretinoin; Tumor Suppressor Proteins; Ubiquitination

Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

Topics: Amyloid beta-Protein Precursor; Axons; Cell Line, Tumor; Culture Media, Serum-Free; Cytosol; High-Temperature Requirement A Serine Peptidase 2; Humans; Interleukin-1beta; Intermediate Filaments; Isothiocyanates; Mitochondria; Mitochondrial Proteins; Mutation, Missense; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Neurogenesis; Neurons; Point Mutation; Pyrimidinones; Serine Endopeptidases; Serine Proteinase Inhibitors; Stress, Physiological; Sulfoxides; Thiones; Transfection; Tretinoin; Tunicamycin; Vimentin

Neuroblastoma is a common solid malignant tumor of the sympathetic nervous system, which contributes to 15% of cancer‑related mortality in children. The differentiation status of neuroblastoma is correlated with clinical outcome, and the induction of differentiation thus constitutes a therapeutic approach in this disease. However, the molecular mechanisms that control the differentiation of neuroblastoma remain poorly understood. The present study aimed to define whether GATA3 is involved in the differentiation of neuroblastoma cells. The results demonstrated that GATA3 is a prognostic marker for survival in patients with neuroblastoma, and that high‑level GATA3 expression is associated with increased self‑renewal and proliferation of neuroblastoma cells. Retinoic acid treatment led to GATA3 downregulation together with neuronal differentiation, suppression of cell proliferation and inhibition of tumorigenecity in neuroblastoma cells. These findings suggest that GATA3 is a key regulator of neuroblastoma differentiation, and provide a novel potential therapeutic strategy for the induction of neuroblastoma differentiation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Databases, Factual; GATA3 Transcription Factor; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Tretinoin; Up-Regulation; Xenograft Model Antitumor Assays

Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation.

Topics: Animals; Apoptosis; Camptothecin; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Humans; Mice; Mice, Transgenic; N-Myc Proto-Oncogene Protein; Neuroblastoma; Prognosis; Proteins; Proto-Oncogene Proteins; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering; Tretinoin; Tumor Cells, Cultured

The alteration of ROS level is frequently observed in the course of morphine addiction, and ROS is proverbially involved in this process. This study aims to explore the relationship among morphine addiction, reactive oxygen species (ROS) and expression of μ-opioid receptor (MOR) in differentiated SH-SY5Y cells.. SH-SY5Y cells were induced to differentiation by treatment with retinoic acid (RA); the activity of lactate dehydrogenase (LDH) and the nitro blue tetrazolium (NBT) reduction were assessed by spectrophotometry. Intracellular reactive oxygen species (ROS) was measured with the 2,7-dichlorofluorescin diacetate (DCFH-DA) assay. Cellular cAMP was determined by using a competitive protein binding kit. The mRNA expression of μ-opioid receptor (MOR) was evaluated by qRT-PCR.. Morphine-induced ROS are generated in a concentration- and time-dependent manner and inhibited by naloxone. Exogenous oxidants increase the level of ROS and aggravate morphine addiction, while the exogenous antioxidants efficiently reverse these effects. Morphine decreases the mRNA level of MOR in a concentration-dependent manner. And the mRNA level of MOR is remarkably reduced in the presence of exogenous oxidants and effectively promoted by antioxidants.. This study indicates that ROS can affect morphine addiction through involving MOR. Treatment with ROS scavenging can serve as a medical therapy for morphine addiction.

Topics: Analgesics, Opioid; Antioxidants; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Humans; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Neuroblastoma; Reactive Oxygen Species; Receptors, Opioid, mu; RNA, Messenger; Time Factors; Tretinoin

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Survival; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Mice; Mice, Nude; Neuroblastoma; Repressor Proteins; Tretinoin; Xenograft Model Antitumor Assays

The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding dopaminergic differentiation.

Topics: Animals; Cell Differentiation; Cell Fractionation; Cell Line, Transformed; Cell Line, Tumor; Dopaminergic Neurons; Glycogen Synthase Kinase 3; Humans; Nerve Tissue Proteins; Neuroblastoma; NFATC Transcription Factors; Phenotype; Protein Serine-Threonine Kinases; Rats; Receptors, Estrogen; Signal Transduction; Tretinoin; Tyrosine 3-Monooxygenase; Up-Regulation

This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling.

Topics: Acrylamide; Butyric Acid; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Janus Kinases; Neurites; Neuroblastoma; Phosphorylation; Signal Transduction; STAT Transcription Factors; Tretinoin

Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Gene Silencing; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neuroblastoma; Neuroglia; Treatment Outcome; Tretinoin

Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo.

Topics: Animals; Apoptosis; Caspase 3; Cell Differentiation; Cells, Cultured; Diarylheptanoids; Embryo, Mammalian; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hippocampus; Male; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurons; Phosphatidylinositol 3-Kinases; Rats; Signal Transduction; Time Factors; Tretinoin

Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines.. NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies.. Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor.. Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

Topics: Cell Differentiation; Cell Line, Tumor; DNA Helicases; Gene Expression Regulation, Neoplastic; Humans; Nerve Growth Factor; Nerve Tissue Proteins; Neuroblastoma; Receptor, trkA; Tretinoin; Up-Regulation

Obtaining a suitable experimental cellular model is a major problem for neuroscience studies. Neuroblastoma cell lines have been often applied in studies related to pathological disorders of nervous system. However, in the search for an ideal model, these cells must be differentiated to cancel their tumor character. The subsequent reactions that are caused by differentiation are not always indifferent to the same model. We evaluated the effect of two well known substances, used for SH-N-SK cell line differentiation, retinoic acid (RA) and phorbol-12-myristate-13-acetate (PMA), on the induction of pro-inflammatory and pro-oxidative reactions in these cells. Cells differentiated with PMA were able to produce significantly higher amounts of pro-inflammatory cytokines whereas the release of nitric oxide radicals was similar to that in undifferentiated cells. On the contrary, in RA-differentiated cells no significant changes in cytokine production were observed and the nitric oxide release was decreased. Additionally, the RA-differentiated neuronal model was more sensible to lipopolysaccharide stimulation, producing pro-inflammatory cytokines abundantly. These results suggest that RA-differentiated SH-N-SK cells provide a more suitable experimental model for the study of molecular and cellular mechanisms of the inflammation and oxidative stress in neuronal cells.

Topics: Antineoplastic Agents; Caspase 3; Caspase 7; Cell Differentiation; Cell Line, Tumor; Cytokines; Free Radicals; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Neuroblastoma; Neurons; Nitric Oxide; Nitrites; Oxidative Stress; Oxygen; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin

Ouabain stimulates activation of various signaling cascades such as protein kinase B (Akt) and Extracellular-signaling-regulated kinase 1/2 (ERK 1/2) in various cell lines. Retinoic acid (RA) is commonly used to induce neuroblastoma differentiation in cultures. Upon RA administration, human neuroblastoma cell line, SK-N-SH demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Here we report that ouabain-induced signaling is altered under the action of 1 μM RA in human neuroblastoma SK-N-SH cells. RA increased the expression of p110α subunit of phosphoinositide 3-kinase (PI3K), Akt and β1 subunit of Na(+)/K(+)-ATPase. Ouabain activated Akt and ERK 1/2 in differentiated SK-N-SH cells; this effect was not observed in non-differentiated SK-N-SH cells. Long-term incubation of non-differentiated SK-N-SH with 1 μM ouabain led to a decrease in the number of cells; this effect was reduced in differentiated SK-N-SH cells. Taken together, these results suggest that ouabain leads to cell death in neuroblastoma cells rather than neuronal cells due to the different response to ouabain manifested by activation of Akt and ERK 1/2.. • RA increases the expression of p110α subunit of PI3K, Akt and β1 subunit of Na(+)/K(+)-ATPase • Ouabain induces activation of Akt and ERK 1/2 in differentiated SK-N-SH cells but not in non-differentiated cells • 1 μM ouabain leads to a decrease in the number of cells in non-differentiated SK-N-SH • Reduction of ouabain-induced cell death in differentiated SK-N-SH.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Humans; Neuroblastoma; Neurogenesis; Neurons; Ouabain; Signal Transduction; Time; Tretinoin

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

Topics: Catecholamines; Cell Differentiation; Cell Line, Tumor; Humans; Neuroblastoma; Staurosporine; Tetradecanoylphorbol Acetate; Tretinoin

Neuroblastoma is the most common and one of the deadliest among pediatric tumors; however, a subset of infants with neuroblastoma display spontaneous regression. The mechanism of spontaneous regression remains to be elucidated. TrkA plays an essential role in the differentiation and functionality of neurons; abundant TrkA expression is associated with favorable prognosis of neuroblastoma. All-trans retinoic acid (ATRA), a first-line drug for acute promyelocytic leukemia (APL) treatment, has been shown to induce differentiation and inhibit cell growth. Neuroblastoma tissues in our hospital inpatient were collected, primary cell culture was performed, and the cells were separated and purified to be cell line. Trypan blue exclusion was used to count the numbers of cells alive, morphological changes were observed under the phase-contrast microscope. RT-PCR was used to determine the expression level of TrkA. In this study, a human neuroblastoma cell line was successfully established; in addition, we demonstrated that ATRA induces growth arrest and promotes the differentiation of neuroblastoma cells. In addition, ATRA was shown to significantly increase the levels of TrkA mRNA expression. Therefore, we concluded that the elevated expression of the TrkA receptor is associated with ATRA-induced growth arrest and differentiation o neuroblastoma cells. The results of this study provide a theoretical basis for the clinical application of differentiation-inducing ATRA for neuroblastoma therapy.

Topics: Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Gene Expression; Humans; Neuroblastoma; Receptor, trkA; RNA, Messenger; Tretinoin

Ellagic acid has previously been reported to induce reduced proliferation and activation of apoptosis in several tumor cell lines including our own previous data from non-differentiated human neuroblastoma SH-SY5Y cells. The aim of this study was now to investigate if in vitro differentiation with the phorbol ester 12-O- tetradecanoylphorbol-13-acetate or the vitamin A derivative all-trans retinoic acid altered the sensitivity to ellagic acid in SH-SY5Y cells. The methods used were cell counting and LDH-assay for evaluation of cell number and cell death, flow cytometric analysis of SubG1- and TUNEL-analysis for apoptosis and western blot for expression of apoptosis-associated proteins. In vitro differentiation was shown to reduce the sensitivity to ellagic acid with respect to cell detachment, loss of viability and activation of apoptosis. The protective effect was phenotype-specific and most prominent in all-trans retinoic acid-differentiated cultures. Differentiation-dependent up-regulation of Bcl-2 and integrin expression is introduced as possible protective mechanisms. The presented data also point to a positive correlation between proliferative activity and sensitivity to ellagic-acid-induced cell detachment. In conclusion, the presented data emphasize the need to consider degree of neuronal differentiation and phenotype of neuroblastoma cells when discussing a potential pharmaceutical application of ellagic acid in tumor treatment.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Survival; Ellagic Acid; Flow Cytometry; Humans; In Situ Nick-End Labeling; Integrins; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Tretinoin; Up-Regulation

This study aimed to detect the protein expression of HA117 in pediatric solid tumors. Immunohistochemistry was performed to detect the expression of HA117 and P-gp in pediatric solid tumors. In Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), nephroblastoma (WT), and neuroblastoma (NB), the positive expression rate of HA117 was 65.4%, 58.3%, 81.3%, and 74.1%, and that of P-gp was 57.7%, 70.8%, 65.6%, and 66.7%, respectively. HA117 expression was closely related to the clinical stage of HL (P=0.004) and to the International Prognostic Index score, mediastinal lesions, and clinical stages of NHL (P=0.01, 0.03, and 0.01). The expression of HA117 in WT was higher than in adjacent normal tissues, but there was no statistical significance (P=0.21). The positive expression of HA117 in NB was markedly higher than that in normal tissues (P=0.002), which closely associated with histologic type and lymph node metastasis (P=0.03 and 0.001). Spearman correlation analysis revealed that HA117 expression was not correlated with P-gp in these 4 tumors. This suggests that HA117 might be an important resistance gene in pediatric solid tumors. The mechanism underlying the resistance to all-trans retinoic acid conferred by HA117 is different from that of P-gp.

Topics: Adolescent; Antineoplastic Agents; Child; Child, Preschool; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Hodgkin Disease; Humans; Kidney Neoplasms; Lymphoma, Non-Hodgkin; Male; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Nervous System Neoplasms; Neuroblastoma; Tretinoin; Wilms Tumor

In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson's disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinson's disease.

Topics: alpha-Synuclein; Aminoimidazole Carboxamide; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Cell Death; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Culture Media, Conditioned; DNA Fragmentation; Embryo, Mammalian; Humans; Hypoglycemic Agents; Metformin; Neuroblastoma; Neurons; Protein Serine-Threonine Kinases; Rats; Ribonucleotides; RNA, Small Interfering; Tretinoin

A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration.. We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics.. In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line.. In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations.. Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Keratolytic Agents; Models, Biological; Neuroblastoma; Receptors, Tumor Necrosis Factor, Type I; Time Factors; Tretinoin

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

Topics: Amyloid beta-Peptides; Brain-Derived Neurotrophic Factor; Cell Communication; Cell Differentiation; Cell Line, Transformed; Coculture Techniques; Extracellular Matrix; Green Fluorescent Proteins; Humans; Lysosomes; Membrane Potential, Mitochondrial; Nerve Growth Factor; Neuregulin-1; Neurites; Neuroblastoma; Peptide Fragments; Protein Isoforms; Time Factors; Tretinoin

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

Topics: Apoptosis; Biomarkers, Tumor; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; N-Myc Proto-Oncogene Protein; Neural Stem Cells; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; RNA Interference; Time Factors; Transfection; Tretinoin

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor with up to 50% of NB patients classified as having high-risk disease with poor long-term survival rates. The poor clinical outcome and aggressiveness of high-risk NB strongly correlates with enhanced angiogenesis, suggesting anti-angiogenic agents as attractive additions to the currently insufficient therapeutics. TL-118, a novel drug combination has been recently developed to inhibit tumor angiogenesis. In the current study, we used the SK-N-BE (2) cell line to generate orthotopic NB tumors in order to study the combinational therapeutic potential of TL-118 with either Gemcitabine (40 mg/kg; IP) or Retinoic acid (40 mg/kg; IP). We show that TL-118 treatment (n = 9) significantly inhibited tumor growth, increased cell apoptosis, reduced proliferation and extended mouse survival. Moreover, the reciprocal effect of TL-118 and Gemcitabine treatment (n = 10) demonstrated improved anti-tumor activity. The synergistic effect of these drugs in combination was more effective than either TL or Gemcitabine alone (n = 9), via significantly reduced cell proliferation (p<0.005), increased apoptosis (p<0.05) and significantly prolonged survival (2-fold; p<0.00001). To conclude, we demonstrate that the novel drug combination TL-118 has the ability to suppress the growth of an aggressive NB tumor. The promising results with TL-118 in this aggressive animal model may imply that this drug combination has therapeutic potential in the clinical setting.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cimetidine; Cyclophosphamide; Deoxycytidine; Diclofenac; Drug Administration Schedule; Drug Combinations; Drug Synergism; Gemcitabine; Gene Amplification; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Male; Mice, Inbred NOD; Mice, SCID; N-Myc Proto-Oncogene Protein; Neovascularization, Pathologic; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Platelet Endothelial Cell Adhesion Molecule-1; Sulfasalazine; Treatment Outcome; Tretinoin; Tumor Burden; Xenograft Model Antitumor Assays

The microtubule (MT) system is important for many aspects of neuronal function, including motility, differentiation, and cargo trafficking. Parkinson's disease (PD) is associated with increased oxidative stress and alterations in the integrity of the axodendritic tree. To study dynamic mechanisms underlying the neurite shortening phenotype observed in many PD models, we employed the well-characterized oxidative parkinsonian neurotoxin, 6-hydroxydopamine (6OHDA). In both acute and chronic sub-lethal settings, 6OHDA-induced oxidative stress elicited significant alterations in MT dynamics, including reductions in MT growth rate, increased frequency of MT pauses/retractions, and increased levels of tubulin acetylation. Interestingly, 6OHDA decreased the activity of tubulin deacetylases, specifically sirtuin 2 (SIRT2), through more than one mechanism. Restoration of tubulin deacetylase function rescued the changes in MT dynamics and prevented neurite shortening in neuron-differentiated, 6OHDA-treated cells. These data indicate that impaired tubulin deacetylation contributes to altered MT dynamics in oxidatively-stressed cells, conferring key insights for potential therapeutic strategies to correct MT-related deficits contributing to neuronal aging and disease.

Topics: Cell Line, Tumor; Cell Proliferation; Complement C1; Histone Deacetylase 6; Histone Deacetylases; Humans; L-Lactate Dehydrogenase; Luminescent Proteins; Microtubule-Associated Proteins; Microtubules; Neurites; Neuroblastoma; Neurons; Neurotoxins; Oxidative Stress; Oxidopamine; Reactive Oxygen Species; Sirtuin 2; Time Factors; Tretinoin

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells.

Topics: Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Tumor; Chromatin Immunoprecipitation; DNA Topoisomerases, Type II; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Keratolytic Agents; Neurites; Neuroblastoma; Neurons; Protein Binding; RNA, Messenger; Sp1 Transcription Factor; Tretinoin

Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.

Topics: Adolescent; Adult; Antimetabolites, Antineoplastic; Azacitidine; Cell Differentiation; Cell Line, Tumor; Child; Child, Preschool; Cyclin-Dependent Kinase Inhibitor p19; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Infant, Newborn; Male; N-Myc Proto-Oncogene Protein; Neoplasm Staging; Nervous System Neoplasms; Neuroblastoma; Neurons; Nuclear Proteins; Oncogene Proteins; Signal Transduction; Survival Analysis; Tretinoin

Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor Curcumin (50 μM), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 μM). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Survival; Ciliary Neurotrophic Factor; Humans; Immunohistochemistry; Neuroblastoma; Neurons; Signal Transduction; Tretinoin

Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Gene Expression; Genes, Homeobox; Hep G2 Cells; Homeodomain Proteins; Humans; Neuroblastoma; Neurons; Trans-Activators; Transcription Factors; Transcription, Genetic; Tretinoin

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

Topics: Animals; Anti-Bacterial Agents; Apoptosis Regulatory Proteins; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Doxycycline; Female; Humans; Membrane Proteins; Mice; Mice, Nude; N-Myc Proto-Oncogene Protein; Neoplasm Metastasis; Neoplasm Staging; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Receptors, Death Domain; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transplantation, Heterologous; Tretinoin; Up-Regulation

We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Amyloid Precursor Protein Secretases; Cell Differentiation; Cell Line, Tumor; Humans; Membrane Glycoproteins; Membrane Proteins; Molecular Sequence Data; Neoplasm Proteins; Neuroblastoma; Peptide Fragments; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteolysis; Tretinoin; Tumor Stem Cell Assay

Ultra-conserved regions (UCRs) are segments of the genome (≥ 200 bp) that exhibit 100% DNA sequence conservation between human, mouse and rat. Transcribed UCRs (T-UCRs) have been shown to be differentially expressed in cancers versus normal tissue, indicating a possible role in carcinogenesis. All-trans-retinoic acid (ATRA) causes some neuroblastoma (NB) cell lines to undergo differentiation and leads to a significant decrease in the oncogenic transcription factor MYCN. Here, we examine the impact of ATRA treatment on T-UCR expression and investigate the biological significance of these changes.. We designed a custom tiling microarray to profile the expression of 481 T-UCRs in sense and anti-sense orientation (962 potential transcripts) in untreated and ATRA-treated neuroblastoma cell lines (SH-SY5Y, SK-N-BE, LAN-5). Following identification of significantly differentially expressed T-UCRs, we carried out siRNA knockdown and gene expression microarray analysis to investigate putative functional roles for selected T-UCRs.. Following ATRA-induced differentiation, 32 T-UCRs were differentially expressed (16 up-regulated, 16 down-regulated) across all three cell lines. Further insight into the possible role of T-UC.300A, an independent transcript whose expression is down-regulated following ATRA was achieved by siRNA knockdown, resulting in the decreased viability and invasiveness of ATRA-responsive cell lines. Gene expression microarray analysis following knockdown of T-UC.300A revealed a number of genes whose expression was altered by changing T-UC.300A levels and that might play a role in the increased proliferation and invasion of NB cells prior to ATRA-treatment.. Our results indicate that significant numbers of T-UCRs have altered expression levels in response to ATRA. While the precise roles that T-UCRs might play in cancer or in normal development are largely unknown and an important area for future study, our findings strongly indicate that the function of non-coding RNA T-UC.300A is connected with proliferation, invasion and the inhibition of differentiation of neuroblastoma cell lines prior to ATRA treatment.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cluster Analysis; Conserved Sequence; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Grading; Neuroblastoma; Reproducibility of Results; RNA Interference; RNA, Untranslated; Transcription, Genetic; Tretinoin

Currently IFN-α is widely used for effective treatment of viral infections and several malignancies. However, IFN-α can cause neuropsychiatric disturbances and mental impairments, including fatigue, insomnia, depression, irritability and cognitive deficits. Molecular and cellular mechanisms leading to such side-effects are still poorly understood. Neurons seem to be an important target in mediating cellular effects induced by exposure to this cytokine, but so far little is known about IFN-α-induced effects on these cells. We have investigated the ability of IFN-α (2-100 ng/ml) to induce damage and toxicity to the human neuroblastoma SH-SY5Y cell line, commonly used for studying such phenomena, and the mechanisms underlying these effects. After 24 h treatment, IFN-α increased mitochondrial activity, whereas cell density was reduced in a dose- and time-dependent manner. This effect did not depend on reduced cell proliferation, but rather the activation of apoptosis, as revealed by an increased Bax:Bcl-2 mRNA ratio after 72-h IFN-α exposure. At this time-point, IFN-α also reduced the expression of the brain-derived neurotrophic factor gene, and induced an increase in reactive oxygen species (ROS). A co-treatment with N-acetyl-cysteine (NAC; 5 mm), a potent antioxidant and mitochondrial modulator, was able to counteract all of these IFN-α-induced effects. These findings demonstrated that IFN-α induces neurotoxicity and apoptosis that is, in part, very likely due to mitochondrial damages and production of ROS. We suggest that NAC, already tested for the treatment of psychiatric disorders, may be useful to prevent IFN-α-induced central side-effects in a safe and effective way.

Topics: Acetylcysteine; Analysis of Variance; Apoptosis; bcl-2-Associated X Protein; Brain-Derived Neurotrophic Factor; Bromodeoxyuridine; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Neuroblastoma; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Time Factors; Tretinoin; Tumor Necrosis Factor-alpha

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG.. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Topics: Antineoplastic Agents; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Ionophores; Cell Differentiation; Cell Line, Tumor; Cell Size; Chemical Warfare Agents; Choline O-Acetyltransferase; Gene Expression Regulation; Glycine; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurotransmitter Agents; Phosgene; Potassium Chloride; Receptors, Cholinergic; Synapses; Synapsins; Tretinoin; Tritium; Tubulin

We screened for a Rab39-specific effector by performing a yeast two-hybrid assay with GTP-locked Rab39A/B as the bait and identified UACA (uveal autoantigen with coiled-coil domains and ankyrin repeats) as a specific Rab39A/B-binding protein. Deletion analysis revealed that a C-terminal coiled-coil domain of UACA functions as a GTP-dependent Rab39-binding domain. shRNA-mediated knockdown of endogenous Rab39A or UACA in mouse neuroblastoma Neuro2A cells resulted in a change in retinoic acid-induced neurite morphology from a multipolar morphology to a bipolar morphology. Taken together, these findings indicate that UACA functions as a Rab39A effector in the retinoic acid-induced differentiation of Neuro2A cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Green Fluorescent Proteins; Immunoblotting; Membrane Proteins; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Monomeric GTP-Binding Proteins; Neurites; Neuroblastoma; Protein Binding; rab GTP-Binding Proteins; RNA Interference; Tretinoin; Two-Hybrid System Techniques

To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Transcriptome; Tretinoin

SH-SY5Y, control, and Parkinson's disease (PD) cybrids prepared from an Indian population were differentiated using retinoic acid (RA) for understanding their dopaminergic characteristics and neuritogenesis. Undifferentiated control and PD cybrids exhibited higher levels of TH mRNA, but lower c-RET expression, short neurites, low neuritic density, and low proportion of cells with neurites compared with the undifferentiated parent cell line, SH-SY5Y. The expression levels of DAT and Ptx3 were similar to SH-SY5Y. PD cybrids showed poor viability and lower differentiating potency than SH-SY5Y or control cybrids. RA treatment for 6 days elevated c-RET expression and corrected the neuritic morphology of the control, but not of PD cybrids. Cell viability was found to be reduced in differentiated control and PD cybrids. TH expression level was significantly elevated in SH-SY5Y following RA treatment, but not in both the cybrids. In differentiated control and PD cybrids, the TH immunofluorescence intensity was significantly lower compared with SH-SY5Y cells. MitoTracker Green fluorescence intensity of the mitochondria was higher in differentiated PD cybrids. Dopamine released into the medium was unaffected in the differentiated SH-SY5Y or in the control cybrids but was significantly elevated in PD cybrids. These results suggest that PD cybrids, differentiated or undifferentiated, maintained morphological and biochemical phenotypes significantly different from those of the control cybrids, or the differentiated SH-SY5Y cells, and therefore could be an ideal cellular model of the disease for pharmacological screening of drugs and for investigation of the pathophysiology of PD.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Survival; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Neurites; Neuroblastoma; Parkinson Disease; RNA, Messenger; Tretinoin; Tyrosine 3-Monooxygenase; Voltage-Dependent Anion Channels

While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Lactams; Neuroblastoma; Tretinoin

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures.

Topics: Antineoplastic Agents; Blotting, Western; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Humans; Neurites; Neuroblastoma; Polymerase Chain Reaction; Spheroids, Cellular; Tretinoin

Retinoic acid (RA) is used pharmacologically to treat neuroblastoma (NB), but its mechanism of action is unclear and it has limited use against refractory disease. This study investigated the expression of LSD1 (also known as KDM1A) in tumors, and assessed the efficacy of combining RA treatment with the inhibition of LSD1 expression.. LSD1 protein expression levels were assessed semi-quantitatively in specimens of NB and ganglioneuroblastoma (GNB), along with the apoptosis markers, Bcl-2 and Bax. The combined effect of RA and LSD1 siRNA inhibition on cell death was then assessed in the human neuroblastoma cell line, SH-SY5Y.. LSD1 expression was higher in NB compared to GNB, and LSD1 overexpression directly correlated with Bcl-2 expression and inversely correlated with Bax expression. RA treatment or LSD1 siRNA inhibition alone inhibited the growth of SH-SY5Y cells, but did not cause significant apoptosis or cell death. Combined treatment led to higher rates of SH-SY5Y cell death, as reflected by an increased Bax/Bcl-2 ratio.. The combined effect of RA and LSD1 siRNA inhibition had a synergistic effect on promoting the apoptosis of NB cells. This novel approach may improve the clinical treatment of NB.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Child; Child, Preschool; Female; Histone Demethylases; Humans; Infant; Male; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Tretinoin

Oxidative stress is an important cause of cellular toxicity in the central nervous system and contributes to the pathology associated with neurodegenerative disorders including Parkinson's disease. As such, elucidation of cellular mechanisms that enhance neuronal resistance to oxidative stress may provide new avenues for therapy. In this study we employed a simple two-state cellular model to identify genes that are associated with resistance to oxidative stress induced by 6-hydroxydopamine (6-OHDA). In this model, undifferentiated neuroblastoma cells display higher sensitivity to 6-OHDA than differentiated cells. By comparing the gene expression between these two states, we identified several genes whose expression is altered concomitant with changes in 6-OHDA sensitivity. This gene set includes cytokine receptor-like factor 1 (CRLF1), which is up-regulated during the differentiation process and has been previously implicated in neuroprotection. We show that the product of this gene is both necessary and sufficient for increased resistance to 6-OHDA in differentiated neuroblastoma cells, and that CRLF1 serves its protective role by a cell autonomous mechanism that is independent from its known role as a co-ligand for the ciliary neurotrophic factor receptor. These data provide an additional role for CRLF1 that could potentially explain its broad expression pattern and effects on cells lacking expression of this receptor.

Topics: Cell Differentiation; Cell Line, Tumor; Cytokine Receptor gp130; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Janus Kinase 1; Janus Kinase 2; Janus Kinases; Microscopy, Fluorescence; Neuroblastoma; Oxidopamine; Receptors, Cytokine; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 μM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neuroblastoma; PPAR alpha; Proto-Oncogene Mas; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tretinoin

Mutations in PTEN-induced putative kinase 1 (PINK1) gene are associated to early-onset recessive forms of Parkinson disease. PINK1 function is related to mitochondria homeostasis, but the molecular pathways in which PINK1 is involved are largely unknown. Here, we report the identification of the embryonic ectoderm development polycomb histone-methylation modulator (EED/WAIT1) as a PINK1-interacting and -regulated protein. The PINK1:EED/WAIT1 physical interaction was mediated by the PINK1 kinase domain and the EED/WAIT1 40 amino acid ending with tryptophan and aspartate (WD40)-repeat region, and PINK1 phosphorylated EED/WAIT1 in vitro. PINK1 associated with EED/WAIT1 in cells and relocated EED/WAIT1 to the mitochondria. This interaction reduced the trimethylation of lysine 27 from histone H3, which affected polycomb-regulated gene transcription during RA differentiation of SH-SY5Y human neuroblastoma cells. Our findings unveil a pathway by which PINK1 regulates histone methylation and gene expression through the polycomb repressor complex.

Topics: Animals; Antineoplastic Agents; Binding Sites; Cell Differentiation; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Gene Expression Regulation; HEK293 Cells; Histones; Humans; Immunoblotting; Lysine; Methylation; Mitochondria; Neuroblastoma; Phosphorylation; Polycomb Repressive Complex 2; Protein Binding; Protein Kinases; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Tretinoin; Two-Hybrid System Techniques

Dihydropyrimidinase-like proteins (DPYSLs) are a family of proteins developmentally regulated during maturation of the nervous system. Recently, members of the DPYSL family have been reported to be involved in cancer with low expression of DPYSL1 correlating with poor clinical outcomes in non-small cell lung cancer and functioning as a metastasis suppressor. Neuroblastoma (NB) is a tumor derived from precursor cells of the sympathetic nervous system and is the most common solid tumor in childhood. So far the biological functions of DPYSLs in NB remain elusive. Studying the potential roles of DPYSLs in NB may give us new insights into NB tumorigenesis. In the present study, using antibodies specific to different members of the DPYSL family, DPYSL1, DPYSL2 and DPYSL3, we investigated regulation of their expression and their subcellular distribution during retinoic acid (RA)-induced differentiation in NB cells. The correlation between DPYSLs and MYCN, a biomarker for poor prognosis of NB, was evaluated. We found that DPYSL3 levels increased during RA-induced cell differentiation. Downregulation of MYCN by small interfering RNA (siRNA) increased DPYSL3 levels, while upregulation of MYCN in non-MYCN NB cells decreased DPYSL3 levels. DPYSL1 and DPYSL2 expression didn't change during RA treatment or under different expression levels of MYCN. Moreover, a high level of DPYSL3 mRNA, but not that of DPYSL1 or DPYSL2 mRNA, was detected in tumors from advanced-stage NB that have a better survival. These data indicated that DPYSL3, not DPYSL1 or DPYSL2, is negatively regulated by MYCN and may be used as a potential biomarker for NB.

Topics: Biomarkers, Tumor; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Muscle Proteins; N-Myc Proto-Oncogene Protein; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Prognosis; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sympathetic Nervous System; Tretinoin; Up-Regulation

Human sialidase NEU4 long (N4L) is a membrane-associated enzyme that has been shown to be localized in the outer mitochondrial membrane. A role in different cellular processes has been suggested for this enzyme, such as apoptosis, neuronal differentiation and tumorigenesis. However, the molecular bases for these roles, not found in any of the other highly similar human sialidases, are not understood. We have found that a proline-rich sequence of 81 amino acids, unique to NEU4 sequence, contains potential Akt and Erk1 kinase motifs. Molecular modeling, based on the experimentally determined three-dimensional structure of cytosolic human NEU2, showed that the proline-rich sequence is accommodated in a loop, thus preserving the typical beta-barrel structure of sialidases. In order to investigate the role of this loop in neuronal differentiation, we obtained SK-N-BE neuroblastoma cells stably overexpressing either human wild-type N4L or a deletion mutant lacking the proline-rich loop. Our results demonstrate that the proline-rich region can also enhance cell proliferation and retinoic acid (RA)-induced neuronal differentiation and it is also involved in NEU4 interaction with Akt, as well as in substrate recognition, modifying directly or through the interaction with other protein(s) the enzyme specificity toward sialylated glycoprotein(s). On the whole, our results suggest that N4L could be a downstream component of the PI3K/Akt signaling pathway required for RA-induced differentiation of neuroblastoma SK-N-BE cells.

Topics: Cell Differentiation; Cell Proliferation; Humans; Models, Molecular; Neuraminidase; Neuroblastoma; Proline; Tretinoin; Tumor Cells, Cultured

Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.. To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.. Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Topics: Apoptosis; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; Leupeptins; Microscopy, Confocal; Neoplastic Stem Cells; Nestin; Neuroblastoma; Octamer Transcription Factor-3; Proteasome Inhibitors; Side-Population Cells; SOXB1 Transcription Factors; Time Factors; Tretinoin

Mammalian genomes are extensively transcribed producing thousands of long non-protein-coding RNAs (lncRNAs). The biological significance and function of the vast majority of lncRNAs remain unclear. Recent studies have implicated several lncRNAs as playing important roles in embryonic development and cancer progression. LncRNAs are characterized with different genomic architectures in relationship with their associated protein-coding genes. Our study aimed at bridging lncRNA architecture with dynamical patterns of their expression using differentiating human neuroblastoma cells model.. LncRNA expression was studied in a 120-hours timecourse of differentiation of human neuroblastoma SH-SY5Y cells into neurons upon treatment with retinoic acid (RA), the compound used for the treatment of neuroblastoma. A custom microarray chip was utilized to interrogate expression levels of 9,267 lncRNAs in the course of differentiation. We categorized lncRNAs into 19 architecture classes according to their position relatively to protein-coding genes. For each architecture class, dynamics of expression of lncRNAs was studied in association with their protein-coding partners. It allowed us to demonstrate positive correlation of lncRNAs with their associated protein-coding genes at bidirectional promoters and for sense-antisense transcript pairs. In contrast, lncRNAs located in the introns and downstream of the protein-coding genes were characterized with negative correlation modes. We further classified the lncRNAs by the temporal patterns of their expression dynamics. We found that intronic and bidirectional promoter architectures are associated with rapid RA-dependent induction or repression of the corresponding lncRNAs, followed by their constant expression. At the same time, lncRNAs expressed downstream of protein-coding genes are characterized by rapid induction, followed by transcriptional repression. Quantitative RT-PCR analysis confirmed the discovered functional modes for several selected lncRNAs associated with proteins involved in cancer and embryonic development.. This is the first report detailing dynamical changes of multiple lncRNAs during RA-induced neuroblastoma differentiation. Integration of genomic and transcriptomic levels of information allowed us to demonstrate specific behavior of lncRNAs organized in different genomic architectures. This study also provides a list of lncRNAs with possible roles in neuroblastoma.

Topics: Cell Differentiation; Cell Line, Tumor; Genomics; Humans; Neuroblastoma; Oligonucleotide Array Sequence Analysis; RNA, Long Noncoding; Transcriptome; Tretinoin

MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Computational Biology; DNA Methylation; Epigenesis, Genetic; Gene Regulatory Networks; Genomics; Humans; MicroRNAs; Neuroblastoma; SOXB1 Transcription Factors; Survival Analysis; Tretinoin

Retinoic acid (RA)-induced differentiation therapy is partially successful in neuroblastoma treatment. We found that a novel combination of vanadium-based PTP inhibitors with RA induced extensive differentiation in neuroblastoma cells. In contrast to RA alone, this led to either permanent differentiation or senescence after 14days of combined treatment followed by chemical removal. Senescence was dependent in part on synergistic AKT and ERK activation. p21 was also strongly induced, but in contrast to oncogene-induced senescence, p53 was not activated. Vanadium-based inhibitors thus serve strongly to enhance RA's ability to drive differentiation and a novel form of senescence in neuroblastoma cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Line, Tumor; Cellular Senescence; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Neuroblastoma; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; Signal Transduction; Tretinoin; Tumor Suppressor Protein p53

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons which leads to progressive paralysis and death by respiratory failure. Although the cause of sporadic ALS is still unknown, oxidative stress is suggested to play a major role in the pathogenesis of this disease and of the rare familial form, which often exhibits mutations of the superoxide dismutase 1 (SOD1) gene. Since enhanced iron levels are discussed to participate in oxidative stress and neuronal death, we analyzed the expression levels of Fe-related mRNAs in a cell culture ALS model with the G93A mutation of SOD1. We observed an increased total iron content in G93A-SOD1 SH-SY5Y neuroblastoma cells compared to wild-type (WT)-SOD1 cells. mRNA expression for transferrin receptor 1 (TfR1) and divalent metal transporter 1 was increased in G93A-SOD1 cells, which was in accordance with higher iron uptake. Experiments with the iron chelator deferoxamine revealed a normal reaction of WT and mutant cells to cytoplasmic iron depletion, i.e. TfR1 upregulation, suggesting a basically conserved function of the iron-responsive element/iron regulatory protein (IRE/IRP) pathway, designed to adapt gene expression to iron levels. Expression levels of mitoferrin 1 and 2, frataxin, and iron-sulfur cluster scaffold protein were also significantly increased in G93A-SOD1 cells, suggesting higher mitochondrial iron import and utilization in biosynthetic pathways within the mitochondria. Moreover, expression of these transcripts was further enhanced, if G93A-SOD1 cells were differentiated by retinoic acid (RA). Since RA treatment increased cytoplasmic reactive oxygen species (ROS) levels in these cells, an IRE/IRP independent, ROS-mediated mechanism may account for dysregulation of iron-related genes.

Topics: Cation Transport Proteins; Cell Differentiation; Cell Line, Tumor; Fluoresceins; Gene Expression Regulation, Neoplastic; Humans; Iron; Iron-Binding Proteins; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species; Receptors, Transferrin; RNA, Messenger; Superoxide Dismutase; Transfection; Tretinoin

Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca(2+) entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca(2+) sensor and store-operated Ca(2+) channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.

Topics: Antineoplastic Agents; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Cell Differentiation; Cell Proliferation; Fluorescent Antibody Technique; Humans; Ion Transport; Membrane Proteins; Neoplasm Proteins; Neuroblastoma; ORAI1 Protein; Stromal Interaction Molecule 1; Tretinoin; TRPC Cation Channels; Tumor Cells, Cultured

We previously reported that intermittent exposure to a 50‑Hz magnetic field (MF) at 100 µT stimulates cell proliferation in the human neuroblastoma cell line NB69. The present study aimed to investigate whether the magnetic field-induced growth promotion also occurs at a lower magnetic flux density of 10 µT. To this purpose, NB69 cells were subjected for 42 h to intermittent exposure, 3 h on/3 h off, to a 50‑Hz MF at a 10 or 100 µT magnetic flux density. The field exposure took place either in the presence or in the absence of the antiproliferative agent retinoic acid. At the end of the treatment and/or incubation period, the cell growth was estimated by hemocytometric counting and spectrophotometric analysis of total protein and DNA contents. Potential changes in DNA synthesis were also assessed through proliferating cell nuclear antigen (PCNA) immunolabeling. The results confirmed previously reported data that a 42-h exposure to a 50‑Hz sine wave MF at 100 µT promotes cell growth in the NB69 cell line, and showed that 10 µT induces a similar proliferative response. This effect, which was significantly associated and linearly correlated with PCNA expression, was abolished by the presence of retinoic acid in the culture medium.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Magnetic Fields; Neuroblastoma; Proliferating Cell Nuclear Antigen; Tretinoin

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells.

Topics: Biomarkers; Blotting, Western; Cell Differentiation; Doublecortin Domain Proteins; Doublecortin Protein; Gene Expression Profiling; Humans; Immunoenzyme Techniques; Membrane Proteins; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neuropeptides; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Schwann Cells; Stathmin; Tretinoin; Tumor Cells, Cultured

We hypothesized that AF/neuron interactions during annular injury were involved in neovascularization and nerve ingrowth, the pathologic hallmarks of symptomatic disc degeneration.. To identify growth factors and inflammatory cytokines related to AF/neuron interactions using in vitro model.. Discogenic pain is the chronic intractable pain initiated by tears in the outer annulus fibrosus (AF); this is a unique structure with free nerve endings at outer one-third, located beside dorsal root ganglia. The relationship between AF and neuron cells in annular injury has not been extensively investigated.. Human AF cells were cocultured with a retinoic acid (RA)-treated SH-SY5Y human neuroblastoma cell line (neuron-like cells). Conditioned media from cells cultured alone or in coculture were assayed for growth factors and inflammatory cytokines using enzyme-linked immunosorbent assays. The responses of the neuron-like cells, the AF cells, and the cocultured group to IL-1β/TNF-α were compared using the same outcome measures.. RA-treated SH-SY5Y cells showed significant neurite outgrowth on the 7th day; this is a typical morphologic finding of neuron-like cells. Neuron-like cells produced vascular endothelial growth factor (VEGF) and IGF-1 under basal conditions and dose-dependently secreted small amounts of IL-8 in response to TNF-α. Coculturing enhanced the secretion of VEGF, TGF-β1, and β-NGF, and suppressed the production of IGF-1. VEGF in the coculture group and the AF cells was downregulated by IL-1β/TNF-α stimulation. IL-1β/TNF-α stimulation enhanced the production of large amounts of IL-6 and IL-8 from AF cells; IL-1β produced a greater response than TNF-α. The neuron-like cells did not produce detectable amounts of IL-6 or IL-8.. These studies suggest that AF cells are involved in an inflammatory reaction and that the interactions between AF and neuron-like cells enhance the production of growth factors responsible for neovascularization and nerve ingrowth. AF injury has the potential to initiate neovascularization/nerve ingrowth and an inflammatory reaction through the interactions of AF and neural tissues.

Topics: Cell Line, Tumor; Coculture Techniques; Culture Media, Conditioned; Cytokines; Female; Humans; Interleukin-1beta; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Neovascularization, Pathologic; Nerve Growth Factors; Neurites; Neuroblastoma; Neurons; Recombinant Proteins; Tretinoin; Tumor Necrosis Factor-alpha

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Topics: Adamantane; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Bromodeoxyuridine; Caspase 3; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cinnamates; DNA Damage; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Models, Biological; Neuroblastoma; Propidium; Receptors, Death Domain; Tretinoin

Despite the successful introduction of 13-cis retinoic acid (13cisRA) therapy for the treatment of neuroblastoma, approximately 50% patients do not respond or experience relapse. A retinoid analogue, fenretinide [N-(4-hydroxyphenyl) retinamide; 4-HPR] can induce apoptosis in neuroblastoma cell lines and could have clinical use after therapy with 13cisRA. However, there are important questions concerning potential retinoid drug interactions which need to be addressed. The aim of this study was to investigate the influence of retinoic acid pre-treatment on fenretinide-induced apoptosis and fenretinide metabolism in neuroblastoma cell lines. Apoptosis was measured by flow cytometry of propidium iodide-stained neuroblastoma cells and a live-cell imaging assay. Intracellular fenretinide metabolism was determined by HPLC analysis. Pre-treatment of neuroblastoma cell lines with retinoic acid (RA) resulted in a significant decrease in the apoptotic response to fenretinide in three of the four lines tested. Comparison between responsive and non-responsive cell lines suggested that RA sensitivity was required to promote fenretinide resistance, and that this was mediated by up-regulation of Bcl-2 and the inhibition of pro-apoptotic fenretinide signalling pathways. Induction of the oxidative metabolism of fenretinide after RA pre-treatment did not significantly impact on intracellular parent drug levels and is unlikely to explain the decreased apoptotic response observed. The interaction between RA and fenretinide could have important implications for the scheduling of fenretinide in therapeutic protocols for neuroblastoma.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Interactions; Fenretinide; Flow Cytometry; Humans; Neuroblastoma; Tretinoin

The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(1)/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Membrane Proteins; Mice; Neurites; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.

Topics: Animals; Base Sequence; Blotting, Western; Cell Differentiation; Cell Line, Tumor; DNA Primers; Gene Knockdown Techniques; Immunohistochemistry; Mice; Neurites; Neuroblastoma; Receptors, Transferrin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Parkinson's disease (PD) is characterized by accumulation of α-synuclein aggregates and degeneration of melanized, catecholaminergic neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD, tTG levels are increased and cross-linking has been identified as an important factor in α-synuclein aggregation. In our quest to link tTGs distribution in the human brain to the hallmarks of PD pathology, we recently reported that catecholaminergic neurons in PD disease-affected brain areas display typical endoplasmic reticulum (ER) granules showing tTG immunoreactivity. In the present study, we set out to elucidate the nature of the interaction between tTG and the ER in PD pathogenesis, using retinoic-acid differentiated SH-SY5Y cells exposed to the PD-mimetic 1-methyl-4-phenylpyridinium (MPP(+)). Alike our observations in PD brain, MPP(+)-treated cells displayed typical TG-positive granules, that were also induced by other PD mimetics and by ER-stress inducing toxins. Additional immunocytochemical and biochemical investigation revealed that tTG is indeed associated to the ER, in particular at the cytoplasmic face of the ER. Upon MPP(+) exposure, additional recruitment of tTG toward the ER was found. In addition, we observed that MPP(+)-induced tTG activity results in transamidation of ER membrane proteins, like calnexin. Our data provide strong evidence for a, so far unrecognized, localization of tTG at the ER, at least in catecholaminergic neurons, and suggests that in PD activation of tTG may have a direct impact on ER function, in particular via post-translational modification of ER membrane proteins.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Calnexin; Calreticulin; Cell Differentiation; Cell Line, Tumor; Cytoplasm; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Drug Interactions; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Electron, Transmission; Neuroblastoma; Neurons; Protein Disulfide-Isomerases; Thapsigargin; Transglutaminases; Tretinoin; Tunicamycin

Through microarray analyses, we identified the Mpped2 gene as differentially expressed in two neuroblastoma cell lines induced to differentiation with all-trans retinoic acid. Mpped2 codes for a new metallophosphodiesterase protein, the expression of which inhibits cell proliferation and soft agar colony formation in SH -SY5Y cells. This inhibition is concomitant to an increased proportion of the cells in G0/G1 phase and enhanced caspase 3 activation, effects not seen for the other phosphodiesterases. A Mpped2-null mutation (H67R) abrogates these functions, which indicates that the biochemical activity of Mpped2 is advantageous for cancer suppression. Expression analyses in the "Los Angeles" and "Essen" neuroblastoma gene-array data sets show that increased expression of Mpped2 is associated with good patient prognosis according to Kaplan-Meier analyses. Tumorigenic assays in mice show that overexpression of Mpped2 improves survival rate, substantially impairs tumor growth and induces neuronal differentiation. Altogether, these data show that Mpped2 expression impairs neuroblastoma tumorigenesis, and they establish a basis for future therapeutic applications.

Topics: Adult; Animals; Antineoplastic Agents; Caspase 3; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; G1 Phase; Humans; Mice; Mice, Nude; Neuroblastoma; Phosphoric Diester Hydrolases; Resting Phase, Cell Cycle; Transplantation, Heterologous; Tretinoin; Up-Regulation

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately sixfold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Respiration; Electron Transport Chain Complex Proteins; Energy Metabolism; Glycolysis; Humans; Mitochondria; Neuroblastoma; Neurons; Oxidative Phosphorylation; Oxygen Consumption; Time Factors; Tretinoin

MECP2 protein binds preferentially to methylated CpGs and regulates gene expression by causing changes in chromatin structure. The mechanism by which impaired MECP2 activity can induce pathological abnormalities in the nervous system of patients with Rett syndrome (RTT) is not clearly understood. To gain further insight into the role of MECP2 in human neurogenesis, we compared the neural differentiation process in mesenchymal stem cells (MSCs) obtained from a RTT patient and from healthy donors. We further analyzed neural differentiation in a human neuroblastoma cell line carrying a partially silenced MECP2 gene. Senescence and reduced expression of neural markers were observed in proliferating and differentiating MSCs from the RTT patient, which suggests that impaired activity of MECP2 protein may impair neural differentiation, as observed in RTT patients. Next, we used an inducible expression system to silence MECP2 in neuroblastoma cells before and after the induction of neural differentiation via retinoic acid treatment. This approach was used to test whether MECP2 inactivation affected the cell fate of neural progenitors and/or neuronal differentiation and maintenance. Overall, our data suggest that neural cell fate and neuronal maintenance may be perturbed by senescence triggered by impaired MECP2 activity either before or after neural differentiation.

Topics: Cell Line, Tumor; Cellular Senescence; Chromatin; CpG Islands; DNA Methylation; Gene Expression Regulation; Humans; Mesenchymal Stem Cells; Methyl-CpG-Binding Protein 2; Neuroblastoma; Neurogenesis; Neurons; Rett Syndrome; RNA Interference; RNA, Small Interfering; Tretinoin

Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells.. Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest/sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells.. Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.

Topics: Biomarkers, Tumor; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neoplastic Stem Cells; Neural Crest; Neuroblastoma; RNA, Small Interfering; Sympathetic Nervous System; Tretinoin

While the biological importance of the cytochrome P450 system in the liver is well established, much less is known about its role in the brain and drug interactions at the level of brain cells have hardly been investigated. Here, we show that modafinil, a well-known inducer of hepatic CYP enzymes, also increases CYP3A4 expression in human-derived neuron-like SH-SY5Y cells. Upregulation of CYP3A4 by modafinil was associated with increased retinoic acid (RA) degradation, which could be blocked by specific CYP3A4 inhibitor erythromycin. In turn, reduced RA levels in culture medium during modafinil treatment resulted in decreased neuronal differentiation of SH-SY5Y cells as assessed by intracellular neurotransmitter concentrations and proliferative activity. Again, this differentiation-impeding effect of modafinil on SH-SY5Y cells was antagonized by erythromycin. Similarly, modafinil treatment of the murine GL261 glioma cell line resulted in increased proliferative activity. This was associated with upregulation of RA-degrading CYP26A1 in GL261 cells. Taken together, our results indicate that psychopharmacological agents such as modafinil may directly act on CYP enzymes in neural tissue. These kinds of drug effects may become highly relevant especially in the context of biomolecules such as RA whose local metabolism in brain is under tight spatial and temporal control.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Antineoplastic Agents; Benzhydryl Compounds; Cell Differentiation; Cell Line, Tumor; Cytochrome P-450 CYP3A; Dopamine; Erythromycin; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Mice; Modafinil; Neuroblastoma; Neurofilament Proteins; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Protein Synthesis Inhibitors; Transfection; Tretinoin

Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All-trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297-304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.

Topics: Cell Differentiation; Cell Proliferation; Down-Regulation; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Neurons; Neutral Ceramidase; Structure-Activity Relationship; Tretinoin

Tumour-induced hem- and lymph-angiogenesis are frequently associated with tumour progression. Vascular endothelial growth factor-C (VEGF-C) is a potent inducer of lymphangiogenesis, while the endogenous soluble splice-variant of VEGF receptor-2, esVEGFR-2, acts as a natural inhibitor. Previously we have shown down-regulation of esVEGFR-2 mRNA in progressed stages of neuro-blastoma (NB), a tumour derived from sympatho-adrenal precursor cells. Here we studied the immunolocalization of esVEGFR-2 in human embryos, infantile adrenal gland and primary NB. We also quantified esVEGFR-2 mRNA in NB cell lines after differentiation-induction by all-trans retinoic acid (ATRA). By immunoperoxidase staining we observed expression of esVEGFR-2 in both the sympathetic trunk and the adrenal medulla. Additionally, esVEGFR-2 was found in spinal ganglia, floor plate of the neural tube, choroid plexus, notochord, arterial endothelium, skeletal muscle, epidermis and gut epithelium. Developing and circulating leukocytes showed the strongest signal. In NB, esVEGFR-2 was considerably stronger in differentiating low grade tumours with neuronal phenotype than in undifferentiated lesions. Differentiation-induction of the NB cell line SMS-Kan with 5-10 µM ATRA resulted in a significant increase of esVEGFR-2 mRNA after 6, 9 and 12 days. We show that esVEGFR-2 is widely expressed in embryonic tissues. Especially, the adrenal medulla and circulating leukocytes seem to be potent inhibitors of lymphangiogenesis. We provide additional evidence for a role of esVEGFR-2 in NB. Thereby, high levels of esVEGFR-2 correlate with a more differentiated phenotype, and may inhibit tumour progression by inhibition of lymphangiogenesis.

Topics: Adrenal Glands; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Gestational Age; Humans; Immunohistochemistry; Lymphangiogenesis; Neuroblastoma; Protein Isoforms; RNA, Messenger; Sympathetic Nervous System; Time Factors; Tissue Array Analysis; Tretinoin; Vascular Endothelial Growth Factor Receptor-2

Mutations of the FERM domain containing protein 7 gene (FRMD7) are associated with X-linked idiopathic congenital nystagmus. Previous studies have shown that FRMD7 plays an important role in neuronal development and is involved in the regulation of F-actin. However, its specific mechanism of action remains undetermined.. Our study used quantitative real-time PCR to assess the levels of neuron-specific genes in a mouse neuroblastoma cell line (Neuro-2a) after transfection with a full-length coding transcript of FRMD7 or a blank control vector. F-actin was detected by rhodamine-phalloidin staining. Neurite number and length were assessed by a confocal laser scanning microscope.. We demonstrated that FRMD7 can promote neurite outgrowth following retinoic acid-induced differentiation in Neuro-2a cells. Neurites were significantly longer in cells transfected with FRMD7, but there was no difference in cell numbers. The mRNA expression of neuron cytoskeletal-related genes (microtubule-associated protein 2 [Mtap2], neurofilament-L and M [NF-L and NF-M] and the microtubule-associated protein tau [MAPT]) were significantly increased compared to controls. Other genes (NF-H, MAPT, neuron-specific class III beta-tubulin (Tuj-1), nestin, and growth-associated protein-43 [GAP-43]) were not obviously altered by FRMD7 overexpression.. Taken together, our data suggest that FRMD7 promotes the extension of neurites and may be involved in regulating the movement of cytoskeletal proteins, which influences not only F-actin, but also NF and microtubule dynamics.

Topics: Actins; Animals; Cell Count; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; GAP-43 Protein; Gene Expression Regulation; Intermediate Filament Proteins; Mice; Microtubule-Associated Proteins; Microtubules; Nerve Tissue Proteins; Nestin; Neurites; Neuroblastoma; Neurofilament Proteins; Plasmids; Protein Transport; RNA, Messenger; tau Proteins; Transfection; Tretinoin; Tubulin

Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27(Kip), which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

Topics: Cell Differentiation; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Tretinoin

Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7, which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumors. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less aggressive clinical phenotype. To test this hypothesis, we investigated the effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a significant inhibition of cell proliferation in vitro and angiogenesis and tumor growth in vivo. DNMT3B7-positive cells had higher levels of total genomic methylation and a dramatic decrease in expression of the FOS and JUN family members that comprise AP1 transcription factors. Consistent with an established antagonistic relationship between AP1 expression and retinoic acid receptor activity, increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all-trans retinoic acid (ATRA) compared to controls. Our results indicate that DNMT3B7 modifies the epigenome in neuroblastoma cells to induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Drug Resistance, Neoplasm; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Mice; Mice, Nude; Neuroblastoma; Tretinoin; Xenograft Model Antitumor Assays

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells--miR-9 and miR-103--restrain ID2 expression by directly targeting the coding sequence and 3' untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development.

Topics: Animals; Antineoplastic Agents; Cattle; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Protein 2; Macaca mulatta; Mice; MicroRNAs; Neoplasm Proteins; Neuroblastoma; Rats; RNA, Neoplasm; Tretinoin; Up-Regulation

Retinoic acid (RA) can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13) that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2)-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1) or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13) on BE(2)-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Homeodomain Proteins; Humans; Multigene Family; Neuroblastoma; Signal Transduction; Tretinoin

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.. Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.. We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.

Topics: Animals; Antibiotics, Antineoplastic; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lamin Type A; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neurites; Neuroblastoma; Proteome; Tretinoin

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

Topics: Cell Line, Tumor; Chromatin; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Enhancer Elements, Genetic; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; Histone Deacetylase 1; Humans; Methyl-CpG-Binding Protein 2; Neuroblastoma; Polycomb Repressive Complex 2; Promoter Regions, Genetic; Proto-Oncogene Proteins c-ret; Receptors, Retinoic Acid; Repressor Proteins; Response Elements; Retinoic Acid Receptor alpha; Sin3 Histone Deacetylase and Corepressor Complex; Transcription Factors; Transcriptional Activation; Tretinoin

The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.

Topics: Cell Differentiation; Cell Line, Tumor; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Humans; Immunoblotting; Intermediate Filaments; Microscopy, Fluorescence; Neuroblastoma; Nuclear Matrix; Nuclear Matrix-Associated Proteins; Prohibitins; Protein Transport; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Repressor Proteins; Reproducibility of Results; Retinoblastoma Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin; Tumor Suppressor Protein p53

MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.

Topics: Alternative Splicing; Animals; Antineoplastic Agents; Cell Differentiation; Cell Movement; Chick Embryo; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Metastasis; Neuroblastoma; Nuclear Proteins; Protein Biosynthesis; Receptors, Immunologic; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Tretinoin

MicroRNAs function as negative regulators of posttranscriptional gene expression, having major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modeling signaling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly overexpressed in SK-N-BE, LAN5 and SHSY-5Y. Ectopic overexpression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted downregulated miR-10a/b targets was nuclear receptor corepressor 2 (NCOR2), a corepressor of gene transcription, which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA-mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic upregulation of NCOR2 using an expression construct lacking the miR-10a/b 3' untranslated region target site. We conclude that miR-10a/b has major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the downregulation of MYCN.

Topics: Cell Differentiation; Cell Line, Tumor; Down-Regulation; Humans; MicroRNAs; N-Myc Proto-Oncogene Protein; Neuroblastoma; Neurons; Nuclear Proteins; Nuclear Receptor Co-Repressor 2; Oncogene Proteins; RNA Interference; RNA, Small Interfering; Transfection; Tretinoin

The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Gamma Rays; Glial Fibrillary Acidic Protein; Humans; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Neural Stem Cells; Neurites; Neuroblastoma; Proteome; Tretinoin

All-trans retinoic acid (ATRA) plays crucial roles in cell survival and differentiation of neuroblastoma cells. In the present study, we investigated the effects of geranylgeranoic acid (GGA), an acyclic retinoid, on differentiation and tropomyosin-related kinase receptor B (TrkB) gene expression in SH-SY5Y human neuroblastoma cells in comparison with ATRA. GGA induced growth suppression and neural differentiation to the same extent as ATRA. Two variants (145 and 95 kD) of the TrkB protein were dramatically increased by GGA treatment, comparable to the effect of ATRA. Following 6- to 8-day GGA treatment, the effect of GGA on TrkB was reversed after 2-4 days of its removal, whereas the effect of ATRA was irreversible under the same conditions. Both GGA and ATRA upregulated the cellular levels of three major TrkB messenger RNA splice variants in a time-dependent manner. Time-dependent induction of cell cycle-related genes, such as cyclin D1 and retinoblastoma protein, and amplification of the neural progenitor cell marker, brain lipid binding protein, were suppressed by GGA treatment and were completely abolished by ATRA. ATRA and GGA induced retinoic acid receptor β (RARβ) expression, whereas the time-dependent expression of both RARα and RARγ was abolished by ATRA, but not by GGA. Our results suggest that GGA may be able to restore neuronal properties of SH-SY5Y human neuroblastoma cells in a similar but not identical way to ATRA.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D; Cyclin E; Diterpenes; Ethanol; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Neuroblastoma; Prealbumin; Protein Kinases; Receptor, trkB; Receptors, Retinoic Acid; Steroids; Time Factors; Tretinoin

To test the hypothesis that Sydenham chorea (SC) immunoglobulin G (IgG) autoantibodies bind to specific neuronal surface proteins, whereas IgG from patients with pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS) or Tourette syndrome (TS) do not bind to neuronal surface proteins.. We used live differentiated SH-SY5Y cells, which have neuronal and dopaminergic characteristics. Using flow cytometry, we measured serum IgG cell surface binding in patients with SC (n = 11), PANDAS (n = 12), and TS (n = 11), and compared the findings to healthy controls (n = 11) and other neurologic controls (n = 11). In order to determine the specificity of binding to neuronal antigens, we also used a non-neuronal cell line, HEK 293.. The mean IgG cell surface binding was significantly higher in the SC group compared to all other groups (p < 0.001). By contrast, there was no difference between the PANDAS or TS groups and the controls. Using the non-neuronal HEK-293 cells, there was no significant difference in IgG cell surface binding between any groups.. Serum autoantibodies that bind to neuronal cell surface antigens are present in SC, but not in PANDAS or TS. These findings strengthen the hypothesis that SC is due to a pathogenic autoantibody, but weaken the autoantibody hypothesis in PANDAS and TS.

Topics: Adolescent; Antineoplastic Agents; Autoimmune Diseases; Cell Differentiation; Cell Line, Tumor; Child; Child, Preschool; Chorea; Female; Flow Cytometry; Humans; Immunoglobulin G; Male; Neuroblastoma; Neurons; Obsessive-Compulsive Disorder; Statistics, Nonparametric; Streptococcal Infections; Tourette Syndrome; Tretinoin

To study the effects of TrkB-BDNF signal pathway on the synthesis and secretion of vascular endothelial growth factor (VEGF) in human neuroblastoma cells (NB).. TrkB protein expression in SY5Y cells before and after all-trans-retinoicacid (ATRA) treatment was detected by Western blot. P-TrkB protein expression in SY5Y cells before and after the treatment of ATRA along with BDNF was also detected by Western blot. VEGF concentrations in the SY5Y cell culture supernatants were measured using ELISA after the treatment with ATRA, BDNF, tyrosine kinase inhibitor K252a and PI3k inhibitor LY294002.. TrkB protein was undetectable in SY5Y cells before ATRA treatment. After the treatment of 1, 10 and 100 nM/L ATRA for five days, TrkB protein was expressed in SY5Y cells and the TrkB protein level increased with the increasing ATRA concentration. P-TrkB protein was not expressed in SY5Y cells treated only with 10 nM/L ATRA, but it was detectable after the treatment of ATRA along with BDNF. VEGF concentrations in the group treated with ATRA+BDNF were significantly higher than those in the untreated control and the ATRA alone treatment groups (P<0.01). VEGF concentrations in the K252a pretreated ATRA+BDNF group were significantly lower than those in the group treated with ATRA+BDNF (P<0.05). VEGF concentrations in the LY294002 treatment group (ATRA+LY294002+BDNF group) were also significantly lower than those in the group treated with ATRA+BDNF (P<0.01).. Activation of TrkB-BDNF signal pathway may increase the synthesis and secretion of VEGF in human NB cells. The synthesis and secretion of VEGF can be inhibited by blocking TrkB-BDNF signal pathway with K252a or blocking the TrkB-BDNF downstream signal pathway PI3K/Akt with LY294002.

Topics: Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Humans; Neuroblastoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, trkB; Signal Transduction; Tretinoin; Vascular Endothelial Growth Factor A

Differentiation status in neuroblastoma strongly affects clinical outcomes and inducing differentiation is a treatment strategy in this disease. However, the molecular mechanisms that control neuroblastoma differentiation are not well understood. Here, we show that high-level HOXC9 expression is associated with neuroblastoma differentiation and is prognostic for better survival in neuroblastoma patients. HOXC9 induces growth arrest and neuronal differentiation in neuroblastoma cells by directly targeting both cell-cycle-promoting and neuronal differentiation genes. HOXC9 expression is upregulated by retinoic acid (RA), and knockdown of HOXC9 expression confers resistance to RA-induced growth arrest and differentiation. Moreover, HOXC9 expression is epigenetically silenced in RA-resistant neuroblastoma cells, and forced HOXC9 expression is sufficient to inhibit their proliferation and tumorigenecity. These findings identified HOXC9 as a key regulator of neuroblastoma differentiation and suggested a therapeutic strategy for RA-resistant neuroblastomas through epigenetic activation of HOXC9 expression.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; G1 Phase; Homeodomain Proteins; Humans; Neuroblastoma; Neurons; Prognosis; Promoter Regions, Genetic; Tretinoin

The repressor element-1 silencing transcription factor (REST) is a repressor of neuronal genes. Its expression is associated with poor neuronal differentiation in many neuroblastoma patient samples and cell lines. Because retinoic acid promotes neuronal differentiation, the authors postulated that it involves modulation of REST expression.. The expression of REST and of an S-phase kinase-associated protein 1/cullin 1/F-box (SCF) protein complex that contains the F-box protein β-transducin repeat-containing protein (β-TRCP) (SCF(β-TRCP) ) in neuroblastoma tumor samples and cell lines was analyzed by immunofluorescence and Western blot analysis. SK-N-SH and SK-N-AS cells were treated with retinoic acid and MG-132 to measure proteasomal degradation of REST by Western blot and quantitative real-time polymerase chain reaction analyses. Immunoprecipitation and coimmunoprecipitation assays were done in SK-N-AS cells that were transfected either with a control plasmid or with an enhanced green fluorescent protein-SCF(β-TRCP) -expressing plasmid.. Several neuroblastoma patient samples and cell lines displayed elevated REST expression. Although, REST transcription increased upon retinoic acid treatment in SK-N-SH and SK-N-AS cells, REST protein levels declined, concomitant with the induction of neuronal differentiation, in SK-N-SH cells but not in SK-N-AS cells. MG-132 treatment countered the retinoic acid-mediated decline in REST protein. SCF(β-TRCP) , a known REST-specific E3-ligase, was poorly expressed in many neuroblastoma samples, and its expression increased upon retinoic acid treatment in SK-N-SH cells but declined in SK-N-AS cells. Ectopic expression of SCF(β-TRCP) in SK-N-AS cells promoted REST ubiquitination and degradation and neuronal differentiation.. The current results indicated that elevated transcription of REST compounded by its impaired degradation by SCF(β-TRCP) may contribute to the failure of these tumors to differentiate in response to retinoic acid.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Neuroblastoma; Proteasome Endopeptidase Complex; Repressor Proteins; RNA Processing, Post-Transcriptional; SKP Cullin F-Box Protein Ligases; Transcription, Genetic; Tretinoin

Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.

Topics: Amino Acid Sequence; Animals; Bradykinin; Cell Differentiation; Cell Line, Tumor; Gene Expression; GPI-Linked Proteins; Humans; Interferon-gamma; Kinetics; Kininogens; Metalloendopeptidases; Mice; Neuroblastoma; Neurons; Protein Binding; Radioligand Assay; Receptor, Bradykinin B1; Recombinant Proteins; Tissue Kallikreins; Tretinoin; Up-Regulation

Neuroblastic tumors account for 9-10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.. In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.. Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.

Topics: Blotting, Western; Calcium-Binding Proteins; Cell Differentiation; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Humans; Mass Spectrometry; Microscopy, Phase-Contrast; Neuroblastoma; NM23 Nucleoside Diphosphate Kinases; Phosphoproteins; Tretinoin

Senataxin is encoded by the SETX gene and is mainly involved in two different neurodegenerative diseases, the dominant juvenile form of amyotrophic lateral sclerosis type 4 and a recessive form of ataxia with oculomotor apraxia type 2. Based on protein homology, senataxin is predicted to be a putative DNA/RNA helicase, while senataxin interactors from patients' lymphoblast cell lines suggest a possible involvement of the protein in different aspects of RNA metabolism. Except for an increased sensitivity to oxidative DNA damaging agents shown by some ataxia with neuropathy patients' cell lines, no data are available about possible functional consequences of dominant SETX mutations and no studies address the function of senataxin in neurons. To start elucidating the physiological role of senataxin in neurons and how disease-causing mutations in this protein lead to neurodegeneration, we analysed the effect of senataxin on neuronal differentiation in primary hippocampal neurons and retinoic acid-treated P19 cells by modulating the expression levels of wild-type senataxin and three different dominant mutant forms of the protein. Wild-type senataxin overexpression was required and sufficient to trigger neuritogenesis and protect cells from apoptosis during differentiation. These actions were reversed by silencing of senataxin. In contrast, overexpression of the dominant mutant forms did not affect the regular differentiation process in primary hippocampal neurons. Analysis of the cellular pathways leading to neuritogenesis and cytoprotection revealed a role of senataxin in modulating the expression levels and signalling activity of fibroblast growth factor 8. Silencing of senataxin reduced, while overexpression enhanced, fibroblast growth factor 8 expression levels and the phosphorylation of related target kinases and effector proteins. The effects of senataxin overexpression were prevented when fibroblast growth factor 8 signalling was inhibited, while exogenous fibroblast growth factor 8 reversed the effects of senataxin silencing. Overall, these results reveal a key role of senataxin in neuronal differentiation through the fibroblast growth factor 8 signalling and provide initial molecular bases to explain the neurodegeneration associated with loss-of-function mutations in senataxin found in recessive ataxia. The lack of effect on neuritogenesis observed with the overexpression of the dominant mutant forms of senataxin apparently excludes a dominant negativ

Topics: Animals; Caspase 3; Cell Death; Cell Differentiation; Cells, Cultured; DNA Helicases; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Fibroblast Growth Factor 8; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Humans; Mice; Multifunctional Enzymes; Mutation; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurons; RNA Helicases; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection; Tretinoin

Jaques Benveniste suggested that electronic transmission of 4-phorbol-12-b-myristate-13-acetate (PMA) activity, carried out using a simple amplifier configured to function as an oscillator when coupled to two electromagnetic coils, demonstrates the same chemical activity as the active molecule. The results obtained suggested that there are associated signals at the PMA molecules that can be transferred to target neutrophils by artificial physical means in a fashion that mimics the original molecules.. Retinoic acid was placed at room temperature on one coil attached to an oscillator (VEGA select 719), while LAN-5 neuroblastoma cells were placed on another coil and incubated under controlled condition. The oscillator was then turned on for 12 hours a day for 5 days, after which cells were counted and morphology studied by contrast microscopy.. The effect of the differentiating agent added to the cell culture by physical means generates a decrease in cell growth, metabolic activity, and the protrusion of a neuritelike structure typical of the differentiated cells.. These preliminary results suggest that retinoic acid molecules emit signals that can be transferred to LAN-5 neuroblastoma cells by artificial physical means in a manner that seems related to the chemical structure of the source molecules.

Topics: Cell Differentiation; Cell Line, Tumor; Electronics; Humans; Neurites; Neuroblastoma; Tretinoin

Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 μM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 μM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.

Topics: Antioxidants; Cell Line, Tumor; Cell Survival; Chromans; Humans; Lipid Peroxidation; Microscopy, Phase-Contrast; Neurites; Neuroblastoma; Neurogenesis; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Tretinoin; Tyrosine 3-Monooxygenase

β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) promotes the formation of GalNAcβ1,4GlcNAc (LacdiNAc or LDN). Drosophila β1,4-N-acetylgalactosaminyltransferase A (B4GALNTA) contributes to the synthesis of LDN, which helps regulate neuronal development. In this study, we investigated the expression and role of B4GALNT3 in human neuroblastoma (NB). We used IHC analysis to examine 87 NB tumors, and we identified correlations between B4GALNT3 expression and clinicopathologic factors, including patient survival. Effects of recombinant B4GALNT3 on cell behavior and signaling were studied in SK-N-SH and SH-SY5Y NB cells. Increased expression of B4GALNT3 in NB tumors correlated with a favorable histologic profile (P < 0.001, χ² test) and early clinical staging (P = 0.041, χ² test) and was a favorable prognostic factor for survival as evaluated by univariate and multivariate analyses. Reexpression of B4GALNT3 in SK-N-SH and SH-SY5Y cells suppressed cell proliferation, colony formation, migration, and invasion. Moreover, B4GALNT3 increased the LacdiNAc modification of β₁ integrin, leading to decreased phosphorylation of focal adhesion kinase (FAK), Src, paxillin, Akt, and ERK1/2. B4GALNT3-mediated suppression of cell migration and invasion were substantially reversed by concomitant expression of constitutively active Akt or MEK. We conclude that B4GALNT3 predicts a favorable prognosis for NB and suppresses the malignant phenotype via decreasing β₁ integrin signaling.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Child; Child, Preschool; Female; Humans; Integrin beta1; Male; N-Acetylgalactosaminyltransferases; Neoplasm Invasiveness; Neuroblastoma; Prognosis; Signal Transduction; Tretinoin

We have developed a stably transfected human cell model for Alzheimer's disease with doxycycline-inducible expression of human misfolded truncated tau protein (AT tau). We have showed that AT tau reduced the metabolic activity of the AT tau cells, slowed down cell proliferation, and induced caspase-3-independent apoptosis-like programmed cell death, tauoptosis. The aim of this study was to test the possible capability of rat mesenchymal stem cells (MSCs) to interfere with AT tau protein-induced cell death. AT tau cells after treatment with 10 μM all-trans retinoic acid were either co-cultivated with MSCs or supplemented with MSC secretome for 6 and 9 days. We found that both MSCs and MSC secretome promoted survival and increased the metabolic activity of the cells. Moreover stem cells induced cell differentiation and formation of neurites with numerous varicosities. Strikingly, treatment had no effect on tau expression suggesting that MSC induced self-protecting mechanism that prevented AT tau cells from tauoptosis. Our results showed that mesenchymal stem cells and their secretome are able to rescue the Alzheimer's disease cell model from cell death induced by misfolded truncated tau. We suggest that cell therapy may represent an alternative therapeutic avenue for treatment of human Alzheimer's disease and related tauopathies.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Antigens, CD; Apoptosis; Bone Marrow Cells; Caspase 3; Cell Differentiation; Cell Line, Transformed; Cell Line, Tumor; Cell Survival; Coculture Techniques; Humans; Male; Mesenchymal Stem Cells; Nerve Growth Factors; Neuroblastoma; Phosphorylation; Rats; Rats, Wistar; tau Proteins; Time Factors; Transfection; Tretinoin

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.

Topics: Adaptation, Physiological; Aluminum Chloride; Aluminum Compounds; Animals; Antineoplastic Agents; Astrocytes; Cell Differentiation; Cell Division; Cell Proliferation; Chlorides; DNA; Information Theory; Neuroblastoma; Neuroepithelial Cells; Neurotoxins; RNA; Tretinoin; Tumor Cells, Cultured

The mainly neuronally expressed protein p42(IP4) (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42(IP4) and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42(IP4) enhances the enzymatic activity of NRD 3-4 times. However, p42(IP4) is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42(IP4) is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42(IP4) showed an enhanced NRD protein expression already at an earlier time point after RA stimulation.

Topics: Adaptor Proteins, Signal Transducing; Brain Chemistry; Cell Line, Tumor; Green Fluorescent Proteins; Humans; Luminescent Agents; Metalloendopeptidases; Nerve Tissue Proteins; Neuroblastoma; Neurons; Organ Specificity; Tretinoin; Up-Regulation

α-Synuclein causes Parkinson's disease if mutated or aberrantly produced in neurons. α-Synuclein-lipid interactions are important for the normal function of the protein, but can also contribute to pathogenesis. We previously reported that deletion of the first 10 N-terminal amino acids dramatically reduced lipid binding in vitro, as well as membrane binding and toxicity in yeast. Here we extend this study to human neuroblastoma SHSY-5Y cells, and find that in these cells the first 10 N-terminal residues do not affect α-synuclein membrane binding, self-association and cell viability, contrary to yeast. Differences in lipid composition, membrane fluidity and cytosolic factors between yeast and neuronal cells may account for the distinct binding behavior of the truncated variant in these two systems. Retinoic acid promotes differentiation and α-synuclein oligomer formation in neuroblastoma cells, while addition of a proteasomal inhibitor induces neurite outgrowth and toxicity to certain wild-type and truncated α-synuclein clones. Yeast recapitulate several features of α-synuclein (patho)biology, but its simplicity sets limitations; verification of yeast results in more relevant model systems is, therefore, essential.

Topics: alpha-Synuclein; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Cell Survival; DNA, Complementary; Humans; Microscopy, Fluorescence; Neuroblastoma; Saccharomyces cerevisiae; Transfection; Tretinoin

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) has been reported to enhance proliferation and invasion in various cancers. The role of IMP3 on neuroblastoma (NB) is unknown. We aimed to clarify the prognostic significance of IMP3 expression in patients with NB. By microarray analysis, high IMP3 expression was found in patients with poor outcome. IMP3 expression in 90 NB samples was analyzed by immunohistochemical staining to correlate with clinical stages, histology, and patient outcome. Positive IMP3 expression was detected in 52 of 90 patients, and was significantly correlated with undifferentiated histology, advanced stages, MYCN amplification, and poor outcome. In subgroups, positive IMP3 expression could predict an even worse prognosis in patients with advanced disease, with normal MYCN status, or with MYCN amplification (P = 0.005, P = 0.001, and P = 0.033, respectively). The IMP3 expression decreased by induction of differentiation with retinoid acid treatment in SK-N-DZ and SK-N-SH cells in vitro. The invasion ability of NB cells also decreased as IMP3 knockdown by using RNA interference in vitro. In summary, high expression of IMP3 in NB might contribute to the undifferentiated phenotype and invasive behaviors, leading to a poor prognosis. Determining IMP3 expression in NB could help to improve a personalized therapy.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Female; Humans; Infant; Infant, Newborn; Male; N-Myc Proto-Oncogene Protein; Neoplasm Invasiveness; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Prognosis; RNA Interference; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Tretinoin

Retinoic acid (RA) has been approved for the differentiation therapy of neuroblastoma (NB). Previous work revealed a correlation between glucose-regulated protein 75 (GRP75) and the RA-elicited neuronal differentiation of NB cells. The present study further demonstrated that GRP75 translocates into the nucleus and physically interacts with retinoid receptors (RARα and RXRα) to augment RA-elicited neuronal differentiation. GRP75 was required for RARα/RXRα-mediated transcriptional regulation and was shown to reduce the proteasome-mediated degradation of RARα/RXRαin a RA-dependent manner. More intriguingly, the level of GRP75/RARα/RXRα tripartite complexes was tightly associated with the RA-induced suppression of tumor growth in animals and the histological grade of differentiation in human NB tumors. The formation of GRP75/RARα/RXRα complexes was intimately correlated with a normal MYCN copy number of NB tumors, possibly implicating a favorable prognosis of NB tumors. The present findings reveal a novel function of nucleus-localized GRP75 in actively promoting neuronal differentiation, delineating the mode of action for the differentiation therapy of NB by RA.

Topics: Animals; Cell Differentiation; Cell Nucleus; Down-Regulation; Gene Dosage; HSP70 Heat-Shock Proteins; Humans; Membrane Proteins; Mice; N-Myc Proto-Oncogene Protein; Neuroblastoma; Neurons; Nuclear Proteins; Oncogene Proteins; Proteasome Endopeptidase Complex; Protein Binding; Protein Multimerization; Protein Transport; Proteolysis; Receptors, Retinoic Acid; Response Elements; Signal Transduction; Treatment Outcome; Tretinoin; Xenograft Model Antitumor Assays

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Cell Differentiation; Cell Line, Tumor; Drug Synergism; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Neuroblastoma; Protease Inhibitors; Pyrazines; Rats; Tretinoin; Xenograft Model Antitumor Assays

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Topics: Adenine; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Apoptosis; Autophagy; Autophagy-Related Protein 5; Cell Differentiation; Cell Nucleus; Cell Survival; Down-Regulation; Humans; Intracellular Space; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Microtubule-Associated Proteins; Mutant Proteins; Neuroblastoma; Oxidants; Oxygen; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Tretinoin; Tumor Cells, Cultured; Vacuoles

Quinolinic acid (QUIN), kynurenine acid (KYNA) and 3-hydroxykynurenine (3-HK) - metabolites of the kynurenine pathway are considered to be associated with many central nervous system diseases. However, in neuroscience research in order to test neurotoxicity or neuroprotection against these compounds only primary cell models are available. In this investigation we aimed to develop a simple, rapid and accurate cellular in vitro model using immortalized human neuroblastoma cell lines, namely SK-N-SH and SH-SY5Y differentiated by treatment with various agents. In order to alter the cell response to the neurotoxins, tumor necrosis factor-alpha and retinoic acid (RA) as differentiating agents and modulation of the cellular metabolism through changing the sugar composition from galactose to glucose in media were used. Our results indicated that although RA-differentiation of both cell lines induced the expression of neuronal features, cell vulnerability after exposure to control neurotoxicants (salsolinol, 6-hydroxydopamine) and 3-HK was decreased in comparison to untreated cells and was not influenced after exposure to QUIN and KYNA. Interestingly, the same observations were done in cells grown in galactose containing media.

Topics: Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Coloring Agents; Fluorescent Antibody Technique; Humans; Hydroxydopamines; Isoquinolines; Kynurenine; Neuroblastoma; Neurotoxins; Quinolinic Acid; Tetrazolium Salts; Thiazoles; Tretinoin

Neuroblastoma is an important pediatric tumor in which Myc-N amplification is a well-known poor prognostic indicator. It has a great diversity in clinical behavior. The effect of pharmacologic agents used in neuroblastoma treatment on Myc-N expression is still unclear.. We analyzed Myc-N expression changes by immunocytochemistry in Myc-N-positive Kelly human neuroblastoma cell line using retinoic acid and cytotoxic drugs (cisplatin, vincristine, cyclophosphamide, etoposide, and doxorubicin) and their combinations compared with control conditions. First, concentration of drugs were determined as LD50 doses. Kelly cells and drugs were incubated for 24 hours in 5% CO2, 37 degrees C in 96-well plates. Myc-N expression was scored semiquantitatively as negative, mild, moderate, or high positive.. Myc-N amplification did not change with any agent or combination. It was higher than 20 copies in all conditions. Myc-N protein expression was high in control and doxorubicin group. It was moderate in retinoic acid, cyclophosphamide, retinoic acid combined with cyclophosphamide and retinoic acid combined with doxorubicin groups. The expression was mild in cisplatin, vincristine, etoposide, retinoic acid combined with etoposide, and retinoic acid combined with cisplatin groups. Myc-N expression was negative in retinoic acid combined with vincristine group.. Myc-N expression is reduced with cytotoxic agents and retinoic acid in neuroblastoma although Myc-N amplification remains the same. Retinoic acid combined with vincristine is the most effective combination to reduce Myc-N expression. Our results suggest that therapeutic applications of these agents as low dose maintenance therapy might be useful.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Child; Gene Amplification; Gene Expression; Humans; Neuroblastoma; Proto-Oncogene Proteins c-myc; Tretinoin; Vincristine

TaqIA, the most widely analyzed genetic polymorphism in addictions, has traditionally been considered a gene marker for association with D2 dopamine receptor gene (DRD2). TaqIA is located in the coding region of the ANKK1 gene that overlaps DRD2 and encodes a predicted kinase ANKK1. The ANKK1 protein nonetheless had yet to be identified. This study examined the ANKK1 expression pattern as a first step to uncover the biological bases of TaqIA-associated phenotypes.. Northern blot and quantitative reverse-transcriptase polymerase chain reaction analyses were performed to analyze the ANKK1 mRNA. To study ANKK1 protein expression, we developed two polyclonal antibodies to a synthetic peptides contained in the putative Ser/Thr kinase domain.. We demonstrate that ANKK1 mRNA and protein were expressed in the adult central nervous system (CNS) in human and rodents, exclusively in astrocytes. Ankk1 mRNA level in mouse astrocyte cultures was upregulated by apomorphine, suggesting a potential relationship with the dopaminergic system. Developmental studies in mice showed that ANKK1 protein was ubiquitously located in radial glia in the CNS, with an mRNA expression pick around embryonic Day 15. This time expression pattern coincided with that of the Drd2 mRNA. On induction of differentiation by retinoic acid, a sequential expression was found in human neuroblastoma, where ANKK1 was expressed first, followed by that of DRD2. An opposite time expression pattern was found in rat glioma.. Spatial and temporal regulation of the expression of ANKK1 suggest an involvement of astroglial cells in TaqIA-related neuropsychiatric phenotypes both during development and adult life.

Topics: Animals; Apomorphine; Astrocytes; Cells, Cultured; Central Nervous System; Dopamine Agonists; Embryo, Mammalian; Glioma; Humans; Mice; Mice, Inbred BALB C; Nerve Tissue Proteins; Neuroblastoma; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptors, Dopamine D2; Sequence Alignment; Sequence Analysis, Protein; Substance-Related Disorders; Sulpiride; Tretinoin; Up-Regulation

Apoptosis is a major mechanism for cell death in the nervous system during development. P2X(7) nucleotide receptors are ionotropic ATP receptors that mediate cell death under pathological conditions. We developed an in vitro protocol to investigate the expression and functional responses of P2X(7) nucleotide receptors during retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y neuroblastoma cells. Neuronal differentiation was examined measuring cellular growth arrest and neuritic processes elongation. We found that SH-SY5Y cells treated for 5 days with RA under low serum content exhibited a neuron-like phenotype with neurites extending more than twice the length of the cell body and cell growth arrest. Concurrently, we detected the abolishment of intracellular-free calcium mobilization and the down-regulation of P2X(7) nucleotide receptor protein expression that protected differentiated cells from neuronal cell death and reduced caspase-3 cleavage-induced by P2X(7) nucleotide receptor agonist. The role of P2X(7) nucleotide receptors in neuronal death was established by selectively antagonizing the receptor with KN-62 prior to its activation. We assessed the involvement of protein kinases and found that p38 signaling was activated in undifferentiated after nucleotide stimulation, but abolished by the differentiating RA pretreatment. Importantly, P2X(7) receptor-induced caspase-3 cleavage was blocked by the p38 protein kinase specific inhibitor PD169316. Taken together, our results suggest that RA treatment of human SH-SY5Y cells leads to decreased P2X(7) nucleotide receptor protein expression thus protecting differentiated cells from extracellular nucleotide-induced neuronal death, and p38 signaling pathway is critically involved in this protection of RA-differentiated cells.

Topics: Brain Neoplasms; Calcium Signaling; Caspase 3; Cell Death; Cell Differentiation; Cytoprotection; Down-Regulation; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; MAP Kinase Signaling System; Neuroblastoma; Neurons; p38 Mitogen-Activated Protein Kinases; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Tretinoin; Tumor Cells, Cultured

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.

Topics: Binding Sites; Cell Death; Cell Proliferation; COUP Transcription Factor I; Electrophoretic Mobility Shift Assay; Exons; Humans; Immunoprecipitation; Molecular Sequence Data; Neurites; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA, Small Interfering; Transcriptional Activation; Transfection; Tretinoin

The clinical prognosis of children with high-stage neuroblastoma is still poor. Therapeutic approaches include surgery and cellular differentiation by retinoic acid, but also experimental interleukin-based immune modulation. However, the molecular mechanisms of all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells are incompletely understood. Herein, we examined the effect of ATRA on the activity of the interleukin-18 (IL-18) system in human SH-SY5Y neuroblastoma cells. It is shown that SH-SY5Y cells express IL-18 receptor (IL-18R) and the secreted antagonist IL-18-binding protein (IL-18BP), but no IL-18. SH-SY5Y cells are highly sensitive to ATRA treatment and react by cellular differentiation from a neuroblastic toward a more neuronal phenotype. This was associated with induction of IL-18 and reduction of IL-18BP expression, while IL-18R expression remained stable. Thereby, we identified the IL-18 system as a novel target of ATRA in neuroblastoma cells that might contribute to the therapeutic properties of retinoids in treatment of neuroblastoma.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-18; Neuroblastoma; Receptors, Interleukin-18; Tretinoin

We investigated the possible modulation by LOX/ COX inhibitors of all-trans retinoic acid (ATRA)-induced cell differentiation in two established neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor of cyclooxygenase-2, were chosen for this study. The effects of the combined treatment with ATRA and LOX/COX inhibitors on neuroblastoma cells were studied using cell morphology assessment, detection of differentiation markers by immunoblotting, measurement of proliferation activity, and cell cycle analysis and apoptosis detection by flow cytometry. The results clearly demonstrated the potential of caffeic acid to enhance ATRA-induced cell differentiation, especially in the SK-N-BE(2) cell line, whereas application of celecoxib alone or with ATRA led predominantly to cytotoxic effects in both cell lines. Moreover, the higher sensitivity of the SK-N-BE(2) cell line to combined treatment with ATRA and LOX/COX inhibitors suggests that cancer stem cells are a main target for this therapeutic approach. Nevertheless, further detailed study of the phenomenon of enhanced cell differentiation by expression profiling is needed.

Topics: Apoptosis; Caffeic Acids; Celecoxib; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Shape; Cyclooxygenase 2 Inhibitors; Flow Cytometry; Humans; Lipoxygenase Inhibitors; Neuroblastoma; Pyrazoles; Sulfonamides; Tretinoin

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Models, Biological; Neuroblastoma; Phosphoproteins; Protein Kinase C-delta; RNA, Messenger; Tretinoin

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

Topics: Aerobiosis; Antigens, CD; Cadherins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Kidney; Neuroblastoma; Phosphofructokinase-1; Phosphofructokinase-2; Tretinoin; Ubiquitin-Protein Ligases

alpha-Secretase cleavage of the amyloid precursor protein (APP) is of great interest because it prevents the formation of the Alzheimer-linked amyloid-beta peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas APLP2 shedding is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP alpha-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate whether the difference in the regulation of APLP2 shedding compared with APP shedding could be due to involvement of different processing enzymes. We focused on the two major alpha-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with small interfering RNAs targeted against TACE. The results clearly demonstrate that different alpha-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.

Topics: ADAM Proteins; ADAM10 Protein; ADAM17 Protein; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Antineoplastic Agents; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Humans; Insulin-Like Growth Factor I; Membrane Proteins; Nerve Tissue Proteins; Neuroblastoma; Phosphorylation; Protein Kinase C; RNA, Small Interfering; Tretinoin; Tumor Cells, Cultured

Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Tretinoin

The molecular mechanisms underlying the cellular lost found in the nigrostriatal pathway during the progression of Parkinson's disease (PD) are not completely understood. Human neuroblastoma cell line SH-SY5Y challenged with 6-hydroxydopamine (6-OHDA) has been widely used as an in vitro model for PD. Although this cell line differentiates to dopaminergic neuron-like cells in response to low serum and retinoic acid (RA) treatment, there are few studies investigating the differences between proliferative and RA-differentiated SH-SY5Y cells. Here we evaluate morphological and biochemical changes which occurs during the differentiation of SH-SY5Y cells, and their responsiveness to 6-OHDA toxicity. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 medium plus 10% of fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 microM RA plus 1% of FBS during 4, 7 and 10 days in culture. We found that SH-SY5Y cells differentiated for 7 days show an increase immunocontent of several relevant neuronal markers with the concomitant decrease in non-differentiated cell marker. Moreover, cells became two-fold more sensitive to 6-OHDA toxicity during the differentiation process. Time course experiments showed loss of mitochondrial membrane potential triggered by 6-OHDA (mitochondrial dysfunction parameter), which firstly occurs in proliferative than neuron-like differentiated cells. This finding could be related to the increase in the immunocontent of the neuroprotective protein DJ-1 during differentiation. Our data suggest that SH-SY5Y cells differentiated by 7 days with the protocol described here represent a more suitable experimental model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD.

Topics: Adrenergic Agents; Animals; Biomarkers; Cattle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Intracellular Signaling Peptides and Proteins; Keratolytic Agents; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Oncogene Proteins; Oxidopamine; Parkinson Disease; Protein Deglycase DJ-1; Tretinoin

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells.. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes.. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines.. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Topics: Brain Neoplasms; Caffeic Acids; Celecoxib; Cell Differentiation; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lipoxygenase Inhibitors; Multigene Family; Neuroblastoma; Oxidative Stress; Pyrazoles; Sulfonamides; Tretinoin

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK-N-SH cells pre- and post-treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two-dimensional gel electrophoresis and MALDI-TOF showed that NPM was a component of the nuclear matrix and its expression in SK-N-SH cells post-treated with RA was down-regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK-N-SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c-myc, c-fos, p53, and Rb in SK-N-SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK-N-SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Humans; Neuroblastoma; Nuclear Matrix; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Nucleophosmin; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Suppressor Protein p53

Dimebon, a drug currently being evaluated in multiple Phase III Alzheimer's disease trials, has previously been shown to have effects on isolated mitochondria at muM concentrations. Here the effects of nM concentrations of Dimebon on mitochondrial function were investigated both in primary mouse cortical neurons and human neuroblastoma cells (SH-SY5Y). Under non-stress conditions nM concentrations of Dimebon increased succinate dehydrogenase activity (MTT-assay), mitochondrial membrane potential (DeltaPsim), and cellular ATP levels. Dimebon treatment had no effect on mitochondria DNA content, implying that mitochondrial biogenesis was not induced. Under stress conditions, mitochondria in Dimebon-treated neurons showed increased resistance to elevated intracellular calcium concentrations, thus, maintaining their DeltaPsim throughout the experiment, in contrast to control neurons, which rapidly lost their DeltaPsim. Moreover, we show that serum-starved differentiated SH-SY5Y cells treated with Dimebon had an increased survival rate as compared to untreated cells. In conclusion, these data demonstrate that Dimebon enhances mitochondrial function both in the absence and presence of stress and Dimebon-treated cells are partially protected to maintain cell viability.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Blood Proteins; Calcium; Cell Differentiation; Cell Line, Tumor; DNA, Mitochondrial; Gene Dosage; Humans; Indoles; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroblastoma; Neurons; Neuroprotective Agents; Tretinoin

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

Topics: Aromatase; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation; Humans; Magnetics; Neurites; Neuroblastoma; RNA, Messenger; Tretinoin

Recently, gene amplification and gain-of-function mutations of ALK have been found in some neuroblastoma cell lines and clinical tumor samples. We have previously reported that knockdown of ALK by RNAi induced apoptosis in neuroblastoma cells with gene amplification of ALK. We report that all-trans retinoic acid (ATRA) downregulates ALK in neuroblastoma cell lines. Downregulation of ALK protein by ATRA was accompanied by apoptosis in neuroblastoma cells with gene amplification or gain-of-function mutation of ALK but not in neuroblastoma cells without these genetic alterations. These results suggest that ALK downregulation by ATRA might lead to apoptosis in neuroblastoma cells with activated ALK.

Topics: Anaplastic Lymphoma Kinase; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Amplification; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neuroblastoma; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; RNA, Messenger; Tretinoin

Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

Topics: Cell Line, Tumor; Cytomegalovirus; Flow Cytometry; Gene Dosage; Genes, Reporter; Green Fluorescent Proteins; Humans; Long Interspersed Nucleotide Elements; Neuroblastoma; Plasmids; Promoter Regions, Genetic; Recombination, Genetic; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin

Retinoic acid (RA) induces differentiation of neuroblastoma cells in vitro and is used with variable success to treat aggressive forms of this disease. This variability in clinical response to RA is enigmatic, as no mutations in components of the RA signaling cascade have been found. Using a large-scale RNAi genetic screen, we identify crosstalk between the tumor suppressor NF1 and retinoic acid-induced differentiation in neuroblastoma. Loss of NF1 activates RAS-MEK signaling, which in turn represses ZNF423, a critical transcriptional coactivator of the retinoic acid receptors. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We find NF1 mutations in neuroblastoma cell lines and in primary tumors. Inhibition of MEK signaling downstream of NF1 restores responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1-deficient neuroblastomas.

Topics: Cell Line, Tumor; DNA-Binding Proteins; Humans; Neuroblastoma; Neurofibromin 1; Prognosis; Proteins; Signal Transduction; Transcriptional Activation; Tretinoin

Retinoids can induce differentiation of neuroblastoma (NB) cells and are in clinical use for the treatment of patients with NB. Despite improvements of standard treatment during the last years, many patients with NB still relapse and new treatment options for these patients are required.. We analyzed NB cells after incubation with retinoids by using Affymetrix HG_U133A microarrays, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry. Sequencing of RT-PCR products was applied for determination of CD117 mRNA sequences from NB cell lines. In addition, we tested sensitivity of NB cells for the kinase inhibitor imatinib mesylate after treatment with retinoids.. Treatment of NB cells with retinoids induced expression of several genes including the retinoid metabolizing enzymes CYP26A1 and CYP26B1. In addition, we observed up-regulation of CD117 (KIT), particularly after long-term treatment with retinoids. Sequencing of CD117 mRNA from NB cell lines revealed heterozygosity for a non-synonymous single nucleotide polymorphism in SH-SY5Y NB cells. Up-regulation of CD117 in NB cells correlated with increased sensitivity for the kinase inhibitor imatinib mesylate.. The combination of retinoids with kinase inhibitors might be worth exploring further for the treatment of NB patients.

Topics: Antineoplastic Agents; Benzamides; Cell Line, Tumor; Cell Survival; Cytochrome P-450 Enzyme System; Flow Cytometry; Gene Expression; Humans; Imatinib Mesylate; Microarray Analysis; Neuroblastoma; Piperazines; Polymorphism, Single Nucleotide; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-kit; Pyrimidines; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Up-Regulation

Retinoids including natural vitamin A, its derivatives and synthetic compounds work as transcription factors through the retinoic acid receptors (RAR, RXR). All-trans retinoic acid (ATRA), a family of retinoids, is an internal ligand of RAR and well known as a useful differentiation inducer to treat acute promyelocytic leukemia (APL). ATRA therapy is now established as an initial treatment for APL. Recently, to improve therapeutic potency and reduce adverse effects of ATRA, a novel synthetic selective agonist for RARalpha and beta, Am80, was developed and applied to APL treatment. In this study, we tested whether Am80 was capable of inducing neuronal differentiation in a human neuroblastoma cell line, NH-12 and compared the differentiation effects between Am80 and ATRA. Morphological studies demonstrated that Am80 induced more potent neurite outgrowth and also proved lesser cell toxicity than ATRA. Am80 up-regulated the expression of tropomyosin-related kinase B as well as ATRA. Moreover, Am80 increased the expression of the neuronal marker, growth-associated protein 43. These findings suggest that Am80 induces neuronal differentiation to a greater extent than ATRA and thus may help establishing therapeutic strategies against neuronal degenerative disorders such as Parkinson's disease.

Topics: Antineoplastic Agents; Benzoates; Cell Differentiation; Cell Line, Tumor; GAP-43 Protein; Humans; Neuroblastoma; Neurons; Receptors, Retinoic Acid; Tetrahydronaphthalenes; Tretinoin

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.

Topics: Cell Differentiation; Cell Division; Colony-Forming Units Assay; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA Methyltransferase 3B; Gene Expression Profiling; Genes, Reporter; Humans; MicroRNAs; Neoplasm Invasiveness; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin

TRH (thyroliberin) and its analogues were reported to possess neuroprotective effects in cellular and animal experimental models of acute and chronic neurodegenerative diseases. In the present study we evaluated effects of TRH and its three stable analogues, montirelin (CG-3703), RGH-2202 and Z-TRH (N-(carbobenzyloxy)-pGlutamyl-Histydyl-Proline) on the neuronally differentiated human neuroblastoma SH-SY5Y cell line, which is widely accepted for studying potential neuroprotectants. We found that TRH and all the tested analogues at concentrations 0.1-50 μM attenuated cell damage induced by MPP(+) (2 mM), 3-nitropropionate (10 mM), hydrogen peroxide (0.5 mM), homocysteine (250 μM) and beta-amyloid (20μM) in retinoic acid differentiated SH-SY5Y cells. Furthermore, we demonstrated that TRH and its analogues decreased the staurosporine (0.5 μM)-induced LDH release, caspase-3 activity and DNA fragmentation, which indicate the anti-apoptotic proprieties of these peptides. The neuroprotective effects of TRH (10 μM) and RGH-2202 (10 μM) on St-induced cell death was attenuated by inhibitors of PI3-K pathway (wortmannin and LY294002), but not MAPK/ERK1/2 (PD98059 and U0126). Moreover, TRH and its analogues at neuroprotective concentrations (1 and 10 μM) increased expression of Bcl-2 protein, as confirmed by Western blot analysis. All in all, these results extend data on neuroprotective properties of TRH and its analogues and provide evidence that mechanism of anti-apoptotic effects of these peptides in SH-SY5Y cell line involves induction of PI3K/Akt pathway and Bcl-2. Furthermore, the data obtained on human cell line with a dopaminergic phenotype suggest potential utility of TRH and its analogues in the treatment of some neurodegenerative diseases including Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; Amyloid beta-Peptides; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Differentiation; Cell Line, Tumor; Convulsants; Cytotoxins; Homocysteine; Humans; Hydrogen Peroxide; MAP Kinase Signaling System; Neuroblastoma; Neuroprotective Agents; Nitro Compounds; Oxidants; Phosphatidylinositol 3-Kinases; Propionates; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Thyrotropin-Releasing Hormone; Tretinoin

Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Adhesion; Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ellagic Acid; Growth Inhibitors; Humans; Neurites; Neuroblastoma; Tretinoin; Tumor Stem Cell Assay

The neurotrophin brain-derived neurotrophic factor (BDNF) is a key survival factor for neural cells. In particular, in neuroblastoma tumour cells, expression of the BDNF/TrkB autocrine signalling system promotes a more malignant phenotype and resistance to chemotherapy. The human BDNF gene contains two clusters of upstream exons encoding the 5'UTR (exon 1 to exon 3 and exon 4 to exon 9a), these are alternatively spliced to a common exon 9, which contains the coding region and the 3'UTR. At least 34 different BDNF mRNA transcripts can be generated, although their physiological role is still unknown. The purpose of this study is to determine which BDNF transcript is involved in cell survival of the human neuroblastoma cell lines SH-SY-5Y (single-copy MYCN) and SK-N-BE (amplified MYCN). Expression of human BDNF mRNAs encoding all possible isoforms was characterised in the two neuroblastoma cell lines. We then investigated whether selective silencing of the different BDNF mRNAs using specific siRNAs could reduce cell survival in response to serum deprivation or the anticancer drugs cisplatin, doxorubicin and etoposide. We found that three isoforms located in the second exon cluster are essential for neuroblastoma cell survival under cytotoxic stress. Notably, promoters of the second exon cluster, but not the first, are controlled by Ca(2+)-sensitive elements.

Topics: Alternative Splicing; Animals; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line; Cell Survival; Exons; Humans; Neuroblastoma; Promoter Regions, Genetic; Protein Isoforms; Receptor, trkB; Stress, Physiological; Tretinoin

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

Topics: 1-Methyl-4-phenylpyridinium; Biomarkers; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Humans; Models, Neurological; Neuroblastoma; Neurons; Oxidopamine; Reactive Oxygen Species; Signal Transduction; Tretinoin

Revealing the molecular changes in chronic ethanol-impaired neuronal differentiation may be of great importance for understanding ethanol-related pathology in embryonic development but also in the adult brain. In this study, both acute and long-term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain-derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal-regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth.. The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT-PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK-cascade, protein kinase C (PKC) isoforms and Raf-Kinase-Inhibitor-Protein (RKIP).. Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF-mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf-1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down-regulated.. Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC-dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf-1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long-term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.

Topics: Biomarkers; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Central Nervous System Depressants; Enzyme Activation; Ethanol; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Neuroblastoma; Phosphatidylethanolamine Binding Protein; Protein Kinase C; Tretinoin

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK-N-SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up-regulated in SK-N-SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK-N-SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation.

Topics: Biomarkers; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Neurons; Nuclear Matrix-Associated Proteins; Tretinoin; Up-Regulation

Neuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously.. Here, neuroblastoma (NB) cell line subtypes were characterized according to embryonic peripheral nervous system development markers (GAP43, Phox2b, Sox10, c-kit, GD2, NF68, vimentin, S100beta, calcyclin and ABCG2), morphological features, gene expression and differentiation potential. I-type cells were investigated as a bipotential (neuronal and glial) differentiation stage.. Positive immunostaining of NCSC (GAP43, c-kit, NF68, vimentin and Phox2b) and undifferentiated cell (ABCG2) markers was observed in all NB subtypes. N- and I-type cells displayed cytoplasmic membrane GD2 staining, while nuclear calcyclin was restricted to S-type. N- and I-type cells showed similar phenotype and immunoreactivity pattern. Differential gene expression was associated with each cell subtype. N- and I-type cells displayed similar differentiation capacity towards neuronal and glial lineage fates. S-type cells, upon induction, did not show a neuronal-like phenotype, despite gene expression changes.. Results suggest that N- and I-type NB cell subtypes represent an immature bilineage stage, able to progress towards neuronal and glial fates upon induction of differentiation. S-type cells appear irreversibly committed to a glial lineage fate.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bromodeoxyuridine; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Lineage; GAP-43 Protein; Homeodomain Proteins; Humans; Immunohistochemistry; N-Acetylgalactosaminyltransferases; Neoplasm Proteins; Neural Crest; Neuroblastoma; Neurofilament Proteins; Proto-Oncogene Proteins c-kit; Reverse Transcriptase Polymerase Chain Reaction; S100 Calcium Binding Protein A6; S100 Proteins; SOXE Transcription Factors; Stem Cells; Transcription Factors; Tretinoin; Vimentin

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, and neuroblastoma. Unfortunately, its poor aqueous solubility hampers its parenteral formulation, whereas oral administration of ATRA is associated with progressively diminishing drug levels in plasma, which is related to induction of retinoic acid-binding proteins and increased drug catabolism by cytochrome P450-mediated reactions. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose, amphiphilic polymers were prepared by polyvinylalcohol (PVA) partial esterification with nicotinoyl moieties and their functional properties evaluated with regard to ATRA complexation. The physicochemical characteristics of the polymers and the complexes were analyzed by 1H-NMR, Dynamic Light Scattering (DLS), Capillary Electophoresis (CE), and were correlated with the complex ability to improve the drug solubilization and release the free drug in an aqueous environment. Subsequently, the best complex, providing the highest ATRA solubilization and release, was evaluated in vitro to test its cytotoxicity towards neuroblastoma cell lines. The PVA substitution degree calculated from 1H-NMR was found to be 5.0%, 8.2%, 15.3% (nicotinoyl moiety:PVA monomer molar ratio), while capillary electrophoresis analysis on the complexes revealed that the drug loadings were 0.95%, 1.20%, 4.76% (ATRA:polymer w:w) for PVA substitution degrees of 5.0%, 8.2%, and 15.3%, respectively. Complexation strongly increased ATRA aqueous solubility, which reached 1.20 +/- 0.25 mg/mL. The DLS measurements of the polymers and the complexes in aqueous solutions revealed mean sizes always below 400 nm, low polydispersity (min 0.202 +/- 0.013, max 0.450 +/- 0.032), and size almost unaffected by concentration. Drug fractional release did not exceed 8% after 48 h. The cytotoxicity studies against neuroblastoma cell lines outlined a significant growth inhibition effect of complexed ATRA with respect to free ATRA. These data suggest that the systems analyzed may be suitable carriers for parenteral administration of ATRA and other hydrophobic antitumor drugs, where the carriers are required to improve drug aqueous solubility and delay drug release almost after the

Topics: Antineoplastic Agents; Cell Proliferation; Chemistry, Pharmaceutical; Drug Administration Routes; Drug Carriers; Esters; Humans; Infusions, Parenteral; Micelles; Neuroblastoma; Polyvinyl Alcohol; Solubility; Tretinoin; Tumor Cells, Cultured

To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Collagen Type XI; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Neoplasm Proteins; Neuroblastoma; Neurons; Niacinamide; Sirtuin 1; Sirtuins; Tretinoin; Tyrosine 3-Monooxygenase; Vitamin B Complex

Properly designed genome-wide screening strategies can provide new insights into biological processes and/or biomarkers for malignant diseases. In this issue of Cancer Cell, Huang et al. demonstrate that the Krüppel zinc-finger protein ZNF423 is critical for retinoic acid signaling and is likely a favorable prognostic marker for neuroblastoma.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Differentiation; Humans; Neuroblastoma; Prognosis; Signal Transduction; Tretinoin; Zinc Fingers

Retinoids play key roles in differentiation, growth arrest, and apoptosis and are increasingly being used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Here, using a large-scale RNA interference-based genetic screen, we identify ZNF423 (also known as Ebfaz, OAZ, or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARalpha/RXRalpha nuclear receptor complex and is essential for transactivation in response to retinoids. Downregulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome in neuroblastoma patients.

Topics: Adolescent; Antineoplastic Agents; Cell Differentiation; Child; Child, Preschool; Chromatin Immunoprecipitation; DNA-Binding Proteins; Female; Humans; Infant; Infant, Newborn; Male; Neuroblastoma; Prognosis; Promoter Regions, Genetic; Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Survival Rate; Teratocarcinoma; Tretinoin; Zinc Fingers

Differentiated histopathology is a favorable prognostic factor in neuroblastic tumors, and molecular pathways underlying neuroblastoma differentiation can be modulated pharmacologically. The calcium-sensing receptor (CaR) and parathyroid hormone-related protein (PTHrP) regulate differentiation processes in some cellular contexts. CaR is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and regulates PTHrP production in astrocytes. The objective of the current study was to assess whether CaR and PTHrP participate in neuroblastoma differentiation pathways.. CaR and PTHrP messenger RNA (mRNA) and protein expression were analyzed in neuroblastic tumors, and correlation with prognostic factors was assessed. CaR and PTHrP expression levels were analyzed in neuroblastoma cell lines treated with all-trans-retinoic acid or 5-bromo-2'-deoxyuridine (BrdU).. CaR expression was correlated with favorable histology, age at diagnosis <1 year, low clinical stage, and low clinical risk. CaR was absent in undifferentiated neuroblasts and was expressed in differentiating neuroblasts. CaR and PTHrP were highly expressed in ganglion and in Schwann-like cells. PTHrP mRNA levels were higher in ganglioneuroblastomas and ganglioneuromas than in neuroblastomas (P < .0001). Both genes were up-regulated in neuroblastomas with treatment-induced maturation features. CaR, but not PTHrP, was up-regulated at early phases of in vitro neuronal differentiation induction. Substrate-adherent, non-neuronal cell lines displayed the highest PTHrP levels among the neuroblastoma cell lines examined. The up-regulation of PTHrP and of 2 glial differentiation markers was observed in 2 cell lines that were treated with BrdU, whereas CaR was induced in only 1 cell line.. CaR and PTHrP were expressed in differentiated, favorable neuroblastic tumors, and both genes were up-regulated by inducing differentiation.

Topics: Bromodeoxyuridine; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Ganglioneuroblastoma; Ganglioneuroma; Humans; Neuroblastoma; Neuroglia; Neurons; Parathyroid Hormone-Related Protein; Receptors, Calcium-Sensing; RNA, Messenger; Schwann Cells; Tretinoin; Up-Regulation

Ethanol is a potent teratogen for the developing central nervous system (CNS), and fetal alcohol syndrome (FAS) is the most common nonhereditary cause of mental retardation. Ethanol disrupts neuronal differentiation and maturation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Using an in vitro neuronal model, mouse Neuro2a (N2a) neuroblastoma cells, we demonstrated that ethanol inhibited neurite outgrowth and the expression of neurofilament (NF) proteins. Glycogen synthase kinase 3beta (GSK3beta), a multifunctional serine/threonine kinase negatively regulated neurite outgrowth of N2a cells; inhibiting GSK3beta activity by retinoic acid (RA) and lithium induced neurite outgrowth, while over-expression of a constitutively active S9A GSK3beta mutant prevented neurite outgrowth. Ethanol inhibited neurite outgrowth by activating GSK3beta through the dephosphorylation of GSK3beta at serine 9. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family rich in many edible berries and other pigmented fruits, enhanced neurite outgrowth by promoting p-GSK3beta(Ser9). More importantly, C3G reversed ethanol-mediated activation of GSK3beta and inhibition of neurite outgrowth as well as the expression of NF proteins. C3G also blocked ethanol-induced intracellular accumulation of reactive oxygen species (ROS). However, the antioxidant effect of C3G appeared minimally involved in its protection. Our study provides a potential avenue for preventing or ameliorating ethanol-induced damage to the developing CNS.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Anthocyanins; Antioxidants; Cell Lineage; Central Nervous System Depressants; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Ethanol; Gene Expression Regulation; Glucosides; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Lithium Chloride; Mice; Neurites; Neuroblastoma; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Tretinoin

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

Topics: Adenosine Triphosphate; Animals; Calcium Signaling; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Survival; Gene Knockdown Techniques; Mice; Neurites; Neuroblastoma; Neurons; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; RNA, Small Interfering; Tretinoin

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental pollutant, is known to induce neurodevelopmental and neurobehavioral deficits. However, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. We have studied TCDD inhibition of neurite outgrowth using human SH-SY5Y neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). TCDD, at concentrations of 3 nM or 5 nM, had no significant effect on the viability of either undifferentiating or differentiated SH-SY5Y cells. However, differentiating SH-SY5Y cells exhibited a distinct decrease of neurite outgrowth 48 h after TCDD treatment in a dose-dependent manner. TCDD treatment 12h or 24h after RA stimulation did not elicit a significant inhibition of neurite outgrowth, whereas TCDD cotreatment with RA or TCDD treatment at 6h after RA stimulation significantly inhibited neurite outgrowth. Western blot analysis of cell extracts of RA-stimulated differentiating SH-SY5Y cells showed increased level of cross reactivities with tissue glutaminase (TGase) antibody compared to control extracts, in a time-dependent manner. By contrast, treatment of differentiating SH-SY5Y cells with 1-5 nM TCDD resulted in decreased level of cross-reactivities with TGase antibody in a dose-dependent manner. The results indicate that TCDD is able to inhibit neurite outgrowth by differentiating SH-SY5Y cells and that this effect might result from reduced levels of TGase.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Environmental Pollutants; Humans; Microscopy, Phase-Contrast; Neurites; Neuroblastoma; Polychlorinated Dibenzodioxins; Transglutaminases; Tretinoin

Inhibitory M(2) muscarinic receptors on airway parasympathetic nerves normally limit acetylcholine release. Viral infections decrease M(2) receptor function, increasing vagally mediated bronchoconstriction. Since retinoic acid deficiency causes M(2) receptor dysfunction, we tested whether retinoic acid would prevent virus-induced airway hyperreactivity and prevent M(2) receptor dysfunction. Guinea pigs infected with parainfluenza virus were hyperreactive to electrical stimulation of the vagus nerves, but not to intravenous acetylcholine, indicating that hyperreactivity was due to increased release of acetylcholine from parasympathetic nerves. The muscarinic agonist pilocarpine, which inhibits vagally mediated bronchoconstriction in control animals, no longer inhibited vagally induced bronchoconstriction, demonstrating M(2) receptor dysfunction. Treatment with all-trans retinoic acid (1 mg/kg) prevented virus-induced hyperreactivity and M(2) receptor dysfunction. However, retinoic acid also significantly reduced viral titers in the lungs and attenuated virus-induced lung inflammation. In vitro, retinoic acid decreased M(2) receptor mRNA expression in both human neuroblastoma cells and primary cultures of airway parasympathetic neurons. Thus, the protective effects of retinoic acid on airway function during viral infection appear to be due to anti-inflammatory and antiviral mechanisms, rather than to direct effects on M(2) receptor gene expression.

Topics: Acetylcholine; Animals; Antineoplastic Agents; Bronchial Hyperreactivity; Bronchoconstriction; Cell Line, Tumor; Cholinergic Agents; DEAD Box Protein 58; DEAD-box RNA Helicases; Female; Gene Expression; Guinea Pigs; Humans; Interleukin-8; Muscarinic Agonists; Neuroblastoma; Neurons; Paramyxoviridae Infections; Pilocarpine; Receptor, Muscarinic M2; Receptors, Immunologic; Respiratory Mucosa; Trachea; Tretinoin; Virus Replication

The amphetamine derivative 3, 4-methylenedioxymethamphetamine (MDMA) has become a popular recreational drug, and has also been shown to cause serotonergic neurotoxicity. This report shows that MDMA impairs brain development in a whole mouse embryo culture. The results of quantitative real-time PCR analysis showed that autophagy-related protein 5 (Atg5) expression is elevated in mouse embryo and neuroblastoma cells after MDMA treatment. This elevated Atg5 expression interferes with the neuronal differentiation of neuroblastoma cells such as SH-SY5Y and PC12 cells. Thus, our results suggest that the use of MDMA during pregnancy may impair neuronal development via an induction of Atg5 expression.

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Cell Differentiation; Embryo Culture Techniques; Female; Fetal Development; Humans; Mice; Microtubule-Associated Proteins; N-Methyl-3,4-methylenedioxyamphetamine; Neuroblastoma; Neurons; PC12 Cells; Pregnancy; Proteins; Rats; Tretinoin

Alpha-synuclein (ASYN) is central in Parkinson's disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson's disease. To elucidate the mechanism underlying wild type (WT) ASYN-mediated neurotoxicity, we have generated a novel Tet-Off SHSY-5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid-differentiated SHSY-5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non-apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo-inositol, an oligomer-stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non-apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN-induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.

Topics: alpha-Synuclein; Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Inositol; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Staurosporine; Time Factors; Transfection; Tretinoin

Neuroblastoma is the childhood malignancy that mainly occurs in adrenal glands and is found also in the neck, chest, abdomen, and pelvis. New therapeutic strategies are urgently needed for successful treatment of this pediatric cancer. In this investigation, we examined efficacy of the retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the isoflavonoid genistein (GST) alone and also in combination for controlling the growth of human malignant neuroblastoma SK-N-BE2 and SH-SY5Y xenografts in nude mice. Combination of 4-HPR and GST significantly reduced tumor volume in vivo due to overwhelming apoptosis in both neuroblastoma xenografts. Time-dependently, combination of 4-HPR and GST caused reduction in body weight, tumor weight, and tumor volume. Combination of 4-HPR and GST increased Bax:Bcl-2 ratio, mitochondrial release of Smac, downregulation of baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins including BIRC-2 and BIRC-3, and activation of caspase-3 and apoptosis inducing factor (AIF). Further, downregulation of nuclear factor-kappa B (NF-kappaB), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2) was also detected. In situ immunofluorescent labelings of tumor sections showed overexpression of calpain, caspase-12, and caspase-3, and also AIF in the course of apoptosis. Combination therapy increased apoptosis in the xenografts but did not induce kidney and liver toxicities in the animals. Results demonstrated that combination of 4-HPR and GST induced multiple molecular mechanisms for apoptosis and thus could be highly effective for inhibiting growth of malignant neuroblastoma in preclinical animal models.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Body Weight; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Genistein; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Nude; Neuroblastoma; NF-kappa B; Transplantation, Heterologous; Treatment Outcome; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The goal of present work was to investigate the use of bioerodible polymeric nanoparticles as carriers of retinoic acid (RA), which is known to induce differentiation of several cell lines into neurons. A novel method, named "Colloidal-Coating", has been developed for the preparation of nanoparticles based on a copolymer of maleic anhydride and butyl vinyl ether (VAM41) loaded with RA. Nanoparticles with an average diameter size of 70 nm and good morphology were prepared. The activity of the encapsulated RA was evaluated on SK-N-SH human neuroblastoma cells, which are known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The activity of RA was not affected by the encapsulation and purification processes.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colloids; Ethers; Humans; Maleic Anhydrides; Nanoparticles; Neuroblastoma; Tissue Engineering; Tretinoin; Vinyl Compounds

The transcription factor Nrf2 has emerged as a master regulator of the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate, two well-studied inducers of neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 in a dose- and time-dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M and microtubule-associated protein 2. Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation, whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to those from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.

Topics: Animals; Antigens, Differentiation; Cell Culture Techniques; Cell Differentiation; Chromatin Immunoprecipitation; Humans; Mice; Mice, Knockout; Microtubule-Associated Proteins; NAD(P)H Dehydrogenase (Quinone); Neuroblastoma; Neurofilament Proteins; Neurons; NF-E2-Related Factor 2; Phorbol Esters; RNA, Small Interfering; Transcriptional Activation; Tretinoin

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.

Topics: Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neuroblastoma; Neurogenesis; Tretinoin

MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.

Topics: Base Sequence; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Cell Movement; Doublecortin Domain Proteins; Doublecortin Protein; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; MicroRNAs; Microtubule-Associated Proteins; Molecular Sequence Data; Neoplasm Invasiveness; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neuropeptides; Reelin Protein; Serine Endopeptidases; Tretinoin

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a-1l, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.

Topics: Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Gene Expression; Gene Expression Regulation; Humans; Immunoprecipitation; Neuroblastoma; Nitric Oxide Synthase Type I; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Retinoic Acid; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Tretinoin

MYCN oncogene is one of the most important regulators affecting the prognosis of neuroblastoma and is frequently amplified in the high-risk subsets. Despite its clinical significance, it remains unclear how the MYCN expression is regulated in human neuroblastomas. Here, we found the presence of a positive auto-regulatory mechanism of MYCN. Enforced expression of MYCN induced endogenous MYCN mRNA expression in SK-N-AS neuroblastoma cells with a single copy of MYCN gene. Luciferase reporter assay revealed that MYCN protein activates its own promoter activity in a dose-dependent manner and the downstream region relative to the transcription start sites is responsible for the activation. Furthermore, ChIP analysis showed that MYCN is directly recruited onto the intron 1 region of MYCN gene which contains two putative E-box sites. Intriguingly, in response to all-trans-retinoic acid (ATRA), MYCN was down-regulated in MYCN-amplified SK-N-BE neuroblastoma cells, and the recruitment of MYCN protein onto its own intron 1 region was reduced in association with an induction of neuronal differentiation. Collectively, our present results suggest that MYCN contributes to its own expression by forming a positive auto-regulatory loop in neuroblastoma cells.

Topics: Cell Line, Tumor; Chromatin Immunoprecipitation; E-Box Elements; Gene Expression Regulation; Genome, Human; Homeostasis; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Neurogenesis; Neurons; Nuclear Proteins; Oncogene Proteins; Promoter Regions, Genetic; RNA, Messenger; Tretinoin

To identify potential biomarkers of therapy response, we have previously done a large-scale gain-of-function genetic screen to identify genes whose expression confers resistance to histone deacetylase inhibitors (HDACI). This genetic screen identified two genes with a role in retinoic acid signaling, suggesting that HDACIs target retinoic acid signaling as part of their anticancer effect. We study here a third gene identified in this genetic screen, UNC45A, and assess its role in retinoic acid signaling and responses to HDACIs using cell-based proliferation and differentiation assays and transcriptional reporter gene assays. The vertebrate Unc45 genes are known for their roles in muscle development and the assembly and cochaperoning of the muscle motor protein myosin. Here, we report that human UNC45A (GCUNC45) can render transformed cells resistant to treatment with HDACIs. We show that UNC45A also inhibits signaling through the retinoic acid receptor alpha. Expression of UNC45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and inhibits the induction of endogenous retinoic acid receptor target genes. These data establish an unexpected role for UNC45A in causing resistance to both HDACI drugs and retinoic acid. Moreover, our data lend further support to the notion that HDACIs exert their anticancer effect, at least in part, through an effect on retinoic acid signaling.

Topics: Amino Acid Sequence; Animals; Bone Neoplasms; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Intracellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Neuroblastoma; Osteosarcoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin

Activated microglia appear to selectively attack dopamine (DA) neurons in the Parkinson's disease (PD) substantia nigra. We investigated potential mechanisms using culture models. As targets, human SH-SY5Y cells were left undifferentiated (UNDIFF) or were differentiated with retinoic acid (RA) or RA plus brain-derived neurotrophic factor (RA/BDNF). RA/BDNF-treated cells were immunoreactive for tyrosine hydroxylase and the DA transporter, took up exogenous DA, and released DA after K(+) stimulation. Undifferentiated and RA-treated cells lacked these characteristics of a DA phenotype. Co-culture of target cells with human elderly microglia resulted in elevated toxicity in DA phenotype (RA/BDNF) cells. Lipopolysaccharide (LPS) plus K(+)-stimulated DA release enhanced toxicity by 500-fold. DA induced microglial chemotaxis in Boyden chambers. Spiperone inhibited this effect. Cultured human elderly microglia expressed mRNAs for D1-D4 but not D5 DA receptors. The microglia, as well as PD microglia in situ, were also immunoreactive for D1-D4 but not D5 DA receptors. These findings demonstrate that activated microglia express DA receptors, and suggest that this mechanism may play a role in the selective vulnerability of DA neurons in PD.

Topics: Aged; Aging; Antineoplastic Agents; Brain-Derived Neurotrophic Factor; Cells, Cultured; Coculture Techniques; Dentate Gyrus; Dopamine; Dopamine Antagonists; Gene Expression Regulation; Humans; Lipopolysaccharides; Microglia; Neuroblastoma; Parkinson Disease; Potassium; Receptors, Dopamine; RNA, Messenger; Spiperone; Tretinoin

The homeodomain transcription factors PHOX2A and PHOX2B are vital for development of the autonomic nervous system. Their spatial and temporal expression at the neural crest is instrumental in determining neuronal precursor fate, and by regulating DbetaH expression, the enzyme catalysing noradrenaline synthesis from dopamine, they also play a role in determination of noradrenergic phenotype. Disturbing this finely regulated process leads to disruption of autonomic development and autonomic dysfunction syndromes such as DbetaH deficiency. As it had previously been shown that the catecholamine system is responsive to ELF-EMF, and as this has also been linked to various pathologies and to certain types of cancer, we wondered whether exposure to this type of radiation could affect the expression of PHOX2A, PHOX2B and DbetaH, also during differentiation triggered by retinoic acid. To investigate this possibility we exposed the human SH-SY5Y neuroblastoma cell line to 50 Hz power-line magnetic field at various flux densities and for various exposure times. We measured gene expression in exposed cells compared to control cells and also investigated any changes at protein level. Using our exposure protocol, we found no changes at either transcript or protein level of these important components of the autonomic nervous system and catecholaminergic system.

Topics: Cell Line, Tumor; Dopamine beta-Hydroxylase; Electromagnetic Fields; Gene Expression; Homeodomain Proteins; Humans; Neuroblastoma; Time Factors; Transcription Factors; Transcription, Genetic; Tretinoin

Therapies aiming at inducing differentiation or apoptosis of neuroblastoma (NB) are an important research topic. Although retinoic acid showed promising antitumoral results, its effects against refractory disease are limited. Putative candidates for combination therapies are nerve growth factor (NGF; Tebu-Bio/Peprotech, Offenbach, Germany) and brain-derived neurotrophic factor (BDNF; Tebu-Bio/Peprotech, Offenbach, Germany) because their receptors are of prognostic clinical value in clinical neuroblastoma. Another clinical prognostic factor is the number of Schwann cells. Substances secreted by Schwann cells proved antitumoral capacities in vitro. The aim of the study was to analyze whether retinoic acid may offer an additional line of attack acting independent from Schwann cells and whether additive treatment with the neurotrophin-receptor ligands NGF/BDNF confers additional benefit.. Human SHSY-5Y NB cells were cultured in vitro. After a 7-day all-trans retinoic acid (ATRA; Sigma-Aldrich Chemie, Taufkirchen, Germany) treatment (15 mumol/L of ATRA), NB proliferation was proportional to extinction in dimethyl-thiazol-diphenyltetrazoliumbromide (MTT) tests. Fluorescence-activated cell sorter (FACS) analysis for annexin and propidium iodide determined the degree of apoptosis and necrosis as well as the expression of the Schwann type cell marker S100. The S100 messenger RNA was assessed by reverse transcriptase polymerase chain reaction. In addition, the effect on NB proliferation was investigated when ATRA was combined with a 7-day treatment with NGF or BDNF (10, 50, 100 ng/mL) either before or after the 7-day ATRA treatment.. All-trans retinoic acid reduced proliferation (0.116 +/- 0.006 SEM vs 0.359 +/- 0.010 SEM in the untreated control group; P < .001). After ATRA treatment, 95% +/- 1.82% SEM were still viable, with only 2.61% +/- 1.17% SEM apoptotic and 2.38% +/- 0.69% SEM necrotic cells. All-trans retinoic acid induced a remarkable decrease in S100 expression in FACS (16.91% +/- 1.72% SEM vs 32.33% +/- 2.54% SEM in controls; P = .009). The S100 messenger RNA levels were not increased by ATRA (DeltaDeltaT values: 1.73, 2.77, and 1.43; n = 3). Both NGF and BDNF had only a modest synergistic effect when given after ATRA treatment. No effect was seen when they were administered before ATRA treatment.. All-trans retinoic proved to be a vigorous inhibitor of NB proliferation in vitro. However, because most NB cells remained viable combination therapies are required. Treatment with NGF and BDNF showed only a modest benefit and did not reflect the strong prognostic impact of tyrosine kinase receptors in clinical NB. The ATRA-induced proliferation arrest is not related to Schwann type subdifferentiation. This suggests that substances secreted by Schwann cells could be possible independent combination partners. We suggest studies using combinations of ATRA and substances secreted by Schwann cells.

Topics: Antineoplastic Agents; Apoptosis; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Nerve Growth Factor; Neuroblastoma; Tretinoin

Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

Topics: alpha-Methyltyrosine; Animals; Antineoplastic Agents; Butyrates; Carbazoles; Cell Differentiation; Cell Line, Tumor; Cyclic CMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Neuroblastoma; Norepinephrine; Phenotype; Pyrroles; Tretinoin; Tyrosine 3-Monooxygenase

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.

Topics: Amines; Cell Line, Tumor; Computer Simulation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Ligands; Models, Chemical; Models, Molecular; Molecular Structure; Neuroblastoma; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Software; Stereoisomerism; Structure-Activity Relationship; Tretinoin

Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.

Topics: Animals; Animals, Genetically Modified; Axons; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Line; Chlorocebus aethiops; Doxycycline; Gene Expression Regulation; Humans; Microfilament Proteins; Mutation; Nerve Growth Factors; Neurites; Neuroblastoma; Neurofilament Proteins; Neurons; RNA Interference; Time Factors; Transfection; Tretinoin; Tubulin

Possible effects of interaction (cross-talk) between signaling pathways is studied in a system of Reaction-Diffusion (RD) equations. Furthermore, the relevance of spontaneous neurite symmetry breaking and Turing instability has been examined through numerical simulations. The interaction between Retinoic Acid (RA) and Notch signaling pathways is considered as a perturbation to RD system of axon-forming potential for N2a neuroblastoma cells. The present work suggests that large increases to the level of RA-Notch interaction can possibly have substantial impacts on neurite outgrowth and on the process of axon formation. This can be observed by the numerical study of the homogeneous system showing that in the absence of RA-Notch interaction the unperturbed homogeneous system may exhibit different saddle-node bifurcations that are robust under small perturbations by low levels of RA-Notch interactions, while large increases in the level of RA-Notch interaction result in a number of transitions of saddle-node bifurcations into Hopf bifurcations. It is speculated that near a Hopf bifurcation, the regulations between the positive and negative feedbacks change in such a way that spontaneous symmetry breaking takes place only when transport of activated Notch protein takes place at a faster rate.

Topics: Cell Line, Tumor; Diffusion; Humans; Models, Biological; Neuroblastoma; Receptors, Notch; Signal Transduction; Tretinoin

Neuroblastoma is a pediatric tumor which can spontaneously regress or differentiate into a benign tumor. MYCN oncogene amplification occurs in 22% of neuroblastomas and is associated with poor prognosis. Retinoic acid (RA), a molecule able to induce differentiation and to decrease MYCN expression, is used in the therapy of neuroblastomas. The neuropeptide vasoactive intestinal peptide (VIP) is known to control proliferation or differentiation of numerous cancer cells. In vitro, VIP induces differentiation of neuroblastoma cells. To determine whether VIP could modulate MYCN expression, we carried out real-time quantitative RT-PCR and Western immunoblot analyses in human neuroblastoma SH-SY5Y and IMR-32 cells. The results indicated that VIP reduced MYCN mRNA and protein expression, especially in the MYCN-amplified IMR-32 cells, with a maximal and transient decrease by approximately 50% after few hours of treatment with VIP at 10(-6) M. This effect was compared to that of RA at 10(-5) M, which induced a diminution of MYCN mRNA expression by approximately 25% after few days of treatment. This indicated that VIP and RA display complementary kinetics. Cotreatments showed that VIP and RA had synergistic effects on regulation of expression of MYCN proteins. VIP and RA cotreatments regulated also expression of two MYCN target genes, SKP2 and TP53INP1. These results suggest that VIP, in combination with RA may have a potential therapeutic benefit in neuroblastomas with MYCN amplification, a genetic abnormality associated with poor prognosis.

Topics: Antineoplastic Agents; Carrier Proteins; Cell Line, Tumor; Cell Shape; Down-Regulation; Drug Synergism; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Kinetics; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; RNA, Messenger; S-Phase Kinase-Associated Proteins; Tretinoin; Vasoactive Intestinal Peptide

Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

Topics: alpha-Synuclein; Analysis of Variance; Benzothiazoles; Cell Count; Cell Differentiation; Cell Fractionation; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Microscopy, Electron, Transmission; Molecular Weight; Neuroblastoma; Neurofilament Proteins; Neurons; Tetracycline; Thiazoles; Time Factors; Transfection; Tretinoin

MYCN amplification marks poor prognosis in neuroblastoma (NB) tumors. In evaluating the mechanisms by which retinoic acid (RA) or nerve growth factor (NGF) decrease cell number in MYCN amplified NB cells, we have identified a number of proteins whose expression either decreases (E2F, CDC2, CDK6, cyclin dependent kinase activity) or increases (p27) in association with a decrease in MYCN expression. However, it was still unclear which were MYCN dependent effects or not.. This study aimed to determine which changes in cell cycle gene expression are modulated as a consequence of the decrease in MYCN. We silenced MYCN expression using siRNA targeted to the coding region of MYCN. Then, by using siRNA transient transfections, we analyzed the change of cell cycle related genes and cell cycle in MYCN amplified NB cell lines.. We demonstrate that expression of MYCN can be suppressed by almost 60% in MYCN amplified NB cell using siRNAs targeted to MYCN. Functionally, the decrease in MYCN leads to a decrease in cells in the S-phase of the cell cycle. Decreases in MYCN are associated with decreases in E2F1-2 and ID2 along with increases in p27 protein levels by post-transcriptional modification. Moreover, we find that a decrease in MYCN is accompanied by a decrease in cdk6 mRNA and protein expression.. These results show that E2F and ID2 expression is associated with MYCN regulation and that cdk6 is a possible new transcriptional target of MYCN.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Gene Silencing; Humans; Mice; N-Myc Proto-Oncogene Protein; Neuroblastoma; NIH 3T3 Cells; Nuclear Proteins; Oncogene Proteins; RNA Interference; RNA, Small Interfering; Transfection; Tretinoin

Use of retinoic acid (RA) has become the standard of care in the treatment of high risk neuroblastoma (NB). In vitro, RA induces growth arrest and differentiation, an effect that likely underlies its activity in the clinical setting. An important event in differentiation is the transcriptional downregulation of the MYCN oncogene, which is frequently activated in aggressive tumors. While it is known that Sp1/Sp3 and E2F are necessary to drive basal MYCN expression, the mechanism for its downregulation by RA remains enigmatic. Changes in E2F binding have been reported, however these occurred after the actual transcriptional response. Here, post-translational modifications of Sp proteins were examined as an alternate mechanism of RA-mediated promoter regulation.. Western blot was used to evaluate steady state levels of nuclear/cytoplasmic Sp1/Sp3. Promoter binding and DNA conformation were determined by gel shift, circular permutation, and chromatin immunoprecipitation assays. Immunoprecipitation/western and (32)P-phosphoamino analyses were used to detect glycosylation, acetylation, sumoylation, and phosphorylation.. RA did not affect the cellular level of Sp1/Sp3 proteins, their nuclear/cytoplasmic distribution, ability to bind the MYCN promoter, degree of Sp-induced DNA bending, or post-translational modifications.. MYCN RA response is not mediated solely though the region controlling basal activity. RA may be exerting its effects via multiple non-adjacent regulatory regions, potentially including basal motifs, either within the MYCN promoter or distally, on the same or even different chromosomes. Such cooperative trans-type DNA-protein interactions could explain the inaccessibility of this mechanism to the locus-specific approaches employed up to this point.

Topics: Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Down-Regulation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulins; Immunoprecipitation; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Protein Processing, Post-Translational; Protein Transport; Sp3 Transcription Factor; Tretinoin

The diversity of neuroblastoma and its clinical course depends on histology, biology and clinical features. We report a male presenting at 4 months of age with an abdominal mass and multiple subcutaneous nodules. The diagnosis was made by histological examination of a subcutaneous nodule and elevated urinary markers. The patient remained well during the subsequent 9 years. During that time no cytostatic treatment was given. Attempt to treat with cis-retinoic acid 10 years later did not result in any significant change of the clinical course. The patient has remained in good clinical condition for a 15-year observation period, having both progressing and regressing distant subcutaneous metastases. Skin nodules are the hallmarks of the indolent clinical course of the disease. We suggest the use of the "chronic neuroblastoma" as a term to describe patients with neuroblastoma showing indolent disease course over a very long period of time, but never achieving complete remission.

Topics: Abdominal Neoplasms; Adolescent; Adrenal Gland Neoplasms; Antineoplastic Agents; Child; Child, Preschool; Chronic Disease; Humans; Immunohistochemistry; Infant; Liver Neoplasms; Male; Neuroblastoma; Skin Neoplasms; Tretinoin

Neuroblastoma is one of the most common cancers in children. Neuroblastoma differentiation is linked to the presence of the promyelocytic leukemia (PML) protein. Retinoic acid, a powerful differentiation-inducer in vitro, is a potent agent for the treatment of neuroblastoma. Using two different human neuroblastoma cell lines, SH-SY5Y and LA-N-5, we show here that PML protein leads to the formation of nuclear bodies (PML-NB) after only 1 h of retinoic acid treatment and that this formation is mediated by the extracellular signal-regulated kinase (ERK) pathway. Inhibition of protein kinase C also leads to formation of PML-NB via the ERK pathway. Both sumoylation and phosphorylation of PML in an ERK-dependent pathway are also required for formation of PML-NB. Finally, we show that PML-NB formation in neuroblastoma cells is associated with neurite outgrowth. These results support the proposal that the formation of PML-NB is correlated with the differentiation of neuroblastoma cells.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Nucleus Structures; Cell Proliferation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Keratolytic Agents; Models, Biological; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Phosphorylation; Promyelocytic Leukemia Protein; Signal Transduction; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

Retinoic acid stimulates alpha-secretase processing of amyloid precursor protein (APP) and decreases beta-secretase cleavage that leads to amyloid-beta formation. Here, we investigated the effect of retinoic acid on the two putative alpha-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the beta-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of alpha-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.

Topics: Amyloid beta-Protein Precursor; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protease Nexins; Protein Kinase C; Receptors, Cell Surface; Signal Transduction; Tretinoin; Up-Regulation

Neuroblastoma (NB) and Ewing's sarcoma (ES) cell lines were analysed by two-dimensional gel electrophoresis (2-DE) searching for new diagnostic/prognostic markers. Protein expression profiles displayed a high degree of similarity with the exception of marked heat shock protein (HSP) 27 and less marked HSP60 and HSP70 family up-modulations in NB cells. HSP27, which showed peculiar variability in different NB cell preparations, responded to all trans-retinoic acid treatment in NB cells but not in ES cells at gene and protein expression levels. Immunohistochemistry studies showed different behaviours of HSP27 and HSP70 expression in NB and ES biopsies. HSP27 was less expressed, whereas HSP70 was more expressed in the immature areas of NB. HSP27 expression showed positive and statistically significant correlation with favourable prognosis, and HSP27 expression also negatively correlated with increasing aggressiveness of histological type. In ES, both chaperones were expressed without characteristic patterns. Our results suggest that HSP27, after further clinical validations, could be used as a marker of neuronal differentiation in vivo for the assessment of the biological behaviour of NB and for the risk stratification of patients.

Topics: Adolescent; Biomarkers, Tumor; Cell Line, Tumor; Cell Transformation, Neoplastic; Child; Child, Preschool; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Infant; Infant, Newborn; Kidney Neoplasms; Neuroblastoma; Prognosis; Proteomics; Sarcoma, Ewing; Tretinoin

Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. In this study, we present evidence that autophagy, a process for bulk degradation of cytoplasm, is activated during retinoic acid-induced neuronal differentiation of neuroblastoma N2a cells. Chemical inhibitors of autophagy, including 3-MA and LY294002, abrogate cell differentiation. RNA interference of autophagy gene beclin 1 markedly delays the process of differentiation. We also find that cell differentiation is accompanied by decreased activity of mTOR, a major controller of cell growth and a negative regulator of autophagy. However, completely inhibiting mTOR by rapamycin decreases neurite outgrowth, cell size and the immunoreactivity for neuronal markers. Our study suggests that an appropriate level of mTOR activity is important in cell differentiation for a balance between macromolecule synthesis and degradation.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Size; Chromones; Dose-Response Relationship, Drug; Down-Regulation; Mice; Morpholines; Neurites; Neuroblastoma; Neurons; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tretinoin

In neuroblastoma cells, retinoic acid induces cell cycle arrest and differentiation through degradation of the F-box protein, Skp2, and stabilization of cyclin-dependent kinase inhibitor, p27. However, the mechanism responsible for retinoic acid-mediated Skp2 destabilization is unknown. Since Skp2 is degraded by anaphase-promoting complex (APC)(Cdh1), here we studied whether retinoic acid promotes differentiation of human SH-SY5Y neuroblastoma cells by modulating Cdh1. We found that retinoic acid induced the nuclear accumulation of Cdh1 that paralleled Skp2 destabilization and p27 accumulation. The mRNA and protein abundance of Rae1-a nuclear export factor that limits APC(Cdh1) activity in mitosis-decreased upon retinoic acid-induced inhibition of neuroblastoma cell proliferation. Furthermore, either Rae1 overexpression or Cdh1 inhibition promoted Skp2 accumulation, p27 destabilization and prevented retinoic acid-induced cell cycle arrest and differentiation. Conversely, inhibition of Rae1 accelerated retinoic acid-induced differentiation. Thus, retinoic acid downregulates Rae1, hence facilitating APC(Cdh1)-mediated Skp2 degradation leading to the arrest of cell cycle progression and neuroblastoma differentiation.

Topics: Anaphase-Promoting Complex-Cyclosome; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Nuclear Matrix-Associated Proteins; Nucleocytoplasmic Transport Proteins; Protein Processing, Post-Translational; S-Phase Kinase-Associated Proteins; Tretinoin; Ubiquitin-Protein Ligase Complexes

Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.. We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.. Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.. a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Movement; Doublecortin Domain Proteins; Doublecortin Protein; Fluorescent Antibody Technique; Humans; Microtubule-Associated Proteins; Neoplasm Invasiveness; Neuroblastoma; Neurofilament Proteins; Neuropeptides; Tretinoin; Vimentin

Retinoids are signaling molecules that are involved in proliferation, differentiation, and apoptosis during development. Retinoids exert their effects, in part, by binding to nuclear receptors, thereby altering gene expression. Clinical use of retinoids in the treatment of neuroblastoma is of interest due to their success in management of acute promyelocytic leukemia. Using the SK-N-SH human neuroblastoma cell line we investigated the effects of the differentiation agent all-trans-retinoic acid (ATRA) on the expression of manganese superoxide dismutase (MnSOD), an enzyme previously shown to enhance differentiation in vitro. Manganese superoxide dismutase mRNA, protein, and activity levels increased in a time-dependent manner upon treatment with ATRA. Nuclear levels of the NF-kappaB proteins p50 and p65 increased within 24 h of ATRA administration. This increase paralleled the degradation of the cytoplasmic inhibitor IkappaB-beta. Furthermore an increase in DNA binding to a NF-kappaB element occurred within a 342-bp enhancer (I2E) of the SOD2 gene with 10 microM ATRA treatment. Reporter analysis showed that ATRA-mediated I2E-dependent luciferase expression was attenuated upon mutation of the NF-kappaB element, suggesting a contribution of this transcription factor to retinoid-mediated upregulation of MnSOD. This study identifies SOD2 as a retinoid-responsive gene and demonstrates activation of the NF-kappaB pathway in response to ATRA treatment of SK-N-SH cells. These results suggest that signaling events involving NF-kappaB and SOD2 may contribute to the effects of retinoids used in cancer therapy.

Topics: Enzyme Induction; Gene Expression Regulation, Enzymologic; Humans; I-kappa B Proteins; Neuroblastoma; NF-kappa B; RNA, Messenger; Superoxide Dismutase; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; Humans; Insulin-Like Growth Factor I; Neuroblastoma; Neurons; PC12 Cells; Protein Binding; Rats; Receptors, Opioid, mu; Repressor Proteins; Transcriptional Activation; Tretinoin; Up-Regulation

Brain-derived neurotrophic factor (BDNF) and its specific tryrosin kinase receptor-B (TrkB) are highly correlated to the chemoresistance of neuroblastoma (NB) cells and poor prognosis. This study observed the changes of the sensibility of NB cells to chemotherapy drug cisplatin (CDDP) before and after blockage of TrkB-BDNF signal pathway by specific tyrosin kinase inhibitor K252a.. Human NB cell line SH-SY5Y (SY5Y) was routinely cultured. Expression of TrkB was induced with nM all trans-retinoid acid (ATRA). Then BDNF, CDDP or K252a were added to the cultured SY5Y cells. Cell livability was assessed by methyl thiazolyl tetrazolium (MTT) assay. TrkB autophosphorylation was determined by Western blot analysis. Cell apoptosis rate was detected by flow cytometry (FCM). The conformation of apoptosis cells was observed by transmission electron microscopy (TEM).. The livability and apoptosis rate in SY5Y cells treated with ATRA, BDNF and CDDP were not different from the blank control group. However, after K252a together with ATRA, BDNF and CDDP treatment, the sensibility of SY5Y cells to chemotherapy drug CDDP increased, the livability decreased and the apoptosis rate increased in SY5Y cells when compared with the blank control group (P <0.01). K252a treatment resulted in blockage of TrkB autophosphorylation.. The blockage of TrkB-BDNF signal pathway by K252a use can increase sensibility of NB cells to chemotherapy and thus decrease the livability of NB cells.

Topics: Apoptosis; Brain-Derived Neurotrophic Factor; Carbazoles; Cell Line, Tumor; Cisplatin; Humans; Indole Alkaloids; Microscopy, Electron, Scanning; Neuroblastoma; Receptor, trkB; Signal Transduction; Tretinoin

Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Small Cell; Caspase 8; Cell Proliferation; Chromatin Immunoprecipitation; Cyclic AMP Response Element-Binding Protein; DNA Methylation; Doxorubicin; Electrophoretic Mobility Shift Assay; Humans; Introns; Luciferases; Lung Neoplasms; Medulloblastoma; Mutation; Neuroblastoma; Phosphorylation; Promoter Regions, Genetic; Response Elements; RNA, Messenger; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

NGF treatment of neuroblastoma cells stimulates outgrowth of neurite processes associated with the expression of TrkA receptor and several differentiation markers. In this study, a 6 DIV exposure to NGF (50 ng/ml) increased immunostaining for alpha-tubulin, and expression of both alpha-tubulin and protein kinase C in the neuroblastoma cell line Neuro2a. Further, up-regulation of transglutaminase 1 and transglutaminase 2 expression, and reduction of transglutaminase 3 levels, were also observed in NGF-treated cells in comparison to untreated cells. Moreover, when Neuro2a cells were treated with the specific NF-kappaB inhibitor SN-50, the strong reduction of NF-kappaB activation was concomitant with a significant decrease of transglutaminase 2 expression, suggesting that NGF-evoked transglutaminase 2 induction could be related to NF-kappaB activation. To characterize the possible transglutaminase 2/NF-kappaB interplay, NGF treatment was carried out in Neuro2a cells which already over-expressed transglutaminase 2 after retinoic acid treatment. An additive effect of NGF was observed on the retinoic acid-induced transglutaminase 2 expression and enzyme activity, and NF-kappaB activation. However, a cystamine-mediated significant inhibition of transglutaminase activity (70%) was accompanied by a drastically reduced NF-kappaB activation only in cells exposed to NGF following retinoic acid treatment. We hypothesize that NF-kappaB activation was dependent on the transamidating activity related to high levels of TG2, and NGF enhanced NF-kappaB activation by a different, synergistically acting, pathway. These data suggest that the combined use of NGF and retinoic acid, or mimicking drugs, may provide the basics for the development of novel strategies in the therapeutic management of neuroblastomas.

Topics: Analysis of Variance; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cystamine; Drug Interactions; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation; GTP-Binding Proteins; Mice; Nerve Growth Factor; Neuroblastoma; NF-kappa B; Peptides; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase C; Time Factors; Transglutaminases; Tretinoin; Tubulin

In the present study, we identified a gene termed Nbla10993 whose expression levels are higher in favorable neuroblastomas versus unfavorable ones. Structural analysis showed that Nbla10993 is a novel splicing variant of the ER-associated protein of 140 kDa (ERAP140), which lacks the central acidic as well as the COOH-terminal Cys/His-rich domain. Similarly, ERAP140 was preferentially expressed in favorable neuroblastomas relative to unfavorable ones. During the all-trans-retinoic acid (ATRA)-mediated neuronal differentiation in neuroblastoma-derived RTBM1 cells, the expression levels of ERAP140/Nbla10993 increased at the mRNA level. Consistent with these observations, the luciferase reporter analysis demonstrated that the ERAP140/Nbla10993 promoter responds to ATRA. In addition, the immunoprecipitation/immunoblotting experiments showed that ERAP140 forms a stable complex with RARalpha but not with RXRalpha in cells, suggesting that ERAP140 is involved in RAR-mediated transcriptional regulation. Furthermore, the quantitative real-time PCR analysis using 109 primary neuroblastoma samples demonstrated that the expression levels of ERAP140/Nbla10993 significantly correlate with a better clinical outcome of neuroblastomas. Taken together, our present findings indicate that ERAP140/Nbla10993 plays an important role in the regulation of ATRA-mediated neuronal differentiation, and is a novel member of prognostic indicators for neuroblastoma.

Topics: Animals; Cell Differentiation; Chlorocebus aethiops; COS Cells; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Luciferases; Neuroblastoma; Nuclear Receptor Coactivators; Prognosis; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Tretinoin

We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca(2+) store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K(+) revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t(1/2) of 2.8 min, and was essentially completely depleted approximately 10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 microM) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca(2+) wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca(2+) signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71+/-8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca(2+) store in 9cRA-differentiated cells, which lack a functional store-operated Ca(2+) entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca(2+) store, and that spontaneous dissipation of Ca(2+) from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca(2+) signal by Ca(2+)-induced Ca(2+) release.

Topics: Antineoplastic Agents; Caffeine; Calcium; Calcium Signaling; Cell Differentiation; Cell Line, Tumor; Humans; Membrane Potentials; Methacholine Chloride; Microscopy, Fluorescence; Neurites; Neuroblastoma; Parasympathomimetics; Phosphodiesterase Inhibitors; Potassium; Tretinoin

E-3-(4'-Hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid (ST1926) is a novel orally available compound belonging to the class of synthetic atypical retinoids. These agents are attracting growing attention because of their unique mechanism of antitumor action that appears different from that of classical retinoic acid. This study aims at investigating the antitumor activity of ST1926 in neuroblastoma (NB) preclinical models. In vitro, ST1926 was more cytotoxic than both its prototype, CD437 and all-trans-retinoic acid (ATRA) and it was active in the SK-N-AS cell line, which is refractory to ATRA. We showed that unlike ATRA, ST1926 does not induce morphological differentiation in NB cells where it produces indirect DNA damage, cell cycle arrest in late S-G2 phases and p53-independent programmed cell death. DNA damage was not mediated by oxidative stress and was repaired by 24h after drug removal. The SK-N-DZ cell line appeared the most sensitive to the proapoptotic activity of ST1926, probably because both the extrinsic and intrinsic pathways appear involved in the process. Studies with Z-VAD-FMK, suggested that ST1926 might also mediate caspase-independent apoptosis in NB cells. In vivo, orally administered ST1926, appeared to inhibit tumor growth of NB xenografts with tolerable toxicity. Overall, our results support the view that ST1926 might represent a good drug candidate in this pediatric tumor.

Topics: Adamantane; Animals; Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cinnamates; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Resistance, Neoplasm; Humans; Male; Mice; Mice, Nude; Molecular Structure; Neuroblastoma; Tretinoin

Our recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity.. Cultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test.. ATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-beta expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells.. These results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.

Topics: Cell Line, Tumor; Cyclooxygenase 2; Dinoprostone; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Protein Kinase Inhibitors; Receptors, Retinoic Acid; Tretinoin

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.

Topics: Antineoplastic Agents; Base Sequence; Cell Differentiation; Cell Line, Tumor; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Nerve Tissue Proteins; Neuroblastoma; Paired Box Transcription Factors; PAX3 Transcription Factor; Promoter Regions, Genetic; RNA Processing, Post-Transcriptional; Sequence Homology, Nucleic Acid; Transcription Factor AP-2; Transcription, Genetic; Tretinoin

Ataxia telangiectasia (A-T) is an autosomal, recessive disorder mainly characterized by neuronal degeneration. However, the reason for neuronal degeneration in A-T patients is still unclear. ATM (A-T, mutated), the gene mutated in A-T, encodes a 370-kDa protein kinase. We measured the levels of the ATM protein found in differentiated neuron-like rat PC12 cells and differentiated neuron-like human SH-SY5Y cells. We found that, in rat PC12 cells, ATM levels decreased dramatically after differentiation, which is consistent with previous results observed in differentiated mouse neural progenitor cells. In contrast, the levels of ATM were similar before and after differentiation in human SH-SY5Y cells. Using an indirect immunofluorescence assay, we showed that ATM translocates from the nucleus to the cytoplasm in differentiated human SH-SY5Y cells. The translocation of ATM was further verified by subcellular fractionation experiments. The constitutive expression and cytoplasmic translocation of ATM in differentiated SH-SY5Y cells suggest that ATM is important for maintaining the regular function of human neuronal cells. Our results further demonstrated that, in response to insulin, ATM protects differentiated neuron-like SH-SY5Y cells from serum starvation-induced apoptosis. These data provide the first evidence that cytoplasmic ATM promotes survival of human neuronal cells in an insulin-dependent manner.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Cell Count; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cell Nucleus; Culture Media, Serum-Free; Cytoplasm; DNA-Binding Proteins; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Nerve Growth Factor; Neural Cell Adhesion Molecules; Neurites; Neuroblastoma; Neurons; Protein Serine-Threonine Kinases; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Subcellular Fractions; Transfection; Tretinoin; Tumor Suppressor Proteins

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.

Topics: Animals; Brain; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Neurites; Neuroblastoma; Phosphoric Diester Hydrolases; Protein Transport; RNA, Messenger; RNA, Small Interfering; Tretinoin

Neuroblastoma accounts for 15% of pediatric cancer deaths, and although a few protein-coding genes, such as MYCN, are involved with aggressive pathogenicity, the identification of novel biological targets for therapeutic intervention is still a necessary prerequisite for improving patient survival. Expression profiling of 157 microRNA (miRNA) loci in 35 primary neuroblastoma tumors indicates that 32 loci are differentially expressed in favorable and unfavorable tumor subtypes, indicating a potential role of miRNAs in neuroblastoma pathogenesis. Many of these loci are significantly underexpressed in tumors with MYCN amplification, which have particularly poor prognoses. Interestingly, we found that miRNA expression levels substantially change in a MYCN-amplified cell line following exposure to retinoic acid, a compound which is well known for causing reductions in MYCN expression and for inducing neuroblastoma cell lines to undergo neuronal differentiation. We also show that small interfering RNA inhibition of MYCN by itself causes similar alterations in the expression of miRNA loci. In vitro functional studies of one locus, miR-184, indicate that it plays a significant role in apoptosis. The association of experimentally induced alterations of miRNA expression in neuroblastoma cell lines with differentiation or apoptosis leads us to conclude that these loci play important roles in neuroblastoma pathogenesis. We further suggest that MYCN may mediate a tumorigenic effect, in part, through directly or indirectly regulating the expression of miRNAs that are involved with neural cell differentiation and/or apoptosis, warranting substantial further studies of miRNAs as potential therapeutic targets.

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Genes, myc; Humans; MicroRNAs; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Prognosis; RNA, Small Interfering; Transfection; Tretinoin

Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Histone Deacetylases; HL-60 Cells; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Rhabdomyosarcoma; RNA Splicing Factors; RNA, Small Interfering; Transcription Factors; Transcriptional Activation; Tretinoin

Geldanamycin (GA) is a specific inhibitor of the 90 kDs heat shock protein (Hsp90) in the cytoplasm of mammalian cells, which binds directly to Hsp90 and promotes proteolytic degradation of its client proteins. As an antitumor drug, GA antagonizes the protecting effects of Hsp90 on cell survival, while its mechanisms remain unclear. Here, we show that GA induces apoptosis in a human neuroblastoma cell line, SH-SY5Y. Treatment of the cells with all trans retinoic acid (RA) generates a neuron-like, morphological change of differentiation, and results in the activation of ERK and Akt pathways, an inhibition of the nuclear translocation of p53 induced by GA, and induces higher resistance to the GA-induced apoptosis. These results provide the first evidence for the requirement of p53 nucleation in SH-SY5Y cells to counteract GA in neuron survival.

Topics: Active Transport, Cell Nucleus; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Ganglia, Sympathetic; Humans; Lactams, Macrocyclic; Neuroblastoma; Neurons; Proto-Oncogene Proteins c-akt; Tretinoin; Tumor Suppressor Protein p53

Advanced glycation end product (AGE) accumulation in brain is believed to contribute to neuronal death in several neurodegenerative diseases. Neurons exposed to AGEs undergo oxidative stress, but the molecular mechanisms able to induce ROS generation and cell death are not yet clear. In this work, we exposed SH-SY5Y neuroblastoma cells to glycated albumin, as a model of AGE-modified protein, and we observed that cells differentiated by retinoic acid died after AGE exposure, through anion superoxide and peroxide generation, while undifferentiated cells resulted resistant. Retinoic acid induced marked increase in p47phox expression and in catalytic activity of PKC delta: the upregulation of a pathway involving NADPH oxidase and PKC delta is likely to be responsible for neuronal susceptibility to AGE. This hypothesis is confirmed by the fact that pre-treatments of differentiated cells with DPI, an inhibitor of NADPH oxidase, or with rottlerin, an inhibitor of PKC delta, were able to prevent AGE-induced neuronal death.

Topics: Analysis of Variance; Cell Death; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Interactions; Glycation End Products, Advanced; Humans; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; NADPH Oxidases; Neuroblastoma; Neurons; Protein Kinase C-delta; Receptor for Advanced Glycation End Products; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons.

Topics: Antibodies; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Fucosyltransferases; Gene Expression Regulation, Enzymologic; Humans; Lewis X Antigen; Lysosomal-Associated Membrane Protein 1; Neurites; Neuroblastoma; Tretinoin; Vesicular Transport Proteins

Alzheimer's disease (AD) is a senile dementia characterized by amyloid plaques, neurofibrillary tangles, and synaptic and cell loss. The "amyloid cascade" hypothesis suggests that amyloid-beta (Abeta), the peptide deposited as amyloid plaques, is the primary insult in AD. However, debate continues over the mechanism of Abeta toxicity and whether fibrillar or oligomeric Abeta is the active species of the peptide that ultimately causes the synaptic loss and dementia associated with AD. Brain-derived neurotrophic factor (BDNF) is required for survival and function of cells compromised in AD. Decreased BDNF causes defects in long-term potentiation and memory and correlates with cognitive decline. We previously demonstrated that BDNF reduction occurs early in the course of AD, suggesting that decreased BDNF may promote neuronal dysfunction in AD. We also demonstrated that three of seven human BDNF transcripts are specifically downregulated in AD. What pathological feature(s) of AD leads to the decreased BDNF is unknown. In this study, we administered both fibrillar and oligomeric conformations of Abeta(1-42) to differentiated SH-SY5Y, a human neuroblastoma cell line, and measured both phosphorylated cAMP response element-binding protein (CREB), a regulator of BDNF transcription, and BDNF total mRNA. We found that oligomeric but not fibrillar preparations of Abeta(1-42) significantly decrease both phosphorylated CREB and total BDNF mRNA. Furthermore, oligomeric Abeta(1-42) decreases BDNF transcripts IV and V in these cells, demonstrating that Abeta(1-42) downregulates the major BDNF transcript decreased in vivo in the AD brain. Thus, oligomeric Abeta(1-42) could compromise neuronal function, causing memory loss and cognitive dysfunction by downregulation of BDNF in AD.

Topics: Amyloid; Amyloid beta-Peptides; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Down-Regulation; Humans; Molecular Conformation; Neuroblastoma; Peptide Fragments; Phosphorylation; RNA, Messenger; Tretinoin

Methylmercury (MeHg) is a ubiquitous environmental toxicant and shows neurotoxicity to central nerve system (CNS) or neuronal cells. It has been known that MeHg has more influence to developing or differentiating CNS/neuronal cells than adult or differentiated CNS/neuronal cells. This study examined the effect of MeHg on differentiation of human neuroblastoma SH-SY5Y cells induced by all-trans-retinoic acid (RA). MeHg caused the impairment of the RA-induced G(1/0) phase arrest; it was induced the reduction of G(1/0) phase and S phase arrest. Extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase C (PKC) are involved in the RA-mediated differentiation and cell cycle progression. Activation of ERK1/2 by RA was increased more in MeHg-treated differentiating cells, comparing with only RA-treated groups. Furthermore, in both cases of inhibition of ERK1/2 with PD98059 or inhibition of PKC with GF109203X, RA/MeHg-induced ERK1/2 phosphorylation was reduced and G(1/0) phase arrest was induced. Thus, it indicates that the neuronal differentiation with RA was mediated by the ERK1/2 and PKC related pathway and MeHg resulted in neurotoxic influences through the disturbance in steps of differentiation by this pathway. These results suggest that MeHg inhibits RA-induced differentiation in SH-SY5Y cells by a pathway dependent ERK1/2 and PKC.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Drug Synergism; Flavonoids; Flow Cytometry; Humans; Indoles; Interphase; Maleimides; Methylmercury Compounds; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Phosphorylation; Protein Kinase C-alpha; Time Factors; Tretinoin

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.

Topics: Amino Acid Substitution; Antineoplastic Agents; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; DNA Damage; DNA Repair; DNA-Binding Proteins; Genes, Dominant; Humans; Mutation, Missense; Neurites; Neuroblastoma; Neurodegenerative Diseases; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Signal Transduction; Tretinoin; Tumor Suppressor Proteins

Differentiation is a key feature in pathologic classification and prognosis of neuroblastic tumors, although the underlying molecular mechanisms are not well defined. To identify key differentiation-related molecules and pathways, we evaluated gene expression during retinoic acid (RA)-induced differentiation of seven neuroblastic tumor cell lines. Transcriptional response to RA was highly variable among cell lines despite the fact that six of seven showed similar morphologic changes. RA consistently altered expression of a small set of genes, some of which are known to play a role in neurogenesis and differentiation. Expression of genes that were regulated by RA was associated with important clinical subgroups of neuroblastic tumors and were differentially expressed by stroma-rich and stroma-poor subtypes. RET, a receptor tyrosine kinase involved with differentiation, was consistently up-regulated throughout the time course of RA treatment in the majority of neuroblastic tumor cell lines. Interference with RET activation abrogated RA-induced transcriptional programs and differentiation, suggesting a key role of RET in this process. The core set of RA-regulated genes includes critical molecular components of pathways necessary for neuroblastic tumor differentiation and have potential as therapeutic targets and molecular markers of response to differentiating agents.

Topics: Cell Differentiation; Cell Line, Tumor; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins c-ret; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Tretinoin

All-trans retinoic acid (RA) causes differentiation of neuroblastoma cells, and retinoids have been used in clinical trials in children with advanced neuroblastoma. Combination of RA with histone deacetylase inhibitors (HDACi) could result in improved antitumorigenic activity. We have examined the effect of the HDACi trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA), alone and in combination with RA in human neuroblastoma SH-SY5Y cells. At concentrations that cause sustained increase of histone H3 acetylation, HDACi produced extensive apoptotic cell death as shown by flow cytometry analysis and induction of poly(ADP-ribose) polymerase proteolysis. HDACi inhibited SH-SY5Y cell growth at a much larger extent than RA. This compound did not cause apoptosis and did not further increase HDACi-mediated cell death. In contrast, both types of drugs cooperated to inhibit cell growth, although synergistic effects were not found. In surviving cells, HDACi repressed cyclin D1 expression and increased the cyclin kinase inhibitors (CKI) p21(Waf1/Cip1) and p27(Kip1). Cyclin D1 was not affected by RA, but this retinoid also increased CKI levels. Induction of p21(Waf1/Cip1) and p27(Kip1) by HDACi was further enhanced in the presence of RA. This effect seems to be at least partially due to transcriptional stimulation of CKI gene expression because both types of drugs cooperated to increase CKI mRNA levels and to activate the CKI promoters in transient transfection assays. These results show the strong antitumorigenic effects of HDACi in neuroblastoma cells and reinforce the idea that combination therapy could be useful to inhibit tumor growth.

Topics: Acetylation; Apoptosis; Caspases; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Neuroblastoma; Transcription, Genetic; Tretinoin

After the treatment of human neuroblastoma SH-SY5Y cells with retinoic acid for 24 h, the expression of c-Ret receptor tyrosine kinase was greatly elevated. Treatment of SH-SY5Y cells with glial cell line-derived neurotrophic factor under serum-free conditions after incubation of cells with retinoic acid resulted in the phosphorylation of c-Ret receptor tyrosine kinase, with subsequent morphological changes that included formation of neurites and rounding of cell bodies within 24-48 h. The number of neurite-bearing cells decreased with increasing concentrations of mitogen-activated protein kinase-specific and phosphatidylinositol 3-kinase inhibitors. These observations suggest that retinoic acid induces the expression of glial cell line-derived neurotrophic factor-responsive c-Ret receptor tyrosine kinase and that a glial cell line-derived neurotrophic factor-c-Ret receptor tyrosine kinase-induced signal transduction system that might be involved in neurite outgrowth via pathways that include phosphatidylinositol 3-kinase and mitogen-activated protein kinase.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor; Humans; Microtubule-Associated Proteins; Neuroblastoma; Phosphorylation; Receptor Protein-Tyrosine Kinases; Signal Transduction; Time Factors; Tretinoin; Tubulin

Large-scale hemizygous loss of chromosome 3p is a common event in neuroblastoma, occurring preferentially in tumors that exhibit loss of chromosome 11q and lack MYCN amplification. Although numerous tumor suppressor genes (TSG) have been mapped to the 3p region, the gene or genes contributing to neuroblastoma pathogenesis have remained elusive. High-resolution oligonucleotide array CGH mapping of chromosome 3p breakpoints relative to the positions of known TSGs indicates that more than one gene may contribute to neuroblastoma pathogenesis. We evaluated the methylation status of semaphorin 3B (SEMA3B), one of the chromosome 3p TSGs, in neuroblastoma tumors with (n = 12) and without (n = 32) 3p deletions. A significantly higher percentage of methylated CpG sites in the SEMA3B promoter was detected in tumors exhibiting 3p loss (95%), relative to tumors without loss (52%), suggestive of a two-hit mechanism of allele inactivation. The involvement of methylation in the control of SEMA3B expression was confirmed by treatment of neuroblastoma cell lines with the demethylating agent 5-aza-2-deoxycytidine. Transcriptional regulation of this locus is complex, however; low levels of SEMA3B expression were also seen in tumors with unmethylated SEMA3B promoters (n = 4). SEMA3B is known to play an important role in the development of normal sympathetic neurons, and interestingly, we found higher levels of SEMA3B expression in differentiated tumors with favorable histopathology (n = 19) than in tumors with unfavorable histology (n = 22). Furthermore, SEMA3B was upregulated in the SK-N-BE neuroblastoma cell line following induction of differentiation with retinoic acid. The association of SEMA3B expression with neuroblastoma differentiation suggests that this TSG may play a role in neuroblastoma pathobiology.

Topics: Adolescent; Antineoplastic Agents; Azacitidine; Base Sequence; Cell Differentiation; Cell Line; Cell Line, Tumor; Child; Child, Preschool; Chromosome Breakage; Chromosome Deletion; Chromosomes, Human, Pair 3; CpG Islands; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Infant; Membrane Glycoproteins; Molecular Sequence Data; Neuroblastoma; Nucleic Acid Hybridization; Reverse Transcriptase Polymerase Chain Reaction; Semaphorins; Tretinoin

We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.

Topics: Animals; Antineoplastic Agents; Antipsychotic Agents; Cell Differentiation; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; Clozapine; COS Cells; Cricetinae; Cricetulus; ErbB Receptors; Hippocampus; Humans; Kidney; Mice; Mice, Knockout; Nerve Tissue Proteins; Neuregulin-1; Neuroblastoma; Neuronal Plasticity; Phosphorylation; Proto-Oncogene Proteins c-fyn; Receptor, ErbB-4; Receptors, N-Methyl-D-Aspartate; Schizophrenia; Signal Transduction; Synapses; Tretinoin

From June 1997 to August 2005, 52 consecutive newly diagnosed stage 4 neuroblastoma patients over 1 year of age were assigned to receive tandem high-dose chemotherapy and autologous stem cell rescue (HDCT/ASCR) as consolidation therapy. Fifty of the 52 patients underwent a first HDCT/ASCR and 44 patients underwent a second HDCT/ASCR. Eight patients (15.4%) died from treatment-related toxicity (seven during the second HDCT/ASCR). Total body irradiation (TBI) in the first HDCT/ASCR and a shorter interval (< 12 weeks) between the first and second HDCT/ASCR were associated with a higher rate of treatment-related death in the second HDCT/ASCR (P = 0.032 and 0.095, respectively). The tumor relapsed or progressed in 11 patients, and 33 patients remained event free with a median follow-up of 53 months (range 19-117) from diagnosis. The 5-year event-free survival (EFS) (+/- 95% confidence interval) for all 52 patients was 62.1+/-13.7%. The application of TBI and local radiotherapy, and a longer interval between the first and second HDCT/ASCR were independently associated with a better EFS (P = 0.026, 0.007 and 0.020, respectively). However, further studies will be needed to decrease the toxic death rate in the second HDCT/ASCR while reducing the relapse rate.

Topics: Antineoplastic Agents; Brain Neoplasms; Combined Modality Therapy; Humans; Immunotherapy; Infant; Interleukin-2; Neoplasm Staging; Neuroblastoma; Retrospective Studies; Survival Rate; Transplantation Conditioning; Transplantation, Autologous; Treatment Outcome; Tretinoin; Whole-Body Irradiation

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg(-1)) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA.

Topics: Animals; Benzothiazoles; Cell Line, Tumor; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Evaluation, Preclinical; Female; Humans; Imidazoles; Isoenzymes; Mice; Mice, Nude; Neuroblastoma; Retinoic Acid 4-Hydroxylase; RNA, Small Interfering; Transplantation, Heterologous; Tretinoin

NMDA receptors exhibit a dichotomy of signaling with excessive stimulation leading to neuronal damage that occurs during neurodegenerative disorders, whereas the normal burst of activity results in plastic responses with the expression of molecular substrates of long-term plasticity, growth and survival. Control of polysialylated neural cell adhesion molecule (PSA-NCAM) expression by NMDA receptor activation has been described in several systems, suggesting a functional link between these two proteins. The coordinated induction of several different transcription factors initiated by NMDA receptor stimulation may be a key mechanism in the orchestration of specific target gene expression that underlies various aspects of CNS function, including plastic responses. We report here the transcriptional regulation of PSA-NCAM expression by subtoxic dose of NMDA in retinoic acid-differentiated SH-SY5Y cell cultures. SH-SY5Y cell cultures differentiated with retinoic acid (10 microM) were exposed to NMDA (100 microM) or to antagonist MK-801 (200 nM) prior to treatment with NMDA and cells were harvested after 24 h of treatment to study the expression of PSA-NCAM, nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) by Western blotting and dual immunocytofluorescence and expression of polysialyltransferase (PST) mRNA by fluorescent in situ hybridization (FISH). We observed the induction of transcription factors NF-kappaB and AP-1 along with PSA-NCAM expression in response to NMDA receptor activation. Also, PSA-NCAM regulation in response to NMDA receptor activity was shown to be transcriptionally controlled, as seen by temporal and spatial changes observed in the expression of PST mRNA in NMDA-treated SH-SY5Y cell cultures. This raises the interesting possibility that NF-kappaB and AP-1 expression is involved in propagating the signals of NMDA receptor activity that leads to downstream strengthening of long-term plasticity changes in differentiated SH-SY5Y neuroblastoma cell cultures. Thus understanding the regulation of PSA-NCAM expression by NMDA receptor-mediated activity may represent a fundamental prerequisite for the development of therapies in order to maintain neuronal plasticity throughout life and functional recovery after brain damage.

Topics: Cell Differentiation; Cell Line, Tumor; Dizocilpine Maleate; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Humans; In Situ Hybridization; Keratolytic Agents; N-Methylaspartate; Neural Cell Adhesion Molecule L1; Neuroblastoma; NF-kappa B; Protein Transport; Receptors, N-Methyl-D-Aspartate; Replication Protein C; Sialic Acids; Sialyltransferases; Tretinoin

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.

Topics: Animals; Cell Differentiation; Cell Line; Cytoskeletal Proteins; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Peptide Mapping; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Tretinoin; Tumor Cells, Cultured

The induction of GRIM-19 has been shown to be essential for interferon-beta (IFN-beta)-induced and retinoic acid (RA)-induced tumor cell death. We have studied the localization and levels of GRIM-19 in IFN/RA-induced cell death in neural cells and in focal cerebral ischemia. Exposure to IFN/RA caused a approximately 15-fold increase in GRIM-19 protein levels and induced >50% cell death in human neuroblastoma SH-SY5Y cells. In rats subjected to permanent focal cerebral ischemia, increased oxidative stress, as well as increased GRIM mRNA levels (32-fold) and increased GRIM-19 (>50%) protein levels were noted in the ipsilateral (affected) hemisphere compared with the contralateral (unaffected) hemisphere. These results suggest that GRIM-19 may play a role in ischemia-induced neuronal cell death.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Ischemia; Cell Line, Tumor; Drug Combinations; Gene Expression Regulation; Humans; Interferon-beta; Male; NADH, NADPH Oxidoreductases; Neuroblastoma; Rats; Rats, Wistar; Time Factors; Tretinoin

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).

Topics: Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Cytosol; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurites; Neuroblastoma; Ouabain; Phosphorylation; Protein Isoforms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Tretinoin; Tumor Suppressor Protein p53

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Cholesterol; Dopamine; Drug Synergism; Growth Cones; Humans; Lysosomal-Associated Membrane Protein 2; Microscopy, Fluorescence; Models, Biological; Neurites; Neuroblastoma; Potassium; Presynaptic Terminals; Pyridinium Compounds; Quaternary Ammonium Compounds; Synapses; Synaptic Vesicles; Synaptophysin; Tretinoin

Neuroblastoma (NB) is an infant tumor which frequently differentiates into neurons. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to analyze the cytosolic and nuclear protein expression patterns of LAN-5 cells following neuronal differentiating agent all-trans-retinoic acid treatment. We identified several candidate proteins, from which G beta2 and Prefoldin 3 may have a role on NB development. These results strength the use of proteomics to discover new putative protein targets in cancer.

Topics: Cell Line, Tumor; Cell Nucleus; Chromatography, Liquid; Computational Biology; Cytosol; Electrophoresis, Gel, Two-Dimensional; Humans; Neoplasm Proteins; Neuroblastoma; Peripheral Nervous System Neoplasms; Protein Array Analysis; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin

There is significant interest in the potential use of telomerase-immortalized cells in transplantation to replace neurons lost to neurodegenerative diseases and other central nervous system injuries. Neural progenitor cells (NPCs) transduced with human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, have the potential both to proliferate indefinitely in vitro and to respond to differentiation signals necessary for generating appropriate cells for transplantation. The purpose of this study was to evaluate the differentiation of neurons from NT2 cells, a model NPC cell line, following hTERT transduction. RT-PCR and telomerase activity data demonstrated that persistent exogenous hTERT expression significantly inhibited the differentiation of neurons from NT2 cells. Following retinoic acid induced differentiation, hTERT-NT2 cells produced only one fourth of the neurons generated by parental and vector-control cells. A differentiation-inhibiting effect of constitutive telomerase activity has not been reported previously in other hTERT-transduced progenitor cell lines, implying a unique role for telomerase in the proliferation and differentiation of NPCs that have tumorigenic potential. Elucidating the mechanism responsible for this effect may aid in understanding the potential role of telomerase activity in the tumorigenicity of NPCs, as well as in optimizing the production of safe, telomerase-engineered, transplantable neurons.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Gene Expression; Humans; Microtubule-Associated Proteins; Neural Inhibition; Neuroblastoma; Neurons; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells; Telomerase; Tretinoin; Tubulin

Causes of retinoid resistance often observed in neuroblastomas are unknown. We studied all trans-retinoic acid (RA) signaling in neuroblastoma cells differing in N-myc levels in terms of neurite formation, expression of tissue transglutaminase, neuronal marker proteins, matrix metalloproteinases (MMPs), and activation of Rac1 and Cdc42. Poor invasiveness observed in SH-SY5Y, LA-N-5, and SMS-KCNR cells was associated with RA-induced neurite formation, Cdc42 activation and N-myc down regulation; expression of constitutively active Cdc42 down regulated N-myc expression and reduced invasion in RA-resistant SK-N-BE(2) and IMR32 cells. RA treatment for 24 h transiently increased invasion and expression of MMP9 in SH-SY5Y, LA-N-5 and MMP2 in SMS-KCNR cells. MMP inhibition prevented RA-induced neurite formation indicating a role in differentiation. Variation in RA signaling thus follows a defined pattern and relates to invasive potential. A defective RA signaling might result in retinoid resistance and unpredictable clinical outcome observed in some neuroblastomas.

Topics: Antineoplastic Agents; cdc42 GTP-Binding Protein; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Matrix Metalloproteinases; Neurites; Neuroblastoma; Signal Transduction; Tretinoin

The retinoic acid receptor beta (RARbeta) is a retinoic acid (RA)-inducible tumor suppressor, which plays an important role in the arrest of neuroblastoma cell growth. Using human neuroblastoma SH-SY5Y cells, we have examined the regulation of RARbeta expression by histone deacetylase inhibitors (HDACi), considered to be promising agents in anticancer therapy. Our results show that HDACi cooperated with RA to increase RARbeta mRNA levels and to activate the RARbeta2 promoter in transient transfection assays. Chromatin immunoprecipitation assays showed that the basal RARbeta2 promoter that contains the RA response element was refractory to acetylation by both HDACi and RA. In addition, HDACi caused a transient increase in acetylation of a downstream RARbeta2 region, even though global histones remain hyperacetylated after a prolonged treatment with the inhibitors. RA potentiated this response and maintained acetylation for a longer period. Despite the cooperation of RA with HDACi to increase transcription of the RARbeta gene, these inhibitors caused a paradoxical reduction of the cellular levels of the RARbeta protein in cells treated with the retinoid. This reduction is secondary to a change in the protein half-life that is decreased by the HDACi due to increased ubiquitin-independent proteasomal degradation. These results show that HDACi regulate expression of the tumor suppressor gene RARbeta by both transcriptional and posttranscriptional mechanisms and might then modulate sensitivity to the retinoid in neuroblastoma cells.

Topics: Cell Line, Tumor; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Neuroblastoma; Receptors, Retinoic Acid; Transcription, Genetic; Tretinoin

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356-5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.

Topics: Antineoplastic Agents; Cell Communication; Cell Differentiation; Humans; Molecular Weight; Neuroblastoma; Proteasome Endopeptidase Complex; RNA-Binding Proteins; Tretinoin; Ubiquitin

The MYCN protooncogene is involved in the control of cell proliferation, differentiation, and survival of neuroblasts. Deregulation of MYCN by gene amplification contributes to neuroblastoma development and is strongly correlated to advanced disease and poor outcome, emphasizing the urge for new therapeutic strategies targeting MYCN function. The transcription factor N-Myc, encoded by MYCN, regulates numerous genes together with its partner Max, which also functions as a cofactor for the Mad/Mnt family of Myc antagonists/transcriptional repressors. We and others have previously reported that IFN-gamma synergistically potentiates retinoic acid (RA)-induced sympathetic differentiation and growth inhibition in neuroblastoma cells. This study shows that combined treatment of MYCN-amplified neuroblastoma cells with RA+IFN-gamma down-regulates N-Myc protein expression through increased protein turnover, up-regulates Mad1 mRNA and protein, and reduces N-Myc/Max heterodimerization. This results in a shift of occupancy at the ornithine decarboxylase N-Myc/Mad1 target promoter in vivo from N-Myc/Max to Mad1/Max predominance, correlating with histone H4 deacetylation, indicative of a chromatin structure typical of a transcriptionally repressed state. This is further supported by data showing that RA+IFN-gamma treatment strongly represses expression of N-Myc/Mad1 target genes ornithine decarboxylase and hTERT. Our results suggest that combined IFN-gamma and RA signaling can form a basis for new therapeutic strategies targeting N-Myc function for patients with high-risk, MYCN-amplified neuroblastoma.

Topics: Acetylation; Antineoplastic Agents; Basic-Leucine Zipper Transcription Factors; Blotting, Northern; Blotting, Western; Cell Cycle Proteins; Cell Differentiation; Chromatin; Chromatin Immunoprecipitation; Dimerization; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; Histones; Humans; Immunoprecipitation; Interferon-gamma; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Ornithine Decarboxylase; Proto-Oncogene Proteins c-myc; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

Topics: Antimitotic Agents; Blotting, Western; Cell Differentiation; Cell Division; Cell Line, Tumor; Dopamine; Gene Expression; Humans; In Vitro Techniques; Microscopy, Fluorescence; Mitotic Index; Nerve Tissue Proteins; Neuroblastoma; Neurons; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.

Topics: Alitretinoin; Antineoplastic Agents; Bexarotene; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Kidney; Neoplasm Invasiveness; Neuroblastoma; Protein Glutamine gamma Glutamyltransferase 2; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; Tetrahydronaphthalenes; Transglutaminases; Tretinoin

One group of proteins that regulates neurite outgrowth and maintains neuronal networks is the immunoglobulin superfamily (IgSF). We previously identified a new member of the IgSF, keratinocyte-associated transmembrane protein-4 (KCT-4), by the signal sequence-trap method from primary cultured human keratinocytes. The KCT-4 mRNA has been found to be highly expressed in the adult human brain, although it is also distributed in various tissues. In the present study, to gain insight into the role of KCT-4 in the nervous system, we examined the expression profile and localization of KCT-4 mRNA in mouse brain. We also evaluated changes in KCT-4 mRNA expression in the differentiation of the neuroblastoma cell line Neuro-2a as the in vitro model of neurite outgrowth. KCT-4 mRNA was detected broadly in various regions of the adult mouse brain by RT-PCR. In situ hybridization revealed that it was expressed highly selectively by neurons but not by glial cells. Moreover, expression of KCT-4 mRNA was induced by neurite outgrowth of Neuro-2a. These data suggest that KCT-4 participates in the regulation of neurite outgrowth and maintenance of the neural network in the adult brain.

Topics: Animals; Antigens, CD; Blotting, Northern; Brain; Cell Line, Tumor; Gene Expression; Glial Fibrillary Acidic Protein; Immunoglobulins; Immunohistochemistry; In Situ Hybridization; Membrane Proteins; Mice; Neurites; Neuroblastoma; Neurons; Phosphopyruvate Hydratase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Proteins; Time Factors; Tretinoin; Up-Regulation

The HEXIM1 protein, in association with 7SK snRNA, binds and inhibits the kinase activity of P-TEFb (CDK9/cyclin T). P-TEFb activity is crucial for efficient transcription elongation of viral and cellular genes. HEXIM1 was originally isolated as a protein up-regulated by hexamethylene bisacetamide (HMBA), a prototypical inducer of differentiation. To determine the causative role of HEXIM1 during cell differentiation we analyzed the biochemical and functional consequences of HEXIM1 protein levels in several in vitro differentiation systems. We found that HEXIM1 mRNA and protein levels are up-regulated during differentiation of murine erythroleukemia cells upon treatment with HMBA or DMSO. Stimulation of HEXIM1 is not restricted to hematopoietic cells, as induction of phenotypic differentiation of neuroblastoma cells by retinoic acid results in up-regulation of HEXIM1. Moreover, ectopic expression of HEXIM1 causes growth inhibition and promotes neuronal differentiation. These findings highlight a crucial role of HEXIM1 protein during cell differentiation.

Topics: Acetamides; Animals; Cell Differentiation; Cell Line, Tumor; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Erythroblastic, Acute; Mice; Neuroblastoma; Positive Transcriptional Elongation Factor B; RNA-Binding Proteins; Transcription Factors; Transcription, Genetic; Tretinoin; U937 Cells

Oxidative stress is considered important for the pathogenesis of Alzheimer disease (AD), which is characterized by the formation of senile plaques rich in amyloid beta-protein (Abeta). Abeta cytotoxicity has been found dependent on lysosomes, which are abundant in AD neurons and are shown to partially co-localize with Abeta. To determine whether oxidative stress has any influence on the relationship between lysosomes and Abeta1-42 (the most toxic form of Abeta), we studied the effect of hyperoxia (40% versus 8% ambient oxygen) on the intracellular localization of Abeta1-42 (assessed by immunocytochemistry) in retinoic acid differentiated SH-SY5Y neuroblastoma cells maintained in serum-free OptiMEM medium. In control cells, Abeta1-42 was mainly localized to small non-lysosomal cytoplasmic granules. Only occasionally Abeta1-42 was found in large (over 1 microm) lysosomal-associated membrane protein 2 positive vacuoles, devoid of the early endosomal marker rab5. These large Abeta1-42 -containing lysosomes were not detectable in the presence of serum (known to suppress autophagy), while their number increased dramatically (up to 24-fold) after exposure of cells to hyperoxia during 5 days. Activation of autophagy by hyperoxia was confirmed by transmission electron microscopy. Furthermore, an inhibitor of autophagic sequestration 3-methyladenine prevented the accumulation of Abeta1-42 -positive lysosomes due to hyperoxia. In parallel experiments, intralysosomal accumulation of Abeta1-40 following oxidative stress has been found as well. The results suggest that Abeta can be autophagocytosed and its accumulation within neuronal lysosomes is enhanced by oxidative stress.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Apoptosis; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cytoplasmic Granules; Humans; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Neuroblastoma; Oxidative Stress; Tretinoin

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Fenretinide; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transfection; Tretinoin

Neuroblastoma (NBL), derived from the sympathetic precursor cells, is one of the most common pediatric solid tumors. The expression of N-acetylglucosaminyltransferase V and IX (GnT-V and GnT-IX) mRNA in 126 primary NBLs were quantitatively analyzed and higher expression levels of GnT-V were found to be associated with favorable stages (1, 2 and 4s). Conversely, the downregulation of GnT-V expression by small interfering RNA resulted in a decrease in the susceptibility to cell apoptosis induced by retinoic acid in NBL cells accompanied by morphological change. These results suggest that GnT-V is associated with prognosis by modulating the sensitivity of NBLs to apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Child; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; N-Acetylglucosaminyltransferases; Neuroblastoma; RNA, Small Interfering; Tretinoin

The aim of this study was to examine whether a modulated radiofrequency of the type used in cellular phone communications at a specific absorption rate (SAR) higher than International Commission on Non-ionizing Radiation Protection (ICNIRP) reference level for occupational exposure, could elicit alterations on proliferation, differentiation, and apoptosis processes in a neuroblastoma cell line. The cell line was exposed for 24, 48, and 72 h to 900 MHz radiofrequency and proliferation and differentiation were tested by WST-I assay and by a molecular analysis of specific markers, two oncogenes and a cytoskeleton protein, in exponential growth phase and in synchronized cell cultures. Apoptosis was evaluated by caspase activation analysis and by molecular detection of Poly (ADP-ribose) polimerase (PARP) cleavage. Combined exposures to radiofrequency and to the differentiative agent retinoic acid or to the apoptotic inducer camptothecin were carried out to test possible interference between electromagnetic field and chemical agents. Overall our data suggest that 900 MHz radiofrequency exposure up to 72 h does not induce significant alterations in the three principal cell activities in a neuroblastoma cell line.

Topics: Antineoplastic Agents; Apoptosis; Camptothecin; Cell Differentiation; Cell Line, Tumor; Cell Phone; Cell Proliferation; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Electromagnetic Fields; Environmental Exposure; Humans; Microwaves; Neuroblastoma; Radiation Dosage; Radio Waves; Tretinoin

Topics: Antineoplastic Agents; Focal Nodular Hyperplasia; Humans; Neuroblastoma; Tretinoin

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-alpha and -beta-targets for STI-571. We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and gamma-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 microM STI-571 for 72h, two cell lines responding already to 10 microM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-alpha and -beta. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and gamma-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed gamma-irradiation therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Line, Tumor; Cell Survival; Chemotherapy, Adjuvant; Gamma Rays; Humans; Imatinib Mesylate; Neuroblastoma; Piperazines; Pyrimidines; Tretinoin

Retinoic acid (RA) has been shown to induce neuronal differentiation and/or apoptosis, and is widely used as a chemotherapeutic agent for treating the patients with neuroblastoma. However, the therapeutic effect of RA is still limited. To unveil the molecular mechanism(s) inducing differentiation and apoptosis in neuroblastoma cells, we compared CHP134 and NB-39-nu cell lines, in which all-trans-RA (ATRA) induces apoptosis, with LA-N-5 and RTBM1 cell lines, in which it induces neuronal differentiation. Here, we found that Bcl-2 was strongly downregulated in CHP134 and NB-39-nu cells, whereas it was abundantly expressed in LA-N-5 and RTBM1 cells. ATRA-mediated apoptosis in CHP134 and NB-39-nu cells was associated with a significant activation of caspase-9 and caspase-3 as well as cytoplasmic release of cytochrome c from mitochondria in a p53-independent manner. Enforced expression of Bcl-2 significantly inhibited ATRA-mediated apoptosis in CHP134 cells. In addition, treatment of RTBM1 cells with a Bcl-2 inhibitor, HA14-1, enhanced apoptotic response induced by ATRA. Of note, two out of 10 sporadic neuroblastomas expressed bcl-2 at undetectable levels and underwent cell death in response to ATRA in primary cultures. Thus, our present results suggest that overexpression of Bcl-2 is one of the key mechanisms to give neuroblastoma cells the resistance against ATRA-mediated apoptosis. This may provide a new therapeutic strategy against the ATRA-resistant and aggressive neuroblastomas by combining treatment with ATRA and a Bcl-2 inhibitor.

Topics: Apoptosis; Base Sequence; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Molecular Sequence Data; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cyclin B; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Humans; Immunosuppressive Agents; Inhibitor of Apoptosis Proteins; Linear Models; Microtubule-Associated Proteins; Neoplasm Proteins; Nerve Growth Factor; Neurites; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survivin; Tacrolimus; Tretinoin

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

Topics: Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Promyelocytic Leukemia Protein; Protein Structure, Tertiary; Receptors, Retinoic Acid; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transcription, Genetic; Tretinoin; Tripartite Motif Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.

Topics: Animals; Autocrine Communication; Cell Differentiation; Cell Line, Tumor; Dimerization; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glial Cell Line-Derived Neurotrophic Factor; Humans; Mice; Neuroblastoma; Proto-Oncogene Proteins c-ret; Tretinoin

Cells require appropriate interaction with extracellular matrix proteins mediated by integrins to grow, differentiate and survive. Many cell types including nervous cells undergo anoikis, a substrate-dependent apoptosis, when adhesion is impaired. Resistance of tumors to cytotoxic drugs is probably due to disturbed apoptosis programs. The proteolytic enzymes caspases are the main executioners of apoptosis. It was reported that caspase-8 expression is deficient in some neuroblastoma cells. We demonstrated that human neuroblastoma cell line SK-B-BE, differentiated with retinoic acid, expressed caspases 3, 8 and 9. Caspases 8 and 3, but not caspase-9 were activated in SK-N-BE cells cultured in suspension or on aspecific adhesive substrate. Cell positive to caspase-8 were classified into four stages, by morphometric and densitometric parameters. The use of the specific caspase-8 inhibitor Z-IETD-FMK dramatically reduced apoptosis, demonstrating that caspase-8 is the upstream initiator caspase during SK-N-BE cells anoikis. Among matrix proteins, type I collagen is the most effective and fibronectin the least in delaying anoikis. The activation of caspases 8 and 3 by unligated integrins was dependent on the state of neuronal differentiation, since the most differentiated cell was the most vulnerable to anoikis. These data show that activation of caspase-8 is specifically required to promote anoikis in SK-N-BE neuroblastoma cells.

Topics: Analysis of Variance; Anoikis; Antineoplastic Agents; Blotting, Western; Caspase 3; Caspase 8; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Activation; Extracellular Matrix Proteins; Gene Expression; Humans; Microscopy, Confocal; Neuroblastoma; Oligopeptides; Time Factors; Tretinoin

Previously, we found that secretin transcript levels were induced by all-trans retinoic acid (RA) in a neuroblastoma cell model, SH-SY5Y. In this article, this RA-dependent upregulation process was further investigated. In the cyclin-dependent kinase 1 (Cdk1) inhibitor-treated cells, the RA-dependent induction of secretin gene expression was inhibited. Together with our previous works, we propose here that the RA responsiveness of the secretin promotor is mediated by two different pathways. The first pathway is by changing the expression levels of NFI-C and Sp proteins while the second pathway is by modifying the phosphorylation status of both NFI-C and Sp proteins via Cdk1.

Topics: CDC2 Protein Kinase; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Protein Kinase Inhibitors; Secretin; Tretinoin

Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

Topics: 3,4-Dihydroxyphenylacetic Acid; alpha-Synuclein; Amino Acid Substitution; Animals; Catechols; Cell Differentiation; Cell Line, Tumor; Cerebral Cortex; Corpus Striatum; Cytosol; Dopamine; Humans; Levodopa; Mice; Mice, Transgenic; Mutation, Missense; Nerve Degeneration; Neuroblastoma; Oxidation-Reduction; Parkinson Disease; Parkinsonian Disorders; Protein Conformation; Recombinant Fusion Proteins; Solubility; Transfection; Tretinoin; Tyrosine 3-Monooxygenase

Neuroblastoma (NB) is a pediatric solid tumor derived from neural crest precursor cells. It is resistant to current therapeutic protocols, including high dose chemotherapy. The mechanisms of chemoresistance are very complex. The recent studies have shown that the levels of tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) are high in NB tumors with poor prognosis. The aim of this research was to explore the effects of TrkB and BDNF levels on the chemotherapeutic sensitivity in neuroblastoma by using the NB cell line SH-SY5Y in vitro.. The expression of TrkB protein was detected with Western-blot after the treatment with different concentrations of all trans-retinoic acid (ATRA). Cell survival rate was analyzed using MTT. Apoptosis was detected using flow cytometry (FCM) and a transmission electron microscope (TEM).. (1) The expression of TrkB protein was undetectable in SY5Y. It was positive, however, after the treatment with ATRA (1, 10, 100 nmol/L) for five days. The level of TrkB protein was increased with adding of ATRA at different concentrations. (2) The difference of the survival and apoptotic rate between the BDNF (10 ng/ml) + ATRA (10 nmol/L) + cisplatin (CP, 5 microg/ml) group (survival rate 46.51% +/- 13.44%, apoptosis rate 11.79% +/- 1.53%) and the CP alone group (survival rate 38.51% +/- 9.66%, apoptosis rate 14.95% +/- 2.06%) was not statistically significant (P > 0.05). The survival rate of the BDNF (50 ng/ml and 100 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (66.85% +/- 18.39%, 94.30% +/- 10.71%) was greatly higher than CP alone group (P < 0.05, P < 0.01), whereas the apoptotic rate (9.36% +/- 1.03%, 5.20% +/- 1.99%) was significantly lower than that of the CP alone group (P < 0.01, P < 0.01). The survival rates of BDNF (100 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group were higher than those of BDNF (50 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (P < 0.01), whereas the apoptotic rates were lower than those of BDNF (50 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (P < 0.05). There were no significant difference between the ATRA (1 nmol/L) + BDNF (50 ng/ml) + CP group (survival rate 45.33% +/- 11.83%, apoptosis rate 12.48% +/- 2.48%) and the CP alone group in the survival and apoptotic rates (P > 0.05). The survival rates of the ATRA (10 nmol/L, 100 nmol/L) + BDNF (50 ng/ml) + CP (61.62% +/- 18.53%, 105.02% +/- 5.55%) group were greatly higher than those of the CP alone group (P < 0.05, P < 0.01), whereas the apoptotic rate (9.36% +/- 1.03%, 5.05% +/- 1.88%) was significantly lower than that of the CDDP alone group (P < 0.05, P < 0.01). The survival rates of the ATRA (100 nmol/L) + BDNF (50 ng/ml) + CP group were higher than those of the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group (P < 0.01), whereas the apoptotic rates were lower than the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group (P < 0.01). (3) Some of the cells showed apoptotic changes in the CP alone group, whereas the intranuclear chromoplasma was well-distributed, the nuclear membrane was clear, and mitochondria, ribosome and solvent were present in the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group.. The sensitivity of SY5Y to CP was affected by TrkB and BDNF. The higher the level of TrkB and BDNF was, the lower the sensitivity of SY5Y to CP.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Microscopy, Electron, Transmission; Neuroblastoma; Receptor, trkB; Tetrazolium Salts; Thiazoles; Tretinoin

The transcription factor Sox11 is expressed at high levels in developing sensory neurons and injured adult neurons but little is known about its transcriptional targets and function. In this study we examined the role of Sox11 using Neuro2a neuroblastoma cells and cultured mouse dorsal root ganglia (DRG) neurons. Results show Sox11 has an essential role in regulation of neuron survival and neurite outgrowth in Neuro2a cells and primary sensory neurons. Neuro2a cells increase expression of Sox11 as they differentiate in culture. Following addition of 20 microM retinoic acid (RA), a stimulus for differentiation that enhances neurite growth and differentiation, Sox11 level rises. RNAi-mediated knockdown of Sox11 in RA-differentiated Neuro2a cells caused a decrease in neurite growth and an increase in the percent of apoptotic cells. RNA expression analysis showed that Sox11 knockdown modulated the level of mRNAs encoding several genes related to cell survival and death. Further validation in the Neuro2a model showed Sox11 knockdown increased expression of the pro-apoptotic gene BNIP3 (BclII interacting protein 1 NIP3) and decreased expression of the anti-apoptotic gene TANK (TNF receptor-associated factor family member-associated NFkappaB activator). Cultured primary DRG neurons also express Sox11 and treatment with Sox11 small interfering RNA (siRNA) caused a significant decrease in neurite growth and branching and a decrease in mRNA encoding actin-related protein complex 3 (Arpc3), an actin organizing protein that may be involved in axon growth. The percent of apoptotic neurons also increased in cultures of DRG neurons treated with Sox11 siRNA. Similar to Neuro2a cells, a decrease in TANK gene expression occurred, suggesting at least some overlap in Sox11 transcriptional targets in Neuro2a and DRG neurons. These data are consistent with a central role for Sox11 in regulating events that promote neurite growth and neuron survival.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Count; Cell Differentiation; Cell Survival; Cells, Cultured; Drug Interactions; Ganglia, Spinal; Gene Expression Regulation; High Mobility Group Proteins; Immunohistochemistry; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mitochondrial Proteins; Neurites; Neuroblastoma; Neurons, Afferent; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sciatic Neuropathy; SOXC Transcription Factors; Time Factors; Transfection; Tretinoin

p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter-gene assays, we demonstrate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1(ICD)) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c-Abl or cisplatin treatment also potently antagonized N1(ICD)/CBF-1-dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split-1 (HES-1) protein levels and antagonized the increase in HES-1 protein induced by exogenous N1(ICD) expression. Evidence of a direct physical interaction between N1(ICD) and TAp73alpha was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73alpha binds the N1(ICD) in a region C-terminal of aa 2094. Interestingly, DeltaNp73alpha and TAp73alpha(R292H) also co-purified with N1(ICD), but neither inhibited N1(ICD)/CBF-1-dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73alpha reversed the N1(ICD)-mediated repression of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73alpha on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Brain Neoplasms; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; Humans; Immunoprecipitation; Luciferases; Microscopy, Confocal; Neuroblastoma; Neurons; Nuclear Proteins; Receptor, Notch1; Repressor Proteins; Signal Transduction; Transfection; Tretinoin; Tumor Suppressor Proteins

Thyroid hormone negatively regulates the amyloid-beta precursor protein (APP) gene in thyroid hormone receptor (TR)-transfected neuroblastoma cells. A negative thyroid hormone response element (nTRE) that mediates this regulation has been identified in the first exon of the APP gene. We demonstrate in an in vivo system that expression of APP mRNA, APP protein, and APP secretase cleavage products in mouse brain is influenced by thyroid status. Adult female mice were made hyperthyroid or hypothyroid for 3 weeks and compared to euthyroid mice. APP gene product expression was increased in hypothyroid mouse brain and reduced in hyperthyroid mouse brain, when compared to euthyroid controls. We observed similar effects of thyroid hormone on endogenous APP gene expression in human neuroblastoma cells. The incidence of hypothyroidism increases with age, and localized hypothyroidism of central nervous system has been reported in some patients with Alzheimer's disease (AD). Reduced action of thyroid hormone on the APP gene may contribute to AD pathology by increasing APP expression and the levels of processed APP products. These findings may be an underlying mechanism contributing to the association of hypothyroidism with AD in the elderly, as well as identifying a potential therapeutic target. Pharmacologic supplementation of thyroid hormone, or its analogs, may reduce APP gene expression and beta amyloid peptide accumulation.

Topics: Amyloid beta-Protein Precursor; Animals; Cell Line, Tumor; Female; Gene Expression Regulation; Humans; Hyperthyroidism; Hypothyroidism; Mice; Neuroblastoma; Thyroxine; Tretinoin

Disseminated forms of neuroblastoma (NB), a tumor derived from neuroectodermal tissue, pose a major therapeutic challenge for pediatric oncology. By performing a comparative cDNA array analysis of metastatic neuroblasts versus primary xenograft from the human IGR-N-91 NB model, we were able to identify a set of downregulated developmental genes in metastatic neuroblasts. One of these genes was Wnt-5a, a member of the Wnt signaling pathway, known to be involved in the development of neural crest cells. Since we also found a significant decrease in Wnt-5a mRNA in unfavorable versus favorable categories in 37 primary NB tumors (P<0.007), we wondered whether retinoic acid (RA), which has a role in neural crest induction and differentiation, might reverse the aberrant negative regulation of Wnt-5a in metastatic malignant neuroblasts. Following treatment with 10 muM RA for 6 days, the MYCN-amplified IGR-N-91 cell lines underwent neuronal differentiation as assessed by reduced MYCN gene expression and neuritic extension. In these conditions, data showed an upregulation of Wnt-5a and PKC-theta; isoform expressions. Our study highlights, for the first time, the involvement of Wnt-5a, which has a role in embryonic and morphogenetic processes, in the response of malignant neuroblasts to RA. In conclusion, we demonstrated that RA, which is used in the treatment of high-risk NB patients with recurrent/residual disease in the bone marrow, is able to upregulate Wnt-5a gene expression.

Topics: Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; N-Myc Proto-Oncogene Protein; Neoplasm Invasiveness; Neural Crest; Neurites; Neuroblastoma; Neurons; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; Prognosis; Protein Kinase C; Protein Kinase C-theta; Proto-Oncogene Proteins; RNA, Messenger; Tretinoin; Up-Regulation; Wnt Proteins; Wnt-5a Protein

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.

Topics: Aldehydes; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Flow Cytometry; Genes, Tumor Suppressor; HL-60 Cells; Humans; Lipid Metabolism; Microscopy, Fluorescence; Neuroblastoma; Nuclear Proteins; Peroxides; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Trans-Activators; Transcription Factors; Tretinoin; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A2 Receptor Agonists; Animals; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Corpus Striatum; Cyclic AMP-Dependent Protein Kinases; Enzyme Activators; Enzyme Inhibitors; G1 Phase; Humans; MAP Kinase Signaling System; Neurites; Neuroblastoma; Neurons; Phosphorylation; Protein Kinase C; Rats; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, trkB; Signal Transduction; Stem Cells; Tretinoin

Lesch-Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch-Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.

Topics: Basal Ganglia; Basal Ganglia Diseases; Cell Cycle Proteins; Cell Differentiation; Cell Enlargement; Cell Line; Enzyme Inhibitors; Guanine Nucleotides; Humans; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; Lesch-Nyhan Syndrome; Models, Neurological; Mycophenolic Acid; Neurites; Neuroblastoma; Neurofilament Proteins; Tretinoin; Up-Regulation

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

Topics: Calcium; Calcium Channels; Calcium Signaling; Cations, Divalent; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Humans; Manganese; Methacholine Chloride; Muscarinic Agonists; Neuroblastoma; Neurons; Thapsigargin; Tretinoin

Bone marrow transplant nephropathy (BMTN) classically presents more than 100 days after transplantation as an acute nephritis with hypertension, azotaemia and anemia that usually results in end stage renal failure (ESRF). The risk of developing BMTN may be greater with the use of more intensive chemotherapy and higher total body and tumor bed irradiation. Cis-retinoic acid (RA) may further increase the risk of developing BMTN. Here, we report the cases of two children who developed typical clinical and biochemical features of BMTN. They were both treated for stage IV neuroblastoma with chemotherapy, bone marrow transplant (BMT) conditioning that included total body irradiation and RA therapy after BMT, although the patient in case 1 had established renal insufficiency prior to the commencement of RA. Renal biopsy of these children showed classical BMTN histology, and the renal manifestations progressed quickly; the patient in case 1 became dialysis dependent by 1 year post-bone marrow transplant. Recently, RA has been added to the post-BMT therapy in children with stage IV neuroblastoma. The occurrence of BMTN in two children treated with RA in our unit is unlikely to be coincidental. Although RA has been shown to confer a significant survival advantage in this disease, animal studies and a previous case report have suggested it could increase the toxic effects of chemotherapy and renal irradiation. It is likely that RA contributed to the deterioration in renal function in these patients.

Topics: Antineoplastic Agents; Bone Marrow Transplantation; Child, Preschool; Female; Humans; Kidney; Kidney Diseases; Male; Neoplasm Staging; Neuroblastoma; Postoperative Care; Stereoisomerism; Tretinoin

ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and -independent functions of ShcC.

Topics: Agar; Animals; Apoptosis; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Genes, Dominant; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Neuroblastoma; Neuropeptides; Phosphorylation; Phosphotyrosine; Plasmids; Protein Binding; Protein Structure, Tertiary; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 3; src Homology Domains; Thymidine; Time Factors; Transfection; Tretinoin; Wound Healing

Neuroblastoma is the second most common pediatric malignancy, characterized by a high rate of unexplained spontaneous remissions. Much progress has been made in understanding neuroblastoma differentiation triggered by certain agents such as retinoic acid. However, little is known about the signalling pathways that lead to differentiation of neuroblastoma cells due to serum withdrawal. We found that in Neuro2a neuroblastoma cells, EGFR, ERK1/2 and Akt showed increased phosphorylation after serum withdrawal, and that the activation of EGFR was necessary for the activation of Akt and ERK1/2. Inhibition of EGFR, ERK1/2 and PI3K blocked neuroblastoma differentiation after serum withdrawal. Interestingly, addition of high-density lipoprotein (HDL) abrogated serum-withdrawal induced neuroblastoma differentiation, as well as the activation of EGFR. Our results demonstrate a novel role for serum-derived lipoproteins in the control of receptor tyrosine kinase activity.

Topics: Cell Differentiation; Cell Line, Tumor; Cholesterol; Culture Media, Serum-Free; Enzyme Inhibitors; ErbB Receptors; Flavonoids; Humans; Lipoproteins, HDL; Membrane Microdomains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Growth Factors; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Signal Transduction; Time Factors; Transfection; Tretinoin

Cdk9 is a member of the Cdc2-like family of kinases. Its cyclin partners are members of the family of cyclin T (T1, T2a and T2b) and cyclin K. The Cdk9/Cyclin T complex appears to be involved in regulating several physiological processes. Recently, Cdk9 has been identified as a regulator of the differentiation program of several cell types, such as muscle cells, monocytes and lymphocytes, suggesting that it may have a function in controlling specific differentiative pathways. We analyzed whether Cdk9 and Cyclin T1 may be involved in the regulation of neuron and astrocyte differentiation. Cdk9 and Cyclin T1 expression levels were monitored during the differentiation program of neuroblastoma and astrocytoma cell lines. Our results suggest that Cdk9/Cyclin T1 complex may be required for neuron differentiation induced by retinoic acid, because the expression level of the complex varies during differentiation, but no significant changes were observed in its expression in the astrocytoma cell line. In addition, the expression of Cdk9 and Cyclin T1 was evaluated by immunohistochemistry in samples of neuroblastoma, PNET (Primary Neuroectodermal Tumor) and astrocytoma tumors of different grades, in order to assess whether there was a correlation between Cdk9 expression and tumor grading. Our results show that in neuroblastoma and PNET tumor samples Cdk9 is more expressed the more differentiated the tumor is. Conversely, no significant alteration of Cdk9 expression was observed in astrocytoma tumor samples of different grades, thus confirming the results obtained for the cell lines.

Topics: Astrocytes; Cell Differentiation; Cell Line, Tumor; Cyclin T; Cyclin-Dependent Kinase 9; Cyclins; Humans; Immunohistochemistry; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; RNA, Messenger; Tretinoin

The biological features and prognosis of neuroblastoma, a neural crest-derived pediatric tumor, are closely associated with expression of the Trk receptor. Because the Shc family proteins (ShcA, ShcB, and ShcC) are adaptors for various receptors, including Trk receptors, and are regulators of neuronal cell development, we speculated that they may play a role in neuroblastoma. Therefore, in this study, we used semiquantitative reverse transcription-PCR to examine the expression of these three genes in 15 neuroblastoma cell lines, an all-trans-retinoic acid-treated neuroblastoma cell line, and 52 tumor samples. In neuroblastoma cell lines and tumor samples, shcA was ubiquitously and highly expressed. Little expression of shcA was observed. Also, shcB was hardly expressed in neuroblastoma cell lines, but its expression in RT-BM-1 cells was enhanced after all-trans-retinoic acid-induced differentiation, and it was highly expressed in low-stage tumors (P = 0.0095). This suggests that ShcB participates in cellular differentiation and may correlate with a favorable prognosis in neuroblastoma. Finally, the expression of shcC was observed in most of the neuroblastoma cell lines and in some stage 4 patients. Patients with a high expression of shcC had a very poor prognosis (P < 0.0001) and amplification of MYCN, and all died within 31 months after diagnosis. Therefore, ShcC seems to be associated with an aggressive tumor phenotype, perhaps by enhancing TrkB signals. Our results suggest that the expressions of shcB and shcC are important biological factors in neuroblastoma and are useful prognostic indicators.

Topics: Adaptor Proteins, Signal Transducing; Cell Differentiation; Cell Line, Tumor; Child; Child, Preschool; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Prognosis; Receptor, trkA; Receptor, trkB; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Src Homology 2 Domain-Containing, Transforming Protein 2; Survival Analysis; Survival Rate; Tretinoin

The spatial orientation of nerve cells plays a pivotal role in nerve regeneration. Here we report a new method for regulating neuronal cell morphology and guiding neurite extension on standard tissue culture dishes. Random copolymers of oligoethyleneglycol methacrylate and methacrylic acid [poly(OEGMA-co-MA)], microcontact printed on standard tissue culture dishes, resist cell attachment and remain intact in serum-containing medium for up to 2 weeks. Cell viability assay of SH-SY5Y cells demonstrated that poly(OEGMA-co-MA) on the substrate or in solution has no cytotoxic effect. When retinoic acid was added to SH-SY5Y cells, they extended neurites along the line patterns that are significantly longer than cells cultured on non-patterned culture dishes. The ability to guide neurite extension with micrometer precision is valuable for guiding directional growth of neurites and path finding of regenerating nerves.

Topics: Actins; Adsorption; Biocompatible Materials; Cell Culture Techniques; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cells, Cultured; Culture Media; Cytoskeleton; Humans; Nerve Regeneration; Neurites; Neuroblastoma; Neurons; Polymers; Substrate Specificity; Time Factors; Tretinoin

The NMDA class of glutamate receptors plays a critical role in CNS, such as synaptic plasticity, axonal sprouting, growth, and migration. NMDA receptor stimulation has been shown to regulate polysialylated neural cell adhesion molecule (PSA-NCAM) expression in glial cell cultures and in hippocampal slice cultures. There is also growing evidence that molecular chaperons and ROS are related to the synaptic plasticity phenomena. We have examined the neuroprotective effect of subtoxic dose of NMDA in retinoic acid differentiated SH-SY5Y neuroblastoma cells. SH-SY5Y cell line differentiated with retinoic acid (10 muM) was exposed to NMDA (100 microM) or to antagonist MK-801 (200 nM) + NMDA and cells harvested after 24 h of treatment for PSA-NCAM, NCAM, and HSP70 expression study and for biochemical analysis. A significant increase was observed in PSA-NCAM, NCAM-180, NCAM-140, and HSP70 expression as seen by Western blotting and immunocytofluorescent studies in NMDA-treated cultures. Biochemical analysis revealed a significant increase in the activities of glutathione peroxidase (GPx) and copper zinc-superoxide dismutase (CuZnSOD) upon exposure to NMDA. No significant change was observed in the level of lipid peroxidation. All the changes observed reverted back to the control values upon pretreatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure indicating the involvement of NMDA receptor in these changes. These results illustrate the neuroprotective role of subtoxic dose of NMDA in SH-SY5Y neuroblastoma cells.

Topics: Brain; Cell Survival; Cytoprotection; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Free Radical Scavengers; Glutathione Peroxidase; HSP70 Heat-Shock Proteins; Humans; Lipid Peroxidation; N-Methylaspartate; Neural Cell Adhesion Molecule L1; Neural Cell Adhesion Molecules; Neuroblastoma; Neuronal Plasticity; Neurons; Neuroprotective Agents; Oxidative Stress; Receptors, N-Methyl-D-Aspartate; Sialic Acids; Superoxide Dismutase; Superoxide Dismutase-1; Tretinoin; Tumor Cells, Cultured

The LIM homeobox family of transcription factors is involved in many processes during the development of the mammalian central nerves system. L3, also called Lhx8 (L3/Lhx8), is a recently identified member of the LIM homeobox gene family and is selectively expressed in the medial ganglionic eminence (MGE). Our previous study demonstrated that L3/Lhx8-null mice specifically lacked cholinergic neurons in the basal forebrain. In this study, we reduced L3/Lhx8 function in the murine neuroblastoma cell line, Neuro2a (N2a), using L3/Lhx8-targeted small interfering RNA (siRNA) produced by H1.2 promoter-driven vector. The levels of cholinergic markers per cell were diminished without a reduction in the number of marker-positive cells. Intriguingly, GABAergic marker expression and the number of GABAergic cells were dramatically increased in the differentiating L3/Lhx8-knockdown N2a. These results suggest the possibility that L3/Lhx8 is involved in the determination of transmitter phenotypes (GABAergic or cholinergic cell fate) in a population of neurons during basal forebrain development.

Topics: Animals; Blotting, Northern; Cell Count; Cell Death; Cell Differentiation; Cell Line, Tumor; Choline O-Acetyltransferase; Cyclic AMP; Drug Interactions; gamma-Aminobutyric Acid; Gene Expression Regulation; Genetic Vectors; Glutamate Decarboxylase; Green Fluorescent Proteins; Homeodomain Proteins; In Situ Nick-End Labeling; Isoenzymes; Ki-67 Antigen; LIM-Homeodomain Proteins; Membrane Transport Proteins; Mice; Nerve Tissue Proteins; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Time Factors; Transcription Factors; Transfection; Tretinoin; Vesicular Acetylcholine Transport Proteins

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.

Topics: Carboxylic Ester Hydrolases; Cell Differentiation; Cell Line, Tumor; Gene Expression; Humans; Nervous System Neoplasms; Neurites; Neuroblastoma; Neurons; Organophosphates; RNA Interference; RNA, Antisense; Transfection; Tretinoin

Neuroblastoma is the most frequent extracranial solid malignancy of childhood with a high mortality in advanced tumour stages. The hallmark of neuroblastoma is its clinical and biological heterogeneity. The molecular mechanisms leading to favourable or unfavourable tumour behaviour are still speculative. However, amplification of the oncogene MYCN and expression of the neurotrophin receptor TrkB are known to contribute to a highly malignant phenotype. To define the mechanisms through which TrkB may mediate neuroblastoma progression, we stably expressed this receptor in the neuroblastoma cell lines SH-SY5Y and SK-N-AS. The transfectants, but not the controls, had an increased invasive potency both, in vitro and in vivo, as demonstrated by Matrigel-invasion and chorioallantoic membrane assays, respectively. The retinoic acid-induced TrkB expression in parental SH-SY5Y cells was also associated with enhanced cell invasiveness. The TrkB mediated invasiveness involved the upregulation of the hepatocyte growth factor (HGF) and its receptor c-Met, resulting in an autocrine loop. Inhibition of HGF activity by anti-HGF neutralizing antibodies or disabling the function of c-Met by small interfering RNA suppressed the TrkB-induced invasiveness. The enhanced TrkB expression was associated with a significant increase in the secretion of various matrix-degrading proteases. Immunostaining and real-time RT-PCR analysis of tumour specimens demonstrated coordinated expression of TrkB and HGF/c-Met in experimental and primary neuroblastomas. We conclude that TrkB expression in neuroblastoma cells results in an increase in their invasive capability via upregulated expression of HGF/c-Met and enhanced activity of proteolytic networks.

Topics: Animals; Chick Embryo; Chorioallantoic Membrane; Collagen; Drug Combinations; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Laminin; Neoplasm Invasiveness; Neovascularization, Pathologic; Neuroblastoma; Phosphorylation; Proteoglycans; Proto-Oncogene Proteins c-met; Quail; Receptor, trkB; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection; Tretinoin; Tumor Cells, Cultured

Neuropathy target esterase (NTE) is inhibited and aged by organophosphorus compounds that induce delayed neuropathy in human and some sensitive animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, to date, there is no direct evidence of the relevance of NTE in neurodifferentiation under physiological conditions. In this study, we have investigated a possible role for NTE in the all-trans retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of NTE by RNA interference indicated that reduction of NTE does not affect process outgrowth or differentiation of the cells, although moderate expression of NTE by expression of the NTE esterase domain accelerates the elongation of neurite processes. Mipafox, a neurotoxic organophosphate, was shown to block process outgrowth and differentiation in cells that have lowered NTE activity due to RNA interference, suggesting that mipafox may interact with other molecules to exert its effect in this context.

Topics: Acetylcholinesterase; Animals; Antineoplastic Agents; Carboxylic Ester Hydrolases; Cell Differentiation; Cell Line, Tumor; Cholinesterase Inhibitors; Gene Silencing; Humans; Isoflurophate; Neuroblastoma; Neurons; Paraoxon; RNA Interference; Tretinoin

The glial cell line-derived neurotrophic factor (GDNF) family of ligands play essential roles in promoting normal neural crest differentiation during embryogenesis, and, may have a therapeutic role in malignancies of neural crest origin, such as neuroblastoma. However, we report here that GDNF and neurturin blocked the growth inhibitory and neuritogenic effects of all-trans-retinoic acid in neuroblastoma cells in vitro. GDNF caused neuroblastoma cells to proliferate in the presence of a range of cytotoxic chemotherapeutic agents at low concentrations. Thus, our findings suggest a role for GDNF signaling in promoting resistance to differentiation or cytotoxic therapy of neuroblastoma, and, preclude their use in this neural crest tumor.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cytotoxins; Drug Resistance, Neoplasm; Glial Cell Line-Derived Neurotrophic Factor; Glial Cell Line-Derived Neurotrophic Factors; Growth Inhibitors; Humans; Ligands; Mice; Mice, Transgenic; Neuroblastoma; Neurturin; Signal Transduction; Tretinoin

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.

Topics: Amyloid beta-Protein Precursor; Blotting, Western; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Drug Interactions; Extracellular Space; Gene Expression Regulation; Humans; Nerve Tissue Proteins; Neuroblastoma; Protein Structure, Tertiary; Time Factors; Tretinoin

mKIAA genes are mouse counterparts of human KIAA genes, which were isolated in our cDNA project and were functionally unknown at the time they were sequenced. Because KIAA/mKIAA genes were isolated mainly from cDNA libraries derived from brain tissues, they are thought to be important for the organization and function of the brain. To investigate the participation of mKIAA genes in neuronal phenomena, we analyzed retinoic acid-induced neurite outgrowth using an mKIAA oligonucleotide microarray. Focusing on the early stage of this outgrowth phenomenon, we analyzed temporal gene expression changes 1-24 h after treatment with retinoic acid and found several change patterns in 38 mKIAA genes. Among them, six were upregulated at 3 h and subsequently returned to the steady state. Supposing that these genes had important roles, we performed semi-quantitative RT-PCR analysis and confirmed the existence of temporal expression patterns in two genes (mKIAA0182 and mKIAA1039). Further computational analysis of the 38 genes enabled us to find the cellular pathway associated with 6 of them with high confidence. These results indicate that some mKIAA genes are apparently relevant to retinoic acid-induced neurite outgrowth.

Topics: Animals; Base Sequence; Cell Line, Tumor; DNA Primers; Genes, Essential; Humans; Mice; Neurites; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence by the alpha-secretase prevents the formation of toxic Abeta peptides. It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as alpha-secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsalpha. Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.

Topics: ADAM Proteins; ADAM17 Protein; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Astrocytoma; Blotting, Western; Cattle; Cell Culture Techniques; Cell Differentiation; Cell Extracts; Cell Line; Cell Line, Tumor; Dipeptides; Disintegrins; Endopeptidases; Gene Expression Regulation; Humans; Metalloproteases; Mice; Mice, Transgenic; Mutagenesis; Nerve Tissue Proteins; Neuroblastoma; Protease Inhibitors; Solubility; Tetradecanoylphorbol Acetate; Tretinoin; Up-Regulation

Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of l-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.

Topics: Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Glial Cell Line-Derived Neurotrophic Factor; Humans; Mitogen-Activated Protein Kinases; Nerve Growth Factors; Neuroblastoma; Phosphorylation; Serine; Synaptophysin; Tretinoin; Tyrosine 3-Monooxygenase

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nervous System; Neurites; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-raf; Signal Transduction; Tretinoin

A 21-year-old patient was admitted to the hospital because of massively enlarged cervical lymph nodes. Additionally, a left-sided facial and brachial edema was visible. Auscultation of the left lung was remarkable for diminished breath sounds.. Diagnostic imaging showed an extensive thoracic tumor and enlarged mediastinal and cervical lymph nodes. The diagnosis of neuroblastoma was established by biopsy.. The patient was treated with a polychemotherapy protocol according to the pediatric neuroblastoma study NB97. Subsequently, the patient underwent partial tumor resection, received two further chemotherapy courses and irradiation of the remaining tumor. Because of residual vital tumor cells, a second surgical tumour reduction followed by high-dose chemotherapy with autologous stem-cell support was performed. Two cycles of high-dose retinoic acid followed. Six months after the end of therapy, the patient is in a good condition despite of the presence of residual tumor.. Neuroblastoma is a very rare tumor in adult patients. Therapy is multimodal and should follow pediatric guidelines for neuroblastoma treatment.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Chemotherapy, Adjuvant; Combined Modality Therapy; Diagnosis, Differential; Humans; Magnetic Resonance Imaging; Male; Neuroblastoma; Radiotherapy, Adjuvant; Reoperation; Stem Cell Transplantation; Thoracic Neoplasms; Transplantation, Autologous; Tretinoin

Amplification of MYCN in neuroblastoma strongly correlates to unfavorable outcome, but little is known of how the high MYCN expression translates into an aggressive tumor phenotype. More aggressive neuroblastomas are generally immature and overexpression of exogenous MYCN in cultured neuroblastoma cells and other neuronal cell types has been reported to inhibit induced differentiation, suggesting a link between high MYCN expression and an immature phenotype. However, we show here that MYCN is expressed in human neuroblasts of sympathetic chain ganglia at fetal week 8.5, a developmental stage at which these neuroblasts express a number of sympathetic neuronal differentiation marker genes. Analyses of 28 neuroblastoma tumor specimens and 27 cell lines for the expression of MYCN and a panel of neuronal differentiation marker genes did not reveal any correlation between MYCN and marker gene expression levels. Finally, we tested five separate differentiation protocols and show that MYCN overexpressing neuroblastoma cells with a neuronal phenotype, derived from the non-MYCN-amplified human neuroblastoma cell line SK-N-SH, retain their capacity to differentiate despite constitutive MYCN overexpression. Our results show that high MYCN expression and sympathetic differentiation are compatible, and indirectly our findings lend support to previously published MYCN neuroblastoma tumor data, which suggest that in single MYCN copy neuroblastomas there is no direct correlation between a high cellular MYCN protein content and aggressive tumor cell behavior.

Topics: Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Genes, myc; Humans; Neuroblastoma; Neurons; Sympathetic Nervous System; Tetradecanoylphorbol Acetate; Tretinoin

Neuroblastoma is the most common solid tumor of infancy and is believed to result from impaired differentiation of neuronal crest embryonal cells. The promyelocytic leukemia protein (PML)-nuclear body is a cellular structure that is disrupted during the pathogenesis of acute promyelocytic leukemia, a disease characterized by impaired myeloid cell differentiation. During the course of studies to examine the composition and function of PML-nuclear bodies, we observed that the human neuroblastoma cell line SH-SY5Y lacked these structures and that the absence of PML-nuclear bodies was a feature of N- and I-type, but not S-type, neuroblastoma cell lines. Induction of neuroblastoma cell differentiation with 5-bromo-2'deoxyuridine, all-trans-retinoic acid, or IFN-gamma induced PML-nuclear body formation. PML-nuclear bodies were not detected in tissue sections prepared from undifferentiated neuroblastomas but were present in neuroblasts in differentiating tumors. Expression of PML in neuroblastoma cells restored PML-nuclear bodies, enhanced responsiveness to all-trans-retinoic acid, and induced cellular differentiation. Pharmacological therapies that increase PML expression may prove to be important components of combined modalities for the treatment of neuroblastoma.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Nucleus Structures; HL-60 Cells; Humans; Immunohistochemistry; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Promyelocytic Leukemia Protein; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

Primary cultures of rat cortical neurons exposed to toxic concentrations of beta-amyloid peptide (betaAP) begin an unscheduled mitotic cell cycle that does not progress beyond the S phase. To analyze possible signal transduction pathways involved in this effect, the action of betaAP has been studied in SH-SY5Y neuroblastoma cells differentiated by a 7-d exposure to 10 microM retinoic acid. Treatment with the betaAP fragment, betaAP(25-35), (25 microM) for 24, 48, or 72 h caused apoptotic cell death, detected by flow cytometry as a prediploid cell population. Cell cycle analysis showed that betaAP(25-35) modified cell cycle profiles by markedly increasing the number of cells in the S phase and reducing the population of the G2/M area. These effects seem to involve activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Inhibition of this pathway by the specific inhibitor PD98059 (2 microM) completely prevented changes of cell cycle distribution induced by betaAP and significantly reduced neuronal death. The data suggest that MAPK cascade can mediate the induction of cell cycle induced by betaAP, thus contributing to the toxicity of the peptide.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Enzyme Inhibitors; Flavonoids; G2 Phase; Gene Expression Profiling; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nerve Degeneration; Neuroblastoma; Peptide Fragments; S Phase; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21(Cip1) at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21(Cip1) promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21(Cip1), whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21(Cip1) promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21(Cip1), and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Helix-Loop-Helix Motifs; Humans; Inhibitor of Differentiation Protein 2; Nerve Tissue Proteins; Neuroblastoma; Promoter Regions, Genetic; Protein Binding; Receptor, trkB; Repressor Proteins; TCF Transcription Factors; Transcription Factor 7-Like 1 Protein; Transcription Factors; Transfection; Tretinoin

Cyclin-dependent kinase-activating kinase (CAK) regulates cell cycle G1 exit, where cells commonly commit either to proliferate or to differentiate. CAK activity in G1 regulation is determined by its assembly factor and targeting subunit, ménage à trois 1 (MAT1). The precise mechanism of how proliferation/differentiation transition is induced from cancer cell G1 arrest remains unknown. We present evidence that in neuroblastoma CHP126 cells, CAK interacts with and phosphorylates retinoblastoma tumor suppressor protein (pRb) and retinoid X receptor alpha (RXRalpha). Retinoic acid (RA)-induced neuroblastoma cell proliferation/differentiation transition is associated with decreased CAK activity, as evidenced by a switch from CAK hyperphosphorylation of pRb and RXRalpha to hypophosphorylation of pRb and RXRalpha. Manipulation of MAT1 abundance shows that MAT1 reduction mimics RA-induced hypophosphorylation of pRb/RXRalpha, proliferation inhibition, and neurite outgrowth, whereas MAT1 overexpression resists these RA actions. Thus, these findings reveal an important mechanism by which MAT1-modulated CAK activity is crucial in the switch from proliferation to differentiation in neuroblastoma cells.

Topics: Alitretinoin; Cell Differentiation; Cell Division; Cell Line, Tumor; Enzyme Activation; G1 Phase; Humans; Neurites; Neuroblastoma; Phosphorylation; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoid X Receptors; Transcription Factors; Tretinoin

Recent findings link increased expression of the structurally complex 'b' pathway gangliosides GD1b, GT1b, GQ1b (CbG) to a favourable clinical and biological behaviour in human neuroblastoma (NB). Seeking a model to probe these observations, we evaluated four human NB cell lines. Very low CbG content (4-10%) in three of the four cell lines (LAN-5, LAN-1, SMS-KCNR) reflected the ganglioside pattern observed in the most aggressive NB tumours. Pharmacological alterations of complex ganglioside synthesis in vitro by a 5-7 day exposure to 5-10 microM retinoic acid, which is employed in maintenance therapy of disseminated NB, included markedly increased (i) relative expression of CbG (6.6+/-2.0-fold increase, P=0.037), (ii) relative expression of the analogous 'a' pathway gangliosides, termed CaG (6.4+/-1.4-fold increase in GM1a and GD1a; P=0.010), and (iii) total cellular ganglioside content (2.0-6.3-fold), which in turn amplified the accumulation of structurally complex gangliosides. Substantial increases (2.7-2.9-fold) in the activity of GD1b/GM1a synthase (beta-1,3-galactosyltransferase), which initiates the synthesis of CbG and CaG, accompanied the all-trans retinoic acid (ATRA)-induced ganglioside changes. Thus, increased CbG synthesis in NB cell lines is attributable to a specific effect of ATRA, namely induction of GD1b/GM1a synthase activity. Since the shift towards higher expression of CbG and CaG during retinoic acid-induced cellular differentiation reflects a ganglioside pattern found in clinically less-aggressive tumours, our studies suggest that complex gangliosides may play a role in the biological and clinical behaviour of NB.

Topics: Antineoplastic Agents; Cell Differentiation; Galactosyltransferases; Gangliosides; Glucosyltransferases; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.

Topics: Apoptosis; Cell Count; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Neurites; Neuroblastoma; Protein Kinase C; Protein Serine-Threonine Kinases; Protein Transport; rho-Associated Kinases; Sphingosine; Transfection; Tretinoin

FOXO3a is a ubiquitously expressed mammalian forkhead transcription factor with a high expression level in adult brain. The activity of FOXO3a is inhibited by growth factors through activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, which phosphorylates FOXO3a and decreases the level of FOXO3a in the nucleus. In the present study, we examined the regulation of FOXO3a by brain-derived neurotrophic factor (BDNF) in retinoic acid (RA)-differentiated human SH-SY5Y neuroblastoma cells. BDNF caused a rapid and time-dependent decrease of nuclear FOXO3a with a corresponding increase of cytosolic FOXO3a. The rate of the BDNF-induced nuclear/cytosolic redistribution was consistent with the time course of BDNF-induced threonine32-phosphorylation of FOXO3a, and was mediated by the PI3K/Akt signaling pathway. Active FOXO3a rapidly increased the level of Bcl-2-interacting mediator (bim) in differentiated SH-SY5Y cells, and BDNF decreased the FOXO3a-induced increase of bim through activation of both PI3K/Akt and Erk signaling pathways. Thapsigargin, an endoplasmic reticulum (ER) stress-inducing agent, significantly decreased threonine32-phosphorylation of FOXO3a, and increased nuclear and decreased cytosolic FOXO3a, suggesting that thapsigargin activates FOXO3a. Treatment with BDNF completely reversed and blocked the thapsigargin-induced dephosphorylation and nuclear accumulation of FOXO3a. In addition, protein phosphatase 1/2A inhibitors increased threonine32-phosphorylation of FOXO3a, decreased nuclear FOXO3a, and blocked thapsigargin-induced activity of FOXO3a. The regulatory effect of BDNF on FOXO3a and its target genes may play a significant role in the BDNF-mediated neuronal survival, differentiation, and plasticity.

Topics: Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Brain-Derived Neurotrophic Factor; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Membrane Proteins; Neuroblastoma; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Fusion Proteins; Serine; Signal Transduction; Subcellular Fractions; Thapsigargin; Threonine; Transcription Factors; Tretinoin

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

Topics: Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Exons; Fetus; Humans; Immunoblotting; Immunohistochemistry; Membrane Proteins; Mutation; Neuroblastoma; Presenilin-1; Tetradecanoylphorbol Acetate; Transfection; Tretinoin

Nerve growth factor (NGF) is a peptide displaying multiple cholinotropic activities. The aim of this work was to explain mechanisms of the positive and negative effects of NGF on phenotypic properties and viability of cholinergic cells. To discriminate these effects we used two p75NTR receptor-positive lines of cholinergic neuroblastoma cells, SN56 and T17 that are devoid of or express high affinity NGF (TrkA) receptors, respectively. cAMP and retinoic acid caused differentiation of both cell lines. In addition to the morphologic maturation, the increase of choline acetyltransferase activity, acetylcholine, Ca and cytoplasmic acetyl-CoA levels and decrease of mitochondrial acetyl-CoA and cell viability were observed. NGF caused similar effects in non-differentiated T17 cells but had no influence on non-differentiated SN56 cells. On the contrary, in both cAMP/all-trans-retinoic acid (RA) differentiated cell lines, NGF resulted in a similar suppression of cholinergic phenotype along with an increase of mitochondrial acetyl-CoA and cell susceptibility to nitric oxide and amyloid-beta25-35. These effects of NGF were prevented by an antibody against the p75NTR receptor. Data indicate that: (i) positive cholinotrophic effects of NGF required activation of both TrkA and p75NTR receptors; (ii) cAMP/RA-evoked differentiation inhibited NGF effects mediated by TrkA receptors and activated its p75NTR-dependent suppressing influences and (iii) a differentiation-evoked decrease of mitochondrial acetyl-CoA and an elevation of mitochondrial Ca could augment impairment of cholinergic neurons by neurotoxic signals.

Topics: Acetyl Coenzyme A; Acetylcholine; Amyloid beta-Peptides; Animals; Antibodies; Bucladesine; Calcium; Cell Differentiation; Cell Line; Cell Survival; Choline O-Acetyltransferase; Cytoplasm; Enzyme Activation; Mice; Mitochondria; Nerve Growth Factor; Neuroblastoma; Neurons; Nitroprusside; Peptide Fragments; Pyruvate Dehydrogenase Complex; Rats; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Tretinoin

The vitamin A metabolite, all-trans retinoic acid (atRA), is required for embryonic development. atRA binds to the nuclear retinoic acid receptors and regulates the transcription of specific target genes. In order to identify atRA-induced genes that play a role in neural development, a subtractive library was created from SH-SY5Y neuroblastoma cells, a human cell line that exhibits changes in cell adhesion and neurite outgrowth after exposure to the vitamin A acid. We report here the identification of 14 genes that are rapidly induced by atRA (retinoic acid induced in neuroblastoma or RAINB), eight of which were previously not known to be atRA responsive (BTBD11, calmin, cyclin M2, ephrin B2, HOXD10, NEDD9, RAINB6 and tenascin R). mRNA regulation by atRA was confirmed in SH-SY5Y cells by Northern blotting, and gene regulation was studied in additional human cell lines using the quantitative polymerase chain reaction. The majority of the atRA-responsive clones revealed in this screen are highly expressed in the nervous system of developing rat embryos. Further, the expression of several of these genes is perturbed in developing rat embryos exposed to excess atRA or conversely, deprived of sufficient retinoid during early development. We propose that a subset of these genes lie downstream of atRA and its receptors in the regulation of neurite outgrowth and cell adhesion in both neural and non-neural tissues within the developing embryo.

Topics: Administration, Oral; Animals; Cell Adhesion; Cell Line, Tumor; Female; Gene Expression Regulation; Gene Expression Regulation, Developmental; Genomic Library; Humans; In Situ Hybridization; Male; Nervous System; Neuroblastoma; Pregnancy; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tretinoin

In the present study the accumulation of protease resistant prion protein (PrPres) in scrapie-infected neuroblastoma cells (ScN2a cells) was shown to be dependent on culture conditions. The highest levels of PrPres were found in slow growing cells. Further increases in PrPres accumulation were observed in ScN2a cells treated with retinoic acid, a compound that is associated with neuronal differentiation. The effects of retinoic acid were dose-dependent with a maximal effect at 200 ng/ml. A similar increase in PrPres was observed in another prion-infected cell line, scrapie-mouse brain (SMB) cells, treated with retinoic acid while retinoic acid increased the amount of PrPC in non-infected cells. Other drugs reported to cause neuronal differentiation, such as phorbol esters, did not increase the PrPres content of ScN2a cells. The survival of retinoic acid-treated ScN2a cells co-cultured with microglia was significantly reduced when compared to untreated ScN2a cells and an inverse correlation was demonstrated between the PrPres content of cells and their survival when co-cultured with microglia. The production of interleukin-6 by microglia cultured with retinoic acid-treated ScN2a cells was significantly higher than that of microglia cultured with untreated ScN2a cells.

Topics: Animals; Animals, Newborn; Antineoplastic Agents; Blotting, Western; Brain; Carcinogens; Cell Count; Cell Line, Tumor; Cell Survival; Cells, Cultured; Coculture Techniques; Dose-Response Relationship, Drug; Drug Interactions; Endopeptidase K; Enzyme-Linked Immunosorbent Assay; Glycosylation; Humans; Interleukin-6; Mice; Microglia; Neuroblastoma; Prions; PrPSc Proteins; Scrapie; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Transfection; Tretinoin

Nitric oxide (NO) has been found to act as an important negative regulator of cell proliferation in several systems. We report here that NO negatively regulates proliferation of neuronal cell precursors and promotes their differentiation by downregulating the oncogene N-Myc. We have studied this regulatory function of NO in neuroblastoma cell lines (SK-N-BE) and in primary cerebellar granule cell cultures. In a neuronal NO synthase (nNOS) overexpressing neuroblastoma cell line exposed to the differentiative action of retinoic acid, NO slowed down proliferation and accelerated differentiation towards a neuronal phenotype. This effect was accompanied by a parallel decrease of N-Myc expression. Similar results could be obtained in parental SK-N-BE cells by providing an exogenous source of NO. Pharmacological controls demonstrated that NO's regulatory actions on cell proliferation and N-Myc expression were mediated by cGMP as an intermediate messenger. Furthermore, NO was found to modulate the transcriptional activity of N-Myc gene promoter by acting on the E2F regulatory region, possibly through the control of Rb phosphorylation state, that we found to be negatively regulated by NO. In cerebellar granule cell cultures, NOS inhibition increased the division rate of neuronal precursors, in parallel with augmented N-Myc expression. Because a high N-Myc expression level is essential for neuroblastoma progression as well as for proliferation of neuronal precursors, its negative regulation by NO highlights a novel physiopathological function of this important messenger molecule.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cyclic GMP; Down-Regulation; Gene Expression Regulation; Genes, myc; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Promoter Regions, Genetic; Rats; Rats, Wistar; Retinoblastoma Protein; Transcription, Genetic; Tretinoin

The present study investigated the effect of three antidepressant drugs (ADs), desipramine (DMI, a noradrenaline reuptake inhibitor), citalopram (CIT, a selective serotonin reuptake inhibitor) and mianserin (MIA, thought to act as an antagonist of pre-synaptic alpha2 adrenoceptor) on the transcriptional activity of the dopamine D2 receptor gene promoter. The fragment of dopamine D2 receptor gene promoter (-850 to +133) was subcloned into pGL3 vector (Promega), which has an insert coding for luciferase used as a reporter gene. Such construct (pGL3-D2R) was used to transiently transfect the neuroblastoma cell lines, Neuro 2a, SH-SY5Y and NB41A3, which endogenously express the dopamine D2 receptor protein. The obtained results indicate that transcriptional activity of dopamine D2 receptor gene promoter was dose-dependently increased by retinoic acid, forskolin, rolipram and phorbol 12 myristate 13-acetate, as well as by DMI, CIT and MIA. In the Neuro 2a cells, the most significant increase was observed after the ADs were present in the incubation medium at a doses of 0.1-1 microM for 72 h. In the SH-SY5Y cells, the significant increase in the transcriptional activity of D2 receptor gene promoter was observed already after 24-h exposure to DMI. Incubation of the Neuro 2a cells in the presence of forskolin (1 microM) or rolipram (50 microM) (but not phorbol 12-myristate 13-acetate at 0.1 microM) in combination with DMI resulted in the further increase in transcriptional activity of the studied promoter, indicating the involvement of protein kinase A pathway in these effects.

Topics: Animals; Antidepressive Agents; Cell Line, Tumor; Colforsin; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Neuroblastoma; Neurons; Promoter Regions, Genetic; Rats; Receptors, Dopamine D2; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tretinoin

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPalpha into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPalpha levels. Pre-incubation with atropine decreased release of sAPPalpha significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.

Topics: Acetylcholinesterase; ADAM Proteins; ADAM10 Protein; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Atropine; Blotting, Western; Brefeldin A; Bungarotoxins; Carbachol; Cell Differentiation; Cell Fractionation; Cell Line, Tumor; Cholinergic Agonists; Cholinesterase Inhibitors; Donepezil; Drug Interactions; Embryo, Mammalian; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Indans; Membrane Proteins; Metalloendopeptidases; Microscopy, Confocal; Muscarinic Antagonists; Neuroblastoma; Physostigmine; Piperidines; Protein Synthesis Inhibitors; Protein Transport; Tretinoin

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; Aged; Aged, 80 and over; Analysis of Variance; Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Dopamine; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Lewy Bodies; Male; Middle Aged; Neuroblastoma; Nuclear Proteins; Parkinsonian Disorders; Postmortem Changes; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tretinoin

The vitamin A metabolite, all-trans retinoic acid (atRA), plays an essential role in vertebrate embryogenesis, including development of the nervous system. In the human neuroblastoma cell line, SH-SY5Y, atRA rapidly induces (within 4 hr) the expression of the Crk-associated substrate (Cas) family member, neural precursor cell-expressed, developmentally down-regulated gene 9 (NEDD9) also called the human enhancer of filamentation (HEF1). NEDD9 is expressed in the developing hindbrain (5-somite stage) in the presumptive rhombomeres 2, 3, and 5 before the onset of overt segmentation. Exposure of rat embryos to excess atRA at times ranging from E9.25 to E12 leads to altered NEDD9 expression in the developing hindbrain within 6 hr. NEDD9 expression is also perturbed in vitamin A-deficient embryos. A putative retinoic acid response element in the 5' region of the NEDD9 promoter binds specifically to a RXR/RAR heterodimer and forms a higher molecular weight complex upon addition of a retinoic acid receptor-specific antibody. Regulation of NEDD9 may be an important means whereby atRA promotes cell spreading and neurite outgrowth in SH-SY5Y human neuroblastoma cells, and NEDD9 represents a new downstream target of atRA and its receptors in the developing hindbrain.

Topics: 5' Untranslated Regions; Adaptor Proteins, Signal Transducing; Animals; Cell Line, Tumor; Dimerization; Electrophoretic Mobility Shift Assay; Embryonic Development; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Kinetics; Nervous System Neoplasms; Neuroblastoma; Phosphoproteins; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Rhombencephalon; RNA, Messenger; Signal Transduction; Tretinoin

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

Topics: Amino Acid Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cloning, Molecular; Gene Expression Regulation, Enzymologic; Gene Library; Humans; Infant; Molecular Sequence Data; Neoplasm Staging; Neuroblastoma; Neurons; Peptide Synthases; Prognosis; Receptor, trkA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Transforming Growth Factor beta; Tretinoin; Tubulin; Tyrosine

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.

Topics: Apoptosis; Cadaverine; Cell Differentiation; Cell Line, Tumor; Cell Survival; Ceramides; Cycloheximide; Cystamine; Cytoprotection; Enzyme Inhibitors; Humans; Immunohistochemistry; Neuroblastoma; Neurons; Protein Synthesis Inhibitors; Transglutaminases; Tretinoin; Tumor Necrosis Factor-alpha

Anti-parkinsonian agents possessing both D(2) and D(3) receptor agonist properties are neuroprotective against 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in a variety of in vitro models. The mechanisms underlying protection by these D(2)/D(3) receptor agonists remain poorly defined. To test if the D(3) receptor preferring agonists S32504 and pramipexole act through D(2) or D(3) receptors and via brain-derived neurotrophic factor (BDNF)-dependent pathways, we utilized a terminally differentiated neuroblastoma SH-SY5Y cell line exhibiting a dopaminergic phenotype. The cytotoxic effects of MPP(+) (LD(50) of 100 microM) were stereospecifically antagonized by S32504 (EC(50) = 2.0 microM) and, less potently, by pramipexole (EC(50) = 64.3 microM), but not by their inactive stereoisomers, R(+) pramipexole and S32601, respectively. Neuroprotective effects afforded by EC(50) doses of S32504 and pramipexole were antagonized by the selective D(3) antagonists S33084, U99194A, and SB269652, and by the D(2)/D(3) antagonist raclopride. However, the preferential D(2) receptor antagonist LY741626 was ineffective as was the D1 antagonist SCH23390. BDNF (1 nM) potently protected against MPP(+)-induced neurotoxicity. Antibody directed against BDNF concentration-dependently blocked both the neuroprotective effects of BDNF and those of pramipexole and S32504 against MPP(+). The protection afforded by BDNF was blocked by the P3K-AKT pathway inhibitor LY249002 and less so by the MEK/MAPKK pathway inhibitor PD98059. LY249002, but not PD98059, blocked the neuroprotective effects of pramipexole and S32504 against MPP(+) toxicity. In conclusion, S32504 and, less potently, pramipexole show robust, stereospecific, and long-lasting neuroprotective effects against MPP(+) toxicity that involve D(3) receptors. Their actions also reflect downstream recruitment of BDNF and via a PK3-AKT pathway.

Topics: 1-Methyl-4-phenylpyridinium; Antibodies; Benzothiazoles; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line; Dopamine Agonists; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Enzyme Inhibitors; Humans; Neuroblastoma; Neurons; Neuroprotective Agents; Oxazines; Pramipexole; Receptors, Dopamine D2; Receptors, Dopamine D3; Tetradecanoylphorbol Acetate; Thiazoles; Tretinoin

Cyclin-dependent kinase 5 (Cdk5), a neuronal Cdc2-like kinase, exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics, as well as neuronal degeneration. However, its role and induction mechanisms in retinoic acid (RA)-induced neuronal differentiation have not been well understood. In this study we newly found that RA treatment of SK-N-BE(2)C, human neuroblastoma cells, increased the expression of Cdk5 and its neuron specific activator p35 through the extracellular-signal-regulated kinase1/2 (ERK1/2) and cAMP-dependent protein kinase A (PKA) pathway. Inhibition of Cdk5 activity either by an inhibitor, roscovitine, or by transfection with a dominant negative form of Cdk5 caused a dramatic decrease in RA-induced differentiation, suggesting the requirement of Cdk5 kinase activity for the RA-induced neurite outgrowth. Furthermore, Cdk5 and p35 expression was decreased by ERK1/2 inhibition with PD98059 and increased by overexpression of a constitutive active mitogen-activated protein kinase kinase 1 (MEK1) mutant, suggesting the critical role of ERK1/2 in the induction of Cdk5 and p35. In addition, a transcription factor early growth response 1 (Egr-1) was induced by RA through the ERK1/2 pathway, suggesting its possible involvement in the p35 induction. RA treatment also induced c-fos mediated AP-1 binding, and cAMP-responsive element binding protein (CREB) mediated CRE binding via ERK1/2 and PKA pathway, respectively, in the Cdk5 promoter region, resulting in the induction of Cdk5. Our results suggest that ERK1/2 and PKA-induced regulation of Cdk5 activity possibly through Egr-1, c-fos, and CREB plays a critical role in the RA-induced neuronal differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Humans; Immediate-Early Proteins; Nerve Tissue Proteins; Neurites; Neuroblastoma; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Signal Transduction; Transcription Factors; Transfection; Tretinoin

To seek a novel therapeutic approach to neuroblastoma (NBL), we used three NBL cell lines (SK-N-DZ, NH12, and SK-N-SH) to examine the underlining molecular mechanisms of cellular reactions and sensitivity to all-trans-retinoic acid (ATRA). SK-N-DZ cells expressed relatively high levels of retinoic acid receptor alpha (RAR-alpha) and underwent ATRA-induced cell death that was blocked by an RAR-alpha antagonist. By contrast, RAR-alpha expression gradually decreased in NH12 and SK-N-SH cells, which did not experience increased cell death in response to ATRA. We report here the ubiquitin-dependent down-regulation of RAR-alpha expression during ATRA treatment. Our data suggest that SK-N-DZ cells have a defect in RAR-alpha down-regulation, resulting in sustained high expression of RAR-alpha that confers high sensitivity to ATRA. Accordingly, treatment with a proteasome inhibitor dramatically increased ATRA-induced cell death in NH12 and SK-N-SH cell lines. Our results reveal the crucial involvement of the RAR-alpha signaling pathway in NBL cell death and show that three NBL cell lines are differentially sensitive to ATRA. These data suggest a potential novel therapy for NBL involving retinoic acid treatment combined with the inhibition of RAR-alpha degradation.

Topics: Benzoates; Cell Division; Cell Line, Tumor; Chromans; Humans; Leupeptins; Neuroblastoma; Proteasome Inhibitors; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Tretinoin; Ubiquitin

We investigated whether peroxisome proliferator-activated receptor gamma (PPARgamma) could be involved in the modulation of the amyloid cascade causing Alzheimer's disease. Inducing expression or activating PPARgamma using synthetic agonists of the thiazolinedione family results in a dramatic decrease in the levels of the amyloid-beta (Abeta) peptide in the conditioned medium of neuronal and non-neuronal cells. PPARgamma does not affect expression or activity of any of the secretases involved in the generation of the Abeta peptide but induces a fast, cell-bound clearing mechanism responsible for the removal of the Abeta peptide from the medium. Although PPARgamma expression is generally low in the CNS, induction of PPARgamma expression during inflammation could be beneficial for inducing Abeta clearance. We confirm that the Abeta clearance mechanism can indeed be induced by PPARgamma activation in primary murine-mixed glia and cortical neuronal cultures. Our results suggest that PPARgamma-controlled mechanisms should be explored further as potential drug targets for Alzheimer's disease treatment.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Anilides; Animals; Aspartic Acid Endopeptidases; Cell Line; Cell Line, Tumor; Cells, Cultured; Cerebral Cortex; Chromans; Culture Media, Conditioned; Endopeptidases; Humans; Kidney; Mice; Neuroblastoma; Neuroglia; Neurons; Peptide Fragments; Pioglitazone; PPAR gamma; Protein Processing, Post-Translational; Receptor, Notch1; Receptors, Cell Surface; Recombinant Fusion Proteins; Retinoid X Receptors; Rosiglitazone; Thiazolidinediones; Transcription Factors; Tretinoin; Troglitazone

Neuroblastoma (NB) is a childhood tumor that depends on insulin-like growth factors (IGFs) for its growth and metastatic spread. Some metastatic NBs acquire independence from the paracrine support of IGF by activating autocrine production of IGF-2. Insulin-like growth factor binding protein-5 (IGFBP-5), a member of the IGF binding protein family, is able to optimize binding between IGF itself and its receptor. NB cell lines retain the ability to differentiate in vitro toward neuronal, schwann-like or melanocytic phenotypes upon treatment with retinoic acid (RA). Retinoids are currently used in NB therapy to achieve a mature postmitotic phenotype. Here, we present evidence that the expression of IGFBP-5 is a common feature of neuroblastoma cell lines and that IGFBP-5 acts in concert with IGF-2 in inducing cell proliferation. RA-induced differentiation causes a sharp increase of IGFBP-5. Functional assays carried out in differentiating conditions demonstrate that IGFBP-5 transcription is sensitive to RA treatment. We show that the effect of RA on the IGFBP-5 promoter is exerted, at least in part, through a proximal 5'-CACCC-3' tandem repeat (-147 bp to -137 bp from the transcription start site) that has previously been described as a cis-acting element involved in the progesterone-mediated response in osteoblasts. Given the relevance of IGF-2 in determining the proliferative and metastatic behavior of NB, the role of IGFBP-5 as a modulator of the IGF signal transduction pathway should be studied further for potential therapeutic applications.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA Primers; Dose-Response Relationship, Drug; Humans; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor II; Luciferases; Mitosis; Models, Genetic; Mutation; Neuroblastoma; Phenotype; Plasmids; Promoter Regions, Genetic; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Tretinoin

Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase-8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN-amplified NB, our results indicate that the transcriptional inactivation of caspase-8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon-gamma (IFN-gamma) induce caspase-8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN-amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN-amplified and MYCN-single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5' terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase-8. The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans-acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Caspase 8; Caspases; Cell Line, Tumor; CpG Islands; DNA; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Neuroblastoma; Promoter Regions, Genetic; RNA; Tretinoin

The role of K(+) channel activity during cell cycle progression has become a research topic of considerable interest. Blocking of K(+) channels inhibits the proliferation of many cell types, although the mechanism of this inhibition is unclear. There is speculation that K(+) channels differentially regulate the electrical potential of the plasma membrane (V(m)) during proliferation. We have demonstrated that in tumor cells the value of V(m) is clamped to rather depolarized values by K(+) channels belonging to the HERG family. We report here that tumor cell lines preferentially express the herg1 gene and a truncated, N-deleted form that corresponds to herg1b. This alternative transcript is also expressed in human primary acute myeloid leukemias. Both HERG1 and HERG1B proteins are expressed on the plasma membrane of tumor cells and can form heterotetramers. The expression of HERG protein isoforms is strongly cell cycle-dependent, accounting for variations in HERG currents along the mitotic cycle. Moreover, the blocking of HERG channels dramatically impairs cell growth of HERG-bearing tumor cells. These results suggest that modulated expression of different K(+) channels is the molecular basis of a novel mechanism regulating neoplastic cell proliferation.

Topics: Base Sequence; Cation Transport Proteins; Cell Cycle; Cell Division; DNA Primers; DNA-Binding Proteins; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neuroblastoma; Potassium Channels; Potassium Channels, Voltage-Gated; Protein Isoforms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Trans-Activators; Transcription, Genetic; Transcriptional Regulator ERG; Transfection; Tretinoin; Tumor Cells, Cultured

Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. The transcription factors driving MYCN expression in neuroblastomas are unknown. In transient-transfection assays, E2F-1, E2F-2, and E2F-3 activate a MYCN reporter construct dependent on the presence of several putative E2F-binding sites. Using chromatin immunoprecipitation, we show that E2F-1, E2F-2, and E2F-3 bind to the proximal MYCN promoter in vivo, specifically in neuroblastoma cell lines expressing MYCN. Inhibition of E2F activity in MYCN-amplified cells by the overexpression of p16(INK4A) reduced MYCN expression. In addition, we provide evidence that E2F proteins are involved in the negative regulation of MYCN by TGF-beta and retinoic acid. These data suggest that E2F transcription factors are critical for both the full activation and the repression of MYCN in neuroblastomas.

Topics: Base Sequence; Binding Sites; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; E2F2 Transcription Factor; E2F3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Promoter Regions, Genetic; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Because of the known property of less aggressiveness of differentiated cells compared to immatured cells all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. We are interested in examining the influence of retinoic acid (RA) on proliferation, apoptosis, cytotoxicity, migration, and invasion in dependence of the differentiation of neuroblastoma cells classified into N-type (SK-N-FI, SH-SY5Y), I-type (SK-PN-DW), and S-type (SK-N-LO, SK-N-MC) cells.. Neuroblastoma cells were exposed to 10(-5) M RA and 200 ng/ml camptothecin (CAM) (control substance for apoptosis). Proliferation, apoptosis, and cytotoxicity were quantified by photometric assays. The influence on migration and invasion of neuroblastoma cells was examined by a scratch-test and by the measurement of the invasion through matrigel coated chamber inserts.. In general, RA treatment induced proliferation inhibition predominantly in the cell lines SK-PN-DW (16%, P < 0.05) and SK-N-MC (8%, (P < 0.001), respectively. In the N-type cell lines SK-N-FI (P > 0.05) and SH-SY5Y (P < 0.001) no proliferation inhibition was determined conforming with no detection of apoptosis. CAM confirmed its capability to induce apoptosis in the cell lines SH-SY5Y (43.6%, P < 0.05), SK-PN-DW (54.8%, P > 0.05), and SK-N-MC (28.9%, P < 0.0 01) except for SK-N-FI with only 9.3% (P > 0.05), but after 24 hr of treatment. Minor signs of restricted migration were observed, while RA treatment reduced significantly the invasion rate through Matrigel of SK-N-FI to 13.3% (P < 0.01), SH-SY5Y to 19.2% (P < 0.05), SK-N-MC to 27.8% (P < 0.05), and SK-N-LO to 17.7% (P < 0.01).. It is demonstrated that RA treatment can interfere with cell growth and in invasion by inducing neuronal differentiation in N-type and apoptosis in S-type neuroblastoma cell lines.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Division; Humans; Neoplasm Invasiveness; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+ exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1. This was confirmed by luciferase reporter assays of pGL3-promoter constructs in transfected SH-SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain-derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+ induced by the partial depolarization of the plasma membrane with KCl down-regulated both the basal and the cAMP-stimulated transcription. The down-regulation of the latter may be mediated by the phosphorylation of the CRE-binding protein by a calmodulin-dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE-binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF-stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE-binding protein.

Topics: Activating Transcription Factors; Base Sequence; Blood Proteins; Brain-Derived Neurotrophic Factor; Calcium; Cell Differentiation; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Down-Regulation; Humans; Membrane Transport Proteins; Molecular Sequence Data; Neuroblastoma; Neurons; Phosphorylation; Promoter Regions, Genetic; Sodium-Calcium Exchanger; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

We have previously shown that brain ferritin H expression, which has been associated with iron utilization, is developmentally regulated. Because retinoic acid (RA) regulates gene expression and is involved in cellular differentiation, we tested the hypothesis that RA regulates ferritin H during brain development and neuronal differentiation. RA, administered to rats on postnatal day 1, produced a 4-fold increase in brain ferritin H mRNA (p < 0.01) after 24 h. To examine whether RA-stimulated neuronal differentiation contributed to this up-regulation, ferritin and ferritin H mRNA were measured in human neuronal precursor cells (NTera-2, NT2) before and after 4-weeks of RA-stimulated differentiation into post-mitotic neurons. Differentiation resulted in a 2-fold increase in both ferritin and ferritin H mRNA (p < 0.05). Immunocytochemistry and Northern analysis showed significant elevations in ferritin expression that began as early as 24 h after RA treatment. While there was also a significant increase in the labile iron pool after RA treatment, this did not occur until 72 h. These data show that RA regulates ferritin H expression during rat brain development and neuronal differentiation and suggests a new role for RA in brain iron metabolism.

Topics: Animals; Animals, Newborn; Blotting, Northern; Brain; Brain Chemistry; Cell Differentiation; Ferritins; Gene Expression; Humans; Immunohistochemistry; Iron; Male; Molecular Sequence Data; Neuroblastoma; Neurons; Rats; Rats, Sprague-Dawley; RNA, Messenger; Stem Cells; Tretinoin; Tumor Cells, Cultured

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.

Topics: Blotting, Western; Cell Nucleus; Dimerization; Humans; Ligands; Neuroblastoma; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Time Factors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The promyelocytic leukemia (PML) protein, whose fusion with retinoic acid receptor alpha is responsible for the tumorigenesis of acute promyelocytic leukemia, acts as a tumor suppressor in various types of human cancers. We analyzed the expression patterns of PML, in both primary neuroblastic tumors ( n=20) and two human neuroblastoma (NB) cell lines, SMS-KCNR (KCNR) and SH-SY5Y (SY5Y). The expression of PML, revealed as speckled or microgranular staining in the nuclei, was positively correlated with the differentiation status of NB cells in vivo, and was upregulated during the differentiation of KCNR and SY5Y cells following retinoic acid treatment. Screening of PML expression in human brain and sympathetic ganglia showed restricted expression of PML in mature neurons and glial cells, a result that was consistent with that in differentiated NB tumors. All these findings strongly suggest that increased PML expression is associated with growth inhibition and differentiation of human NB cells, and that it is of critical significance in the biology of NBs and in human nervous system development.

Topics: Adolescent; Cell Differentiation; Cell Division; Cell Nucleus; Child; Child, Preschool; Female; Fluorescent Antibody Technique; Humans; Infant; Male; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Promyelocytic Leukemia Protein; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology. Our results raise the intriguing clue that the neurotoxic effect of AICA riboside on the developing brain might contribute to the neurological manifestations of syndromes related to purine dismetabolisms.

Topics: 5'-Nucleotidase; Acetylcholine; Aminoimidazole Carboxamide; Apoptosis; Brain; Caspase 3; Caspases; Cell Differentiation; Cells, Cultured; Dipyridamole; Enzyme Inhibitors; Humans; Lesch-Nyhan Syndrome; Neuroblastoma; Neurons; Norepinephrine; Phorbol Esters; Proto-Oncogene Proteins c-bcl-2; Purines; Ribonucleosides; Ribose-Phosphate Pyrophosphokinase; Tretinoin

Somatostatin (SS) is an inhibitory regulator of secretory and proliferative responses that activates a group of receptors in the plasma membrane termed SSR1-5. SSR2 is one of the most abundant SSR, which also is expressed in high numbers in many neuroendocrine tumor types. Here, we describe a study of the presence and intracellular localization of the spliced variant SSR2(a) and its endogenous ligand SS in the cultured human neuroblastoma (NB) cell line, SH-SY5Y, by immunohistochemistry and confocal laser scanning. The integral neuronal synaptic vesicle membrane proteins synaptophysin (p38) and SV2 were studied, as well as the IR of catecholaminergic and cholinergic markers. RA treatment was used as an inducer of neuronal-like differentiation in our SH-SY5Y cell line. After the treatment, the presence of catecholaminergic markers (including NPY) decreased while the cholinergic markers (including VIP) increased. p38 and SV2 as well as VIP were shifted into the rather long neuritic processes, indicating efficient intracellular transport. The SSR2(a) protein was significantly increased by RA treatment, but only minor increases in mRNA for this receptor protein could be seen. No subcellular co-localization between p38/SV2 and the cytoplasmic granular receptor material was demonstrated. The SSR2(a) receptor ligand SS was found to be present not only in the cytoplasm but also in the nucleus, and more strongly so after RA treatment. The possible reason for this may be that this peptide, like other small peptides, may serve as transcription factor, or cofactor.

Topics: Biomarkers; Catecholamines; Cell Differentiation; Choline O-Acetyltransferase; Gene Expression; Humans; Immunohistochemistry; Membrane Proteins; Neuroblastoma; Receptors, Somatostatin; Somatostatin; Synaptic Vesicles; Synaptophysin; Tretinoin; Tumor Cells, Cultured

Activation of glial cells has been proposed to contribute to neuronal dysfunction and neuronal cell death in Alzheimer's disease. In this study, we attempt to determine some of the effects of secreted factors from activated murine N-11 microglia on viability and morphology of neurons using the differentiated neuroblastoma cell line Neuro2a. Microglia were activated either by lipopolysaccharide (LPS), bacterial cell wall proteoglycans, or advanced glycation endproducts (AGEs), protein-bound sugar oxidation products. At high LPS or AGE concentrations, conditioned medium from microglia caused neuronal cell death in a dose-dependent manner. At sublethal LPS or AGE concentrations, conditioned media inhibited retinoic acid-induced neurite outgrowth and stimulated retraction of already extended neurites. Among the many possible secreted factors, the contribution of NO or NO metabolites in the cytotoxicity of conditioned medium was investigated. Cell death and changes in neurite morphology were partly reduced when NO production was inhibited by nitric oxide synthase inhibitors. The results suggest that even in the absence of significant cell death, inflammatory processes, which are partly transmitted via NO metabolites, may affect intrinsic functions of neurons such as neurite extension that are essential components of neuronal morphology and thus may contribute to degenerative changes in Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Cell Death; Cell Differentiation; Cell Survival; Culture Media, Conditioned; Dose-Response Relationship, Drug; Gliosis; Glycation End Products, Advanced; Inflammation; Lipopolysaccharides; Mice; Microglia; Neurites; Neuroblastoma; Nitric Oxide; Proteoglycans; Tretinoin; Tumor Cells, Cultured

Poor prognosis neuroblastoma (NB) tumors are marked by amplification and overexpression of N-myc. Retinoic acid (RA) decreases N-myc levels and induces cell cycle arrest in vitro and increases event-free survival in advanced stage NB patients. In this study, we investigated the mechanism(s) by which RA regulates cell cycle and how N-myc affects NB cell cycle progression. Constitutive N-myc overexpression stimulates increases in cyclin E-dependent kinase activity and decreases in p27 resulting in increased DNA synthesis. N-myc regulates p27 levels through an increase in targeting of p27 to the proteasome via cyclin E kinase-dependent phosphorylation of p27 and its ubiquitination. N-myc also stimulates an increase in proteasome activity. In RA-treated cells in which N-myc levels decline as p27 levels increase, degradation of p27 is also decreased. However, RA does not affect the activity of proteasome. The decrease in the degradation of p27 in RA-treated cells is due in part to a decrease in the N-myc stimulated phosphorylation of p27. However, RA also decreases Skp2 levels thus impairing the ability of p27 to be ubiquitinated. Thus, RA induces both N-myc-dependent and -independent mechanisms to minimize the degradation of p27 and arrest NB cell growth.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin E; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Gene Expression Regulation, Neoplastic; Humans; Microfilament Proteins; Multienzyme Complexes; Muscle Proteins; Neuroblastoma; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-myc; S-Phase Kinase-Associated Proteins; Tretinoin; Ubiquitins

Earlier studies showed that treatment of LA-N-1 cells with TPA, a tumoral promoter, leads to the stimulation of a G protein-regulated phospholipase D (PLD) in the nuclei. Now we demonstrate that retinoic acid, a cellular differentiation inducing agent, activates a nuclear oleate-dependent PLD in LA-N-1 cells. Treatment of the nuclei with retinoic acid induces the breakdown of phosphatidylcholine (PtdCho). Our results indicate that PLD is regulated differentially depending on the nature of the stimulatory agent. These results strongly suggest the existence of two nuclear PLD isoforms in LA-N-1 nuclei that hydrolyze PtdCho.

Topics: Cell Nucleus; Diglycerides; Enzyme Activation; Glycerophospholipids; Humans; Neuroblastoma; Oleic Acid; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Tretinoin; Tumor Cells, Cultured

Extracellular nucleotides exert a variety of biological actions through several kinds of P2 receptors in many tissues and cell types. We found that treatment with nucleotides increases intracellular Ca2+ concentration ([Ca2+]i) in SK-N-BE(2)C human neuroblastoma cells with a following order of potency: UDP > UTP > ADP >> ATP. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that specific mRNAs coding for human P2Y1, P2Y4, and P2Y6 receptors were expressed in the cells, but Northern blot analysis revealed that P2Y6 receptors were the predominant type. Activation of protein kinase C-alpha by treatment with 1 micro m phorbol 12-myristate 13-acetate dramatically inhibited both the UDP-induced [Ca2+]i rise and inositol 1,4,5-trisphosphate (IP3) generation, whereas incubation with pertussis toxin had little effect on the responses. The UDP-induced [Ca2+]i rise and IP3 production were maintained up to 30 min after stimulation, while bradykinin-induced responses rapidly decreased to the basal level within 5 min of stimulation. Pretreatment of cells with the maximal effective concentration of UDP reduced the subsequent carbachol- or bradykinin-induced [Ca2+]i rise without inhibition of IP3 generation. Neuronal differentiation of the cells by treatment with retinoic acid for 7 days did not change the expression level of P2Y6 receptors. Taken together, the data indicate that P2Y6 receptors highly responsive to diphosphonucleotide UDP are endogenously expressed in the human neuroblastoma SK-N-BE(2)C cells and that they are involved in the modulation of other phospholipase C-coupled receptor-mediated Ca2+ mobilization by depleting the IP3-sensitive Ca2+ stores.

Topics: Bradykinin; Calcium; Cell Differentiation; Cholinergic Agonists; Enzyme Activation; Humans; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Nucleotides; Pertussis Toxin; Protein Kinase C; Protein Kinase C-alpha; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Uridine Diphosphate; Uridine Triphosphate

Nuclear retinoid receptors mediate retinoid effects through tissue-specific, ligand-receptor interactions and subsequent transcriptional regulation of secondary target genes. Retinoic acid receptor beta (RARbeta) is itself a retinoid target gene with a retinoic acid response element (betaRARE) in the 5' untranslated region of the RARbeta2 gene. Altered transcriptional regulation of RARbeta may play a role in human carcinogenesis and the retinoid-responsiveness of malignant cells. Here we used retinoid X receptor-specific antibodies in electrophoretic mobility shift assays to show that the retinoid X receptor beta (RXRbeta) protein was recruited to the betaRARE, after retinoid treatment of retinoid-sensitive neuroblastoma (NB), lung and breast cancer cell lines, but not retinoid-resistant lung and breast cancer cell lines. RXRbeta selectively enhanced retinoid-induced transcriptional activation of the betaRARE. Stable overexpression of RXRalpha and RXRbeta in NB cells resulted in marked growth inhibition and cell death, which increased after retinoid treatment. However, only proteins from the RXRbeta transfectants exhibited specific RXRbeta binding to the betaRARE in vitro and in vivo, enhanced histone acetylation and increased endogenous RARbeta expression. These data indicate that recruitment of RXRbeta to the betaRARE, and consequent induction of endogenous RARbeta expression, is an important component in the retinoid anticancer signal. RXRalpha may also participate in the retinoid signal, but through mechanisms that do not involve RARbeta.

Topics: Acetylation; Antineoplastic Agents; Breast Neoplasms; Cell Division; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Histones; Humans; Lung Neoplasms; Nervous System Neoplasms; Neuroblastoma; Promoter Regions, Genetic; Protein Transport; Receptors, Retinoic Acid; Response Elements; RNA, Messenger; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Neurite outgrowth is a central event of neuronal differentiation that proceeds in multiple processes requiring various cellular factors. Here we demonstrated that c-Jun N-terminal kinase 1 (JNK1) plays an essential role in RA-induced neurite outgrowth of SH-SY5Y cells. Treatment of SH-SY5Y cells with RA induced a strong activation of JNK1 within 10 min, and the immediate increase of JNK1 activity returned to the basal level in an hour. The second surge of JNK1 activity was observed around 1 day after RA treatment, which coincided with the period of extensive neurite outgrowth. Interestingly, phospho-JNK was concentrated in the nucleus of cells during the early induction, whereas it was distributed into neurite processes during the delayed second activation period. In SH-SY5Y carrying a dominant negative form of SEK1, an upstream kinase of JNK1, both early and late inductions of JNK1 activity were repressed along with RA-induced neurite outgrowth. These results suggest that JNK1 plays an essential role in RA-induced neuronal differentiation of SH-SY5Y cells.

Topics: Cell Differentiation; Enzyme Activation; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145(trkB) ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non-MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF-treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients.

Topics: Base Sequence; Brain-Derived Neurotrophic Factor; DNA Primers; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Proto-Oncogene Proteins c-myc; Receptor, trkB; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Neuroblastoma (NB) tumors uniquely exhibit heterogeneous cellular differentiation. Advanced histologic differentiation portends favorable clinical outcome, and differentiation therapy with retinoic acid has shown promise clinically. Although cellular hypoxia is a common feature of solid tumors, how this influences differentiation in NB is not known. The authors' hypothesis contends that induced differentiation may alter the susceptibility of NB cells to hypoxic injury.. Human NB cells (SH-SY5Y) were subjected to a self-generated diffusion gradient of oxygen and nutrient deprivation in an in vitro "sandwich" model as previously established. Differentiation was induced by the addition of either all-trans-retinoic acid (RA; 5 micromol/L) or the phorbol ester 12-0-tetradeconoylphorbol-13-acetate (TPA; 16 nmol/L). Cellular survival is quantified as the viable cell border width, the viability of which is confirmed by substrate adherence, MTT assay, and trypan blue exclusion. Differentiation and proliferation were evaluated by tyrosine hydroxylase and proliferating cell nuclear antigen (PCNA) immunocytochemistry, respectively.. Addition of RA or TPA improved NB cell survival under conditions of hypoxia/metabolite deprivation (8.4 +/- 0.3 mm v 4.8 +/- 0.2 mm; RA v control at 24 hours, P <.05). This improvement was evident at all time-points examined (2 hours to 4 days) for both NB cell lines tested. Proliferation was diminished with addition of either TPA or RA. Treated cells displayed advanced differentiation by morphologic and immunocytochemical criteria. Nontreated NB cells did not differentiate under hypoxic conditions alone. Neither RA nor TPA treatment affected survival rate in non-NB cell lines (ie, hepatocyte HepG2 cells).. Pharmacologic differentiation protects NB cells from hypoxia and metabolite deprivation in vitro. Hypoxic conditions do not induce differentiation. Although clinical differentiation therapy may well limit cell proliferation, it also may afford resistance against hypoxia-induced injury and death, antiangiogenesis therapy notwithstanding.

Topics: Cell Differentiation; Cell Division; Cell Hypoxia; Cell Survival; Humans; Models, Biological; Neuroblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

We have investigated the effects of the low-toxic retinoid, all-trans retinoyl beta-glucuronide (RAG) alone and in combination with the phenylacetate (PA) derivative 4-chloro-phenylacetate (4-CPA) on the human neuroblastoma cell line, LA-N-5. In vitro studies demonstrated that RAG and 4-CPA treatments alone showed differentiation-inducing activity on LA-N-5 cells, with 4-CPA found to be about three-fold more potent than the PA parent compound in inducing morphologic differentiation and growth inhibition. As previously reported for retinoic acid (RA) and PA, RAG and 4-CPA were significantly more effective in their antiproliferative effects on the cells than either agent alone. Pharmacologic studies of 4-CPA in mice demonstrated that blood plasma levels reached peak concentrations 4 h after bolus administration of the compound and showed slow clearance characteristics with an apparent half-life of 4-8 h. As opposed to PA, 4-CPA was found to be essentially odourless and readily consumed in drinking water, giving rise to steady-state blood plasma levels of 4-CPA in the near mM range. Continuous consumption of 4-CPA in this manner for up to 5 months demonstrated no apparent adverse effects on the mice. Long-term RAG- and/or 4-CPA-treatment of nude mice injected with LA-N-5 cells demonstrated that both compounds alone exhibit potent antitumour activity. Together, RAG plus 4-CPA was the most effective treatment for inhibiting established tumour growth. In contrast, 4-CPA alone was equally as effective as the combination for preventing tumour development. The potent in vivo antitumour effects of 4-CPA could not be accounted for by the known ability of PA compounds to induce expression of the RA nuclear receptor beta (RARbeta) suppressor gene. Taken together, these findings demonstrate the possibility that RAG and/or 4-CPA may serve as effective, less-toxic alternatives to 13-cis RA, which is presently being utilised for nb therapy.

Topics: Administration, Oral; Animals; Cell Division; Disease Models, Animal; Drug Therapy, Combination; Female; Humans; Mice; Mice, Nude; Neuroblastoma; Phenylacetates; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

LMO4 is a transcription regulator interacting with proteins involved, among else, in tumorigenesis. Its function in the nervous system, and particularly in the adult nervous system, has however still to be elucidated. We decided to modify its expression in a neuronal model, human SH-SY5Y neuroblastoma cells, by permanent transfection of sense or anti-sense Lmo4 cDNAs. Generated clones overexpressing the Lmo4 transcript in sense orientation tended to aggregate. They showed significantly reduced average number of neurites per cell and average neuritic length per cell. The opposite was observed with clones overexpressing the anti-sense Lmo4 transcript. Furthermore, selected clones were subjected to 72 h long-term treatments with retinoic acid and phorbol ester (TPA), two biochemicals known to stimulate differentiation of non-transfected SH-SY5Y cells and other neuroblastoma cells. Neuritogenesis occurred after retinoic acid stimulation in all cases. The inhibitory effect of sense Lmo4 RNA overexpression on neuritic outgrowth was indeed prevented. The protein kinase C activator TPA could not induce neuritogenesis in SH-SY5Y cells overexpressing sense Lmo4 RNA. Thus, sense Lmo4 RNA overexpression, not Lmo4 endogenous transcription, overrides the stimulatory effect of TPA upon neuritic outgrowth. We also showed that Lmo4-dependent neuritic retraction and outgrowth correspond to altered phosphorylation of cytoskeletal proteins. Overall, Lmo4 RNA overexpression interferes with neuritic outgrowth, whereas anti-sense Lmo4 RNA expression favors neuritogenesis in SH-SY5Y cells. Consequently, changes in Lmo4 RNA expression levels might alter the rate of neuritic outgrowth in the developing and adult nervous system.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Blotting, Western; Carcinogens; Cell Aggregation; Cell Differentiation; Cell Line; Cloning, Molecular; Cytoskeletal Proteins; Gene Expression; Homeodomain Proteins; Humans; LIM Domain Proteins; Models, Neurological; Neurites; Neuroblastoma; Phorbol Esters; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors; RNA, Antisense; RNA, Messenger; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The LA-N-1 cell nucleus contains Ca2+-independent phospholipase A2 (PLA2) activity hydrolyzing plasmenylethanolamine (PlsEtn) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn). These enzymes hydrolyze glycerophospholipids to produce arachidonic acid and lysoglycerophospholipids. The treatment of LA-N-1 cell cultures with all-trans retinoic acid (atRA) results in time- and dose-dependent stimulation of PlsEtn-PLA2 and PtdEtn-PLA2 activities in the nuclear fraction. PLA2 activities in the non-nuclear fraction (microsomes) are not affected by atRA, whilst the pan retinoic acid receptor (RAR) antagonist, BMS493, blocks the PLA2 activities in the nuclear fraction. This indicates that the stimulation of PLA2 activities is a receptor-mediated process. Treatment of LA-N-1 cell cultures with cycloheximide has no effect on basal PLA2 activities. However, atRA-mediated stimulation of PLA2 activities in LA-N-1 cell nuclei is partially inhibited by cycloheximide indicating that this decrease in PLA2 activity is due to a general decreased protein synthesis. Our results also support earlier studies in which atRA induces morphologic differentiation through the stimulation of PLA2-generated second messengers such as arachidonic acid and eicosanoids.

Topics: Antineoplastic Agents; Arachidonic Acid; Cell Nucleus; Cycloheximide; Cytosol; Dose-Response Relationship, Drug; Drug Interactions; Humans; Neuroblastoma; Phospholipases A; Phospholipases A2; Protein Synthesis Inhibitors; Receptors, Retinoic Acid; Subcellular Fractions; Time Factors; Tretinoin; Tumor Cells, Cultured

For better understanding of functions of the Calcyclin Binding Protein (CacyBP) and exploring its possible roles in neuronal differentiation, the subcellular localization of human CacyBP was examined in retinoic acid(RA)-induced and uninduced neuroblastoma SH-SY5Y cells. Immunostaining indicated that CacyBP was present in the cytoplasm of uninduced SH-SY5Y cells, in which the resting Ca(2+) concentration was relatively lower than that of RA-induced cells. After the RA induction, immunostaining was seen in both the nucleus and cytoplasm. In the RA-induced differentiated SH-SY5Y cells, CacyBP was phosphorylated on serine residue(s), while it existed in a dephosphorylated form in normal (uninduced) cells. Thus, the phosphorylation of CacyBP occurs when it is translocated to the nuclear region. The translocation of CacyBP during the RA-induced differentiation of SH-SY5Y cells suggested that this protein might play a role in neuronal differentiation.

Topics: Active Transport, Cell Nucleus; Calcium; Calcium-Binding Proteins; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Humans; Immunohistochemistry; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphorylation; Tretinoin

Retinoic acid (RA) is a potent regulator of morphogenesis, growth and cell differentiation. Incubation with RA causes arrest of proliferation and neurite extension in SH-SY5Y cells, a neuroblastoma cell line of human origin. In these cells, RA regulates the expression of the beta-amyloid precursor protein. The retinoid increases the levels of intracellular and secreted forms of APP (amyloid precursor protein), APP-mRNA levels and the activity of the APP promoter in transient transfection studies. These responses require long periods of exposition to the ligand, thus suggesting a nondirect effect of the RA receptors on the APP gene. Also in these cells, RA induces the expression of TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), and 4 days of pretreatment with retinoic acid confers BDNF responsiveness to the APP promoter.

Topics: Amyloid beta-Protein Precursor; Autoradiography; Blotting, Northern; Blotting, Western; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Division; Chloramphenicol O-Acetyltransferase; Drug Interactions; Gene Expression Regulation; Humans; Neuroblastoma; Receptor Protein-Tyrosine Kinases; Receptor, trkB; RNA, Messenger; Thymidine; Time Factors; Transfection; Tretinoin; Tritium; Tumor Cells, Cultured

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.

Topics: Animals; Butadienes; Cell Differentiation; Cell Membrane; Choline-Phosphate Cytidylyltransferase; Cytosol; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Isoenzymes; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mitogen-Activated Protein Kinase Kinases; Nerve Growth Factor; Neurites; Neuroblastoma; Neurons; Nitriles; PC12 Cells; Phosphatidylcholines; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

High susceptibility of cholinergic neurons to neurotoxic signals may result from their utilization of acetyl-CoA for both energy production and acetylcholine synthesis. SN56 cholinergic cells were transfected stably with cDNA for choline acetyltransferase. Transfected cells (SN56ChAT2) expressed choline acetyltransferase activity and acetylcholine content, 17 times and 2 times higher, respectively, than did nontransfected cells. Transfection did not change pyruvate dehydrogenase but decreased the acetyl-CoA level by 62%. Differentiation by cAMP and retinoic acid caused an increase of choline acetyltransferase activity and decrease of acetyl-CoA levels in both cell lines. Negative correlation was found between choline acetyltransferase activity and acetyl-CoA level in these cells. SN56ChAT2 cells were more susceptible to excess NO than were native SN56 cells, as evidenced by the thiazolyl blue reduction assay. Thus, the sensitivity of cholinergic neurons to pathologic conditions may depend on the cholinergic phenotype-dependent availability of acetyl-CoA.

Topics: Acetyl Coenzyme A; Acetylcholine; Animals; ATP Citrate (pro-S)-Lyase; Cell Differentiation; Cell Survival; Choline O-Acetyltransferase; Cyclic AMP; Hybrid Cells; Mice; Neuroblastoma; Neurons; Nitric Oxide; Oxidative Stress; Phenotype; Pyruvate Dehydrogenase Complex; Rats; Septum Pellucidum; Transfection; Tretinoin; Tumor Cells, Cultured

Experiments were carried out to assess whether a magnetic field of 50 Hz and 1 mT can influence apoptosis and proliferation in the human neuroblastoma cell line LAN-5. TUNEL assays and poly-ADP ribose polymerase (PARP) expression analysis were performed to test apoptosis induction, and the WST-1 assay was used to calculate the proliferation index in a long term exposure. No alterations were found in cellular ability to undergo programmed cell death, but a small increase in the proliferation index was evidenced after 7 days of continuous exposure. Also, a slight and transient increase of B-myb oncogene expression was detected after 5 days of exposure. Combined exposures of cells to EMF and to chemical agents which interfere with proliferation, such as the differentiative agent retinoic acid and the apoptotic inducer camptothecin, showed an antagonistic effect of magnetic fields against the differentiation of the LAN-5 cells and a protective effect towards apoptosis.

Topics: Apoptosis; Camptothecin; Cell Differentiation; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Radiation; Drug Tolerance; Electricity; Electromagnetic Fields; Humans; Neuroblastoma; Tretinoin

Stanniocalcin (STC) is a new mammalian polypeptide hormone and appears to be a regulator of neuronal function. We have already shown that the induction of STC mRNA and protein expression by cAMP is integral to neuroblastoma cell differentiation, particularly neurite outgrowth. In this study, we examined the cAMP pathway in greater detail. Some common neuritogenic agents, euxanthone (PW1) and trans-retinoic acid (RA), were studied for possible interactions with the dibutyryl cAMP (dbcAMP)-mediated response. Our results showed that STC mRNA induction by dbcAMP was mediated by protein kinase A-cAMP response element binding protein (CREB) pathway, accompanied with phosphorylation of CREB and a reduction of p50, p65, and phosphorylated inhibitor kappaBalpha levels. Using a synthetic peptide nuclear factor-kappaB SN50, stimulation of dbcAMP-mediated STC expression was observed; indicating the nuclear translocation of nuclear factor kappaB might possibly repress STC expression. dbcAMP-induced STC mRNA expression was enhanced by PW1. In contrast, RA had highly suppressive effects. Cotreatment of cell with PW1 and cAMP provoked an increase in phosphorylated CREB (pCREB). Conversely, cotreatment with RA suppressed pCREB. The results highlighted the importance of phosphorylation of CREB in mediating STC gene expression. Taking a step further to dissect the possible regulatory pathways involved, with the aid of phorbol 12-myristate 13-acetate or ionomycin, additive effects on STC gene expression were observed. The induction was aided by further elevation of pCREB, which was completely abolished by Gö 6976, a Ca2+-dependent protein kinase C (PKC) alpha and PKCbeta1 inhibitor. Our results indicated that cross-talk with PKC and/or Ca2+ signaling pathways might sensitize cAMP-mediated effects, on CREB phosphorylation and STC gene expression.

Topics: Animals; Antineoplastic Agents; Bucladesine; Calcium; Cell Differentiation; Drug Interactions; Gene Expression Regulation; Glycoproteins; Hormones; Mice; Neuroblastoma; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Xanthenes; Xanthones

In the retinoic acid-differentiated neuroblastoma SH-SY5Y cells, IL-1 induced binding activity of NFkappaB and up-regulated the expression and activity of MnSOD. The IL-1-elicited effects were partly reversed by IL-4 and IL-6. It is proposed that IL-4 and IL-6 may participate in the regulation of the imbalanced oxidant status induced by IL-1 in differentiated neuroblastoma cells. In the SH-SY5Y cell line, TNFalpha neither activated NFkappaB nor induced MnSOD expression and activity, but was capable of modulating the IL-1 effects. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NFkappaB activation, down-regulated the expression and activity of MnSOD, which may suggest that the regulation of MnSOD by IL-1 in retinoic acid-differentiated neuroblastoma cells was mediated by the nuclear factor kappaB.

Topics: Brain Neoplasms; Cell Differentiation; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-4; Interleukin-6; Neuroblastoma; NF-kappa B; Oxidative Stress; Proline; Protein Binding; Superoxide Dismutase; Thiocarbamates; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

A human neuroblastoma cell line IMR-32 was used as an in vitro model to examine three naturally occurring retinoic acid (RA) isomers, 9-cis (9c), 13-cis (13c) and all-trans (AT) RA, in mediating growth differentiation and neuronal differentiation. All RA isomers inhibited cellular proliferation, with 13c-RA being most effective. Cyclic AMP-responsive-element-binding-protein (CREB) was activated during RA treatment. AT-RA was a better differentiating agent in inducing the highest expression of the neurotrophic factor receptor TrkA. After prolonged RA treatment, the expression of RA receptors (RARs) was comparable for the three isomers, but retinoid X receptors (RXRs) were differentially regulated. These results imply that distinctive molecular pathways might be involved in the in vitro differentiation of neuroblastoma with different RA isomers.

Topics: beta-Galactosidase; Biomarkers; Blotting, Western; Brain Neoplasms; Cell Differentiation; Cell Division; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Humans; Neuroblastoma; Neurons; Receptor, trkA; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; Stereoisomerism; Transcription Factors; Transfection; Tretinoin

In this study, we examined the role of endogenous ceramide in the inhibition of telomerase and induction of morphologic differentiation in response to all-trans-retinoic acid (ATRA) in the SK-N-SH and SK-N-AS human neuroblastoma cell lines. The results showed that ATRA inhibited the activity of telomerase significantly in a time- and dose-dependent manner, as determined by telomere repeat amplification protocol (TRAP). The inhibition of telomerase by ATRA was maximum (about 50-80% of untreated controls) at 5-10 microM for 4-8 days. Treatment of cells with ATRA (5 microM) also resulted in the inhibition of growth by about 30-70% after 4 and 8 days of treatment, respectively, which was measured by trypan blue exclusion method. Measurement of accumulation of endogenous ceramide by high pressure liquid chromatography coupled with mass spectroscopy (LC/MS) showed that treatment of cells with ATRA resulted in increased levels of mainly C24:0 and C24:1 ceramides at days 2, 4, and 8, respectively. Also, treatment of cells with ATRA in the presence of myriocin blocked the accumulation of ceramide significantly, and more importantly, presence of myriocin partially prevented the inhibition of telomerase. Mechanistically, inhibition of telomerase by endogenous ceramide in response to ATRA treatment involves, at least in part, down-regulation of the expression of telomerase reverse transcriptase (hTERT) mRNA, as determined by semi-quantitative RT-PCR, in these cells. In addition, the modulation of telomerase activity by ATRA correlated with the induction of morphologic differentiation, which was also blocked by myriocin, as determined by extension of neurites using phase-contrast microscopy. These results, therefore, reveal an important effect of ATRA on telomerase inhibition and induction of morphologic differentiation in human neuroblastoma cells. These data also demonstrate that endogenous ceramide is one of the upstream regulators of telomerase activity in human neuroblastoma cells in response to ATRA.

Topics: Cell Differentiation; Cell Line, Tumor; Ceramides; Dose-Response Relationship, Drug; Enzyme Activation; Fatty Acids, Monounsaturated; Humans; Neuroblastoma; Telomerase; Tretinoin

Amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. The function of these three proteins is not yet fully understood. One of the proposed roles of APP is to promote neurite outgrowth. The aim of this study was to investigate the regulation of the expression levels of APP family members during neurite outgrowth. We observed that retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y cells resulted in increased expression of APP, APLP1 and APLP2. We also examined the effect of the NFkappaB, AP-1 and c-Jun N-terminal kinase inhibitor curcumin (diferuloylmethane) on the RA-induced expression levels of these proteins. We found that treatment with curcumin counteracted the RA-induced mRNA expression of all APP family members. In addition, we observed that curcumin treatment resulted in neurite retraction without any effect on cell viability. Surprisingly, curcumin had differential effects on the APLP protein levels in RA-differentiated cells. RA-induced APLP1 protein expression was blocked by curcumin, while the APLP2 protein levels were further increased. APP protein levels were not affected by curcumin treatment. We propose that the sustained levels of APP and the elevated levels of APLP2, in spite of the reduced mRNA expression, are due to altered proteolytic processing of these proteins. Furthermore, our results suggest that APLP1 does not undergo the same type of regulated processing as APP and APLP2.

Topics: Amyloid beta-Protein Precursor; Cell Differentiation; Cell Line, Tumor; Central Nervous System; Curcumin; Enzyme Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Neurites; Neuroblastoma; NF-kappa B; RNA, Messenger; Transcription Factor AP-1; Tretinoin

The current therapeutic modalities achieve low response rates in human neuroblastoma, a frequent extracranial malignancy of the early childhood. We have assessed the effect of retinoids, used presently for the treatment of neuroblastoma, on the discrete steps of the MHC class I processing machinery and susceptibility of neuroblastoma cells to CTL-mediated killing. We demonstrate that retinoic acid derivatives induce the expression of proteolytic and regulatory subunits of the immunoproteasome, increase the half-life of MHC class I complexes, and enhance the sensitivity of neuroblastoma cells to both MHC class I-restricted peptide-specific and HLA nonrestricted lysis by CTLs. Importantly, effects of retinoids on the MHC class I pathway appear to be independent of IFN-gamma and/or TNF-alpha as intermediate messengers. To our knowledge, this is the first demonstration of inflammation-unrelated biological molecules that induce systemic modulation of antigen presentation in nonprofessional antigen presenting cells. Our findings suggest that the application of retinoids and T cell-based immunotherapy may be an effective combination for the treatment of neuroblastoma.

Topics: Antigen Presentation; Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; Histocompatibility Antigens Class I; Humans; Immunotherapy, Adoptive; Interferon-gamma; Neuroblastoma; T-Lymphocytes, Cytotoxic; Tretinoin; Tumor Necrosis Factor-alpha; Up-Regulation

Beta-Cryptoxanthin (beta Cx) was investigated for cell functions in neuroblastoma Neuro2a cells. The following results were obtained. 1. Beta-Cx induced neurite outgrowth. 2. Beta-Cx inhibited the etoposide-induced activation of caspase-3 activity in a dose-dependent manner. These data suggest a bioregulatry function of beta Cx in the control of differentiation and apoptosis in Neuro2a cells.

Topics: Anticarcinogenic Agents; Apoptosis; beta Carotene; Cell Differentiation; Cell Line, Tumor; Cryptoxanthins; Humans; Neuroblastoma; Tretinoin; Xanthophylls

Retinoic acid (RA) modulates differentiation and apoptosis of neural cells via RA receptors (RARs) and retinoid X receptors (RXRs). Neuroblastoma cells are potentially useful models for elucidating the molecular mechanisms of RA in neural cells, and responses to different isomers of RA have been interpreted in terms of differential homo- and heterodimerization of RXRs. The aim of this study was to identify the RXR types expressed in neuroblast and substrate-adherent neuroblastoma cells, and to study the participation of these RXRs in RAR heterodimers. RXRbeta was the predominant RXR type in N-type SH SY 5Y cells and S-type SH EP cells. Gel shift and supershift assays demonstrated that RARbeta and RARgamma predominantly heterodimerize with RXRbeta. In SH SY 5Y cells, RARgamma/RXRbeta was the predominant heterodimer binding to the DR5 RARE in the absence of 9-cis RA (9C), whereas the balance shifted in favor of RARbeta/RXRbeta in the presence of ligand. There was a marked difference between the N- and S-type neuroblastoma cells in retinoid receptor-DNA interactions, and this may underlie the differential effects of retinoids in these neuroblastoma cell types. There was no evidence to indicate that 9C functions via RXR homodimers in either SH SY 5Y or SH EP neuroblastoma cells. The results of this study suggest that interactions between retinoid receptors and other nuclear proteins may be critical determinants of retinoid responses in neural cells.

Topics: Alitretinoin; Dimerization; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Receptor for advanced glycation end products (RAGE) mediates neurite outgrowth and cell migration upon stimulation with its ligand, amphoterin. We show here that RAGE-dependent changes in cell morphology are associated with proliferation arrest and changes in gene expression in neuroblastoma cells. Chromogranin B, a component of secretory vesicles in endocrine cells and neurons, was found to be up-regulated by RAGE signaling during differentiation of neuroblastoma cells along with the two other members of the chromogranin family, chromogranin A and secretogranin II. Ligation of RAGE by amphoterin lead to rapid phosphorylation and nuclear localization of cyclic AMP response element-binding protein (CREB), a major regulator of chromogranin expression. Furthermore, inhibition of ERK1/2-Rsk2-dependent CREB phosphorylation efficiently inhibited up-regulation of chromogranin gene expression upon RAGE activation. To further study the effects of RAGE and amphoterin on cellular differentiation, we stimulated embryonic stem cells expressing RAGE or a signaling-deficient mutant of RAGE with amphoterin. Amphoterin was found to promote RAGE-dependent neuronal differentiation of embryonic stem cells characterized by up-regulation of neuronal markers light neurofilament protein and beta-III-tubulin, activation of CREB, and increased expression of chromogranins A and B. These data suggest that RAGE signaling is capable of driving neuronal differentiation involving CREB activation and induction of chromogranin expression.

Topics: Animals; Cell Differentiation; Cell Line; Cell Size; Chromogranins; Cyclic AMP Response Element-Binding Protein; Gene Expression Regulation; Glycation End Products, Advanced; HMGB1 Protein; Membrane Proteins; Mice; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Phosphorylation; Rats; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Signal Transduction; Stem Cells; Synaptophysin; Tretinoin; Up-Regulation

A comparison between retinoic acid (RA) differentiation-resistant and differentiation-sensitive SK-N-BE neuroblastoma (NB) cell lines revealed an association between resistance to differentiation, exhibited by N-myc stable transfected SK-N-BE 9N cells, with sensitivity to RA induction of p50/p65 nuclear factor kappaB (NF-kappaB) transcription factor activity and induction of matrix metalloproteinase (MMP)-9 expression leading to enhanced invasive behavior in vitro. These effects were not observed in differentiation-sensitive parental SK-N-BE or control-transfected SK-N-BE 2N counterparts. RA activated a MMP-9 promoter reporter gene construct in SK-N-BE 9N but not parental SK-N-BE or SK-N-BE 2N cells through a NF-kappaB element (-600) in association with enhanced p50 mRNA expression, reduced cytoplasmic inhibitor of nuclear factor kappaBalpha protein levels, and the induction of nuclear p50/p65 containing MMP-9 NF-kappaB site binding activity. RA activation of the MMP-9 promoter was inhibited by transient overexpression of a dominant-negative inhibitor of nuclear factor kappaBalpha protein and stimulated by transient p50 but not p65 overexpression in the absence of RA. A limited, nonessential function for activator protein 1 (-74), Ets (-540), and SP1 (-560) elements within the MMP-9 promoter was revealed by point mutation but was not associated with changes in the binding or position of complexes constitutive to differentiation-sensitive or -resistant cells. Our data indicates that in this model of NB resistance to differentiation that results from uncoupled RA regulation of N-myc expression, RA stimulates malignant NB cell behavior by inducing nuclear NF-kappaB transcription factor activity, which in turn induces MMP-9 expression and stimulation of basement membrane invasive capacity involving MMP-9 activity.

Topics: Antineoplastic Agents; Basement Membrane; Biocompatible Materials; Cell Differentiation; Child; Collagen; Drug Combinations; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neuroblastoma; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Promoter Regions, Genetic; Proteoglycans; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

In this paper, it is demonstrated that all-trans, 9-cis and 13-cis retinoic acid (RA) decreased the sensitivity of SK-N-BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine-5'-triphosphate synthetase. Retinoic acid attenuated CPEC-induced apoptosis as reflected by a decreased caspase-3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [(3)H]uridine showed a strongly decreased biosynthesis of CTP via CTP synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP synthetase.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Division; Cytidine; Cytidine Triphosphate; Drug Interactions; Humans; Neuroblastoma; Polyphosphates; Tretinoin; Tumor Cells, Cultured

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.

Topics: Alitretinoin; Bexarotene; Dose-Response Relationship, Drug; Gene Expression; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Signal Transduction; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation.

Topics: Antineoplastic Agents; Biomarkers; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cyclin A; Cyclin D1; DNA-Binding Proteins; Humans; Neuroblastoma; Pregnancy-Specific beta 1-Glycoproteins; rho GTP-Binding Proteins; Trans-Activators; Tretinoin

Recent research into depression has focused on the involvement of long-term intracellular processes, leading to abnormal neuronal plasticity in brains of depressed patients, and reversed by antidepressant treatment. Given a suggested decrease in noradrenergic transmission in depression, and an antidepressant induced increase in norepinephrine (NE) level, a possible role for NE in mediating alterations in neuronal morphology and plasticity was examined. Human neuroblastoma SH-SY5Y cells treated with 10-5 m NE presented an elongated granule-rich cell-body and increased number of neurites, when compared with non-treated cells. Moreover, cell survival was enhanced in the presence of NE, while proliferation was inhibited. The above effects suggest a role for NE in cell differentiation. Indeed similar effects on cell survival and neurite outgrowth were induced in SH-SY5Y cells by retinoic acid (RA), an established differentiating agent. Finally, NE treatment resulted in a progressive decrease in the pluripotent marker Oct4 and an increase in the neuronal growth cone marker, growth-associated-protein 43 (GAP-43). Alongside these effects, NE-treated cells presented alterations in the expression of 44 genes as observed in a neurobiology cDNA microarray. Among the altered genes, an increase in the expression level of two neurite-outgrowth promoting genes, neural cell adhesion molecule L1 and laminin, was confirmed by RT-PCR. Taken together, the results support a role for NE in processes of synaptic connectivity, and may point to a role for this neurotransmitter in mediating the suggested neuronal plasticity in depression and in antidepressant treatment.

Topics: 3,4-Dihydroxyphenylacetic Acid; Antigens, Differentiation; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Depressive Disorder, Major; DNA-Binding Proteins; GAP-43 Protein; Gene Expression; Gene Expression Profiling; Humans; Laminin; Neural Cell Adhesion Molecule L1; Neurites; Neuroblastoma; Neuronal Plasticity; Neurons; Norepinephrine; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tretinoin

Accumulation of vitronectin protein increased in the conditioned medium of mouse neuroblastoma Neuro2a cells during retinoic acid-induced differentiation. To study the regulatory mechanism of the increase in vitronectin expression during the differentiation, the activity of the -527/+95 vitronectin promoter was observed in Neuro2a cells with or without retinoic acid treatment. The result showed that the -527/+95 promoter activity increased 2.7-fold with retinoic acid, and despite deletion of regions from -527 to -49 and +54 to +95 base pairs (bp), the -48/+53 promoter preserved the retinoic acid response. We recently showed that the -48/+53 region has two transcription factor Foxa (HNF3)-binding sites (site A from -34 to -25 bp and site B from +15 to +26 bp), suggesting that Foxa may up-regulate the vitronectin expression. Therefore, we examined the change of Foxa expression in Neuro2a cells during the differentiation. The expression of Foxa1 protein was increased during the differentiation, but the expression of Foxa2 protein was not detected. In addition, overexpression of Foxa1 increased the amount of vitronectin protein in the conditioned medium of Foxa1-overexpressed Neuro2a cells, but overexpression of Foxa2 only weakly increased it. The site-A and -B double mutation of the -527/+95 promoter remarkably reduced the promoter activity induced by Foxa overexpression, indicating that Foxa-binding sites in the -527/+95 region are located only on sites A and B. The mutation of site A in the -48/+53 promoter did not affect the retinoic acid response, but the site-B mutation abolished the constitutive promoter activity and remarkably reduced the promoter activity with retinoic acid. These results demonstrate that Foxa up-regulates the vitronectin expression during the retinoic acid-induced differentiation in Neuro2a cells.

Topics: Animals; Base Sequence; Binding Sites; Cell Differentiation; Culture Media, Conditioned; DNA-Binding Proteins; Gene Expression Regulation; Hepatocyte Nuclear Factor 3-beta; Mice; Mutagenesis, Site-Directed; Neuroblastoma; Neurons; Nuclear Proteins; Point Mutation; Promoter Regions, Genetic; Sequence Deletion; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Vitronectin

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.

Topics: Acetylcholinesterase; Animals; Cell Differentiation; Cell Membrane; Genetic Engineering; Immunoblotting; Isoenzymes; Mice; Neurites; Neuroblastoma; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK-N-BE) was evaluated measuring growth rate, motility, neurite extension and GAP-43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor-mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP-2, whereas MMP-9 expression is up-regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co-treatment with the two drugs.

Topics: Cell Differentiation; Cell Division; Cell Movement; Dehydroepiandrosterone; Drug Synergism; Humans; Matrix Metalloproteinases; Neurites; Neuroblastoma; Neurons; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Von Hippel-Lindau (VHL) tumor suppressor protein is expressed in neurons of the central nervous system and plays an important role during the neuronal differentiation of central nervous system progenitor cells. To elucidate the neuronal differentiating potential of VHL protein in neuroblastoma cells, we overexpressed or inhibited VHL protein in human neuroblastoma cells (SY-SH5Y), and examined the morphological change, expressions of neuronal markers, and electrophysiological functions. Here we show that with VHL gene transduction SY-SH5Y cells stably expressing the VHL protein had neurite-like processes with varicosities, showed the distinct expression of the neuronal markers neuropeptide Y and neurofilament 200, acquired regulated neurosecretion competence in response to depolarizing and cholinergic stimuli, and had large voltage-gated fast sodium currents and delayed rectifier potassium (Kv) currents compatible with those of functional neurons. In addition, they displayed inactivated ether-á-go-go potassium channels related to the promotion of the cell cycle and to the termination of differentiation. Also, by treatment with retinoic acid, they rapidly underwent cell death related to apoptosis. These findings suggest that the induction of neuronal function by VHL protein is associated with down-regulation of the cell cycle. In contrast, the inhibition of endogenous expression of VHL protein with antisense-orientated VHL gene transduction reduced such neuronal properties inherent to these cells, including the capacity for activation of ether-á-go-go channels. In conclusion, VHL protein has a neuronal differentiating potential to transform neuroblastoma cells into functional neuron-like cells. Our finding of the neuronal differentiation of neuroblastoma cells under the control of the VHL gene may contribute to the development of clinical techniques for neuronal regeneration in the case of intractable neuronal diseases and for differentiation therapy against neuroblastomas.

Topics: Apoptosis; Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Ether-A-Go-Go Potassium Channels; Genetic Therapy; Humans; Ion Channel Gating; Ligases; Neuroblastoma; Neurofilament Proteins; Neurons; Neuropeptide Y; Potassium Channels; RNA, Messenger; Sodium Channels; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Double-stranded (ds) RNA-induced sequence-specific interference with gene expression, RNA interference (RNAi), has been extensively used in invertebrates, allowing for efficient and high-throughput gene silencing and gene function analysis. In vertebrates, however, use of RNAi to study gene function has been limited due to non-specific effects induced by double-stranded RNA (dsRNA)-dependent protein kinase and interferon activation. dsRNA-induced specific inhibition of vertebrate gene expression has only been shown in embryonic and non-differentiated mammalian cells. In this report, we demonstrate dsRNA-induced specific interference of gene expression and gene function in partially as well as fully differentiated mouse neuroblastoma cells. Specific silencing was observed in the expression of an integrated transgene coding for green fluorescent protein and a variety of endogenous genes. Moreover, we show that RNAi-mediated inhibition of poly (ADP-ribose) polymerase (PARP) expression induced cellular resistance to oxygen-glucose deprivation, consistent with the role of PARP in ischemia-induced brain damage. Our results indicate that RNAi can be used as a powerful tool to study gene function in neural cells.

Topics: Animals; Cell Culture Techniques; Gene Silencing; Green Fluorescent Proteins; Luminescent Proteins; Mice; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering; Time Factors; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma SH-SY5Y cells were used to study the effects of transforming growth factor beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2) on neuronal differentiation and acquisition of a catecholaminergic phenotype. SH-SY5Y cells express the intracellular factors activated through the receptors of the TGFbeta superfamily members, Smad1 and Smad4, as in basal conditions or after differentiation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA). TGF-beta1 and BMP-2 induce differentiation in SH-SY5Y cells by different pathways: the effect of TGF-beta1 is potentiated by TPA and the effect of BMP-2 is blocked by RA. Cell differentiation due to TGF-beta1 treatment is accompanied by an increase in tyrosine hydroxylase (TH) expression, more pronounced in the presence of TPA or RA and counteracted by BMP-2. BMP-2 and RA both induce noncatecholaminergic cell differentiation, and together they may induce choline acetyltransferase expression in serum-cultured cells. In conclusion, our results suggest that TGF-beta1 and BMP-2 may contribute, in opposite ways, to regulation of the neuronal catecholaminergic phenotype.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Catecholamines; Cell Differentiation; Cell Size; Central Nervous System; Culture Media, Serum-Free; DNA-Binding Proteins; Humans; Inhibitor of Differentiation Protein 1; Neuroblastoma; Neurons; Phenotype; Repressor Proteins; Smad Proteins; Smad1 Protein; Smad4 Protein; Sympathetic Nervous System; Tissue Plasminogen Activator; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Up-Regulation

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.

Topics: Acetylcholine; Calcium Signaling; Cell Differentiation; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Humans; Immunohistochemistry; Magnesium Deficiency; Neuroblastoma; Neurons; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Tretinoin; Tumor Cells, Cultured

The receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, is known to interact with amphoterin. This interaction has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, there is as yet no direct evidence of the relevance of this pathway to neurodifferentiation under physiological conditions. In this study we have investigated a possible role of RAGE and amphoterin in the retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of RAGE by dominant negative and antisense strategies showed that RAGE is not required for process outgrowth or differentiation, although overexpression of RAGE accelerates the elongation of neuritic processes. Using the antisense strategy, amphoterin was shown to be essential for process outgrowth and differentiation, suggesting that amphoterin may interact with other molecules to exert its effect in this context. Interestingly, the survival of the neuroblastoma cells treated with retinoic acid was partly dependent on the expression of RAGE, and inhibition of RAGE function partially blocked the increase in anti-apoptotic protein Bcl-2 following retinoic acid treatment. Based on these results we propose that a combination therapy using RAGE blockers and retinoic acid may prove as a useful approach for chemotherapy for the treatment of neuroblastoma.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Line; Cell Survival; Cloning, Molecular; Coloring Agents; DNA Fragmentation; DNA, Complementary; Electrophoresis, Agar Gel; Genes, Dominant; Glycation End Products, Advanced; HMGB1 Protein; Humans; Immunohistochemistry; Mice; Neuroblastoma; Neurons; Oligonucleotides, Antisense; Plasmids; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Hypoxia-hypoglycemia has played an important role in inducing both phospholipase A2 activation and the expression of the early gene c-fos, in the neuroblastoma cell line SK-N-BE, after it has been differentiated by retinoic acid. Under hypoxic-hypoglycemic conditions, arachidonic acid release has found to be significant after 30 min, whereas c-fos expression has required at least 4 h. This model has been obtained by adding glycolytic inhibitor 2-deoxyglucose to the culture and by placing cells in an atmosphere containing 100% N2 for different time periods. This condition has been compared with two different models: NaCN and nitrogen have been used as hypoxic stimuli, without inhibiting the glycolytic pathway, but the same cell cultures have been used. Cell viability and the fall of cellular ATP levels have been evaluated in all the models, in order to monitor and compare the hypoxic cellular damage. Phospholipase A2 activation has been found to be significant in all conditions, even if to a different extent; but only hypoxia combined with the inhibition of the glycolytic pathway, has induced a significant expression of c-fos. It is very difficult to study hypoxic stimuli in 'in vitro' systems. Our study has compared three different models and the one combining gaseous hypoxia and hypoglycemic conditions seems to be very effective in stimulating early events involved in hypoxic phenomena such as phospholipase activation and the expression of the early gene c-fos.

Topics: Arachidonic Acid; Cell Differentiation; Enzyme Activation; Glycolysis; Humans; Hypoglycemia; Hypoxia; Neuroblastoma; Nitrogen; Phospholipases A; Phospholipases A2; Proto-Oncogene Proteins c-fos; Sodium Cyanide; Time Factors; Tretinoin; Tumor Cells, Cultured

Treatment with 13-cis retinoic acid (13-cis RA) has been shown to significantly improve the clinical outcome of children with high-risk neuroblastoma. Despite the large number of studies investigating the cellular effects of retinoids in neuroblastoma cells, the influence of RA isomerisation and the factors that determine the extent of RA isomerisation and uptake are unknown. The aim of this study was to establish the extent of extra- and intracellular isomerisation of 13-cis RA and all-trans retinoic acid (ATRA) in neuroblastoma cell lines, and to investigate the influence of isomerisation on their growth inhibitory effects and on the regulation of expression of cellular retinoic acid binding protein II (CRABP II) and RAR-beta. Limited extracellular isomerisation was observed up to 72 hr after incubation of four neuroblastoma cell lines with 10 microM 13-cis RA or ATRA. The retinoic acid isomer present initially in the medium accounted for >75% of extracellular retinoid exposure. By contrast, incubation with 13-cis RA resulted in intracellular levels of ATRA comparable to those of 13-cis RA. This degree of intracellular isomerisation was not observed after ATRA incubations, with 13-cis RA accounting for <10% of total intracellular retinoids. No differences were observed in the sensitivity of three N-type neuroblastoma cell lines to either 13-cis RA (IC(50): 11.2-13.9 microM) or ATRA (IC(50): 12.9-14.4 microM), despite 10-fold differences in intracellular retinoid levels. A decrease in sensitivity to 13-cis RA (IC(50)=137 microM), as compared to ATRA (IC(50)=41 microM), was observed in the S-type cell line SH S EP. RAR-beta was induced in a dose-dependent manner in SH SY 5Y cells following incubation with ATRA, whereas a weaker and delayed induction was observed with 13-cis RA. Similarly, incubation with ATRA resulted in a greater induction of CRABP II in these cells. In summary, these results indicate either an intracellular conversion of 13-cis RA to ATRA or a selective uptake of ATRA and suggest that this may mediate the differential activity of 13-cis RA in neuroblastoma cell subtypes.

Topics: Antineoplastic Agents; Cell Division; Gene Expression; Humans; Isotretinoin; Neuroblastoma; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Vitamin A is required for a number of developmental processes and for the homeostatic maintenance of several adult differentiated tissues and organs. In human neuroblastoma (NB) cells as well as some other tumor types, pharmacological doses of retinoids are able to control growth and induce differentiation in vitro and in vivo. In a search for new genes that are regulated by retinoids and that contribute to the biological effects retinoids have on NB cells, we have isolated five differentially expressed transcripts. Here we report on the characterization of one of them (DD83.1) in NB cell lines. DD83.1 is identical to the human retSDR1, a short chain dehydrogenase/reductase that is thought to regenerate retinol from retinal in the visual cycle. Its expression is strongly, but differently, regulated by retinoids in NB cell lines, and it is widely expressed in human tissues, which suggests that it is involved in a more general retinol metabolic pathway. Both the retinoic acid-dependent and the exogenous expression of retSDR1 in SK-N-AS cells induce the accumulation of retinyl esters, which indicates that it is involved in generating storage forms of retinol in tissues exposed to physiological retinol concentrations. We also show that the human retSDR1 gene, which maps on chromosome 1p36.1, is contained in the DNA fragment deleted in many NB cell lines bearing MYCN amplification but is conserved in a cell line with a small 1p deletion and normal MYCN. Our observations suggest that retSDR1 is a novel regulator of vitamin A metabolism and that its frequent deletion in NB cells bearing MYCN amplification could compromise the sensitivity of those cells to retinol, thereby contributing to cancer development and progression.

Topics: Alcohol Oxidoreductases; Chromosomes, Human, Pair 1; Enzyme Induction; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Receptors, Retinoic Acid; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured; Vitamin A

Retinoic acid (RA) induced differentiation of SH-SY5Y cells increases the expression of mu opioid receptors (HMOR) and inhibitory G proteins, as well as the efficacy of opioids to inhibit forskolin-induced adenylyl cyclase activity. We examined the time course of the effects of all-trans retinoic acid (RA) on HMOR and c-fos mRNA levels as determined by solution hybridization (using HMOR and rat c-fos riboprobes) in RNA extracts from SH-SY5Y cells. Electrophoretic Mobility Shift Assay (EMSA) and Western blot analysis were used to assess the changes AP-1 DNA binding and the presence of fos-related proteins in nuclear extracts from untreated, vehicle (ethanol) or RA-treated SH-SY5Y cells. Exposure to RA for 0.5 h had no effect on HMOR while after 6-18 h of exposure HMOR in mRNA levels were decreased by 50% and then after 168 h of RA exposure, HMOR mRNA levels were doubled. In contrast, c-fos mRNA levels were unchanged at 0.5 h, but increased by 50% after 18 and 168 h of RA exposure. RA increased AP-1 binding after 18 and 168 h and a pan fos-FRA antibody produced a supershift. Western analysis indicates that RA activates a 45-kDa protein corresponding to the size of the fos B protein. These results identify two signal transduction targets that are regulated by RA during differentiation.

Topics: Binding Sites; Central Nervous System; DNA; DNA-Binding Proteins; Gene Expression Regulation; Humans; Neuroblastoma; Neurons; Proto-Oncogene Proteins c-fos; Receptors, Opioid, mu; RNA, Messenger; Time Factors; Transcription Factor AP-1; Tretinoin; Tumor Cells, Cultured

The vitamin A metabolite, all-trans retinoic acid (atRA), plays an important role in neuronal development, including neurite outgrowth. However, the genes that lie downstream of atRA and its receptors in neuronal cells are largely unknown. By using the human neuroblastoma cell line, SH-SY5Y, we have identified an atRA-responsive gene (RAINB1: retinoic acid inducible in neuroblastoma cells) that is induced within 4 h after exposure of SH-SY5Y cells to atRA. RAINB1 mRNA is highly expressed in the nervous system (10.5- to 11-kb transcript) in both developing embryos and adults. Its expression is perturbed in developing rat embryos exposed to excess or insufficient atRA. RAINB1 is present on chromosome 11 and is spread over 38 exons, resulting in a putative ORF of 2,429 amino acids. The RAINB1 protein shows high similarity to a gene in Caenorhabditis elegans, unc-53, that is required for axonal elongation of mechanosensory neurons, suggesting that these proteins are orthologs. Thus, RAINB1 may represent a critical downstream gene in atRA-mediated neurite outgrowth.

Topics: Aging; Amino Acid Sequence; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromosomes, Human, Pair 11; Cloning, Molecular; Embryo, Mammalian; Embryo, Nonmammalian; Exons; Gene Expression Profiling; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Molecular Sequence Data; Nerve Tissue Proteins; Nervous System; Neuroblastoma; Physical Chromosome Mapping; Rats; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Prenylcysteine carboxymethyltransferase (pcCMT) is an enzyme that catalyzes the post-translational carboxymethylation of isoprenylated proteins ensuring a more efficient membrane attachment and proper guiding to a specific target membrane. In this paper, we report on modulation of pcCMT activity in retinoic acid (RA)-treated SH-SY5Y neuroblastoma cells using N-acetyl-S-farnesyl-L-cysteine (AFC) as artificial methyl acceptor. In addition, the methylation of endogenous proteins was followed by the vapor phase equilibrium assay and the storage phosphor screen (P-screen) technique with S-adenosyl-[3H-methyl] methionine (AdoMet) as methyl donor. Methylation of AFC was reduced to 75% of that of the control, the most prominent decrease being observed with the post-nuclear membrane fraction as enzyme source. With regard to protein methylation both screening methods yielded analogous results showing the [3H]-labeling of endogenous proteins in the 21-25kDa molecular mass (MM) range to be diminished by nearly 50%. This questions the role of protein carboxymethylation as an essential component of the differentiation process in SH-SY5Y neuroblastoma cells. The P-screen technique revealed that the methylation of other molecular mass proteins was also affected. Both S-adenosylhomocysteine (AdoHcy) and AFC (AdoHcy being the most effective) inhibited endogenous methylation. An interesting feature was that AFC inhibited the protein methylation proportionally more effective in RA-treated cells. Finally, the levels of three small guanosine-5'-triphosphate (GTP) binding proteins were screened upon differentiation showing rab3A to be increased while rhoA and H-ras were decreased.

Topics: Acetylcysteine; Antineoplastic Agents; Cell Differentiation; Genes, ras; Humans; Membrane Proteins; Methylation; Neuroblastoma; Norepinephrine; Nuclear Envelope; Protein Isoforms; Protein Methyltransferases; rab3A GTP-Binding Protein; rhoA GTP-Binding Protein; S-Adenosylhomocysteine; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces neural differentiation of SH-SY5Y neuroblastoma cells. We show that the mRNA levels of the differentiation-inhibiting basic helix-loop-helix transcription factors ID1, ID2, and ID3 are down-regulated during RA-induced differentiation of SH-SY5Y cells. The levels of ID proteins decreased in parallel to the observed transcriptional repression. The expression of other basic helix-loop-helix genes changed during RA-induced differentiation: expression of neuroblast-specific ASCL1 (HASH-1) gene was promptly reduced after RA treatment, whereas expression of differentiation-promoting genes NEUROD6 (NEX-1, HATH-2) and NEUROD1 was increased. Treatments with 12-O-tetradecanoylphorbol-13-acetate, another inducer of neuroblastoma cell differentiation, also resulted in coordinated down-regulation of ID gene expression, underscoring the role of ID genes in differentiation. Down-regulation of ID gene expression by RA involves a complex mechanism because full transcriptional repression required newly synthesized proteins and signaling by phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, resulting in increased PI3K activity in extracts from RA-treated cells and a rapid increase in phosphorylation of Akt in Ser-473. Inhibition of PI3K by LY294002 impaired RA-induced differentiation, as assessed by morphological and biochemical criteria. We propose that RA, by activating the PI3K/Akt signaling pathway, plays an important role in the regulation of neuronal cell survival.

Topics: Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; DNA-Binding Proteins; Enzyme Activation; Humans; Molecular Sequence Data; Neuroblastoma; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Brain-derived neurotrophic factor (BDNF) is a major neurotrophin in the brain and abnormal regulation of BDNF may contribute to the pathophysiology of mood disorders. In the present study, we examined if alterations in the activity of glycogen synthase kinase-3-beta (GSK3beta) or treatment with mood stabilizers modulated BDNF-mediated signal transduction pathways in differentiated human neuroblastoma SH-SY5Y cells. BDNF increased the phosphorylation of the forkhead transcription factor FKHRL1 through activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and the phosphorylation of the cyclic AMP response element binding protein (CREB) through activation of extracellular signal-regulated kinase1/2 (ERK1/2). BDNF also increased serine(9) -phosphorylation of GSK3beta, which inhibits GSK3beta activity. Overexpression of GSK3beta did not affect BDNF-induced phosphorylation of Akt, ERK1/2, or FKHRL1, but abolished CREB phosphorylation induced by BDNF. This inhibition of BDNF-induced CREB phosphorylation in GSK3beta-overexpressing SH-SY5Y cells was blocked by treatment with lithium. In contrast to lithium, sodium valproate and lamotrigine did not affect BDNF-mediated signaling, whereas carbamazepine induced a rapid and prolonged phosphorylation of ERK1/2 and CREB in the absence or the presence of BDNF. Therefore, increased GSK3beta selectively attenuates BDNF-induced CREB phosphorylation, and lithium and carbamazepine can facilitate activation of CREB.

Topics: Anticonvulsants; Antimanic Agents; Brain-Derived Neurotrophic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cyclic AMP Response Element-Binding Protein; DNA-Binding Proteins; Forkhead Box Protein O1; Forkhead Box Protein O3; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Lithium; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Polysialic acid (PSA) is a dynamically regulated carbohydrate modification of the neural cell adhesion molecule NCAM, which is implicated in neural differentiation and cellular plasticity. The cloning and characterization of two polysialyltransferases, termed ST8SiaII (STX) and ST8SiaIV (PST), opened up new perspectives in the search for factors that control this unique cell surface glycosylation. In vitro and transfection approaches revealed that ST8SiaII and ST8SiaIV are independently capable of synthesizing PSA on NCAM with slightly different specificities towards the major NCAM isoforms and glycosylation sites. Their overlapping but distinct expression patterns during brain development point towards an independent transcriptional regulation. However, the factors driving their joint or distinct expression, as well as the significance of divergent expression patterns in vivo, are not yet understood. In the present study, the mRNA expression of ST8SiaII and ST8SiaIV was comparatively analyzed in neuronal differentiation of PSA-positive human neuroblastoma cell lines induced by retinoic acid (RA), phorbolester, or growth factors. Using a semiquantitative RT-PCR strategy, we demonstrated a general decrease in the mRNA level of ST8SiaII upon differentiation of SH-SY5Y and LAN-5 cells. In contrast, a drastic increase of ST8SiaIV was specifically induced by RA-treatment of SH-SY5Y cells. To explore the significance of these changes, the cellular capacity to perform PSA synthesis and the degree of NCAM polysialylation were analyzed. Our data indicate that the increased expression of ST8SiaIV enables an accelerated polysialylation of NCAM, which, however, is not converted into higher amounts of PSA.

Topics: Antigens, Surface; Cell Differentiation; Cell Size; Gene Expression Regulation; Humans; Molecular Weight; Neural Cell Adhesion Molecules; Neuroblastoma; Neuronal Plasticity; RNA, Messenger; Sialic Acids; Sialyltransferases; Tretinoin; Tumor Cells, Cultured

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization. These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.

Topics: Carbachol; Cell Differentiation; Gene Expression; Gene Silencing; GTP-Binding Proteins; Humans; Muscarinic Agonists; Mutagenesis, Site-Directed; Neurites; Neuroblastoma; Neurons; Oligonucleotides, Antisense; Protein Glutamine gamma Glutamyltransferase 2; Transfection; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wildtype but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.

Topics: Agar; Apoptosis; Blotting, Western; Cell Adhesion; Cell Differentiation; Cell Division; DNA; Down-Regulation; Fibroblasts; Fluorescent Antibody Technique, Indirect; Humans; Neuroblastoma; Neurofilament Proteins; Oncogene Proteins, Viral; Phenotype; Plasmids; Radiation, Ionizing; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection; Tretinoin; Tubulin; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vimentin

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.

Topics: Androstadienes; Apoptosis; Bacterial Proteins; Bacterial Toxins; bcl-X Protein; Biomarkers; Cell Differentiation; Cell Size; DNA Fragmentation; Gene Expression Regulation; Humans; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphopyruvate Hydratase; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; rhoA GTP-Binding Protein; Signal Transduction; Staurosporine; Synaptophysin; Tetradecanoylphorbol Acetate; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Wortmannin

The plasma and nuclear membranes of neural cells have been shown to express gangliosides to a limited extent before, and at increasing levels during, differentiation. Recent studies employing qualitative cytochemistry have shown that GM1 expression in particular is significantly elevated in both membranes by specific neuritogenic agents. The present study provides a more complete description of ganglioside patterns of the 2 membranes of NG108-15 cells and a mutated form of the latter lacking gangliotetraose gangliosides. Nuclei of wild type NG108-15 cells were found to contain predominantly GM1 and GD1a, whereas whole cells had those in addition to substantial amounts of GM2 and GM3. GM1 and GD1a levels increased 2--3.5-fold in both whole cells and nuclei following axonogenic stimulation, but changed little in response to dendritogenic agents. GM2 expression, limited to the plasma membrane, showed little if any change with axonogenic stimuli but a 1.5--2-fold increase following treatment with dendritogenic agents. GM3 resembled GM2 in being virtually absent from the nuclear membrane, while its presence in the plasma membrane showed only modest change at most with any of the stimuli. The gangliotetraose ganglioside-deficient mutant cell line, NG-CR72, had significantly higher basal levels of GM2 in the plasma membrane compared to wild type NG108-15 cells, and this level increased significantly on treatment with dendritogenic agents. Basal GM3 levels were greatly reduced in the mutant cells and changed little with any of the stimuli. As expected, nuclei of NG-CR72 cells were virtually devoid of gangliosides. These mutant cells were previously shown to extend well defined dendritic neurites but were incapable of forming stable axonal processes. This study thus demonstrates major differences in the ganglioside content of wild type and mutated NG108-15 cells and their nuclei, and in their response to different neuritogenic stimuli.

Topics: Animals; Axons; Bucladesine; Cell Differentiation; Cell Membrane; Cell Nucleus; Dendrites; Gangliosides; Ionomycin; Mice; Mutation; Neurites; Neuroblastoma; Nuclear Envelope; Oligosaccharides; Potassium Chloride; Rats; Tretinoin; Tumor Cells, Cultured

Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents.. We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA.. Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged.. This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.

Topics: Bucladesine; Cell Communication; Connexin 43; Gap Junctions; Glutamate-Cysteine Ligase; Glutathione; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Cyclooxygenases-1 and -2 are both expressed in neuronal cells in vivo. In the neuroblastoma cell lines NG108 and N2a, however, only cyclooxygenase-1 was detectable. Differentiation of the cells with retinoic acid increased cyclooxygenase-1 mRNA and protein expression within 24 and 48 h, respectively. A further increase was observed when the cells were concomitantly treated with the glucocorticoid dexamethasone (a 2-3-fold increase compared with retinoic acid alone). In the absence of retinoic acid, dexamethasone only slightly up-regulated cyclooxygenase-1 expression. The inhibitor of protein synthesis cycloheximide abrogated the effect of dexamethasone, indicating the involvement of newly synthesised proteins. Retinoic acid increased the transcription of cyclooxygenase-1 mRNA, determined with a luciferase-coupled promoter construct. Dexamethasone only slightly augmented cyclooxygenase-1-promoter activity but increased cyclooxygenase-1 mRNA stability. Other corticosteroids, hydrocortisone and aldosterone, also up-regulated cyclooxygenase-1 whereas neurosteroids or oestrogen were ineffective. Up-regulation was mediated primarily by the glucocorticoid receptor, because the receptor antagonist RU486 strongly reduced the effects of all corticosteroids. This indicated that in NG108 cells, the mineralocorticoid aldosterone may bind to the glucocorticoid receptor. Treatment of NG108 or N2a cells with corticosteroids did not alter the morphological phenotype obtained during differentiation. We thus show that corticosteroids, which down-regulate cyclooxygenase expression in most cell types, up-regulate cyclooxygenase-1 during neuronal differentiation.

Topics: Adrenal Cortex Hormones; Aldosterone; Animals; Benzimidazoles; Bucladesine; Calcimycin; Cell Differentiation; Cycloheximide; Cyclooxygenase 1; Cyclooxygenase 2; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Dexamethasone; Dinoprostone; Drug Synergism; Enzyme Induction; Estradiol; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioma; Hybrid Cells; Hydrocortisone; Ionophores; Isoenzymes; Luciferases; Membrane Proteins; Mice; Mifepristone; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Protein Synthesis Inhibitors; Receptors, Glucocorticoid; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.

Topics: Acetylation; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Cinnamates; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Female; Growth Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Mice; Mice, SCID; Neuroblastoma; Tretinoin; Tumor Cells, Cultured; Weight Loss; Xenograft Model Antitumor Assays

KIF3A, KIF3B and KIF3C are kinesin-related motor subunits of the KIF3 family that associate to form the kinesin-II motor complex in which KIF3C and KIF3B are alternative partners of KIF3A. We have analysed the expression of Kif3 mRNAs during prenatal murine development. Kif3c transcripts are detectable from embryonic day 12.5 and persist throughout development both in the CNS and in some peripheral ganglia. Comparison of the expression patterns of the Kif3 genes revealed that Kif3c and Kif3a mRNAs colocalize in the CNS, while only Kif3a is also present outside the CNS. In contrast, Kif3b is detectable in several non-neural tissues. We have also performed immunocytochemical analyses of the developing rat brain and have found the presence of the KIF3C protein in selected brain regions and in several fibre systems. Using neuroblastoma cells as an in vitro model for neuronal differentiation, we found that retinoic acid stimulated the expression of the three Kif3 and the kinesin-associated protein genes, although with different time courses. The selective expression of Kif3c in the nervous system during embryonic development and its up-regulation during neuroblastoma differentiation suggest a role for this motor during maturation of neuronal cells.

Topics: Animals; Blotting, Northern; Brain; Brain Chemistry; Cell Differentiation; Gene Expression; Gestational Age; Humans; Immunoblotting; Immunoenzyme Techniques; Immunohistochemistry; In Situ Hybridization; Kinesins; Kinetics; Mice; Neuroblastoma; Neuroglia; Neurons; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Neuroblastomas are heterogeneous tumors arising from sympathetic precursors in the neural crest. Growth factor stimulation of neuroblastomas promote diverse biological responses (mitogenesis, differentiation, cell death) depending on the particular tumor studied. Here we show that brief treatment with retinoic acid (RA) rendered the human neuroblastoma lines SY5Y, NGP, SMS-KCNR, and SK-N-SH dependent on brain-derived neurotrophic factor (BDNF) for survival. The BDNF- and trkB-expressing line SMS-KCN was dependent on an autocrine BDNF/trkB survival without exposure to RA. We conclude that the BDNF/trkB pathway plays an important role in neuroblastoma survival and speculate on a possible role in tumor pathogenesis.

Topics: Antibodies; Antineoplastic Agents; Apoptosis; Brain-Derived Neurotrophic Factor; Dose-Response Relationship, Drug; Humans; In Situ Nick-End Labeling; Mutation; Neuroblastoma; Receptor, trkB; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, the most common extracranial solid tumor in children, arises from precursors of the sympathetic nervous system. Neuroblastoma cell lines are responsive to the differentiation agent retinoic acid, which induces its effects by altering transcription rates of specific target genes. We identified autotaxin (ATX), which encodes an autocrine tumor motility-stimulating factor, as a gene whose expression is significantly induced by retinoic acid in neuroblastoma cells. ATX induction was specific for neuroblastoma cell lines that contain N-myc amplification, a cytogenetic feature commonly associated with aggressive neuroblastomas. Although ATX expression was associated with amplification of the N-myc locus, N-myc itself was neither sufficient nor required for ATX expression, suggesting that a coamplified gene is responsible. ATX induction by retinoic acid was due to increased transcription and required new protein synthesis.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Amplification; Gene Expression; Genes, myc; Glucose-6-Phosphate Isomerase; Glycoproteins; Humans; Multienzyme Complexes; Neuroblastoma; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Repressor Proteins; Retroviridae; RNA, Messenger; Tretinoin

LA-N-1 neuroblastoma cell cultures contain Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.

Topics: Calcium; Cell Nucleus; Cytosol; Humans; Hydrolysis; Neuroblastoma; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Plasmalogens; Tretinoin; Tumor Cells, Cultured

The HNK-1 carbohydrate epitope is expressed in neural and natural killer cells and is a mediator of cell adhesion. It is well documented that acetylcholinesterase has a secondary function in cell adhesion and differentiation. The presence of HNK-1 on isoforms of Torpedo and Electrophorus acetylcholinesterase, as well as isoforms from the bovine central nervous system has been described. In this paper, we have investigated the association of the epitope with acetylcholinesterase from human neuroblastoma cells. Acetylcholinesterase was extracted, with or without detergent, purified on immunoaffinity columns and the isoforms separated by sucrose density gradient sedimentation. Secreted acetylcholinesterase, from spent serum-free culture medium, was similarly treated. The presence of the HNK-1 epitope was determined by ELISA using the anti-HNK-1 and Elec 39 monoclonal antibodies. The epitope was found to be associated with the detergent-soluble G4 isoform, but not with the hydrophilic G1 nor the secreted hydrophilic G4 isoforms. Likewise, no HNK-1 was observed associated with human erythrocyte acetylcholinesterase. These results indicate that acetylcholinesterase-G4, anchored in the extracellular membrane, is capable of mediating cell-substrate adhesion through HNK-1.

Topics: Acetylcholinesterase; Animals; Antibodies, Monoclonal; CD57 Antigens; Cell Adhesion; Cell Differentiation; Detergents; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Humans; Isoenzymes; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Octoxynol; Organ Specificity; Protein Structure, Tertiary; Solubility; Torpedo; Tretinoin; Tumor Cells, Cultured

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD.

Topics: Antineoplastic Agents; Cell Survival; Gene Expression; Humans; Huntingtin Protein; Huntington Disease; Immunohistochemistry; Mutagenesis; Nerve Tissue Proteins; Neuroblastoma; Neurons; Nuclear Proteins; Peptides; Polyamines; Precipitin Tests; Transfection; Transglutaminases; Tretinoin; Tumor Cells, Cultured

This study examines the effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)(2)D(3) or its derivatives, but significantly decreased in the presence of the two retinoids (0.001--10 microM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060, and 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.

Topics: Acetylcholinesterase; Alitretinoin; Antineoplastic Agents; Cell Division; Drug Synergism; Gene Expression; Humans; Neuroblastoma; Proto-Oncogene Proteins c-myc; Steroid Hydroxylases; Tretinoin; Tumor Cells, Cultured; Vitamin D

The expression of several genes is modulated during neuroblastoma differentiation. The retinoblastoma family proteins, pRb, p107 and pRb2/p130, act in the repression of proliferation genes, interacting mainly with the E2F transcription factors.. In this study, we found that, in neuroblastoma cell lines, pRb and p107 proteins decreased, undergoing progressive dephosphorylation, whereas pRb2/p130 increased at late stages of differentiation. B-myb expression was down-regulated in association with the up-regulation of pRb2/p130, the major partner of E2F on the E2F site of the B-myb promoter in differentiated cells. Transfection of each of the retinoblastoma family genes in neuroblastoma cells was able to induce neural differentiation, to inhibit 3H-thymidine incorporation, and to down-regulate B-myb promoter activity.. In conclusion, our data suggest a major contribution of retinoblastoma proteins, and especially of pRb2/p130, in B-myb promoter regulation and demonstrate the induction of neural differentiation by p107 and pRb2/p130, suggesting a role of these proteins in triggering differentiation-specific genes.

Topics: Animals; Cell Cycle Proteins; Cell Differentiation; Dimethyl Sulfoxide; DNA-Binding Proteins; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Genes, myb; Genes, Reporter; Genes, Retinoblastoma; Humans; Luciferases; Mice; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Phosphoproteins; Promoter Regions, Genetic; Proteins; Recombinant Fusion Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; Trans-Activators; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may undergo spontaneous differentiation or regression due to apoptosis after no or minimal therapy. However, the majority of neuroblastomas are diagnosed as metastatic tumours with a poor prognosis in spite of intensive multimodal therapy. Vitamin A and its analogues (retinoic acid, RA) play an important role in normal cel lular differentiation and programmed cell death. RA regulates neuroblastoma growth and differentiation in vitro, and has shown activity against human neuroblastoma in vivo.. Recently, 9-cis RA was shown to induce apoptosis in vitro in neuroblastoma using a 5 days short-term treatment and subsequent washout. In the present study, nude rats with human neuroblastoma SH-SY5Y xenografts were treated with 13-cis RA (4 mg po daily), 9-cis RA (5 mg po daily) or the novel analogue Ro 13-6307 (0.3 mg po daily) using either a continuous or short-term schedule.. ALL three different retinoids decreased neuroblastoma growth significantly in terms of tumour weight after 8-12 days when compared to untreated controls (P < 0.05). Minor signs of toxicity in 13-cis RA treated rats were observed. However, severe toxicity with significant weight loss was seen in all rats treated with 9-cis RA and Ro 13-6307. Toxicity was more pronounced with the continuous regimen.. We conclude that different retinoids reduce neuroblastoma tumour growth in vivo. Drug scheduling and dosage may affect both therapeutic efficacy and toxic side effects. Further in vivo studies are warranted, including pharmacokinetic and molecular analyses, before clinical trials with promising retinoids like 9-cis RA and Ro 13-6307 can be started in children with neuroblastoma.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Body Weight; Cell Differentiation; Cell Division; Diarrhea; Drug Administration Schedule; Fatty Acids, Unsaturated; Female; Humans; Isotretinoin; Male; Mice; Neoplasm Transplantation; Neuroblastoma; Rats; Rats, Nude; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear.. Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6.. TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells.. The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.

Topics: Active Transport, Cell Nucleus; Apoptosis; Caspase 3; Caspases; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drosophila Proteins; Enzyme Induction; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor; Glial Cell Line-Derived Neurotrophic Factor Receptors; Humans; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurturin; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

MASH1, a transcription factor with basic helix-loop-helix domain, has a pivotal function to promote differentiation of neural crest cells into autonomic neurons.. To investigate the functional significance of human MASH1 (hASH1) in the pathogenesis of neuroblastoma, which is originated from autonomic precursor cells, we studied hASH1 gene expression in primary neuroblastomas and human nueroblastoma cell lines.. The follovving results were obtained: (i) hASH1 was expressed in 40 out of 61 (66%) primary neuroblastomas, (ii) hASH1 transcripts were downregulated in several cell lines prior to differentiation induced by all-trans retinoic acid (RA), (iii) a neuroblasotma cell line without expression of endogenous hASH1 did not respond to RA at all, and (iv) the analysis of the hASH1 genomic DNA revealed two possible transcription initiation sites, which may correspond to 3.0 kb and 3.5 kb transcripts.. Our observations suggest that, although hASH1 may not preserve the growth capacity of neuroblastomas, downregulation of hASH1 may be necessary to promote neuronal differentiation of neuroblastoma.

Topics: 5' Untranslated Regions; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; DNA-Binding Proteins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes; Helix-Loop-Helix Motifs; Humans; Neoplasm Proteins; Neural Crest; Neuroblastoma; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; RNA, Messenger; RNA, Neoplasm; Sequence Analysis, DNA; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

To investigate the molecular mechanisms by which retinoic acid (RA) alters cell growth, the expression and activity of components of the cell cycle machinery were analyzed.. Within 2 days of RA treatment, and prior to the arrest of NB cells in the G1 phase of the cell cycle, there was a complete downregulation of GI cyclin/cdk activities. Protein levels for the G1 cyclin/cdk were essentially unchanged during this time, although there was a decrease in the steady state levels of hyperphosphorylated Rb and p60N-MYC proteins. The cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR, while p15 INK4B and p16 INK4A mRNA were undetected. Within 24 hr of RA treatment, there was a 4-fold increase in the expression of p27Kip1, although p27 mRNA levels were unchanged. Levels of p21Cip1 were unaltered. Coincident with the decrease in kinase activity there was an increase in p27 bound to G1 cyclin/cdk. The increase in p27 was not due to an increase in transcription. In other cell systems, increased expression of c-MYC has been shown to lead to a decrease in p27 levels that is regulated at the post-transcriptional level (sequestration). To determine whether increased levels of N-MYC could affect the level of p27, we evaluated the expression of p27 in a series of N-MYC transfected cells and found that constitutive overexpression of N-MYC led to a decrease in the steady-state levels of p27 and in p27 bound to G1 cyclin/cdk complexes. Using adenoviral vectors expressing p27, we found that infection leads to increased p27 expression, which causes a decrease in cdk activity and an accumulation of cells in G1.

Topics: Adenoviridae; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Retinoblastoma; Genetic Vectors; Humans; Neoplasm Proteins; Neuroblastoma; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Recombinant Fusion Proteins; Retinoblastoma Protein; RNA, Messenger; RNA, Neoplasm; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, "flat" cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16(INK4A), responds to RA with elevation of p18(INK4C), marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21(Cip1). These changes result in sustained cdk activity. Accordingly, overexpression of p21(Cip1) but not p16(INK4A) induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.

Topics: Cell Cycle; Cell Differentiation; Cell Line; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Neuroblastoma; RNA, Messenger; Tretinoin

A microarray system is a powerful and very useful technology for analyzing the expression profile of thousands of genes. In this study, we made a cDNA microarray system carrying 2007 cDNAs obtained from primary neuroblastoma cDNA library and identified retinoic acid (RA)-regulated genes in a RTBM1 neuroblastoma cell line. We repeated independent hybridization experiment twice and found that 7 genes were up-regulated, and 5 genes were down-regulated on the cDNA microarray. The semi-quantitative reverse transcriptase (RT)-PCR analysis to confirm the results showed that 4 genes which included amyloid precursor-like protein 2 (APLP2), P311, dihydropyrimidinase related protein3 (DRP3) and RGP4 were up-regulated, while 2 genes, Id-2 and vimentin, were down-regulated. Thus, our neuroblastoma cDNA microarray system is useful to screen the neuronal differentiation- and growth-related genes regulated by RA with high efficiency.

Topics: Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Mice; Nerve Growth Factor; Nerve Growth Factors; Neuroblastoma; Neurons; Neurotrophin 3; Tretinoin; Tumor Cells, Cultured

The prognosis of patients with advanced stages of neuroblastoma with N-myc amplification remains poor despite escalated therapy, a situation that has called for alternative therapeutic approaches. Neuroblastoma cells, which represent immature peripheral neuronal cells, treated with certain physiologic and nonphysiologic agents such as retinoic acid (RA), phorbol esters and interferons (IFN) in vitro undergo cellular differentiation and stop to divide, a process that mimics normal neuronal development. Such "differentiation therapy" using RA after autologous bone marrow transplantation has recently given encouraging results in neuroblastoma patients with advanced disease. Considering approaches for improved differentiation therapy, we investigated possible synergistic effects of combining agents known to influence neuroblastoma growth and differentiation in vitro. Our results show that combined treatment with IFN-gamma and RA or the phorbol ester 12-O-tetradecanoyl-phorbol acetate (TPA) had synergistic or enhancing effects on morphologic differentiation and neurite outgrowth in 5 of 5 neuroblastoma cell lines, 3 of which expressed very high levels of N-myc mRNA due to N-myc amplification. The combinations RA+IFN-gamma or TPA+IFN-gamma also enhanced induced growth inhibition in all 5 cell lines, in several cases resulting in complete growth arrest under conditions where cells stimulated with either agent alone continued to grow. The phenotypic effects of the combined RA+IFN-gamma or TPA+IFN-gamma treatments were in most, but not all, investigated cases accompanied by moderate reductions in N-myc expression, suggesting that the cooperative signals may counteract N-Myc activity at several levels. The cooperativity between IFN-gamma and other differentiation signals may be relevant for approaches to improve the therapy for high-risk neuroblastoma with N-myc-amplification.

Topics: Cell Differentiation; Drug Synergism; Genes, myc; Humans; Interferon-gamma; Neuroblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.

Topics: Alprostadil; Cell Differentiation; Cyclic AMP; Dinoprostone; Dopamine beta-Hydroxylase; Humans; Neuroblastoma; Neurons; Osmolar Concentration; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue. In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS). Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression. Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls. Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid. Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death.

Topics: Apoptosis; Cell Differentiation; Cell Division; Clone Cells; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression; Humans; In Situ Nick-End Labeling; Microscopy, Fluorescence; Multidrug Resistance-Associated Proteins; Neuroblastoma; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Transfection; Tretinoin; Tumor Cells, Cultured

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10(-6) M) or NGF (50-100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10(-6) M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75(NGFR) mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75(NGFR) mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis than undifferentiated cells.

Topics: Alitretinoin; Animals; Apoptosis; Cell Differentiation; Cell Division; Flow Cytometry; Kinetics; Nerve Growth Factor; Neurites; Neuroblastoma; Neurons; Receptor, Nerve Growth Factor; Receptor, trkA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, the most common extracranial solid tumour in children, may undergo spontaneous differentiation or regression, but the majority of metastatic neuroblastomas have poor prognosis despite intensive treatment. Retinoic acid regulates growth and differentiation of neuroblastoma cells in vitro, and has shown activity against human neuroblastomas in vivo. The retinoid 9-cis RA has been reported to induce apoptosis in vitro, and to inhibit the growth of human neuroblastoma xenografts in vivo. However, at given dosage, the treatment with 9-cis RA caused significant toxic side effects. In the present study we investigated the bioavailability of 9-cis RA in rat. In addition, we compared two different dose schedules using 9-cis RA. We found that a lower dose of 9-cis RA (2 mg day(-1)) was non-toxic, but showed no significant effect on tumour growth. The bioavailability of 9-cis RA in rat was 11% and the elimination half-life (t1/2) was 35 min. Considering the short t1/2, we divided the toxic, but tumour growth effective dose 5 mg day(-1) into 2.5 mg p.o. twice daily. This treatment regimen showed no toxicity but only limited effect on tumour growth. Our results suggest that 9-cis RA may only have limited clinical significance for treatment of children with poor prognosis neuroblastoma.

Topics: Administration, Oral; Alitretinoin; Animals; Antineoplastic Agents; Biological Availability; Cell Differentiation; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Administration Schedule; Half-Life; Humans; Male; Neuroblastoma; Rats; Rats, Sprague-Dawley; Tretinoin; Xenograft Model Antitumor Assays

To facilitate the characterization of compounds that have positive growth factor mimetic effects on neuritogenesis, we have implemented a high-throughput functional assay which measures, in a multiparametric manner, the proliferation and differentiation characteristics of cells in a microtiter plate. Conditions were established using chronic incubation of SH-SY5Y human neuroblastoma cells with retinoic acid (RA) and/or nerve growth factor (NGF) in which discernible alterations in proliferation, growth, and differentiation of cells were induced. SH-SY5Y cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on Cellomics ArrayScanII was utilized to quantify the effects of these treatments on cell characteristics. NGF and retinoic acid were found to increase multiple parameters of SH-SY5Y differentiation, including an increased proportion of cells having neurites and increased extent of branching. However, marked differences in the effects of these compounds on SH-SY5Y growth and differentiation were also detected: whereas NGF increased cell number, RA treatment decreased cell number, and RA but not NGF caused significant elongation of neurites. This study quantifies and characterizes the effects of differentiating and proliferating agents on a human-derived neuroblastoma cell line. The high-content, rapid-throughput nature of this assay makes it ideal for functional identification and characterization of compounds regulating cell behavior.

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Fluorescence; Humans; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Nerve Growth Factors; Neurites; Neuroblastoma; Tretinoin

The human SH-SY5Y cell line is an established model for retinoic acid (RA)-induced neural differentiation. We employed a broad human 15K microarray (15,000 genes) and focused Neuroarray (1152 genes) to examine changes in gene expression early in the process of differentiation (6 hr), before morphology or growth changes are observed.. 33 P-labeled CDNA probes prepared from RNA extracts of RA-treated and control cultures were hybridized to array membranes, and levels of expression were quantified and compared.. In the 15K array, 19 % of the genes were decreased (0.4 % were named genes and the remainder were expressed sequence tags (ESTs) or unknowns), and 9 % were increased (4.2 % named genes). In the Neuroarray, 3 % were decreased and 8 % were increased.. Summary gene profiles are presented, which include transcription factors, genes associated with cell cycle, cell shape, neurotransmission, intermediary filaments, and others. The prevalence of down-regulated genes in the broad 15K array and up-regulated genes in the neuro-focused array suggests a pattern shift in gene expression associated with differentiation. The predominance of ESTs among the down-regulated genes indicates a great number of as-yet-unidentified genes are repressed in early stage neural differentiation.

Topics: Antineoplastic Agents; Blotting, Western; Cell Differentiation; Contractile Proteins; Gene Expression; Humans; Microfilament Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Oligonucleotide Array Sequence Analysis; Profilins; Tretinoin; Tumor Cells, Cultured

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Humans; Microtubule-Associated Proteins; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Tretinoin; Tumor Suppressor Proteins

Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth-associated protein-43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT-PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion-like clusters of neuronal cells induced upon RA exposure. MMP-2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor-alpha (TNFalpha), or phorbol 12-myristate 13-acetate (PMA) treatment. In contrast, MMP-9 was inducible by RA, TNFalpha, or PMA. MMP-9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFalpha or PMA potentiated RA-induced MMP-9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP-9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP-9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP-9 could participate in the neurite growth process and cell migration and organization into ganglion-like clusters.

Topics: Cell Differentiation; Enzyme Induction; GAP-43 Protein; Humans; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neural Cell Adhesion Molecules; Neuroblastoma; Neurons; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Differentiation; Cell Division; Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The aim of this study was to clarify retinoid receptor mechanisms mediating the effects of 9-cis retinoic acid (RA) and investigate the ability of RAR- and RXR-specific analogues to induce differentiation and inhibit proliferation in neuroblastoma cells. Differentiation and the inhibition of proliferation by 9-cis RA, but not all-trans RA, were inhibited by the RXR-homodimer antagonist LG745. The RXR-specific agonist LGD1069 was ineffective at inducing differentiation or inhibiting proliferation, but showed marked synergism with RAR-specific agonists with respect to inhibiting proliferation. These data suggest that the effects of 9-cis RA are mediated via both RXR-homodimers and heterodimers. However, combinations of RAR- and RXR-selective analogues were not as effective at promoting differentiation. This study indicates that different receptor mechanisms are involved in retinoid-induced differentiation and inhibition of proliferation in neuroblastoma cells.

Topics: Alitretinoin; Bexarotene; Cell Differentiation; Cell Division; Dimerization; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Survivin (SVV) is a family member of inhibitor of apoptosis proteins (IAPs) and its expression is cell cycle regulated. The gene is mapped to chromosome 17q25, the region of which is frequently gained in advanced stages of neuroblastoma (NBL). However, the role of SVV in NBL is poorly understood. Here we studied the clinical and biological role of SVV in NBL. A 1.9 kb SVV transcript was expressed in all of 9 NBL cell lines at higher levels than those in adult cancer cell lines. In 34 primary NBLs, high levels of SVV expression was significantly associated with age greater than 12 months (two sample t-test: P= 0.0003), advanced stages (P = 0.0136), sporadic tumors (P= 0.0027) and low levels of TrkA expression (P = 0.0030). In NBL cell lines, SVV mRNA expression was dramatically down-regulated in CHP134 and IMR32 cells undergoing apoptosis after treatment with all-trans retinoic acid (RA) or serum deprivation. It was only moderately decreased in cells (SH-SY5Y and CHP901) undergoing RA-induced differentiation. On the other hand, in proliferating NBL cells or RA-treated SK-N-AS line which is refractory to RA, the SVV mRNA remained at steady state levels or rather up-regulated. Furthermore, transfection of SVV into CHP134 cells induced remarkable inhibition of the RA-induced apoptosis. Collectively, our results suggest that high expression of SVV is a strong prognostic indicator for the advanced stage neuroblastomas, and that it could be one of the candidate genes for the 17q gain.

Topics: Apoptosis; Caspase 3; Caspases; Cell Survival; Chromosomes, Human, Pair 17; Enzyme Activation; Enzyme Induction; Humans; Infant; Infant, Newborn; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Neuroblastoma; Prognosis; Protein Biosynthesis; Proteins; Risk Factors; Survivin; Transfection; Tretinoin; Tumor Cells, Cultured

Some neuroblastoma cell lines change their neurotransmitter phenotype from noradrenergic to cholinergic under retinoic acid treatment. Such "neurotransmitter switch" seems to be a consequence of changes in the expression and activity of the biosynthetic machinery for both neurotransmitters. In this study, we have characterized this "neurotransmitter switch" induced by retinoic acid in a human neuroblastoma cell line (NB69) showing catecholaminergic characteristics. Retinoic acid treatment reduced tyrosine hydroxylase activity and noradrenaline levels in NB69 cells but did not modify the expression of this enzyme. Moreover, the calcium-dependent release of [(3)H]noradrenaline in control cells was highly reduced by retinoic acid treatment. On the other hand, NB69 cells treated with retinoic acid enhanced the expression of choline acetyltransferase and acquired the capability to release [(3)H]acetylcholine in a calcium-dependent way. In addition, we found that the expression of the vesicular monoamine transporter 2 (VMAT2) and the vesicular acetylcholine transporter (VAChT) was increased in those cells treated with retinoic acid. Immunostaining revealed that retinoic acid treatment changed the cellular distribution of both vesicular monoamine transporter 2 and vesicular acetylcholine transporter. In conclusion, retinoic acid induces a noradrenergic to cholinergic switch in NB69 cells by acting at several levels of the neurotransmitter phenotypic expression.

Topics: Acetylcholine; Antineoplastic Agents; Brain Neoplasms; Carrier Proteins; Cell Differentiation; Cell Line; Humans; Immunohistochemistry; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Neuroblastoma; Neuropeptides; Norepinephrine; Parasympathetic Nervous System; Phenotype; Potassium; Sympathetic Nervous System; Tretinoin; Tyrosine 3-Monooxygenase; Vesicular Acetylcholine Transport Proteins; Vesicular Biogenic Amine Transport Proteins; Vesicular Monoamine Transport Proteins; Vesicular Transport Proteins

The CNS is enriched in phosphoinositide-specific phospholipase C (PLC) and in the G proteins linked to its activation. Although the regional distributions of these signaling components within the brain have been determined, neither their cell type-specific localizations (i.e., neuronal versus glial) nor the functional significance of their high expression has been definitively established. In this study, we have examined the expression of phosphoinositide signaling proteins in human NT2-N cells, a well characterized model system for CNS neurons. Retinoic acid-mediated differentiation of NT2 precursor cells to the neuronal phenotype resulted in five- to 15-fold increases in the expression of PLC-beta1, PLC-beta4, and Galpha(q/11) (the prime G protein activator of these isozymes). In contrast, the expression of PLC-beta3 and PLC-gamma1 was markedly reduced following neuronal differentiation. Similar alterations in cell morphology and in the expression of PLC-beta1, PLC-beta3, and Galpha(q/11) expression were observed when NT2 cells were differentiated with berberine, a compound structurally unrelated to retinoic acid. NT2-N neurons exhibited a significantly higher rate of phosphoinositide hydrolysis than NT2 precursor cells in response to direct activation of either G proteins or PLC. These results indicate that neuronal differentiation of NT2 cells is associated with dramatic changes in the expression of proteins of the phosphoinositide signaling system and that, accordingly, differentiated NT2-N neurons possess an increased ability to hydrolyze inositol lipids.

Topics: Animals; Antineoplastic Agents; Berberine; Brain; Cell Differentiation; Glioma; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Humans; Hydrolysis; Isoenzymes; Neuroblastoma; Neurons; Phenotype; Phosphatidylinositols; Phospholipase C beta; Rats; Signal Transduction; Stem Cells; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 microM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3.

Topics: Acetylcholine; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Choline O-Acetyltransferase; DNA Fragmentation; Free Radicals; Gene Expression Regulation, Enzymologic; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Mice; Neuroblastoma; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidative Stress; Penicillamine; Phenotype; Receptors, Cholinergic; RNA, Messenger; Septal Nuclei; Tretinoin; Tumor Cells, Cultured

Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neuronal)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against beta-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all-trans-RA as well as 13-cis-RA was more profound in S-type cells (up to 40% in primary cell lines). To elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32: p < 0.001; JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-beta/1, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGF-beta/1: p < 0.001; NF68: p < 0.01; PECAM-1 and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-beta/1: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.

Topics: Animals; Antigens, Surface; Cell Adhesion; Cell Differentiation; Cell Division; Humans; Mice; Mice, SCID; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Apoptosis Associated Tyrosine Kinase (AATYK), a novel protein recently isolated from differentiating 32D mouse myeloid cells, contains a putative tyrosine kinase domain and several binding motifs for src homology 2 (SH-2) and src homology 3 (SH-3) domain containing proteins. We observed that AATYK is expressed in different regions of the brain. Although it might play a role in normal nervous system development by modulating apoptosis, little is known regarding its function in the brain or its intracellular localization and kinase activity. Recognizing its homology with Insulin like growth factor-I (IGF-I) receptor (IGF-IR) and the critical role of IGF-I in neuronal survival, we hypothesized that AATYK plays an important role in neuronal differentiation/apoptosis. To test this hypothesis, we transfected the human adrenergic neuroblastoma (NB):SH-SY5Y cells with AATYK cDNA under a tetracycline-repressible promoter and established stable cell lines that readily express AATYK on removal of tetracycline. AATYK immunoprecipitated from these cell lysates is an active kinase. Indirect immunofluorescent staining of the clones revealed AATYK to be localized in the cytoplasm. By itself, AATYK overexpression for short duration (2-3 days) did not induce differentiation in the stable SH-SY5Y clones. On the other hand, overexpression for longer periods (7-8 days) per se, significantly (P<0.05-0.001) increased the percent of differentiated cells as well as the neurite length. AATYK-induced differentiation was in the same range as the differentiation induced by agents like all-trans retinoic acid (RA), 12-O-Tetradecanoyl phorbol 13-acetate (TPA) and IGF-I. In addition, AATYK significantly promoted the neuronal differentiation induced by these agents. Our results demonstrate for the first time that AATYK is an active, non-receptor, cytosolic kinase which induces neuronal differentiation and also promotes differentiation induced by other agents in the SH-SY5Y cells.

Topics: Animals; Apoptosis Regulatory Proteins; Brain; Cell Differentiation; Cell Size; Cytoplasm; Enzyme Induction; Humans; Insulin-Like Growth Factor I; Mice; Neurites; Neuroblastoma; Neurons; Nuclear Envelope; Protein-Tyrosine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids are involved in the regulation of development and differentiation in many tissues, including the nervous system, where they have been associated with some neurotransmitter systems. In the present study, we evaluated the effects of all-trans retinoic acid (RA) on the biosynthesis and secretion of neuropeptide Y (NPY), a widely expressed neuroregulatory peptide. The SH-SY5Y human neuroblastoma cell line has been used as the in vitro model system. Treatment with 10 microM RA induced a marked decrease in NPY gene expression after as little as 3-6 h of incubation and resulted in its almost complete suppression at 12-24 h and after a 6-day differentiating treatment. The NPY content in cell extracts and the NPY secreted and accumulated in the culture medium were also reduced by exposure to 10 microM RA at 12 and 24 h and at 6 days. Moreover, RA treatment for 6 days, but not for 24 h, resulted in a marked stimulation of proNPY processing to mature NPY. The presence of negative retinoic acid-response elements in the human NPY promoter (up to -1078 bp) was excluded by a computer search. When SH-SY5Y cells were treated simultaneously with 20 nM TPA and 10 microM RA for 24 h, the marked stimulatory effect of TPA alone was completely suppressed. These observations suggest that the expression of NPY in SH-SY5Y human neuroblastoma cells is negatively regulated by RA at the level of gene expression, probably by mechanisms involving the interaction of activated RARs with transcription factors (such as AP-1).

Topics: Blotting, Northern; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Neuropeptide Y; Protein Precursors; Protein Processing, Post-Translational; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Cellular adhesion to the extracellular matrix is mediated by a diverse class of alpha/beta heterodimeric receptors known as integrins, which transduce signals to activate multiple intracellular signal transduction pathways within the cells. The signaling pathway linking integrins to mediate neuronal process outgrowth is not well understood. Here, we have provided evidence that intracellular signaling by the alpha(1)beta(1) integrin-induced activation of cyclin-dependent kinase 5 (cdk5) is involved in neurite outgrowth and human neurofilament protein H (hNF-H) Lys-Ser-Pro (KSP) tail domain phosphorylation in differentiated human SH-SY5Y cells. The integrin alpha(1) and beta(1) monoclonal antibodies and BL-1, a specific cdk5 inhibitor, inhibited these effects. We also demonstrated that cdk5 activity and hNF-H KSP tail domain phosphorylation were increased in cdk5/p35 and hNF-H tail domain co-transfected HEK293 cells grown on laminin. This increased hNF-H tail domain phosphorylation was triggered by cdk5 activation. Taken together, these results indicated that cdk5 may play an important role in promoting neurite outgrowth and hNF-H tail KSP domain phosphorylation through the integrin alpha(1)beta(1) signaling pathway.

Topics: Antibodies; Bacterial Outer Membrane Proteins; Cell Differentiation; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Extracellular Matrix; Humans; Integrin alpha1beta1; Integrins; Laminin; Lipoproteins; Neurites; Neuroblastoma; Neurofilament Proteins; Peptide Fragments; Phosphorylation; Protein Structure, Tertiary; RNA, Messenger; Signal Transduction; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Topics: Apoptosis; Brain-Derived Neurotrophic Factor; Caspases; Cell Cycle; Cell Differentiation; Cell Division; Cell Survival; Humans; In Situ Nick-End Labeling; Kinetics; Microtubule-Associated Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; tau Proteins; Tretinoin; Tumor Cells, Cultured

The effect of tumor necrosis factor-alpha (TNF-alpha) on neuronal viability has been investigated in the SK-N-BE neuroblastoma cell line. These cells undergo differentiation upon chronic treatment with retinoic acid. Exposure of SK-N-BE cells to TNF-alpha produced a proliferative response in undifferentiated cells, whereas a reduced cell number was observed in retinoic acid (RA)-differentiated cultures. This biphasic response may be related to the different expression of TNF-alpha receptors (TNFRs); a significant increase in the density of TNFR1 was in fact observed following RA-induced differentiation. Under these conditions, a pronounced increase in the formation of ceramide-1-phosphate (which was prevented by the selective inhibitor of phosphatidylcholine-specific phospholipase C, D609) and an activation of caspase-3 upon TNF-alpha challenge were evident. Selective blockade of each TNFR subtype allowed a more detailed analysis of the effect observed. Preincubation with an anti-TNFR1 antibody prevented the cytotoxic effect of TNF-alpha in RA-differentiated SK-N-BE cells, whereas the anti-TNFR2 antibody blocked the proliferative activity of the cytokine in undifferentiated cultures.

Topics: Antigens, CD; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cell Differentiation; Cell Division; Ceramides; Gene Expression Regulation; Humans; Kinetics; Neuroblastoma; Neurons; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell's proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA alpha-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell's proliferative status.

Topics: Acetylcholine; Animals; Aphidicolin; Carrier Proteins; Cell Differentiation; Cell Division; Choline; Choline O-Acetyltransferase; DNA Polymerase III; Drug Synergism; Enzyme Inhibitors; Membrane Transport Proteins; Mice; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Telomerase is the enzyme that is responsible for maintaining telomere length in human germ cells, tumor cells, and immortalized cells. Its specific role in the immortilization process is unknown. This study was performed to determined whether the level of telomerase activity in human neuroblastoma cell lines correlated with their level of differentiation. We proposed that as neuroblastoma cells differentiated into more mature or benign cells, the levels of telomerase expression would decrease.. Two human neuroblastoma cells lines, SK-N-AS and SK-N-DZ, were differentiated using retinoic acid. These cells were assayed for telomerase activity by the telomere repeat amplification protocol (TRAP) before, during, and after treatment with retinoic acid for 8 days. Untreated cells were used for control and were compared to the retinoic acid-treated cells. Differentiation of the cell lines was confirmed by assaying expression of ret mRNA using the reverse-transcriptase polymerase chain reaction (RT-PCR) and gel electrophoresis of the radiolabeled products.. No statistical difference in telomerase activity was noted between control and treated groups.. While telomerase activity has been shown by others to correlate with tumor aggressiveness in human neuroblastoma cells, the mechanism that is involved appears to be separate from cellular differentiation.

Topics: Cell Differentiation; Drosophila Proteins; Humans; Neuroblastoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Reference Values; RNA, Messenger; Telomerase; Tretinoin; Tumor Cells, Cultured

Recent clinical studies have shown that inorganic arsenic trioxide (As(2)O(3)) at low concentrations induces complete remission with minimal toxicity in patients with refractory acute promyelocytic leukemia (APL). Preclinical studies suggest that As(2)O(3) induces apoptosis and possibly differentiation in APL cells. Like APL cells, neuroblastoma (NB) cells are thought to be arrested at an early stage of differentiation, and cells of highly malignant tumors fail to undergo spontaneous maturation. Both APL and NB cells can respond with differentiation to retinoic acid (RA) treatment in vitro and probably also in vivo. For that reason we investigated the effect of As(2)O(3) alone and in combination with RA on NB cell lines. In vitro, the number of viable NB cells was reduced at As(2)O(3) concentrations around 1 microM after 72 h exposure. The IC50 in six different cell lines treated for 3 days was in the 1.5 to 5 microM concentration interval, the most sensitive being SK-N-BE(2) cells derived from a chemotherapy resistant tumor. The combined treatment with RA (1 and 3 microM) showed no consistent additional effect with regard to induced cell death. The effect of As(2)O(3) on NB cell number involved As(2)O(3)-induced apoptotic pathways (decreased expression of Bcl-2 and stimulation of caspase-3 activity) with no clear evidence of induced differentiation. The in vivo effect of As(2)O(3) on NB growth was also investigated in nude mice bearing tumors of xenografted NB cells. Although tumor growth was reduced by As(2)O(3) treatment, complete remission was not achieved at the concentrations tested. We suggest that As(2)O(3), in combination with existing treatment modalities, might be a treatment approach for high risk NB patients.

Topics: Antigens, Differentiation; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase 3; Caspase Inhibitors; Caspases; Cell Differentiation; Cell Division; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Neoplasm Transplantation; Neuroblastoma; Oxides; RNA, Messenger; Tetrazolium Salts; Thiazoles; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours.

Topics: Antineoplastic Agents; Blotting, Western; Cell Differentiation; Child, Preschool; Female; Ganglioneuroblastoma; Ganglioneuroma; Gene Expression Regulation, Neoplastic; High Mobility Group Proteins; HMGA1a Protein; Humans; Immunohistochemistry; Infant; Male; Neoplasm Proteins; Neuroblastoma; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carboplatin; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Etoposide; Fenretinide; Flow Cytometry; Free Radicals; Humans; Neuroblastoma; Retinoids; Time Factors; Tretinoin; Tumor Cells, Cultured

Neuroblastoma is a common childhood cancer with a poor overall prognosis. Retinoic acids (RAs) have been studied as a potential therapy, showing promise in recurrent disease. The histone deacetylase inhibitor (HDACI) M-carboxycinnamic acid bishydroxamide (CBHA) is another potential therapy, which we recently described. Combinations of RAs and HDACIs currently under investigation display synergy in certain neoplasms. In this study, we evaluate the effect of combinations of RAs and HDACIs on human neuroblastoma cells.. Established cell lines were cultured in increasing concentrations of HDACIs, RAs, and combinations thereof. Following exposure, viable cell number was quantified by trypan blue dye exclusion on a hemacytometer. Cell cycle analysis was performed by propidium iodide staining and FACS.. All assayed HDACIs and RAs decreased viable cell number. Lower concentrations of each agent were effective when the two were combined. The primary reason for decreased cell number appears to be apoptosis following HDACI exposure and G1 arrest following RA exposure. Both effects are seen with cotreatment. Caspase inhibition abrogates the apoptotic response.. CBHA causes apoptosis of human neuroblastoma in vitro, an effect that can add to the effects of RA. HDACIs and RAs inhibit neuroblastoma in significantly lower concentrations when used together than when used individually. Combination therapy may improve the ultimate efficacy while reducing the side effects of these agents in clinical use.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase Inhibitors; Cell Division; Cinnamates; G1 Phase; Histone Deacetylase Inhibitors; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

High-dose, pulse-13-cis-retinoic acid (13-cis-RA) given after intensive cytotoxic therapy improves event-free survival for high-risk neuroblastoma (NB), but more than 50% of patients have tumor recurrence.. We conducted multistep selection for resistance to all-trans-retinoic acid (ATRA) in NB cell lines with (SMS-KCNR and LA-N-5) or without (SMS-LHN) MYCN genomic amplification.. After 12 exposures to 10 microM ATRA, the two MYCN-amplified cell lines (KCNR 12X RR and LA-N-5 12X RR) showed partial resistance to the cytostatic/differentiation effects of ATRA; complete resistance was seen in LHN 12X RR. ATRA-selected cells showed general RA resistance (cross-resistance to 13-cis-RA). Transient (KCNR 12 X RR, LA-N-5 12X RR) or sustained (LHN 12X RR) novel overexpression of c-myc was associated with RA resistance. RA-insensitive overexpression of MYCN by transduction in SMS-LHN also conferred RA resistance. Both parental and RA-resistant lines showed 2-4 logs of cell kill in response to N-(4-hydroxyphenyl)retinamide (4- HPR, fenretinide). Compared to parental lines, 4-HPR achieved 1-3 log greater cell kills in RA-resistant LHN 12X RR, LA-N-5 12X RR, KCNR 12X RR, and MYCN-transduced SMS-LHN or SK-N-RA. NB cell lines (n = 26) from 21 different patients showed that 16 of 26 (62%) were sensitive to 4-HPR (LC(90) < 10 microM), including lines established at relapse after myeloablative and/or 13-cis-RA therapy.. Thus, RA-resistant NB cell lines can be sensitive (and in some cases collaterally hypersensitive) to 4-HPR.

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Fenretinide; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Neuroblastoma LAN-5 cells exposed to retinoic acid cease to multiply and extend neurite outgrowths acquiring a neuronal phenotype. We now report that protein kinase C-theta; (PKC-theta;) isozyme is involved in this differentiation process due to the following findings: (i) PKC-theta; is expressed by LAN-5 cells as a nuclear and perinuclear protein; (ii) cell stimulation with retinoic acid promotes in a large increase in the expression level of the kinase and its intracellular redistribution; and (iii) a PKC-theta; antisense oligonucleotide reduces at the same time the expression level of the kinase and the cell response to retinoic acid. Altogether these data are consistent with a specific role played by PKC-theta; in the differentiation program of neuronal cells.

Topics: Cell Differentiation; Cell Nucleus; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Neuroblastoma; Oligodeoxyribonucleotides, Antisense; Polymerase Chain Reaction; Protein Kinase C; Protein Kinase C-theta; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Previous work has shown that neuroblastoma cells secrete IGFBP-2, -4 and -6 and that expression of these proteins is regulated by retinoic acid (at-RA) which promotes differentiation in these cells. Other agents also induce differentiation of neuroblastoma cells: these include the 9- cis and 13- cis isomers of at-RA, 1,25 dihydroxy- vitamin D3 (VD3), triidothyronine (T3) and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Nine- cis and 13- cis isomers of at-RA increased IGFBP-6 expression, but decreased IGFBP-2 and IGFBP-4. VD3 stimulated IGFBP-6 and IGFBP-2 expression, whereas T3 inhibited IGFBP-6 expression without affecting IGFBP-2. TPA markedly enhanced expression of all three IGFBPs produced by SK-N-SH cells. Since IGFBP-6 secretion is associated with the arrest of proliferation in neuroblastoma cells and is regulated by the combined actions of differentiation factors, we subcloned the proximal promoter of human IGFBP-6 (nt -766/+1) into a pCAT expression vector so as to examine modulation of its transcriptional activity. VD3 and TPA were capable of stimulating promoter activity, T3 depressed it and at-RA and its 9- cis and 13- cis isomers had no effect. These results confirm the high sensitivity of IGFBP-6 expression to these differentiation agents, essentially at transcriptional level.

Topics: Antineoplastic Agents; Base Sequence; Blotting, Northern; Blotting, Western; Carcinogens; Cell Division; Chloramphenicol O-Acetyltransferase; Cholecalciferol; Cloning, Molecular; Down-Regulation; Humans; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 6; Isomerism; Luciferases; Models, Genetic; Molecular Sequence Data; Neuroblastoma; Plasmids; Promoter Regions, Genetic; RNA; Sequence Analysis, DNA; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Up-Regulation

In order to determine how glycosylation changes associated with cellular differentiation may be influenced by the basal cellular sialylation potential, the effect of retinoic acid (RA)-induced differentiation was investigated in neuroblastoma cells expressing differing levels (and activities) of the alpha2,6(N) sialyltransferase (ST6N) enzyme. The increase in ST activity was proportional to the basal cellular sialylation potentials with the high activity clones showing the greatest increase. This was paralleled by an up-regulation of the level of overall sialoglycoprotein glycosylation level. An increase in the levels of the polysialic acid (PSA) epitope was associated with a parallel increase in the levels of the neural cell adhesion molecule (NCAM) protein backbone although there was no overall change in the PSA:NCAM ratio following RA treatment.

Topics: Animals; beta-D-Galactoside alpha 2-6-Sialyltransferase; Cell Differentiation; Enzyme Induction; Epitopes; Neural Cell Adhesion Molecules; Neuroblastoma; Rats; Recombinant Proteins; Sialic Acids; Sialyltransferases; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Brain Neoplasms; Canada; Child; Chromosomes, Human, Pair 1; Congenital Abnormalities; Gene Deletion; Humans; Leukemia, Promyelocytic, Acute; Neoplasms; Neuroblastoma; Occupations; Tretinoin

The mammalian achaete-scute homologue, MASH-1, is crucial for early development of the sympathetic nervous system and is transiently expressed in sympathetic neuroblasts during embryogenesis. Here we report that the human homologue (HASH-1) was expressed in all analyzed cell lines (6/6) derived from the sympathetic nervous system tumor neuroblastoma. The majority of small-cell lung carcinoma (4/5) cell lines tested expressed HASH-1, while other nonneuronal/non-neuroendocrine cell lines were negative. Induced differentiation of neuroblastoma cells resulted in HASH-1 downregulation. This occurred concomitant with induction of neurite outgrowth and expression of the neuronal marker genes GAP-43 and neuropeptide Y. Constitutive expression of exogenous HASH-1 did not alter the capacity of the neuroblastoma cells to differentiate in response to differentiation-inducing agents. It is concluded that moderate HASH-1 expression does not compromise the capacity of these cells to differentiate.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Northern; Carcinoma, Small Cell; Carrier Proteins; Cell Differentiation; Cell Size; DNA-Binding Proteins; Down-Regulation; GAP-43 Protein; Growth Substances; Humans; Lung Neoplasms; Membrane Proteins; Neurites; Neuroblastoma; Neurons; Neuropeptide Y; Receptor, trkA; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

Topics: Adaptor Proteins, Signal Transducing; ATPases Associated with Diverse Cellular Activities; Carrier Proteins; DNA-Binding Proteins; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Isomerism; LIM Domain Proteins; Neuroblastoma; Nuclear Proteins; Nuclear Receptor Co-Repressor 2; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; Repressor Proteins; Retinoid X Receptors; Transcription Factors; Tretinoin; Tripartite Motif-Containing Protein 28; Tumor Cells, Cultured

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.

Topics: Antineoplastic Agents; Astrocytoma; Biological Transport; Cell Differentiation; Cells, Cultured; Humans; Inositol; Myo-Inositol-1-Phosphate Synthase; Neuroblastoma; Neurons; Phlorhizin; Stem Cells; Teratocarcinoma; Tretinoin; Water; Water-Electrolyte Balance

Ras-GRF, a neuron-specific Ras exchange factor of the central nervous system, was transfected in the SK-N-BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras-GRF expression with a concomitant high increase in the level of active (GTP-bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal-like phenotype more rapidly than the parental ones. Cells expressing Ras-GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras-GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras-GRF and/or Ras-pathway may have a modulatory effect on IRK1 channel activity.

Topics: Antineoplastic Agents; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle Proteins; Cell Differentiation; Cell Division; Drosophila Proteins; Electrophysiology; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; Humans; Ion Channel Gating; Neuroblastoma; Neurons; Phosphoprotein Phosphatases; Potassium Channels; Potassium Channels, Inwardly Rectifying; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; ras Guanine Nucleotide Exchange Factors; ras-GRF1; Receptor Protein-Tyrosine Kinases; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 (ERK1) and extracellular signal-regulated kinase 2 (ERK2), induced by resveratrol, a natural antioxidant present in grapes and wine, has been studied in vitro on undifferentiated and differentiated (induction by retinoic acid) SH-SY5Y human neuroblastoma cells. In undifferentiated cells resveratrol 1 microM induced phosphorylation of ERK1 and ERK2, which was already evident at 2 min, peaked at 10 min and persisted at 30 min. A wide range (from 1 pM to 10 microM) of resveratrol concentrations were able to induce phosphorylation of ERK1 and ERK2, while higher concentrations (50-100 microM) inhibited MAP kinases phosphorylation. In retinoic acid (RA) differentiated cells resveratrol (1 microM) induced an evident increase in ERK1 and ERK2 phosphorylation. This study demonstrates that resveratrol, even at very low concentrations, may have a biological effect on neuron-like cells.

Topics: Antioxidants; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Enzyme Activation; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neuroblastoma; Osmolar Concentration; Phosphorylation; Resveratrol; Stilbenes; Time Factors; Tretinoin; Tumor Cells, Cultured

Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti-tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP-1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors. TSP-1-adherent U-251MG cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins alpha3beta1 and alpha5beta1 in clusters in lamellipodia and filopodia. The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36. Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins alpha3beta1 and alpha5beta1 mediated TSP-1 attachment of SK-N-SH cells, and integrins alpha3beta1, alpha5beta1, and alphavbeta3 mediated TSP-1 attachment of U-251MG cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1. Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells.

Topics: Astrocytes; Astrocytoma; Brain; CD36 Antigens; Cell Adhesion; Cell Differentiation; Chondroitin ABC Lyase; Chondroitin Sulfates; Cytoskeleton; Endothelium; Ganglioneuroblastoma; Ganglioneuroma; Glioblastoma; Heparin; Humans; Integrin alpha3beta1; Integrins; Neuroblastoma; Neurons; Oligopeptides; Peptide Fragments; Polysaccharide-Lyases; Receptors, Fibronectin; Recombinant Proteins; Thrombospondin 1; Tretinoin; Tumor Cells, Cultured

This study examines the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on SH-SYSY human neuroblastoma cells cultured in the presence of medium containing varying concentrations of calcium (0.1, 0.9, 1.4, 1.8 mM). Pyruvate kinase activity was assayed in SH-SY5Y cells incubated in variable calcium medium with or without 1, 10 or 100 nM 1,25(OH)2D3 for 48 h. The enzyme levels showed a significant increase in comparison with control, when the cells were incubated with 100 nM hormone in the presence of 0.1 mM calcium, while pyruvate kinase activity decreased, when the cells were treated with 100 nM 1,25(OH)2D3 in the presence of 1.8 mM calcium. The proliferative activity of SH-SY5Y was dependent on the extracellular concentration of calcium, being the highest at 1.8 mM calcium and completely absent at 0.1 mM calcium. In the presence of 1,25(OH)2D3, at the three concentrations used and after 48 h incubation, a significant decrease in cell number was always observed, without a direct correlation between 1,25(OH)2D3 effect and calcium concentration in the medium. [3H]Thymidine incorporation in SH-SY5Y cells significantly increased in comparison with control, when the 48 h incubation with 1, 10 or 100 nM 1,25(OH)2D3 was carried out in the presence of 0.1 mM calcium, while, at the other calcium concentrations, the hormone did not cause any significant change in this parameter. The treatment of SH-SYSY cells with 1 nM 1,25(OH)2D3 for 48 h did not affect cell morphology, when 0.1 mM calcium was present, while, in the medium containing 1.8 mM calcium, the treated cells showed a slight trend to differentiation. The differentiating effect of 10 microM all-trans retinoic acid, even if incomplete after 48 h treatment, was only observed in the cultures grown in 1.8 mM calcium, in comparison with those maintained in 0.1 mM calcium.

Topics: Calcitriol; Calcium; Cell Differentiation; Cell Division; DNA; Dose-Response Relationship, Drug; Humans; Neuroblastoma; Pyruvate Kinase; Tretinoin; Tumor Cells, Cultured

HMGI-C and HMGI(Y) are architectural DNA-binding proteins that participate in the conformational regulation of active chromatin. Their pattern of expression in embryonal and adult tissues, the analysis of the "pygmy" phenotype induced by the inactivation of the HMGI-C gene, and their frequent qualitative or quantitative alteration in experimental and human tumors indicate their pivotal role in the control of cell growth, differentiation, and tumorigenesis in several tissues representative of the epithelial, mesenchymal, and hematopoietic lineages. In contrast, very little information is available on their expression and function in neural cells. Here, we investigated the expression of the HMGI(Y) and HMGI-C genes in neuroblastoma (NB), a tumor arising from an alteration of the normal differentiation of neural crest-derived cells and in embryonal and adult adrenal tissue. Although HMGI(Y) is constitutively expressed in the embryonal and adult adrenal gland and in all of the NB cell lines and ex vivo tumors examined, its regulation appears to be associated to growth inhibition and differentiation because we observed that HMGI(Y) expression is reduced by retinoic acid (RA) in several NB cell lines that are induced to differentiate into postmitotic neurons, whereas it is up-regulated by RA in cells that fail to differentiate. Furthermore, the decrease of HMGI(Y) expression observed in RA-induced growth arrest and differentiation is abrogated in cells that have been made insensitive to this drug by NMYC overexpression. In contrast, HMGI-C expression is down-regulated during the development of the adrenal gland, completely absent in the adult individual, and only detectable in a subset of ex vivo NB tumors and in RA-resistant NB cell lines. We provide evidence of a causal link between HMGI-C expression and resistance to the growth arrest induced by RA in NB cell lines because exogenous HMGI-C expression in HMGI-C-negative and RA-sensitive cells is sufficient to convert them into RA-resistant cells. Therefore, we suggest that HMGI-C and HMGI(Y) may participate in growth- and differentiation-related tumor progression events of neuroectodermal derivatives.

Topics: Adrenal Glands; Adult; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; High Mobility Group Proteins; HMGA1a Protein; HMGA2 Protein; Humans; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and protection against neuronal damage. We addressed whether BDNF might promote survival and chemoprotection in neuroblastoma (NB) using a drug-sensitive human NB cell line. All-trans-retinoic acid (ATRA) induces a striking phenotypic differentiation of NB1643 cells, and exogenous BDNF treatment promotes survival of these differentiated cells. ATRA induces TRKB expression, and exogenous BDNF stimulates both autophosphorylation of TRKB and induction of the immediate early gene, FOS, in these cells. BDNF mRNA is expressed in NB1643 cells. Because the time course of TRKB induction closely parallels phenotypic differentiation of these cells, it seems probable that ATRA induces differentiation of NB1643 cells by establishing an autocrine loop involving BDNF and TRKB. Exogenous BDNF treatment resulted in a further increase in neurite outgrowth, which again suggests that an autocrine loop is involved in differentiation of NB1643 cells in response to ATRA. We then tested whether BDNF might afford drug resistance in NB and found that BDNF does indeed protect in this NB model against cisplatin, a DNA-damaging agent actually used in the treatment of NB.

Topics: Antineoplastic Agents; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Survival; Cisplatin; Drug Interactions; Humans; Neuroblastoma; Oncogene Proteins v-fos; Phenotype; Phosphorylation; Tretinoin; Tumor Cells, Cultured

We established a unique parental neuroblastoma cell line, NUB-7, which mimics the bipotentiality of neuroblastoma in vivo along neuronal and Schwann cell lineages following dibutyryl cAMP and retinoic acid treatments, respectively. Differential display identified a putative novel zinc finger gene as a potential differentiation-responsive gene coincident with retinoic acid treatment of NUB-7. This cDNA clone, now designated zf5-3, was mapped to chromosome 19 using somatic cell hybrids, and a larger cDNA clone further localized this gene to band 13.1-13.2 by fluorescent in situ hybridization. zf5-3 possesses 4 characteristic zinc finger DNA-binding motifs as determined by its nucleic acid and proposed amino acid sequence. Expression of zf5-3 is restricted to fetal neuronal, hepatic and renal tissues and their tumor-derived cell lines, including 8/9 neuroblastomas and 2/2 malignant rhabdoid tumors of kidney. The restricted expression in the kidney of zf5-3 to collecting tubules and ureter epithelium is suggestive of an ectodermal histogenesis of malignant rhabdoid tumors of kidney. During development of the fetal human brain, high levels of zf5-3 mRNA are restricted to the mitotically active, undifferentiated neuroblasts. Morphological evidence of overt differentiation was generally accompanied by a marked loss in zf5-3 expression. Therefore, the neuronal tissue expression profile and the down-regulation coincident with retinoic acid-induced neuroblastoma maturation implicate zf5-3 as a potential mediator of their differentiation.

Topics: Amino Acid Sequence; Base Sequence; Brain; Bucladesine; Cell Differentiation; Chromosome Mapping; Chromosomes, Human, Pair 19; DNA-Binding Proteins; Fetus; Gene Expression Regulation; Gene Expression Regulation, Developmental; Gene Library; Humans; Kidney; Kidney Neoplasms; Molecular Sequence Data; Neuroblastoma; Neurons; Organ Specificity; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Zinc Fingers

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Apoptosis; DNA Fragmentation; Drug Resistance; Etoposide; Fluorescent Antibody Technique; Gamma Rays; Humans; Neuroblastoma; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Cyclin-dependent kinase-5 (CDK-5) has been shown to play important roles in neuronal development and neurogenesis. In vitro studies indicate a role of CDK-5 in phosphorylation of neurofilaments (NFs). In this study, we have chosen the human neuroblastoma cell line SHSY5Y as a model system to study the in vivo phosphorylation of NF proteins by CDK-5. Upon differentiation of SHSY5Y cells with retinoic acid, we found that the phosphorylation of high molecular mass (NF-H) and medium molecular mass (NF-M) NFs increased, whereas the CDK-5 protein level and kinase activity were unaffected. The role of CDK-5 in the phosphorylation of cytoskeletal proteins was studied by using antisense oligonucleotides (ONs) to inhibit the expression of the CDK-5 gene. We found that inhibition of CDK-5 levels by antisense ON treatment resulted in a decrease in phosphorylation of NF-H that correlated with a decline in neurite outgrowth. These results demonstrate that CDK-5 is a major proline-directed kinase phosphorylating the human NF-H tail domain.

Topics: Cell Differentiation; Cell Line; Cyclin-Dependent Kinases; Fluorescent Antibody Technique; Humans; Isoenzymes; Microscopy, Confocal; Molecular Weight; Neuroblastoma; Neurofilament Proteins; Oligonucleotides, Antisense; Phosphoproteins; Phosphorylation; Tretinoin

The modulation of herg gene and HERG currents (I(HERG)) was studied in SH-SY5Y neuroblastoma (NB) cells treated with all-trans-retinoic acid (RA) in the absence or presence of the neurotrophin brain-derived neurotrophic factor (BDNF). Both treatments produced a strong increase in the percentage of cells differentiated along the neuronal pathway, with an orientation to a cholinergic phenotype, while a minority of cells displayed a glial phenotype particularly evident after long-term exposure to the inducers. Differentiation of NB cells was accompanied by an increase in herg gene transcription, which attained its maximum after 6 days of treatment with RA and was not further increased by BDNF. This effect evidently reflected on HERG currents: In fact, RA produced an increase in HERG current density which was strongly potentiated by BDNF. Moreover, RA treatment affected the biophysical properties of I(HERG), inducing an increase in the deactivation time constant and a left shift of the activation curve. These effects were not substantially affected by BDNF. This modulation of I(HERG) influenced the value of the resting potential (V(REST)), which resulted significantly hyperpolarized in (RA with or without BDNF)-treated cells. Interestingly, these effects were absent in the glial population, which prevailed in cultures after long-term exposure to the inducers. On the whole, we demonstrate that besides expressing IRK currents, NB cells display another strategy to hyperpolarize their V(REST), based on the appropriate modulation of HERG currents. Different from what happens in normal neuroblast development, the latter are never lost by cancer cells despite the progression of these cells along the neuronal differentiative pathway, raising intriguing questions about the role of HERG currents in tumour behavior.

Topics: Acetylcholine; Brain-Derived Neurotrophic Factor; Cation Transport Proteins; Cell Differentiation; DNA-Binding Proteins; Drug Synergism; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Humans; Ion Transport; Neoplasm Proteins; Neuroblastoma; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Voltage-Gated; Trans-Activators; Transcriptional Regulator ERG; Tretinoin; Tumor Cells, Cultured

The expression of the endogenous CRF gene was examined in the human neuroblastoma cell line, BE(2)-M17. In this cell line, treatment with retinoic acid induces CRF mRNA transcription. We examined the requirement for the POU transcription factor, Bm-2, for this response. We confirmed that Bm-2 is expressed in retinoic acid-induced BE(2)-M17 cells. Expression of antisense Bm-2 message aborted the retinoic acid-mediated induction of CRF transcription. However, overexpression of Bm-2 was not sufficient for CRF expression in the absence of retinoic acid. These experiments support the hypothesis that Bm-2 is an intermediary for retinoic acid-induced CRF expression.

Topics: Binding Sites; Blotting, Southern; Corticotropin-Releasing Hormone; DNA; Gene Expression; Genes, Reporter; HeLa Cells; Homeodomain Proteins; Humans; Mutagenesis, Site-Directed; Neuroblastoma; Neurons; POU Domain Factors; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactose is an experimental model commonly used for in vitro evaluation of typical neuronal alterations observed in diabetes mellitus. In the present study, we observed that 2 weeks of exposure to high carbohydrate concentrations caused both a significant impairment in neurite formation induced by supplementation of retinoic acid or by subtraction of fetal calf serum to the culture medium and a marked reduction in Na(+)-K(+)-ATPase activity. However, only the exposure to high millimoles of glucose caused an enhancement of mono-ADP-ribosylation, typical of diabetes mellitus, affecting at least five proteins. The concomitant exposure to high glucose and to silybin, a mono-ADP-ribosylation inhibitor, normalized the extent of ADP-ribosylation of the five proteins and counteracted the inhibitory effects of high glucose on Na(+)-pump activity and on neuritogenesis. Conversely, the supplementation of silybin did not prevent fructose and galactose inhibitory effects on Na(+)-pump activity and neurite formation. These data confirm those of previous reports suggesting a link between excessive protein mono-ADP-ribosylation and the onset of diabetic complications such as diabetic neuropathy.

Topics: Adenosine Diphosphate Ribose; Culture Media, Serum-Free; Diabetes Mellitus; Glucose; Humans; Kinetics; NAD; Neurites; Neuroblastoma; Sodium-Potassium-Exchanging ATPase; Tretinoin; Tumor Cells, Cultured

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

Topics: Astrocytes; Astrocytoma; Blotting, Western; Brain Neoplasms; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Glioblastoma; Humans; Membrane Proteins; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Presenilin-1; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Teratocarcinoma; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The retinoic acid (RA) receptor beta isoform (RARbeta) plays an important role in RA-induced differentiation of human neuroblastoma. In this study we show that insulin-like growth factor 1 (IGF-1) and tetradecanoyl phorbol acetate (TPA) induce RARbeta gene expression in neuroblastoma SH-SY5Y cells. IGF-1 and TPA caused a marked induction of RARbeta2 promoter activity and had a synergistic effect with RA that also upregulates transcription. The effect of RA is mediated by two RA responsive elements (RAREs), whereas the IGF-1 and TPA actions are independent of the RAREs and map to sequences that overlap the TATA box. These results suggest that the signaling pathways stimulated by TPA and IGF-1 could modify the components assembled at the core RARbeta2 promoter and activate transcription. Expression of RasVal12 mimics the effect of IGF-1 and TPA on the promoter, and a dominant negative Ras mutant abrogates activation. A dominant negative Raf also blocks activation showing that the Ras-Raf pathway mediates stimulation of the RARbeta2 promoter. Our results show that neuronal differentiation induced by non-retinoid agents that activate Ras is accompanied by increased transcription of the RARbeta gene.

Topics: Carcinogens; Cell Differentiation; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Neuroblastoma; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH-SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA-predifferentiated SH-SY5Y cells with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), or neurotrophin 4/5 (NT-4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal-regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT-3, or NT-4/5, but not NGF, promotes cell survival and neurite outgrowth in serum-free medium. The mitogen-activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF-induced neurite outgrowth and growth-associated protein-43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3-kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.

Topics: Brain-Derived Neurotrophic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Survival; Chromones; Enzyme Inhibitors; Flavonoids; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Nerve Growth Factors; Neurites; Neuroblastoma; Neuroprotective Agents; Neurotrophin 3; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, Ciliary Neurotrophic Factor; Receptor, trkA; Receptor, trkC; Receptors, Nerve Growth Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Human catecholaminergic neuroblastoma cells (SH-SY5Y) have been widely used in different neurochemical investigations. Quite often these cells are induced to differentiation by various agents, such as staurosporine and retinoic acid. Interestingly, even though both staurosporine and retinoic acid induce similar morphological differentiation in SH-SY5Y cells, we found that these two groups of differentiated cells exhibited opposite vulnerability to harmful chemicals and physical insults. In the present study, cisplatin, 5-fluorouracil (5-FU), N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), 6-hydroxydopamine (6-OHDA), and gamma-radiation were used to assess the tolerance of the differentiated cells. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Staurosporine-treated SH-SY5Y cells were more sensitive to these toxic insults than the untreated controls. In contrast, retinoic acid-treated cells became more resistant to the same treatments. The expression of the proteins of the protooncogene Bcl-2 and the tumor suppressor gene p53 following staurosporine or retinoic acid treatment was assessed by Western blot and immunocytochemistry. Retinoic acid increased Bcl-2 and decreased p53 levels, whereas staurosporine decreased Bcl-2 and increased p53 levels. The opposite alteration of Bcl-2 (anti-apoptotic) and p53 (apoptotic) contents in SH-SY5Y cells with retinoic acid and staurosporine are attributed to the changes in cell vulnerability. These observations also indicate that caution should be taken when chemically induced differentiated neuroblastoma cells are to be used as an in vitro model for studying neuronal survival.

Topics: Benzylamines; Cell Differentiation; Cell Survival; Fluorouracil; Gamma Rays; Humans; Neuroblastoma; Neurotoxins; Oxidopamine; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The expression, cellular localization, and activation of the NF-kappaB/Rel transcription factors are altered during neuronal differentiation, but the significance is unclear. Here we investigate the requirement for NF-kappaB/Rel proteins in neuronal differentiation. SH-SY5Y neuroblastoma cells were induced to differentiate with retinoic acid (RA) or 12-O-tetradecanoylphorbol 13-acetate (TPA), and differentiation was demonstrated by morphological criteria and the enhanced expression of Bcl-2. NF-kappaB was transiently activated after the addition of the differentiation inducers before the morphological signs of differentiation and the enhanced Bcl-2 synthesis. The onset of NF-kappaB activation coincided with a significant reduction in the amount of only one of four NF-kappaB-inhibitory proteins examined (I-kappaBbeta). In contrast, NF-kappaB activation and the reduction in I-kappaBbeta failed to occur in SH-SY5Y cells transformed with I-kappaBalphaM, a dominant-negative inhibitor of NF-kappaB/Rel proteins. These I-kappaBalphaM-expressing cells failed to differentiate into neuronal cell types when treated with RA or TPA, and the increased Bcl-2 synthesis was blocked. Therefore, NF-kappaB/Rel proteins are required for neuronal differentiation of SH-SY5Y neuroblastoma cells.

Topics: Cell Differentiation; Gene Expression Regulation; Humans; I-kappa B Kinase; Neuroblastoma; Neurons; NF-kappa B; Nuclear Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-rel; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.

Topics: Antineoplastic Agents; Base Sequence; DNA Primers; Gene Expression; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Neuroblastoma; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma CHP100 cells were forced into apoptosis (programmed cell death, PCD) or necrosis by treatment with calcium chloride or sodium nitroprusside (a nitric oxide donor), respectively. Cellular luminescence, a marker of membrane lipid peroxidation, was increased by calcium but not by nitroprusside, and reached a maximum of 4-fold the control value 2 hours after treatment. The increase in luminescence was paralleled by increased 5-lipoxygenase (up to 250% of the control value) and decreased catalase (down to 50%) activity within the same time window. Consistently, incubation of CHP100 cells with inhibitors of 5-lipoxygenase (5,8,11,14-eicosatetraynoic acid and MK886) reduced light emission and PCD, whereas inhibition of catalase by 3-amino-1, 2,4-triazole enhanced both processes. Treatment of CHP100 cells with retinoic acid or cisplatin, unrelated PCD inducers reported to activate the lipoxygenase pathway, also gave enhanced light emission parallel to PCD increase. Altogether, these results suggest that cellular luminescence is an early marker of apoptotic, but not necrotic, program(s) involving generation of hydrogen peroxide and activation of 5-lipoxygenase.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Amitrole; Apoptosis; Arachidonate 5-Lipoxygenase; Catalase; Cisplatin; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Indoles; Leukotrienes; Lipid Peroxidation; Lipoxygenase Inhibitors; Luminescent Measurements; Necrosis; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The human neuroblastoma cell line SK-N-BE, after incubation with 10 microM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10-14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [3H]arginine into [3H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor NG-nitro-L-arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells.

Topics: Cell Differentiation; Enzyme Inhibitors; Humans; Keratolytic Agents; Neuroblastoma; NG-Nitroarginine Methyl Ester; Nitric Oxide; Tretinoin; Tumor Cells, Cultured

The electrophysiological properties of voltage-dependent outward currents were investigated under voltage-clamp conditions in the human neuroblastoma cell line SH-SY5Y before and after in vitro differentiation with retinoic acid, by using the whole cell variant of the patch-clamp technique. Voltage steps to depolarizing potentials from a holding level of -90 mV elicited, in both undifferentiated and differentiated cells, outward potassium currents that were blocked by tetraethylammonium, but were unaffected by 4-aminopyridine, cadmium, and by shifts of the holding potentials to -40 mV. These currents activated rapidly and inactivated slowly in a voltage-dependent manner. In undifferentiated cells the threshold for current activation was about -30 mV, with a steady-state half activation potential of 19.5 mV. Maximum conductance was 4.3 nS and mean conductance density was 0.34 mS/cm2. Steady-state half inactivation potential was -13.8 mV and approximately 10% of the current was resistant to inactivation. Both activation and inactivation kinetics were voltage dependent. In differentiated cells the threshold for current activation was about -20 mV, with a half potential for steady-state activation of 37.0 mV. Maximum conductance was 15.2 nS and mean conductance density was 0. 78 mS/cm2. Steady-state half inactivation potential was -9.7 mV and approximately 37% of the current was resistant to inactivation. Both activation and inactivation kinetics were voltage dependent. This diversity in potassium channel properties observed between undifferentiated and differentiated cells was related to differences in cell excitability. Under current-clamp conditions, the action potential repolarization rate in differentiated cells was about threefold faster than that of the abortive action potentials elicitable in undifferentiated cells. Furthermore, during prolonged stimulation, trains of spikes could be generated in some differentiated cells but not in undifferentiated cells.

Topics: Action Potentials; Cell Differentiation; Delayed Rectifier Potassium Channels; Humans; Ion Channel Gating; Ion Transport; Kinetics; Neoplasm Proteins; Neuroblastoma; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Voltage-Gated; Tetraethylammonium; Tretinoin; Tumor Cells, Cultured

1. SH-SY5Y human neuroblastoma cells were investigated with whole-cell and perforated patch recording methods. 2. Besides a quickly activating delayed rectifier channel and a HERG-like channel, a slowly activating potassium channel with biophysical properties identical to those of rat eag (r-eag) channels was detected, here referred to as h-eag. 3. h-eag shows a marked Cole-Moore shift, i.e. the activation kinetics become very slow when the depolarization starts from a very negative holding potential. In addition, extracellular Mg2+ and Ni2+ strongly slow down activation. 4. Application of acetylcholine induces a fast block of the current when recorded in the perforated patch mode. This block is presumably mediated by Ca2+, as about 1 microM intracellular Ca2+ completely abolished h-eag outward current. 5. When cells were grown in the presence of 10 microM retinoic acid in order to synchronize the cell line in the G1 phase of the cell cycle, h-eag current was reduced to less than 5 % of the control value, while the delayed rectifier channel was expressed more abundantly. Down-regulation of h-eag by long-term exposure to retinoic acid was paralleled by a right shift in the activation potential of HERG-like channels. 6. Acute application of 10 microM retinoic acid blocked the delayed rectifier channel but enhanced the h-eag current. 7. Thus, our results show that human neuroblastoma cells express in a cell cycle-dependent manner an [Mg2+]o-dependent potassium channel (h-eag) which is blocked by submicromolar concentrations of intracellular Ca2+.

Topics: Acetylcholine; Animals; Cation Transport Proteins; Cell Cycle; DNA-Binding Proteins; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Humans; Kinetics; Membrane Potentials; Models, Biological; Neuroblastoma; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Voltage-Gated; Rats; Time Factors; Trans-Activators; Transcriptional Regulator ERG; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) plays a major role in neuronal cell differentiation. Neuroblastoma cells differentiate in vitro by extending neurites and forming ganglion-like aggregates in response to RA. In the present study, we have examined a biological role(s) of DAN in the regulation of RA-mediated cellular differentiation in neuroblastoma cells. RTBM1 and SH-SY5Y cells undergo marked morphological changes associated with a remarkable induction of DAN gene expression when exposed to RA. By transfecting an expression vector harboring a rat DAN cDNA into SH-SY5Y cells, we have obtained two independent transfectants which express a large amount of DAN. The forced expression of DAN gene enhanced the neurite extension in the presence of RA, suggesting that DAN gene product might contain some regulatory role(s) in the RA-induced cellular differentiation in neuroblastoma cells.

Topics: Animals; Blotting, Northern; Cell Cycle Proteins; Cell Differentiation; Cytomegalovirus; Gene Expression; Genes, Tumor Suppressor; Humans; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurons; Promoter Regions, Genetic; Proteins; Rats; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

The use of model systems derived from cell lines has been a valuable tool in understanding the molecules and cellular processes that govern differentiation processes (T.R. Breitman, S.E. Selonick, S.J. Collins, Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid, Proc. Natl. Acad. Sci. USA 77 (1980) 2936-2940 [2]; N. Gomez, S. Traverse, P. Cohen, Identification of a MAP kinase in phaeochromocytoma (PC12) cells, FEBS Lett. 314 (1992) 461-465 [4]). The use of such systems provides an inexpensive, quick and simple way to identify and test molecules that can be further studied in more complex in vivo experiments. Some cell lines such as embryonic stem cells can be induced to differentiate in vitro, however, the differentiation is difficult to control and most often leads to the generation of a wide variety of cell types. Cell lines derived from sources committed to a restricted cell fate provide an opportunity to examine cell growth and differentiation within a specific cell type (G.M. Keller, In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol. 7 (1995) 862-869 [10]). In this article we describe a simple system for the differentiation of the human neuroblastoma cell line LA-N-5 into cholinergic neurons using all-trans retinoic acid (G. Han, B. Chang, M.J. Connor, N. Sidell, Enhanced potency of 9-cis versus all-trans retinoic acid to induce the differentiation of human neuroblastoma cells, Differentiation, 59 (1995) 61-69 [5]; D.P. Hill, K.R. Robertson, Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid, Dev. Brain Res. 102 (1997) 53-67 [6]; J.A. Robson, N. Sidell, Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid, Neuroscience 14 (1985) 1149-1162 [12]; N. Sidell, C.A. Lucas, G.W. Kreutzberg, Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line, Exp. Cell Res. 155 (1984) 305-309 [14]). These cells provide a setting for the study of cholinergic neuronal differentiation and of the factors that influence that process. We also discuss procedures that can be used to study gene expression in LA-N-5 cells by immunohistochemistry and reporter gene analysis.

Topics: Cell Differentiation; Cell Division; Genes, Reporter; Humans; Immunohistochemistry; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Sodium phenylacetate (NaPA) has been shown to synergize with retinoic acid (RA) in inducing the differentiation of human neuroblastoma cells. Our studies indicated that NaPA can impact on the RA differentiation program by upregulating nuclear retinoic acid receptor-beta (RAR beta) expression. We have found that NaPA does not alter the half-life of RAR beta mRNA; thus, increased stability of mRNA levels does not contribute to NaPA induction. In contrast, NaPA was able to specifically activate a reporter gene construct (delta SV beta RE-CAT) which contains a retinoic acid response element (RARE beta) that is located in the RAR beta promoter. Activation of delta SV beta RE-CAT by NaPA also occurred in neuroblastoma cells cotransfected with a nuclear retinoic acid receptor expression vector, demonstrating the independence of this activation on cellular RAR levels. Taken together, our findings suggest that induction of RAR beta by NaPA is regulated at the level of transcription and mediated through the retinoic acid response element, RARE beta. This effect may account, at least in part, for the strong synergy between NaPA and RA in promoting neuroblastoma differentiation.

Topics: Antimetabolites, Antineoplastic; Binding Sites; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Genes, Reporter; Humans; Neuroblastoma; Phenylacetates; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Combined sialyltransferase (ST) activities were induced in the HN9 hippocampal cell line following treatment with the synthetic glucocorticoid dexamethasone (dex) for 24 hr. Induction occurred in a dose-dependent manner, with the maximum induction of a 2-fold increase over control enzyme levels occurring at a concentration of 10(-8) M dex. A minimum of 6 hr pretreatment with drug was required before significant induction could be detected and elevated enzyme levels persisted for up to 48 hr post-treatment. The induced form of the enzyme showed an increase in reaction maximum velocity (Vmax) while showing no change in affinity (Km) for the acceptor substrate asialofetuin. The alpha2,6 ST enzyme was demonstrated to be the primary enzyme induced and there was no change in expression of the alpha2,3 ST enzyme. Lectin blot analysis demonstrated an increase in the levels of the alpha2,6-linked cellular sialoglycoproteins and a parallel decrease in the alpha2,3 sialoglycoprotein levels.

Topics: Animals; Antineoplastic Agents; beta-D-Galactoside alpha 2-6-Sialyltransferase; Cell Differentiation; Dexamethasone; Glucocorticoids; Kinetics; Lectins; Mice; Neuroblastoma; Neurons; Sialyltransferases; Tretinoin; Tumor Cells, Cultured

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.

Topics: Animals; Axons; Bucladesine; Cholera Toxin; Dendrites; G(M1) Ganglioside; Ionomycin; Membrane Glycoproteins; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neuraminidase; Neuroblastoma; Neurofilament Proteins; Neurotransmitter Agents; Potassium Chloride; Rats; Tretinoin; Tumor Cells, Cultured

The modulation of inward K+ conductances was studied during neuronal differentiation of human SH-SY5Y neuroblastoma cells. Under standard culture conditions, these cells express the herg gene, and the HERG current is the main inward K+ current regulating their Vrest. After 10-20 days exposure to Retinoic Acid (RA), SH-SY5Y cells showed, in addition to HERG currents, a novel current characterized by inward rectification, dependence on the extracellular K+ concentration, and blockade by Cs+ and Ba2+, the main features of the IRK1 current. The appearance of this current is accompanied by a strong hyperpolarisation of Vrest. RT-PCR experiments confirmed that a transcript of the IRK1 (Kir 2.1) gene actually appears in SH-SY5Y cells treated for 10-20 days with RA. On the whole, data here presented demonstrate that RA-induced neuronal differentiation of neuroblastoma cells is accompanied by the switch from a HERG-driven to a IRK1-driven control of Vrest, similarly to what happens in normal differentiating neurons; however, in tumor cells, this switch does not imply the abolition of HERG channel expression.

Topics: Cation Transport Proteins; Cell Differentiation; DNA-Binding Proteins; Electric Conductivity; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Gene Expression; Humans; Neuroblastoma; Neurons; Patch-Clamp Techniques; Potassium Channels; Potassium Channels, Inwardly Rectifying; Potassium Channels, Voltage-Gated; RNA, Messenger; Time Factors; Trans-Activators; Transcriptional Regulator ERG; Tretinoin; Tumor Cells, Cultured

Alzheimer disease is a progressive neurodegenerative disorder that is characterized by a loss of cognitive and memory functions. Amyloid fibrils deposited in neuritic plaque is mainly beta-amyloid protein (Abeta) that is derived from amyloid precursor protein (APP). The secreted form of APP, which is corresponded to N-terminal portion of APP, shows neurotrophic activities. On the other hand, Abeta and cytoplasmic domains of APP are thought to be neurotoxic. In order to investigate the effect of C-terminal fragment of APP covering Abeta and the cytoplasmic domain upon cell growth and differentiation, we established a stably transfected cell line producing the C-terminal 100 amino acid peptide of APR The transfected clones stained positively with anti-Abeta monoclonal antibody, TB-1. The growth rate of the transfected cells was not significantly different from that of mock-transfected cells or native NB39 cells. After treatment with all-trans retinoic acid (ATRA), mock-transfected cells extended neurite processes and showed neuronal-like differentiation, while a transfected clone overexpressing C-terminal fragment did not present neuronal-like morphology. These results suggest that ATRA-induced neurite extension may be suppressed by overexpression of the C-terminal fragment of APP.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Brain; Cell Differentiation; Cell Division; Humans; Immunohistochemistry; Neuroblastoma; Transfection; Tretinoin; Tumor Cells, Cultured

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

Topics: Antineoplastic Agents; Cell Differentiation; Child; Child, Preschool; DNA-Binding Proteins; Drosophila Proteins; Early Growth Response Protein 1; Genes, fos; Glial Cell Line-Derived Neurotrophic Factor; Glial Cell Line-Derived Neurotrophic Factor Receptors; Humans; Immediate-Early Proteins; Infant; Neoplasm Proteins; Neoplasm Staging; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neuroprotective Agents; Neurturin; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Ras protooncogenes encode small guanine nucleotide binding proteins (p21ras) activated by phosphorylation. Phosphorylation of p21ras is predominantly regulated by the GTPase activating proteins type 1 GAP120 and neurofibromin. Increased levels of p21ras-GTP (active) have been associated with increased cell growth and malignant transformation. In this study the relationship between p21ras, type 1 GAP120 and neurofibromin with growth and differentiation has been examined in neuroblastoma and peripheral primitive neuroectodermal tumour (pPNET) cell lines. The level of p21ras protein in neuroblastoma and pPNET cells was the same. However, the amount of p21ras-GTP bound was higher in pPNET than in neuroblastoma cells. This most likely reflects the absence of neurofibromin. Retinoic acid (RA)-induced differentiation and growth inhibition of neuroblastoma cells was associated with an increase in type 1 GAP120 and neurofibromin mRNA, and a decrease in p21ras-GTP. In pPNET cells levels of type 1 GAP120 but not neurofibromin mRNA were increased to similar levels to those in neuroblastoma cells. This was not associated with decreased p21ras-GTP, modulation of growth or change in morphology. In summary, constitutive activation of p21ras may have a role in the biology of pPNET cells. This may reflect abnormalities in neurofibromin expression, and could inpart explain why RA did not induce morphological differentiation and growth inhibition in pPNETs.

Topics: Cell Differentiation; Cell Division; Enzyme Activation; Genes, ras; GTPase-Activating Proteins; Humans; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurofibromin 1; Phosphorylation; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; RNA, Messenger; Sarcoma, Ewing; Sequence Analysis, DNA; Tretinoin; Tumor Cells, Cultured

Tuberous sclerosis is an autosomal dominant disorder. Besides the development of benign growths (hamartomas) in different tissues, one hallmark of this disease is the presence of highly epileptogenic dysplastic lesions in the cerebral cortex (tubers) composed of abnormal shaped neurones. Patients often show evidence of severe mental retardation. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid putative tumour suppressor protein, tuberin, that functions as a GTPase-activating protein. Here we show that tuberin expression is upregulated upon induction of neuronal differentiation in the neuroblastoma cell lines SK-N-SH and LAN-1. This upregulation occurs at post-transcriptional level and is independent of the proliferation status. TSC2 expression is unaffected during differentiation of C2C12 myoblasts into myotubes and of F9 embryonal carcinoma cells into cells resembling parietal endoderm. Antisense inhibition of tuberin expression in SK-N-SH or LAN-1 cells inhibits neuronal differentiation, but does not affect the differentiation of F9 cells. Ectopic overexpression of TSC2 not only reverts the antisense-associated phenotype but furthermore accelerates the neuronal differentiation process. Our data show for the first time that tuberin plays a critical role in neuronal differentiation. Such role is consistent with the phenotype of tuberous sclerosis patients, who inherit one defective TSC2 allele, and frequently lose the remaining normal allele in many of the tubers/hamartomas which develop in the central nervous system of these patients.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Humans; Mice; Neuroblastoma; Neurons; Oligonucleotides, Antisense; Repressor Proteins; Transfection; Tretinoin; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured; Tumor Suppressor Proteins

The aim of this study was to investigate in vitro the effects of all-trans retinoic acid (RA), 9-cis RA and the RXR-selective analogue, LG69, on the morphological differentiation, proliferation and gene expression of neuroblastoma cells. Three different cell lines were cultured with the retinoid for either 9 continuous days or for 5 days followed by 4 days without the retinoid and morphological differentiation was assessed both qualitatively and quantitatively. SH SY 5Y cell proliferation was examined by measuring cell numbers after exposure to the retinoids and RAR-beta gene expression was examined by Northern blot analysis. Morphological differentiation was more effectively induced by all-trans and 9-cis RA than by LG69. SH SY 5Y cells, when treated with 9-cis RA for only 5 of the 9 days of culture, underwent apoptosis, but this was not seen with 9 days continuous exposure nor with LG69. Inhibition of SH SY 5Y cell proliferation by all-trans or 9-cis RA was dose-dependent, but LG69 had little effect. Conversely, LG69 induced higher expression of RAR-beta than all-trans RA, but less than that produced by 9-cis RA. These data suggest that 9-cis RA as a single agent is the most effective modulator of neuroblastoma behaviour and may be the most appropriate therapeutic agent.

Topics: Antineoplastic Agents; Bexarotene; Blotting, Northern; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Gene Expression; Humans; Neuroblastoma; Tetrahydronaphthalenes; Tretinoin

Monoamine-activated alpha2-macroglobulin (alpha2M) was shown to reduce the dopamine concentration in corpus striatum of adult rat brains and inhibit other neuronal functions in vivo and in vitro. As brain-derived neurotrophic factor, neurotrophin-4, and neurotrophin-3 are important neurotrophic factors for dopaminergic neurons, the effect of monoamine-activated alpha2M on signal transduction by trkB and trkC was investigated. The results show that monoamine-activated alpha2M binds to trkB and inhibits brain-derived neurotrophic factor/neurotrophin-4-promoted autophosphorylation of trkB in a dose-dependent manner in both trkB-expressing NIH3T3 (NIH3T3-trkB) and human neuroblastoma SH-SY5Y cells. Monoamine-activated alpha2M also blocks tyrosine phosphorylation of phospholipase C-gamma1 and extracellular signal-regulated protein kinase (ERK)-1, which are key intracellular proteins involved in trkB signal transduction. Similarly, monoamine-activated alpha2M inhibits tyrosine phosphorylation of neurotrophin-3-induced trkC and its signal transduction in a dose-dependent manner in NIH3T3 cells expressing trkC (NIH3T3-trkC). In contrast to monoamine-activated alpha2M, normal alpha2M has little or no significant inhibitory effect on the phosphorylation of trkB and trkC. In addition, the retinoic acid-promoted tyrosine phosphorylation of phospholipase C-gamma1, ERK-1, and/or ERK-2 in SH-SY5Y cells was unaffected by monoamine-activated alpha2M; this suggests that the inhibitory effect of activated alpha2M on the neurotrophin-stimulated phosphorylation of intracellular signalling proteins may be specific. Taken together, the data indicate that activated alpha2M is a pan-trk inhibitor, which by virtue of its binding to trk receptors may block trk-mediated signal transduction in dopaminergic neurons and lead to reduction of dopamine concentration in corpus striatum.

Topics: 3T3 Cells; alpha-Macroglobulins; Animals; Antineoplastic Agents; Brain-Derived Neurotrophic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Humans; Isoenzymes; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nerve Growth Factors; Neuroblastoma; Neuroprotective Agents; Neurotrophin 3; Phospholipase C gamma; Phosphorylation; Receptor Protein-Tyrosine Kinases; Receptor, Ciliary Neurotrophic Factor; Receptor, trkC; Receptors, Nerve Growth Factor; Serotonin; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

Tau isoforms migrating at 46-68 and 97-115 kDa were prominent within heat-stable Triton-soluble material, and were present in lesser concentration with Triton-insoluble cytoskeletons, derived from undifferentiated SH-SY-5Y human neuroblastoma cells. Conversely, a 26-30 kDa tau isoform was enriched in the cytoskeleton and detected at relatively minor levels within cytosolic fractions. Pulse labeling with 35S-methionine indicated that this 26-30 kDa "small tau" did not represent a breakdown product of larger isoforms. Since the nucleus is retained within the Triton-insoluble cytoskeleton, additional cultures were fractionated onto sucrose to obtain purified nuclei. The vast majority of small tau was recovered within purified nuclei. Small tau was reactive with tau antibodies directed towards N-terminal, C-terminal and central epitopes, further confirming that this small isoform was not derived from proteolytic cleavage of larger tau isoforms. Small tau demonstrated alkaline phosphatase-sensitive reactivity with multiple phospho-dependent tau antibodies. Small tau was depleted within 3 days of retinoic acid-induced differentiation, suggesting that the putative function of this isoform may be obsolete following terminal differentiation of neurons.

Topics: Blotting, Western; Brain Neoplasms; Cell Differentiation; Cell Nucleus; Humans; Mitosis; Molecular Weight; Neuroblastoma; Precipitin Tests; tau Proteins; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) treatment of SMS-KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal differentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR while p15INK4B and p16INK4A were not detected. RA induced an increase in the expression of p27Kip1 but not p21Cip1. Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27Kip1. These results indicate that changes in the level of p27Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.

Topics: Antineoplastic Agents; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Gene Expression; Genes, Tumor Suppressor; Humans; Microtubule-Associated Proteins; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have previously shown that ectopic overexpression of retinoic acid receptor (RAR) subtypes alpha, beta and gamma in human neuroblastoma cells had different effects on growth and retinoid sensitivity. Only overexpressed RAR beta induced profound growth inhibition in the absence of additional retinoid, and increased retinoid sensitivity. In this study, we measured mRNA expression levels of RAR alpha, beta, and gamma in 50 primary neuroblastoma tumor samples, and found a strong correlation between favorable patient prognosis and high-level RAR beta expression. Human neuroblastoma cells transfected with a vector expressing RAR beta demonstrated irreversible growth arrest following a 1 week exposure to all-transretinoic acid, whereas control cells continued to proliferate. In the absence of additional retinoid, RAR beta transfectants demonstrated a higher proportion of cells in the G0/G1 phase of the cell cycle, increased p21WAF1/CIP1 expression and specific binding to a retinoic acid response element. These were changes which we also observed in control neuroblastoma cells following retinoid treatment. Our data indicate that RAR beta is an important factor mediating the growth inhibitory effects of retinoids in neuroblastoma cells. The favorable effect of high-level RAR beta expression on prognosis in primary tumor tissue may occur through RAR beta effects on p21 expression and consequent G0/G1 cell cycle arrest.

Topics: Cell Cycle; Child; Child, Preschool; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Infant; Neoplasm Staging; Neuroblastoma; Prognosis; Receptors, Retinoic Acid; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; RNA, Messenger; Signal Transduction; Transfection; Tretinoin

Vitamin A and its derivatives (retinoids) have profound effects on the proliferation and differentiation of many cell types and are involved in a diverse array of developmental and physiological regulatory processes, including those responsible for the development of the mature nervous system. Retinoid signals are mediated by retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), which show distinct spatio-temporal patterns of expression during development and in adult tissues. We have used SK-N-BE2(c) neuroblastoma cells to study the effects of reciprocal regulation of expression of various RARs. We show that in these cells RARgamma1 acts as a repressor of RARbeta2 transcription in the absence of an agonist. In the presence of RA, the expression of RARgamma1 is reduced and that of RARbeta2 is induced. Overexpression of RARgamma1 neutralizes the effects of RA on RARbeta induction. Expression of an RARgamma1-specific antisense construct leads to the constitutive expression of RARbeta2. Although both overexpression of RARgamma1 and its reduction of expression can result in inhibition of cell proliferation, they induce different morphological changes. Reduction of RARgamma1 (and induction of RARbeta) leads to increased apoptosis, whereas RARgamma1 overexpression leads to differentiation in the absence of apoptosis. Thus, RARgamma1 appears to control a differentiation-apoptosis switch in SK-N-BE2(c) neuroblastoma cells.

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neuroblastoma; Oligonucleotides, Antisense; Receptors, Retinoic Acid; Repressor Proteins; Retinoic Acid Receptor gamma; Retinoid X Receptors; Retinoids; RNA, Messenger; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factors (IGF-I and IGF-II) stimulate proliferation and differentiation in many cell types. In biological fluids, they associate non-covalently with high-affinity binding proteins (IGFBPs) which control their bioavailability and modulate their action. We previously demonstrated that IGFBP-2, -4 and -6 are intimately involved in the growth of cells derived from human neuroblastomas. Here, we have investigated the effects of retinoic acid (RA), which induces differentiation in these cells, on the expression of IGFBPs secreted by SK-N-SH neuroblastoma cells. Analysis of transcriptional activity of the IGFBP-2, -4 and -6 genes in isolated nuclei (run-on experiments) showed that RA increased the transcriptional activity of the IGFBP-6 gene, reduced that of the IGFBP-4 gene and had no effect on that of the IGFBP-2 gene. Northern blot analysis following treatment with actinomycin D showed that RA increased the stability of IGFBP-6 mRNA by a factor of 2.6, decreased that of IGFBP-2 mRNA by a factor of 2.3 and failed to affect IGFBP-4 mRNA. Treatment of cells with cycloheximide indicated the involvement of labile proteins in the stabilization of these mRNAs the expression of which could be under the control of RA. The transcriptional and/or post-transcriptional mechanisms by which RA regulates each of the IGFBPs produced by SK-N-SH cells are therefore different. Such regulation may also reflect the state of differentiation of the neuroblastoma cells. With RA-induced differentiation, IGFBP-6 is strongly stimulated, whereas IGFBP-2 and IGFBP-4 are severely depressed, which would suggest that each IGFBP plays a specific role. Moreover, this regulation seems tissue-specific because it is different in other cell types.

Topics: Blotting, Northern; Cycloheximide; Dactinomycin; Humans; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor Binding Proteins; Neuroblastoma; Protein Synthesis Inhibitors; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Functional nerve growth factor (NGF) responsiveness was investigated in human neuroblastoma (NB) cell lines in vitro and retinoic acid (RA) was found to transcriptionally enhance expression of the trkA, but not the trkB gene in GOTO cells, while the reverse was found in HTLA230 NB cells. Ciliary neurotrophic factor (CNTF) specifically induced trkA gene transcription in both cell lines. Transcriptional activation of the trkA gene increased total trkA protein and surface bound receptor, which was tyrosine phosphorylated upon NGF stimulation inducing immediate early response gene transcription (i.e. c-fos, Egr-1). Newly synthesized trkA protein had a molecular weight of 110 kDa and was post-translationally modified. Rapid down regulation of the receptor protein occurred upon NGF stimulation, despite the presence of high levels of trkA mRNA, due to an increased rate of receptor degradation. Transient DNA synthesis and cell proliferation upon NGF treatment occurred in GOTO cells with elevated trkA expression. In contrast, NGF induced neuronal differentiation in HTLA230 cells expressing the endogenous trkA receptor gene, despite the lack of p75 expression. Hence, transcriptional activation of trkA gene expression can be achieved by different signal pathways in human NB cells, but NGF can act either as mitogen or inducer of cell differentiation, depending on the tumor from which cells are derived.

Topics: Ciliary Neurotrophic Factor; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, Ciliary Neurotrophic Factor; Receptor, Nerve Growth Factor; Receptor, trkA; Receptors, Nerve Growth Factor; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

NG108-15 neuroblastoma cells differentiated with 0.1 M of all-trans retinoic acid (RA) were processed for immunohistochemical analysis using polyclonal antisera against the delta opioid receptor (DOR) and the N-Methyl-D-Aspartate receptor (NMDAR1) to determine the cellular sites for possible functional associations between DOR and NMDAR1 receptors. In this study, 6 days of RA treatment resulted in prominent morphological differentiation characterized by the appearance of numerous axon- and dendrite-like processes and formation of networks between the cell clusters. An immunocytochemical approach allowed the demonstration of antibody concentration-dependent differences, not evident in ligand binding studies, in the distribution of DOR and NMDA receptor protein between cell soma and processes. RA-differentiated cultures showed positive DOR-like immunostaining (DOR-LI) throughout the cell bodies as well as on the newly acquired processes. In contrast, NMDAR1-like immunoreactivity (NMDAR1-LI) in the RA-treated cells was detected in the cell soma and processes only with the higher concentration of the antiserum. With the lower concentration of the antibody the NMDAR1-LI was not detected in the processes and was limited to a punctuate subcellular distribution in the soma. The DOR-LI pattern of distribution in NG108-15 cells differentiated with RA appeared to be consistent with the DOR-LI detected in the CNS. The NMDAR1-LI distribution in these cells is similar to brain tissue with respect to its presence on the newly acquired processes. However, it differed from brain in that a much higher abundance of NMDAR1 receptors was observed in the cell soma. This differential distribution of DOR and NMDAR1 receptors in the RA-treated NG108-15 cells could provide a basis for future studies of drug-induced changes in these two receptors.

Topics: Animals; Antibody Specificity; Antineoplastic Agents; Axons; Cell Differentiation; Cell Size; Dendrites; Glioma; Hybrid Cells; Immunoenzyme Techniques; Mice; Neuroblastoma; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, delta; Tretinoin

We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.

Topics: Antineoplastic Agents; Binding Sites; Carcinogens; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Neuroblastoma; Phosphorylation; Protein Biosynthesis; Protein Kinase C; Protein Processing, Post-Translational; Receptor, Ciliary Neurotrophic Factor; Receptors, Nerve Growth Factor; RNA, Messenger; Sensitivity and Specificity; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate; Trans-Activators; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Gangliosides are believed to play a critical role in cellular differentiation. To test this concept, we determined the effect of inhibition of endogenous ganglioside synthesis upon neurite formation induced by retinoic acid in LAN-5 human neuroblastoma cells. Ganglioside synthesis and content of LAN-5 cells exposed for 6 days to 10 microM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (an inhibitor of glucosylceramide synthase) were reduced by >90%. However, these ganglioside-depleted cells were not blocked from forming neurites when exposed to 10 microM retinoic acid. Even more extensive treatment of LAN-5 cells with 20 microM D-PDMP (6 day pretreatment followed by 6 days together with 10 microM retinoic acid) still did not block the retinoic acid-induced neurite formation. An element of neuroblastoma tumor cell differentiation, neurite formation, is therefore dependent neither on an intact cellular ganglioside complement nor on new ganglioside synthesis.

Topics: Down-Regulation; Enzyme Inhibitors; Gangliosides; Glucosyltransferases; Humans; Morpholines; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate IGF action at cellular level through inhibition or, alternatively, potentiation, where their limited proteolysis is a contributory mechanism. Under basal conditions, neuroblastoma cells secrete IGFs (essentially IGF-II), IGFBPs (IGFBP-4 and predominantly IGFBP-2 that is partially proteolysed), and proteases, including tissue-type plasminogen (PLG) activator, whose activity is inhibited by PLG activator inhibitor-1. Neuroblastoma cells were used to investigate the influence of the plasmin system, transforming growth factor-beta retinoic acid on cell growth and the IGF system. In cells treated with 5 micrograms/ml PLG, proliferation was stimulated, an effect that was inhibited in the presence of either alpha IR-3 (which blocks the type 1 IGF receptor) or anti-IGF-II antibodies. There was a parallel increase in IGFBP-2 proteolysis, which resulted in a 5-fold loss of affinity for IGF-II. In the presence of 1 ng/ml transforming growth factor-beta, PLG-induced mitogenesis and IGFBP-2 proteolysis were reduced, and Northern blot analysis revealed increased PLG activator inhibitor-1 mRNA. Conversely, with 2 microM retinoic acid, the mitogenic effect of PLG, IGFBP-2 proteolysis, and tissue-type PLG activator mRNAs were increased. Therefore, IGF-II mediates autocrine proliferation in neuroblastoma cells under the control of IGFBPs secreted by the cells, its bioavailability being enhanced as a result of plasmin-induced IGFBP-2 proteolysis.

Topics: Blotting, Northern; Cell Division; Fibrinolysin; Humans; Immunoblotting; Insulin-Like Growth Factor Binding Protein 2; Neuroblastoma; Plasminogen Activator Inhibitor 1; RNA, Messenger; Tissue Plasminogen Activator; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Midkine (MK), a neurotrophic polypeptide of which expression is developmentally regulated in embryogenesis, is expressed in malignant tumor tissues including neuroblastoma (NB). A retinoic acid analogue, E5166, and dibutyryl cyclic AMP (dbcAMP) are known to induce differentiation in NB cells. This study showed that MK mRNA expression increased in association with differentiation by E5166, but not by dbcAMP in SK-N-SH and KP-N-RTBM1 human NB cell lines. We concluded that MK could be an important factor in differentiation of NB cells, and further, that there could be at least two pathways in differentiation of NB cells at molecular mechanism.

Topics: Antineoplastic Agents; Bucladesine; Carrier Proteins; Cell Differentiation; Cytokines; Humans; Midkine; Neoplasm Proteins; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.

Topics: Animals; Axons; Bucladesine; Dendrites; Microtubule-Associated Proteins; Microtubules; Mitosis; Neuroblastoma; Rats; Tretinoin; Tumor Cells, Cultured

To date, the clinical success of 13-cis or all-trans retinoic acid in the treatment of neuroblastoma has been disappointing. In vivo, 13-cis will isomerise to both all-trans and 9-cis retinoic acid, believed to be the main biologically-active isomers. In vitro studies with an N-type neuroblastoma cell line, SH SY 5Y, show that 9-cis is better than other isomers at both inducing morphological differentiation and inhibiting proliferation. RAR-beta, a gene which may mediate retinoic acid responsiveness and be of prognostic significance, is also more-effectively induced by 9-cis retinoic acid. 9-cis and all-trans retinoic acid do not have synergistic effects on SH SY 5Y cell proliferation and gene expression. A retinoid X receptor (RXR)-specific analogue of 9-cis retinoic acid had similar effects on gene expression to 9-cis retinoic acid alone. In view of these results, 9-cis retinoic acid or stable analogues of this retinoid may have potential for the treatment of neuroblastoma.

Topics: Alitretinoin; Blotting, Northern; Cell Differentiation; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Neurofibromin, the gene product of the NF-1 gene is expressed in two isoforms. The m-RNA of NF-1 type II contains an insertion of 63 bases in the so called GAP-related domain, that distinguishes it from the type I transcript. By sequence homology neurofibromin is supposed to have a similar function in regulating activity of ras in intracellular signal transduction as the GTPase activating protein (GAP). Both transcripts of NF-1 are simultaneously expressed in different molar ratio in neuroblastoma tumors. 1) We examined 9 different neuroblastoma cell lines for the ratio of expression of NF-1 type I and type II. For quantification of the two transcripts we performed RT-PCR of the m-RNA of the neuroblastoma cells using primers designed to cover the GAP-related domain. We found values ranging from a more than 3-fold excess of type I transcript in the cell line Kelly to a slight excess of the type II transcript (I/II = 0.6) in the cell line IMR 5. 2) As there are indications that expression of NF-1 type II is related to the state of differentiation, we tried to shift expression of NF-1 from type I to type II by treatment of the neuroblastoma cells with retinoic acid. Treatment of Kelly cells with 5 microM retinoic acid for 24 h already lowered the excess of the type I transcript from 3-fold to an only 1.6-fold excess. An inversion of the molar ratio from an excess of the type I transcript to an excess of type II transcript would enable to investigate the different role of the two transcripts in the regulation of ras-activity and differentiation.

Topics: Antineoplastic Agents; Cell Differentiation; Gene Expression Regulation, Neoplastic; Neuroblastoma; Neurofibromin 1; Polymerase Chain Reaction; Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Preliminary data have shown that IL-6 may act as an autocrine growth factor to control proliferation. We further characterised the role of IL-6 in tumour growth as an autocrine/paracrine growth factor in neuroectodermal tumours. We evaluated the production and secretion of IL-6 by seven human melanoma, five neuroblastoma and one glioblastoma cell lines. Moreover, we determined their IL-6-dependent growth in serum free-medium or under minimal growth-supplement conditions: IL-6 dependent growth was observed in two non-IL-6 producing melanoma and in one neuroblastoma cell lines. In addition, expression of IL-6 mRNA and peptide was increased by retinoic acid. The data support the hypothesis that IL-6 contributes to neuroectodermal tumour growth, even though it shows a less potent effect than other reported growth factor such as IGF-II.

Topics: Antineoplastic Agents; Blotting, Northern; Cell Division; Culture Media, Serum-Free; Gene Expression Regulation, Neoplastic; Glioblastoma; Growth Substances; Humans; Interleukin-6; Melanoma; Neuroblastoma; Neuroectodermal Tumors; Radioimmunoassay; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The overall survival rate for patients with neuroblastoma has improved over the past two decades, but long-term survival for the subgroup of patients with high-risk disease remains low. In recent years, there has been interest in the potential clinical use of drugs able to induce differentiation of neuroblastoma cells. Since 9-cis-retinoic acid induces better and more sustained differentiation of neuroblastoma in vitro than other retinoic acid isomers, this may be a more appropriate retinoid for use in neuroblastoma therapy.. The purpose of this work was to compare the long-term effects of all-trans- and 9-cis-retinoic acid on neuroblastoma differentiation using an N-type (neuroblastic) cell line, SH SY 5Y, as an in vitro model. In addition, we wanted to find out whether 9-cis-retinoic acid would induce programmed cell death (apoptosis) in these N-type neuroblastoma cells and to determine whether the effects of either 9-cis- or all-trans-retinoic acid are dependent on their continued presence in the culture medium.. SH SY 5Y cells were incubated in either the continued presence of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic acid followed by culture in the absence of retinoid for up to 13 days. Morphologic changes were observed using phase-contrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodide-stained cells and by using terminal deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells.. Culture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. These effects were maintained in the continuous presence of each retinoic acid isomer but were more profound in cells treated with 9-cis-retinoic acid. The differentiation of cells treated with all-trans-retinoic acid was reversible once retinoic acid was removed from the medium. Conversely, apoptosis was induced in cells treated with 9-cis-retinoic acid for 5 days and cultured for 9 days (4 days after washout) but not in cells cultured in the continuous presence of 9-cis-retinoic acid. This effect was specific to 9-cis-retinoic acid.. Previous studies have demonstrated differential responses to all-trans-retinoic acid in N- and S-type (substrate-adherent or Schwann-like) neuroblastoma cells: Apoptosis is induced in S-type cells, whereas differentiation occurs in N-type cells. The present results show that, unlike all-trans-retinoic acid, 9-cis-retinoic acid induces both differentiation and apoptosis in N-type SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9-cis-retinoic acid from the culture medium.. Since both differentiation and apoptosis are involved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apoptosis on treatment and subsequent removal of 9-cis-retinoic acid implies that drug scheduling may be an important parameter affecting therapeutic efficacy.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; DNA Nucleotidylexotransferase; Flow Cytometry; Immunoenzyme Techniques; Neuroblastoma; Time Factors; Tretinoin; Tumor Cells, Cultured

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Cell Adhesion; DNA Fragmentation; DNA, Neoplasm; Down-Regulation; Fenretinide; Flow Cytometry; Humans; Integrin beta1; Integrins; Neuroblastoma; Oligonucleotides, Antisense; Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

A cDNA, 7G1, was isolated from retinoic acid (RA) differentiated neuroblastoma cells whose expression was high in human fetal brain and spinal cord mRNA but undetectable in adult brain or non-neuronal tissues. Sequence analysis indicates that 7G1 is homologous to the Caenorhabditis elegans gene unc-33. A 5.5-kilobase pair full-length cDNA from a human fetal brain cDNA library contains an 1710-base pair open reading frame. Because the predicted 570 amino acid sequence of 7G1 shares 98% identity with the murine Ulip gene product, an unc-33-like-phosphoprotein, we refer to 7G1 as the human Ulip (hUlip). hUlip is also similar to the bacterial enzyme D-hydantoinase and the recently described vertebrate gene products CRMP62, TOAD-64, CRMP1, CRMP2, and mUNC. RA stimulates an increase in hUlip mRNA that is transcriptionally regulated. RA stimulates an increase in polypeptides of 58, 60, 65, and 70 kDa with the 58- and 65-kDa species being dephosphorylated forms of the 60- and 70-kDa species. This study presents a model in which to study the regulation and expression of the hUlip gene, a member of an emerging family of molecules that potentially mediates signals involved in axonal outgrowth.

Topics: Adult; Amino Acid Sequence; Animals; Base Sequence; Brain; Caenorhabditis elegans Proteins; Cell Differentiation; Fetus; Gene Expression Regulation, Neoplastic; Gene Library; Helminth Proteins; Humans; Mice; Molecular Sequence Data; Muscle Proteins; Nerve Growth Factors; Neuroblastoma; Neurons; Peptide Fragments; Phosphoproteins; RNA, Messenger; Sequence Homology, Amino Acid; Spinal Cord; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Vertebrates

beta-Amyloid precursor protein (APP) belongs to a family of homologous beta-amyloid precursor-like proteins (APLPs) including APLP1 and APLP2. Previously it has been shown that APP is subject to regulation by retinoic acid (RA). In this paper we show that APLP1 and APLP2 mRNA expression is upregulated during RA-induced differentiation of human SH-SY5Y neuroblastoma cells. The cells were treated with RA (10 microM) for 3 and 6 days and mRNA levels were analysed by a non-radioactive Northern blot assay. RA induced a 2- to 3-fold increase in the gene expression of both APLP2 and APP, whereas the increase in APLP1 mRNA expression was significantly higher. Our results support a role for APLPs during neuronal differentiation.

Topics: Amyloid beta-Protein Precursor; Blotting, Northern; Brain Neoplasms; DNA Probes; Gene Expression; Humans; Molecular Sequence Data; Nerve Tissue Proteins; Neuroblastoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The metastatic potential of undifferentiated neuroblastomas is typically lost when differentiation into ganglioneuroblastomas occurs spontaneously or is induced. Cell adhesion may play a role in metastasis, and we have shown recently that expression of integrin alpha v beta 5 protein and mRNA is up-regulated in ganglioneuroblastomas in vivo. To investigate whether interactions of alpha v beta 5 with matrix components play a role in the loss of metastatic potential, we used immunohistochemical and in situ hybridization to analyze neuroblastic tumors at various stages of differentiation for expression of the alpha v beta 5 ligands, vitronectin and osteopontin, and determined the ability of vitronectin to promote attachment and neurite outgrowth in vitro in a retinoic-acid-differentiated neuroblastoma cell model. We found that vitronectin, but not osteopontin, was expressed in 5 of 5 ganglioneuroblastomas but was absent or weakly expressed in 6 of 6 undifferentiated neuroblastomas. Neuronal cell vitronectin was detected in 7 of 9 ganglioneuromas, 5 of 8 peripheral ganglia, and 14 of 21 adrenal gland medullae, confirming expression of vitronectin in mature peripheral neurons. In vitro, vitronectin promoted attachment of both undifferentiated and retinoic-acid-differentiated neuroblastoma cells, which was inhibited 20 and 60%, respectively, by monoclonal antibody anti-integrin alpha v beta 5. Vitronectin-promoted neurite outgrowth of retinoic-acid-differentiated neuroblastoma cells was not inhibited by monoclonal antibody anti-alpha v beta 5. These data suggest that the synthesis of vitronectin and the ability of integrin alpha v beta 5 to mediate vitronectin adhesion on retinoic-acid-differentiated neuroblastoma cells may promote differentiation of neuroblastoma cells in vivo.

Topics: Adrenal Gland Neoplasms; Cell Adhesion; Cell Differentiation; Ganglia, Sensory; Ganglioneuroma; Humans; Integrins; Neuroblastoma; Pheochromocytoma; Receptors, Vitronectin; RNA, Messenger; Schwann Cells; Tretinoin; Vitronectin

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Topics: Brain; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cysteine Proteinase Inhibitors; Humans; Isomerism; Molecular Weight; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Retinoids play fundamental roles in CNS development, but their distribution, metabolism, and function within the mature human CNS are unknown. In these studies, extracts of autopsy tissues recovered from histopathologically confirmed control and Alzheimer diseased brains were tested for their ability to synthesize retinoic acid. Retinaldehyde dehydrogenase (RLDH), the enzyme that forms retinoic acid from retinaldehyde, was present in hippocampus, frontal cortex, and parietal cortex. The RLDH activity of hippocampus and parietal cortex from Alzheimer diseased brains was 1.5- to 2-fold higher (p < 0.05) compared to the controls. In contrast, the RLDH activity of frontal cortex was the same for both Alzheimer diseased and control groups. A cultured human glioblastoma (U251) and neuroblastoma (LA-N-5) cell line synthesized retinoic acid from retinaldehyde or retinol, suggesting that a variety of neural cell types possess this activity. LA-N-5 cells grown in vitamin A-depleted medium had higher (p < 0.05) RLDH activity (0.35 +/- 0.04 nmol/mg/h) than LA-N-5 cells grown in vitamin A-replete media (0.15 +/- 0.02 nmol/mg/h). This difference was lost when retinol was added back to the medium, confirming that a reduction in vitamin A supply can induce RLDH activity in neural cells. However, this feedback mechanism does not appear to explain the higher RLDH activity of Alzheimer diseased hippocampus and parietal cortex, because the overall vitamin A status as indicated by serum retinol and carotenoid levels and by hippocampal retinoid content was similar for the Alzheimer diseased and control groups. These studies establish the presence of retinoids and RLDH activity in human brain tissues, and indicate that retinoic acid synthesis is modulated in some regions of Alzheimer diseased brain.

Topics: Alzheimer Disease; Brain; Frontal Lobe; Hippocampus; Humans; Neuroblastoma; Neurons; Parietal Lobe; Retinoids; Tretinoin; Tumor Cells, Cultured; Vitamin A

Retinoic acid (RA) induces growth inhibition, differentiation or cell death in many human neuroblastoma cell lines. Recently, the transactivation activity of nuclear retinoids receptors has been shown to be modulated through physical association with other proteins that act as co-activators or as co-repressors. We investigated the expression of the co-repressor (SMRT) and co-activator (Trip 1) for retinoid and thyroid-hormone receptors in several neuroectodermal tumour cell lines, and its modulation by all-trans-retinoic acid, as well as by synthetic agonists, for RAR alpha, RAR beta, RAR gamma and RXR. We demonstrate that (i) SMRT and Trip-1 mRNAs are expressed in many human neuroblastoma and melanoma cell lines in basal conditions, (ii) SMRT mRNA expression in human neuroblastoma cell line SK-N-BE(2) increases after 48 hours of incubation with 1 microM RA and RARs specific agonists, (iii) Trip-1 mRNA in the same cell line does not change during incubation with RA or selective synthetic agonists for RARs and RXR.

Topics: Chromatography, High Pressure Liquid; Dimerization; DNA Primers; DNA-Binding Proteins; Electrophoresis, Agar Gel; Eukaryotic Initiation Factor-3; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Neuroblastoma; Nuclear Receptor Co-Repressor 2; Polymerase Chain Reaction; Proteins; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Repressor Proteins; RNA, Messenger; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

p55 tumour necrosis factor receptors in neuroblastoma SKNBE cells were localized by immunofluorescence microscopy. They were detected at the surface on non-permeabilized cells and, after differential permeabilization, in the Golgi area as diffuse staining and in the perinuclear region as clusters and punctuations. Intracytoplasmic punctuate forms were detected after permeabilization with Triton X-100, suggesting their location within organelles. Treatment with the differentiation-inducing agent all-trans retinoic acid (RA) greatly increased the cellular density of the immunoreactive receptors and receptors were detected in the newly formed neurites, suggesting that RA promoted their transport from the cell body. The results suggest direct influences on neuronal functions of tumour necrosis factor alpha released from adjacent or from target cells.

Topics: Antigens, CD; Fluorescent Antibody Technique; Humans; Microscopy, Fluorescence; Neuroblastoma; Neurons; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Tretinoin; Tumor Cells, Cultured

Human SH-SY5Y neuroblastoma cells were induced to neuronal differentiation by using 12-0-tetradecanoylphorbol-13-acetate (TPA) and retinoic acid (RA). Both treatments rapidly induced long neurites and increased the content of neurofilaments as shown by immunocytochemistry and immunoblotting. Immunoprecipitation and immunoblotting of the culture medium with monoclonal antibodies demonstrated a rapid onset of synthesis and secretion of Mr 280,000 tenascin (Tn) polypeptide with TPA and both Mr 280,000 and 190,000 Tn polypeptides with RA and an increased secretion of extradomain A cellular fibronectin (EDA-Fn) upon both treatments. Upon RA treatment both Tn polypeptides were also found in extracellular matrix preparations of the differentiated cells. A diffuse extracellular Tn immunoreactivity and a distinct cytoplasmic reaction were seen in differentiated cells especially after exposure to monensin to inhibit cellular secretion. Instead, immunoprecipitation experiments suggested that laminin was synthesized by the cells but was not upregulated upon differentiation. Experiments with purified Tn, used to coat the culture substratum, demonstrated that the undifferentiated cells were unable to adhere or spread on Tn but rapidly acquired the spreading capacity upon differentiation with the inducing agents. In immunofluorescence and immunoblotting the undifferentiated cells presented only a faint heterogenous reaction for beta1 integrin (Int) subunit, whereas cells exposed to RA presented a strong reaction for the Int alpha1 and beta1 subunits, hence suggestive of Int alpha1beta1, and for Int alpha(v) subunit. Cells exposed to TPA showed an enhanced immunoreaction for Int alpha2 and beta1 subunits, suggestive of Int alpha2beta1, and for Int alpha(v) subunit. Immunoreactivity for Int alpha(v) located to distinct punctate plaques in the differentiated cells after both inducing agents. The results suggest that Tn is produced by cultured neuronally differentiating cells, and it is accompanied by the acquitance of an adhesion receptor for Tn.

Topics: Antigens, CD; Cell Differentiation; Fibronectins; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Humans; Integrin alphaV; Microscopy, Fluorescence; Neoplasm Proteins; Neuroblastoma; Neurons; Tenascin; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The neural cell adhesion molecule (N-CAM) plays a significant role in the development of the nervous system. Three different isoforms of the molecule have been described, with molecular masses of 180, 140 and 120 kDa, whose differential expression in neurons seems to be related to their state of differentiation. We took advantage of the use of the human neuroblastoma cell line LAN-5, which can be differentiated in vitro by retinoic acid (RA) into neuronal cells, for studying the expression of N-CAM isoforms, and their polysialic acid (PSA) content, at the protein and mRNA levels. Anti-N-CAM polyclonal antibodies recognizing all the N-CAM isoforms and a monoclonal antibody recognizing PSA were used in Western blot experiments with extracts from undifferentiated and RA-differentiated cells. We found that undifferentiated cells express very little of the 180 kDa N-CAM isoform and a large amount of the 140 kDa isoform. A 4-fold increase in the expression of the 180 kDa N-CAM isoform was obtained when LAN-5 cells were differentiated by RA for 8 days, whereas a 1.8-fold increase in the expression of the 140 kDa N-CAM isoform was observed upon differentiation. Similarly, the levels of the 7.4 kb mRNA coding for N-CAM 180 kDa, determined by Northern blot analysis, were barely detectable in undifferentiated cells, and showed a 3.8-fold increase upon differentiation. By contrast, only a 1.3-fold increase in the 6.7 kb mRNA, coding for the 140 kDa N-CAM isoform, was observed. N-CAM was always found in its polysialylated form in both undifferentiated and RA-differentiated cells. This indicates that, in LAN-5 cells, the expression and activity of the polysialytransferase enzyme precedes the acquisition of a neuronal phenotype.

Topics: Antibody Specificity; Antineoplastic Agents; Blotting, Western; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Polysaccharides; RNA, Messenger; Sialic Acids; Tretinoin; Tumor Cells, Cultured

The use of chemically differentiated neuroblastoma cells in the study of neuronal function has become a common alternative to primary neuronal cell cultures in recent years, particularly in the area of cell death. Staurosporine, a nonselective protein kinase inhibitor, has been demonstrated to be a particularly strong inducer of differentiation in the SH-SY5Y human neuroblastoma cell line. However, at present, no data exist on the long-term effects of this compound. We have compared the effects of staurosporine with 12-O-tetradecanoyl phorbol-13 acetate and retinoic acid in terms of long-term cell viability and neuronal function in the SH-SY5Y cell line. In the presence of serum, staurosporine-treated cells underwent apoptosis, which ultimately resulted in total cell loss. In contrast, when cultured in defined serum-free medium, a cessation of apoptosis occurred after approximately 1 week, at which point viability could be maintained in excess of 1 month. The addition of aurintricarboxylic acid, which has been demonstrated to prevent apoptosis in a variety of cell models, completely prevented both apoptosis and differentiation in staurosporine-treated cells both under serum-supplemented and serum-free conditions. Apoptosis was not prevented by the protein synthesis inhibitor, cycloheximide. The removal of staurosporine from the culture medium after 3 weeks had no effect on cellular morphology, function, or proliferation, indicating that the attained neuronal phenotype was terminal. Voltage-gated calcium channel sensitivity, used as a measurement of neuronal function, was highest in staurosporine-treated cells. On the basis that apoptosis and neurotrophin independence are hallmarks of the maturation of dorsal root ganglion neurons, results suggest that staurosporine-differentiated SH-SY5Y cells may bear a similar phenotype to that found in vivo. Furthermore, this model may provide for an excellent means of obtaining a stable and homogenous population of postmitotic monoaminergic neurons for investigating neuronal function and differentiation.

Topics: Apoptosis; Aurintricarboxylic Acid; Calcium Channels; Cell Differentiation; Cell Survival; Electrophysiology; Enzyme Inhibitors; Humans; Ion Channel Gating; Nerve Growth Factors; Neuroblastoma; Neurons; Norepinephrine; Phenotype; Staurosporine; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (RA) reduces human neuroblastoma growth by inducing either differentiation or apoptosis. The apoptotic program in these cells is regulated by RA and is paralleled by the transcriptional induction of "tissue" transglutaminase (tTG). tTG is a protein cross-linking enzyme, which specifically accumulates in cells undergoing apoptosis in various in vivo and in vitro systems. In neuroblastoma cells, tTG is detected exclusively in the cells expressing the S-type phenotype and showing an increased apoptosis. The present study was undertaken to identify the retinoid receptors which are involved in the regulation of tTG and apoptosis as well as in the in vitro neuronal differentiation of the human SK-N-BE(2) neuroblastoma cell line. We have previously characterized the retinoid acid receptors expressed in this cell line. In the present study, by using synthetic retinoids selectively activating RAR/RXR isoforms, we have identified the RAR/RXR receptors involved in the induction of either apoptosis or differentiation. We have also studied the effect of the selective RA analogs on tTG activity. We observed that while RARalpha- and RARgamma-selective retinoids alone were able to induce tTG activity, only the combined stimulation of both RARalpha and RARgamma induced apoptosis. Conversely, several combinations of RAR/RXR closely mimicked the differentiation effects observed with all-trans retinoic acid. These results indicate that, at variance with differentiation, the induction of apoptosis in human SK-N-BE(2) neuroblastoma cells is under the specific control of RARalpha and RARgamma. These data seem relevant for the reported ability of RARgamma to suppress the clinically malignant tumor phenotype in patients.

Topics: Apoptosis; Cell Differentiation; DNA Fragmentation; Enzyme Induction; Humans; Neurites; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; Retinoids; Transcription Factors; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Neuroendocrine tumors, neuroblastoma in particular, commonly express the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) and their receptors. Retinoic acid (RA) has been shown to induce differentiation of neuroblastoma cell lines, possibly by augmenting or interfering with neuropeptide autocrine loops. We sought to determine which receptor gene subtypes are expressed in selected human neuroblastoma cell lines (SH-SY5Y, IMR-32, and LA-N-5), and the effect of RA on the VIP/PACAP ligand/receptor system. Expression of both PACAP1 and VIP1/PACAP2 receptor genes was detected by Northern analysis, which characteristically encode Type I (PACAP-preferring), and Type II (bivalent VIP/PACAP) receptors, respectively. Binding experiments carried out on IMR-32 cells, using 125I VIP and 125I PACAP-27 as tracers, corroborated that both receptor subtypes were expressed. In contrast to RA upregulation of VIP binding (confirmed here in IMR-32 cells), levels of both receptor mRNAs were reduced after RA treatment. VIP mRNA in each cell line was increased by RA, whereas PACAP mRNA, detected in IMR-32 cells only, was reduced. The studies indicate that several components of the VIP/PACAP autocrine system are regulated in neuroblastoma cell lines during RA differentiation.

Topics: Autocrine Communication; Gene Expression; Humans; Ligands; Neuroblastoma; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation; Vasoactive Intestinal Peptide

Analysis of the molecular factors that control cellular differentiation in mammalian embryos is difficult due to the small amount of material available from embryos and their inaccessibility during gestation. One way to circumvent these limitations is to use model systems that allow the study of differentiation in vitro. In this study we have characterized the response of a human neuroblastoma cell line, LA-N-5, to the differentiation-inducing agent, all-trans retinoic acid (RA) using 23 markers that are characteristic of neural crest cells and some of their derivatives. Following induction with RA, the neural crest-like LA-N-5 cells undergo differentiation into cholinergic neurons with increased expression of a variety of neural-specific markers including neurofilaments, growth associated protein-43, tetanus toxin binding sites, receptors for neurotrophic factors, neuropeptides, choline acetyl transferase, vesicular acetylcholine transporter, and acetylcholinesterase with a concomitant decrease in the expression of non-neuronal markers. These results provide the basis for the use of retinoic acid-induced differentiation of LA-N-5 cells as a model system to study molecular events associated with the differentiation of cholinergic neurons.

Topics: Acetylcholine; Antineoplastic Agents; Apoptosis; Cell Differentiation; Gene Expression; Humans; Neural Crest; Neuroblastoma; Neurons; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydro-derivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

Topics: Animals; Cell Differentiation; Ceramides; Cytosol; Kinetics; Mice; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Sphingosine; Tretinoin; Tumor Cells, Cultured

DNA synthesis and postreplication mismatch repair were measured in vitro using cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases delta, alpha, and possibly epsilon, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either alpha- or beta-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T . G and G . G mismatches were repaired most efficiently; C . A, A . A, A . G and a five-base loop were repaired with intermediate efficiency; repair of G . A, C . C, and T . T mismatches was extremely inefficient.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Extracts; DNA Ligases; DNA Polymerase beta; DNA Polymerase I; DNA Repair; DNA Replication; DNA, Viral; Fibroblasts; HeLa Cells; Humans; Neuroblastoma; Replication Origin; Simian virus 40; Tretinoin

Levels of acylphosphatase isoenzymes and free intracellular calcium have been investigated in cultured SH-SY5Y human neuroblastoma cells under stimulation with all-trans retinoic acid and phorbol-12-myristate-13-acetate. Under these conditions morphological and functional characteristics demonstrated the differentiation of SH-SY5Y cells towards neuronal phenotype. Retinoic acid treatment caused a progressive and synchronous increase of the organ common-type acylphosphatase and of free intracellular calcium but not of the muscle-type acylphosphatase. Phorbol-12-myristate-13-acetate treatment gave rise to a peak of the muscle-type acylphosphatase levels during the early differentiation stage whereas organ common-type isoenzyme and free calcium levels show a pattern similar to that observed in retinoic acid-treated cells. These evidences indicate that the two acylphosphatase isoenzymes play different roles in SH-SY5Y differentiation and that during this process the expression of organ common-type acylphosphatase increases in a synchronous way with intracellular free calcium concentration.

Topics: Acid Anhydride Hydrolases; Acylphosphatase; Calcium; Carcinogens; Cell Differentiation; Cell Division; Humans; Neoplasm Proteins; Neuroblastoma; Phorbol Esters; Time Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) plays a major role in embryogenesis of the nervous system and has been reported to induce differentiation in neuroblastoma cell lines. To identify RA signaling pathways involved in such differentiation processes, two RA-sensitive neuroblastoma cell lines (LA-N-5 and SH-SY5Y) were extensively studied. Northern blot experiments determined that of the three RAR mRNAs, only RARalpha was significantly expressed, with respectively weak or undetectable levels of RARgamma and RARbeta. RXRs (alpha and beta) receptors were weakly expressed. Western blotting analysis confirmed the constitutive expression of RARalpha and absence of RARbeta and weak levels of RXRalpha. Treatment with all-trans-RA up-regulated RARalpha and induced a drastic increase of RARbeta (both at the RNA and protein level). To further characterize the function of RARalpha, RARbeta and RXRalpha in NB cells, nuclear extracts from LA-N-5 cells were analysed by EMSA studies. Three specific retarded complexes were observed which were significantly decreased or shifted in the presence of monoclonal antibodies to RARalpha, RARbeta and RXRalpha. RA treatment dramatically induced a DR5-binding RXRalpha-RARbeta heterodimer. Treatment with combinations of RARalpha or RARbeta agonists with a RXRalpha agonist or with a RARalpha agonist alone, induced neurite-outgrowth supporting the probability that both RXRalpha-RARalpha or RXRalpha-RARbeta heterodimers are involved in RA-mediated differentiation of NB cells. The availability of novel synthetic RA-specific receptor ligands should provide the possibility of tissue specific therapeutic regimes.

Topics: Cell Differentiation; Dimerization; Humans; Interferon-alpha; Neuroblastoma; Receptors, Retinoic Acid; RNA, Messenger; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A unique structure and in situ localization of E proteins were demonstrated in cultured neurons infected with neurovirulent and aneurovirulent strains of local Japanese encephalitis virus (JEV). Dilated rough endoplasmic reticulum (rER) containing smooth membrane structures (SMS) was continuous with the outer membrane of the nuclear envelope. These membranes were found to be connected to unique dense bodies, membrane vesicle structures (MVS). The de novo formation of SMS, annulate lamellae, and the appearance of MVS indicated proliferation of the membranous system in response to JEV infection. E proteins were possibly assembled in the virions in the nuclear envelope or rER or on the plasma membrane. The interconnections between MVS, rER, and the nuclear envelope and immunogold labeling of E proteins on the MVS provided strong evidence that MVS serve as a reservoir of JEV components during virus assembly.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Encephalitis Virus, Japanese; Humans; Mice; Microscopy, Immunoelectron; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Replication

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation.

Topics: Antineoplastic Agents; Basement Membrane; Biocompatible Materials; Brain-Derived Neurotrophic Factor; Cell Division; Collagen; Drug Combinations; Humans; Laminin; Metalloendopeptidases; Neoplasm Invasiveness; Neuroblastoma; Phenotype; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Proteoglycans; Receptor Protein-Tyrosine Kinases; Receptor, trkB; Receptors, Nerve Growth Factor; Tissue Inhibitor of Metalloproteinases; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These result

Topics: 3T3 Cells; Animals; Brain-Derived Neurotrophic Factor; Coculture Techniques; Kinetics; Mice; Neurites; Neuroblastoma; Receptor Protein-Tyrosine Kinases; Receptor, Ciliary Neurotrophic Factor; Receptors, Nerve Growth Factor; Recombinant Proteins; Sequence Deletion; Transfection; Tretinoin; Tumor Cells, Cultured

Activin, a member of the transforming growth factor-beta superfamily, regulates various physiological functions. In the present study, we investigated the effect of activin on neuronal differentiation, particularly the functional activity of voltage-dependent Ca2+ channels, in murine neuroblastoma C1300 cells. A slight K(+)-induced increase in the intracellular free Ca2+ ([Ca2+]i) was observed in C1300 cells untreated and treated with either activin A or all-trans-retinoic acid, while treatment with both agents significantly enhanced the increase. The [Ca2+]i increases potentiated by activin A and all-trans-retinoic acid were nearly abolished in the presence of 1.0 mM nickel or in the absence of extracellular Ca2+. Nifedipine (0.1 microM) and omega-conotoxin (1.0 microM), inhibitors of L- and N-type Ca2+ channels, respectively, partially inhibited these responses, however the inhibitory effects of these compounds were not additive. In addition, Bay K 8644, an activator of L-type Ca2+ channels, enhanced the K(+)-induced [Ca2+]i increase. These findings indicated that depolarization evoked the Ca2+ influx, at least in part, through L-type Ca2+ channels in C1300 cells treated with both activin A and all-trans-retinoic acid.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Activins; Animals; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Channels, L-Type; Cell Differentiation; Drug Synergism; Growth Substances; Inhibins; Isomerism; Mice; Nerve Tissue Proteins; Neuroblastoma; Neurons; Potassium; Tretinoin; Tumor Cells, Cultured

The RET proto-oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up-regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5' to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.

Topics: Base Sequence; Drosophila Proteins; Gene Expression Regulation; HeLa Cells; Humans; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Proto-Oncogenes; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Restriction Mapping; RNA, Messenger; Sequence Analysis, DNA; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

The low density lipoprotein receptor-related protein (LRP) has been localized in human brain at the level of neurons, astrocytes and along capillary membranes. It is a multifunctional receptor responsible for binding and internalization of lipoproteins enriched with apoliprotein E, lipoprotein lipase, protease-alpha 2 macroglobulin complexes and plasminogen activator-inhibitor complexes. LRP expression is observed in cells involved in Alzheimer's disease, neoplastic transformation and tissue repair. Moreover, its synthesis is modulated during brain development. In this study we used the SK-N-AS human neuroblastoma cell line as a model system to study LRP expression during cellular differentiation induced by phorbol esters, retinoic acid and interferon gamma. Since LRP plays a major role in the regulation of lipid metabolism, the decreased levels of LRP measured by immunofluorescence, western blot and PCR on differentiated neuroblastoma cells may be the consequence of the lower requirements of cholesterol and lipids of differentiated cells in relation to their reduced mitotic index.

Topics: Blotting, Western; Cell Differentiation; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Low Density Lipoprotein Receptor-Related Protein-1; Neuroblastoma; Phorbol Esters; Receptors, Immunologic; Receptors, LDL; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Actins; Cell Differentiation; Cell Nucleus; Choline; Choline-Phosphate Cytidylyltransferase; Cytosol; Humans; Kinetics; Neuroblastoma; Phosphatidylcholines; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma cells contain a 260 kDa surface-associated antigen (NB-p260) that is recognised by natural cytotoxic IgM antibodies. In this study we demonstrate that NB-p260 is expressed in vivo in a neuroblastoma tumour specimen but not in normal human tissues of neuronal origin. Since MYCN amplification is a clinical marker of neuroblastoma disease progression, we analysed the expression of NB-p260 in human neuroblastoma cell lines with different MYCN amplification status. However, both amplified and non-amplified neuroblastoma cell lines exhibited comparable NB-p260 expression. Treatment of neuroblastoma cells with the differentiation-inducing agent retinoic acid (RA) also had no effect on the expression of NB-p260. Collectively, the data suggest that expression of NB-p260 on human neuroblastoma cells is independent of malignancy and differentiation status of neuroblastoma.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Transformation, Neoplastic; Gene Amplification; Gene Expression; Genes, myc; Humans; Immunoblotting; Immunoglobulin M; Immunohistochemistry; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

We investigated the potential for 9-cis-retinoic acid in the differentiation therapy of neuroblastoma using an N-type neuroblastoma cell line, SH SY 5Y, as an experimental model. In these cells, 9-cis-retinoic acid is more effective than other isomers at inducing the expression of RAR-beta. An RAR-alpha-specific antagonist inhibited the induction of RAR-beta in response to all-trans-but not to 9-cis-retinoic acid. This indicates that the mechanism of gene induction by 9-cis-retinoic acid differs markedly from all-trans-retinoic acid. 9-cis-retinoic acid is also better than all-trans at producing sustained morphological differentiation and inhibition of proliferation of SH SY 5Y cells. Although N-type neuroblastoma cells are not thought to undergo apoptosis in response to all-trans-retinoic acid, we observed a significant degree of apoptosis in SH SY 5Y cells treated with 9-cis-retinoic acid for 5 days and then cultured in the absence of retinoid, an effect not observed in cells treated with the all-trans isomer. These results suggest that 9-cis- and all-trans-retinoic acid have distinct biological properties and that 9-cis retinoic acid may be clinically effective in neuroblastoma by inducing both differentiation and apoptosis under an appropriate treatment regimen.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Benzoates; Cell Differentiation; Cell Division; Chromans; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.

Topics: Animals; Follow-Up Studies; Ganglioneuroma; Gene Amplification; Genes, myc; Humans; Infant; Neoplasm Staging; Neuroblastoma; Octreotide; Rats; Rats, Nude; Receptors, Somatostatin; Somatostatin; Survival Rate; Transplantation, Heterologous; Tretinoin; Vasoactive Intestinal Peptide

The N-myc oncogene plays a key role in the biology of neuroblastoma and the differentiation process. N-myc expression is associated with metastatic disease, as well as the undifferentiated state of normal neuroblasts migrating from the neural crest during embryogenesis. Its down-regulation is a pivotal event in the differentiation of neuroblastoma cells by retinoic acid (RA). Our previous work has shown that RA works synergistically with other agents, such as interferon-gamma (IFN-gamma), to down-regulate N-myc expression and induce differentiation. The present study demonstrates that IFN-gamma, like RA, decreases N-myc transcription. However, functional analysis of N-myc upstream regulatory sequences using 5' deletion mutants of a promoter-CAT construct containing germ line sequences from nucleotide position -887 to +151 showed that IFN-gamma and RA act through different sites on the N-myc promoter. In addition to its transcriptional effect, IFN-gamma was also found to shorten the half-life of N-myc mRNA. Taken together, these findings provide a mechanistic basis for the synergistic action of IFN-gamma and RA in inducing neuroblastoma differentiation and a rationale for the possible development of combination differentiation therapy for clinical use.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, myc; Half-Life; Humans; Interferon-gamma; Neuroblastoma; Promoter Regions, Genetic; RNA Processing, Post-Transcriptional; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Cholinergic hybrid mouse septal neurons SN56 were differentiated by separate and combined application of 0.001 mM all-trans-retinoic acid and 1 mM dibutyryl cAMP. Each of agents caused about twofold increase of choline acetyltransferase activity. These activatory effects were additive. Dibutyryl cAMP resulted in twofold increase of ATP-citrate lyase and acetylcholinesterase activities. Retinoic acid did not affect these enzyme activities but partially abolished activatory effects of dibutyryl cAMP. Pyruvate dehydrogenase and other enzymes of acetyl-CoA metabolism were not affected by this treatment. This work demonstrates that it is possible to rise cholinergic neurons of different expression of cholinergic and acetyl-CoA metabolism.

Topics: Acetyl Coenzyme A; Acetylcholine; Acetylcholinesterase; Animals; ATP Citrate (pro-S)-Lyase; Bucladesine; Cell Differentiation; Choline O-Acetyltransferase; Cytoplasm; Enzyme Induction; Hybrid Cells; Mice; Nerve Tissue Proteins; Neuroblastoma; Septum Pellucidum; Tretinoin

An early event in the pathogenesis of neuroblastoma (NB), a tumor derived from embryonal neural crest tissue, appears to be the arrested differentiation of neuroblasts. However, NB cells can be induced to differentiate in vitro with numerous chemicals including retinoic acid (RA) and dibutyryl cyclic AMP (db-cAMP). One family of transcription factors, encoded by the homeobox (HOX) genes, plays a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development. We have previously identified six HOX genes (HOXC6, HOXC8, HOXD1, HOXD4, HOXD8, and HOXD9), by a sensitive PCR-based approach, in a cDNA library prepared from the human LA-N-5 NB cell line induced to differentiate with RA. In this report, we studied the regulation of these six HOX genes in a series of NB cell lines chemically induced to differentiate. Untreated NB cells express low or undetectable levels of HOX mRNA, and HOXC8 remains undetectable in the induced cells. However, a significant induction of HOXC6, HOXD1, and HOXD8 expression is seen in the RA-treated NB cell lines, albeit with different patterns and degree of up-regulation. db-cAMP treatment also induced HOXC6 and HOXD8 expression in two of the three NB cell lines analyzed. Low levels of HOXD4 and HOXD9 induction were observed in two and one RA-treated NB cell line, respectively. Up-regulation of HOXC6, HOXD1, and HOXD8 expression in human NB cells, chemically induced to differentiate, appears to be associated with maturation toward a differentiated neuronal phenotype.

Topics: Cell Differentiation; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Neuroblastoma; Phenotype; Tretinoin; Tumor Cells, Cultured; Up-Regulation

N-myc expression is negatively regulated by retinoic acid (RA) which induces the growth arrest and differentiation of neuroblastoma (NB) cells. However, it has not been completely defined whether N-Myc promotes growth and/or antagonises neuronal differentiation of NB cells or whether the down regulation of N-myc occurs as a consequence of the onset of differentiation. By transfecting an N-myc gene construct into these cells, we found that the constitutive overexpression of N-myc stimulated proliferation in RA containing medium and, although these cells were still responsive to RA, they were no longer able to differentiate. Since N-Myc functions appear to be mediated by heterodimerization with Max, the ectopic overexpression of max in NB cells was also investigated. In contrast to N-Myc, Max strongly induced the differentiation by enhancing the effects of RA. Max-transfected cells rapidly arrested growth and differentiated fully within a few days of RA treatment. These findings suggest that the relative levels of N-Myc compared to Max appears to be crucial in stimulating neuroblastoma growth or differentiation, and may contribute to explain the remarkable clinical behaviour of neuroblastomas.

Topics: Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Cell Differentiation; Cell Division; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Molecular Sequence Data; Neuroblastoma; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, involved in de novo cholesterol synthesis and cell-cycle progression, was identified as a potential mediator of the growth inhibitory effects of retinoic acid on human neuroblastoma. Lovastatin, a nonreversible inhibitor of HMG-CoA reductase, induced extensive cytotoxicity that was restricted to drug-resistant P-glycoprotein-expressing neuroblastoma cell lines. This response was potentiated by dibutyryl cyclic AMP but not retinoic acid. Patients with advanced-stage metastatic neuroblastoma often display an acquired chemoresistant phenotype, which may in part be mediated by P-glycoprotein. Our studies support the application or use of HMG-CoA reductase inhibitors as potential therapeutic agents in the treatment of these patients who are refractory to chemotherapy.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Base Sequence; Bucladesine; DNA Primers; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Molecular Sequence Data; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

We examined the effects of 2-methoxyestradiol, a metabolite of estradiol, on cell death in retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cell cultures. Cell death was induced by 2-methoxyestradiol in a concentration-dependent manner. Estradiol and 2-methoxyestradiol failed to induce cell death. The cell death response to 2-methoxyestradiol was sensitive to the protein synthesis inhibitor cycloheximide and the apopain inhibitor Ac-Asp-Glu-Val-Asp-H(aldehyde). 2-Methoxyestradiol also induced internucleosomal for and endogenous neuroactive steroid metabolite in the etiology of some neurodegenerative diseases.

Topics: 2-Methoxyestradiol; Amino Acid Sequence; Apoptosis; Cell Differentiation; Cycloheximide; Estradiol; Humans; L-Lactate Dehydrogenase; Molecular Sequence Data; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Growth in neuroblastoma cells is regulated by insulin-like growth factors (IGFs) whose action is modulated by IGF binding proteins (IGFBPs). In this study, SK-N-SH neuroblastoma cells were shown to produce IGF-II, IGFBP-2, IGFBP-4 and small quantities of IGFBP-6. We have studied the effects of a natural morphogen, retinoic acid (RA), on growth and IGFBP expression in these cells. In all experiments, cells were cultured in serum-free medium and treated with 1 mumol/l RA for 12 h. Cell number increased by almost 50% during the first 24 h after the beginning of treatment. This stimulation was inhibited by 80% or more in the presence of the anti-type 1 IGF receptor antibody alpha-IR3 and anti-IGF-II antibody. The IGF-II concentrations in the culture media, measured after acidic gel filtration, increased about 1.5-fold and Northern blotting showed a concomitant increase in IGF-II mRNA levels. The mitogenic effect of RA therefore reflects its stimulation of IGF-II production. The availability of IGF-II to the cells may also be enhanced because of the proteolysis of IGFBP-2 to which it is bound. After this initial phase, proliferation ceased despite continued IGF-II production between 24 and 72 h. Both IGFBP-2 and IGFBP-4 production decreased, whereas that of IGFBP-6 increased. These changes appeared both in the protein quantities and in their mRNAs. Insulin-like growth factor binding protein 6 has a strong affinity for IGF-II, 5-10 times that of IGFBP-2 and at least 10 times that of the type I IGF receptor, and the arrested proliferation may result, at least in part, from sequestration by IGFBP-6 of the IGF-II secreted.

Topics: Blotting, Western; Cell Division; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

We have investigated the relevance of actin rearrangement to neurotransmitter release, in neuroblastoma cell line SH-SY5Y stimulated with carbachol (1 mM). Carbachol reversibly polymerizes actin and releases norepinephrine in undifferentiated SH-SY5Y cells as well as in tetradecanoylphorbol 13-acetate (16 nM) and retinoic acid (5 microM) differentiated SH-SY5Y cells. Microscopic analysis of F-actin distribution indicates polymerization of cortical actin. Prior treatment with iota toxin E from Clostridium perfringens inhibits both carbachol-induced actin polymerization and norepinephrine release, slightly affecting the basal actin network. These data suggest that actin polymerization is associated with norepinephrine release.

Topics: Actins; Carbachol; Cell Differentiation; Cell Line; Humans; Kinetics; Neuroblastoma; Norepinephrine; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured

Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.

Topics: Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Brain-Derived Neurotrophic Factor; Drug Resistance; Humans; Nerve Tissue Proteins; Neuroblastoma; RNA, Messenger; Tretinoin; Tubulin; Tumor Cells, Cultured; Vinblastine

In a cultured hybrid neuronal cell line (BIM) which was produced between human neuroblastoma cells (IMR32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103), the mRNAs encoding ciliary neurotrophic factor (CNTF) and neurotrophins were detected by the polymerase chain reaction method. The conditioned medium of BIM cells enhanced choline acetyltransferase (ChAT) activity in septal neurons and survival of ciliary ganglion neurons. The mRNA expression of CNTF and neurotrophins in BIM cells was differently regulated by the stimulation with cAMP, FGF and retinoic acid. These data suggest multiple regulation and collaboration of neurotrophic factors.

Topics: Animals; Base Sequence; Brain-Derived Neurotrophic Factor; Cell Line; Chick Embryo; Choline O-Acetyltransferase; Ciliary Neurotrophic Factor; Cyclic AMP; Fibroblast Growth Factors; Humans; Hybrid Cells; Molecular Sequence Data; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.

Topics: Animals; Arachidonic Acid; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Fatty Acids, Unsaturated; Humans; Lipid Metabolism; Nerve Tissue Proteins; Neuroblastoma; Neurons; Rats; Tretinoin; Tumor Cells, Cultured

Physiologically, the action of insulin-like growth factors (IGFs) is controlled at different levels, from its transcription start by tissue-specific and development-specific transcriptional factors to its degradation by peptidases such as insulin-degrading enzyme (IDE). Since IGF-II is the major autocrine/paracrine growth factor for neuroblastoma cells, we studied the expression and the role of IDE in this system. Here, we show that (a) IDE is expressed in several human neuroectodermal tumor cell lines, including neuroblastoma cell lines; (b) in a neuroblastoma cell line, IDE expression is up-regulated by retinoic acid, a well-known inducer of neuronal differentiation and/or programmed cell death; (c) IDE is probably not the only IGF-degrading enzyme present in these cells, since the activity of a novel thermolysin-like metalloendopeptidase, clearly distinct from IDE, is also detected. The TME activity is inhibited by IGF-I, Des-IGF-I, and IGF-II, and it is down-regulated by retinoic acid. Since retinoic acid plays a relevant role in controlling the growth of these cells and affects the expression of IDE, we have also: (a) identified the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) expressed in these cell lines and (b) by means of synthetic retinoid analogues identified the RAR/RXR isoforms whose activation may be sufficient to induce the expression of the IDE gene. These results provide evidence that complex posttranslational molecular mechanisms participate in the autocrine/paracrine growth control of the IGF-II loop in neuroblastomas involving proteolytic systems.

Topics: Endopeptidases; Humans; Insulysin; Molecular Structure; Neuroblastoma; Neuroectodermal Tumor, Melanotic; Thermolysin; Tretinoin; Tumor Cells, Cultured

We have investigated the effects of several neurokine/cytokine family members on the level of alpha-bungarotoxin-binding to neuronal nicotinic acetylcholine receptors. Exposure of human neuroblastoma cells (SH-SY5Y and IMR-32) to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or oncostatin-M resulted in a 30-40% decline in alpha-bungarotoxin receptors on the cells with no decrease seen in either muscarinic acetylcholine receptors or in L-type Ca2+ channels. The level of nicotinic receptor was not affected by the related cytokine, interleukin-6. Treatment of IMR-32 cells with 40 pM CNTF produced a half-maximal decrease of alpha-bungarotoxin binding which compared well with the affinity estimated from binding of 125I-CNTF (Ki approximately 40 pM) and the concentration causing c-fos activation in SH-SY5Y cells, as detected by nuclear run-on assays (60-120 pM). Previous results have indicated that the differentiating agents, phorbol esters and retinoic acid, also decrease nicotinic receptor numbers. Here the effects of CNTF, which did not induce neural differentiation, were enhanced by differentiation with 12-O-tetradecanoylphorbol 13-acetate (10 nM) and prevented by retinoic acid (10 microM). Therefore, the response of neuroblastoma cells to cytokines may be under developmental control. These cells offer a system to examine cytokine responses and signal transduction mechanisms during neural development.

Topics: Bungarotoxins; Ciliary Neurotrophic Factor; Dose-Response Relationship, Drug; Gene Expression Regulation; Genes, fos; Humans; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Receptors, Nicotinic; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Widespread proliferation of dystrophic neurites in the cerebral cortex represents an important neuroanatomical correlate of dementia in Alzheimer's disease (AD). Increased CNS expression of the 21-kDa neuronal thread protein (NTP) species is also correlated with dementia in AD. Pilot in vitro experiments provided evidence that high-level NTP expression might be linked to neuritic growth. The present study examines retinoic acid (RA) modulation of NTP expression during neurite outgrowth and neuronal differentiation in SH-Sy5y neuroblastoma and PNET2 CNS-derived cells. In both cell lines, RA-induced neuronal differentiation resulted in increased synthesis, expression, and phosphorylation of several NTP species, with high steady-state levels and stepwise hyper-phosphorylation of 21-kDa NTP molecules. With neurite outgrowth, NTP molecules were translocated from the perikarya to long, slender, unbranched cell processes (axons) and growth cones. RA-mediated changes in NTP expression were independent of DNA synthesis. The findings suggest that high-level expression of 21-kDa, and closely related phosphorylated NTP molecules correlates with neuritic growth. Therefore, over-expression of 21-kDa NTP molecules in AD probably reflects the widespread cortical neuritic sprouting associated with dementia. In view of the rapid phosphorylation and cell process translocation of NTP that occurs during neurite outgrowth in vitro, the accumulation of NTP in AD cortical neuronal perikarya suggests a further problem related to post-translational processing and transport of NTP molecules in AD neurodegeneration.

Topics: Alzheimer Disease; Calcium-Binding Proteins; Cell Differentiation; DNA; Humans; Lithostathine; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; Phosphoproteins; Tretinoin; Tumor Cells, Cultured

The promoter region of the alpha-subunit of the calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) gene was inserted into a beta-galactosidase (beta-gal) reporter plasmid, and beta-gal activities were examined in neuroblastoma (NB2a) and pheochromocytoma (PC12) cells after transient or stable transfections. The alpha-CaMKII promoter was 12- to 45-fold more active in NB2a compared with PC12 cells after transient or stable transfections. All-trans retinoic acid (RA) stimulated reporter gene expression at both protein and mRNA levels in transfected PC12 cells. RA increased the level of endogenous alpha-CaMKII mRNA in untransfected PC12 cells by 4.4-fold. The transcription initiation site(s) (TIS) of the alpha-CaMKII gene in PC12 cells and rat brain was examined by RNase protection assays (RPA) and reverse transcriptase PCRs. The TIS for the alpha-CaMKII/beta-gal reporter gene in transfected PC12 cells was indistinguishable from the TIS+1 in rat hippocampus. In contrast, the only detectable TIS for the alpha-CaMKII gene in untransfected PC12 cells was located near the ATG translation start codon, 147 nucleotides 3' to TIS+1 in hippocampus. This unusual TIS was also the predominant TIS in rat cerebellum. These results suggest that the alpha-CaMKII promoter may contain sequences that respond directly or indirectly to RA. In addition, the unusual TIS of the alpha-CaMKII gene in PC12 cells and rat cerebellum may contribute to the very low expression of this gene compared with that in hippocampus.

Topics: Animals; Base Sequence; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Gene Expression; Mice; Molecular Probes; Molecular Sequence Data; Neuroblastoma; PC12 Cells; Promoter Regions, Genetic; Rats; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Mitochondrial dysfunction and attendant bioenergetic defects are increasingly recognized as playing an important role in neurodegenerative disorders. The increased attention on mitochondrial involvement points to the need for developing cell lines that have neuron-like characteristics for the genetic analysis and modeling of these diseases. We describe the creation of respiratory-deficient SH-SY5Y neuroblastoma cell lines (rho zero 64/5) by selectively depleting mitochondrial DNA through prolonged exposure to ethidium bromide. Oxygen consumption in these cells and activities of the electron transport chain enzyme complexes I and IV that contain subunits encoded by the mitochondrial genome are eliminated. In contrast, the function of complex II, a nuclear-encoded electron transport chain component, is largely intact in these cells. The rho zero 64/5 cells retain the ability to differentiate into cells with neuron-like phenotypes following treatment with phorbol ester or retinoic acid. Normal respiratory function is recovered by repopulation of rho zero 64/5 cells with exogenous human platelet mitochondria. The rho zero 64/5 cell line serves as a valuable model for the study of neurologic diseases suspected of involving mitochondrial dysfunction.

Topics: Blood Platelets; Cell Differentiation; Cell Line; Cell Line, Transformed; Citrate (si)-Synthase; DNA, Mitochondrial; Electron Transport; Electron Transport Complex II; Ethidium; Humans; Mitochondria; Multienzyme Complexes; NAD(P)H Dehydrogenase (Quinone); Neuroblastoma; Neurons; Oxidoreductases; Oxygen Consumption; Phorbol Esters; Reactive Oxygen Species; Succinate Dehydrogenase; Tretinoin; Tumor Cells, Cultured

We determined the effects of all-trans retinoic acid (RA) on the levels of delta opioid receptor (DOR) mRNA and N-Methyl-D-Aspartate receptor (NMDAR1) mRNA in neuroblastoma x glioma hybrid cells (NG108-15) by use of quantitative solution hybridization assays. The assays utilized riboprobes complementary to major portions of the coding region of the DOR and NMDAR1 cDNAs. At 10 microM RA a 3-fold increase in DOR mRNA at 48 h, and later (144 h) alterations were observed in NMDAR1 mRNA levels. Northern blot analysis revealed six transcripts for DOR mRNA ranging in size from 8.7 to 2.0 Kb, and three transcripts for NMDAR1 mRNA ranging in size from 4.1 to 3.5 Kb. Neither the size nor the fractional band intensity was affected by RA treatment. The delayed induction of DOR mRNA suggests an indirect mechanism by which RA acts on transcription of this gene. A surprising induction of DOR mRNA by the protein synthesis inhibitor cycloheximide (CHX) suggests that either a repressor molecule or degrading enzymes/proteases may regulate basal levels of this mRNA. Treatment with RA resulted in a concentration- and time-dependent morphological differentiation characterized by increased size of the cell body and the appearance of numerous short and long processes.

Topics: Animals; Brain Neoplasms; Cells, Cultured; Glioma; Mice; Neuroblastoma; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, delta; RNA, Messenger; Time Factors; Tretinoin

The presence of luteinizing hormone-releasing hormone (LHRH) in SK-N-SH human neuroblastoma cells was investigated by immunocytochemistry and enzyme-linked immunosorbent assays of whole cell extracts and culture medium. In addition, ribonuclease protection assays were utilized to quantitate LHRH messenger RNA. The expression of LHRH mRNA and LHRH protein level was correlated with neuronal differentiation induced by retinoic acid (RA). In differentiated SK-N-SH cells the LHRH mRNA level as well as the amount of LHRH in cell extracts and cell medium were significantly lower than in differentiated cells. These results suggest that RA affects the expression of LHRH mRNA and the level of LHRH protein in SK-N-SH cells. These data show that altering the growth state of the human neuroblastoma SK-N-SH cells toward more neuronal phenotype results in a significant decrease in expression of LHRH mRNA and the protein. The ability of RA to induce changes in LHRH at the mRNA and at the peptide levels will allow further study of RA regulation of LHRH at the neuronal level.

Topics: Blotting, Northern; Brain Neoplasms; Cell Differentiation; Densitometry; DNA Probes; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Gonadotropin-Releasing Hormone; Humans; Neuroblastoma; Oligonucleotides, Antisense; Phenotype; Ribonucleases; RNA Probes; Tretinoin; Tumor Cells, Cultured

SK-N-BE cells were differentiated (D) to neurons with retinoic acid. Total n-6 polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA), were increased in D versus ND cells. AA synthesis from linoleic acid (LA) and phospholipid (PL) synthesis from glycerol were initially elevated, whereas both processes were reduced approaching differentiation. At this stage, the incorporation of glycerol in triglycerides was enhanced. Formation of long chain PUFA and synthesis of acceptors (PL) for esterification, during RA-induced SK-N-BE differentiation, appear to be associated.

Topics: Arachidonic Acid; Cell Differentiation; Humans; Keratolytic Agents; Lipid Metabolism; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Expression of neuronal nitric oxide synthase (n-NOS) was investigated during neuronal cell differentiation. Trace amounts of n-NOS mRNA were detected in the primary culture of neural precursor cell (NPC) at embryonic day 10, and a high level of n-NOS mRNA was observed after a further 7 days cultivation of NPC. Both n-NOS activity and its mRNA level were also increased in the human neuroblastoma cell line, TGW, following trans-retinoic acid (RA)-induced neuronal differentiation. These findings indicate that there is positive regulation of n-NOS mRNA during neuronal cell differentiation. However TGW cell differentiation with trans-RA could not be reversed by treatment with specific inhibitors of NOS. Thus, the signal transduction mechanism which is involved in the induction of neuronal cell differentiation by trans-RA appears to be distinct from the NO-mediated pathway.

Topics: Animals; Base Sequence; Cell Differentiation; Cells, Cultured; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Molecular Sequence Data; Neuroblastoma; Neurons; Nitric Oxide Synthase; Rats; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Two-dimensional (2-D) electrophoretic methods have been available that allow separation of the protein constituents of a cell population. It has also become feasible to electrophoretically separate in two dimensions and to display DNA fragments derived from genomic digests. Through the appropriate choice of restriction enzymes, the functional component of the genome that encompasses CpG islands can be preferentially visualized in 2-D gels. The same computerized approach for the analysis of 2-D patterns can be applied to investigations at either the protein or DNA levels. Our group has utilized 2-D electrophoresis to investigate both protein and DNA changes in cancer. The emphasis to date has been on the identification of proteins, the abundance of which is related to specific biological features of the tumors analyzed and of DNA fragments encompassed in genomic amplifications, as the latter commonly contain growth-related genes. Findings derived from our analysis of neuroblastoma tumors and cell lines using 2-D approaches are reviewed. Data for four proteins observed in 2-D gels are presented because of our demonstrated association of these proteins with differentiation and proliferation properties of neuroblastoma. At the genomic level, the detection of amplifications using 2-D gels has necessitated an understanding of the variability displayed by multi-copy genomic fragments, which we have accomplished to a large part and which we present. An important benefit of 2-D approaches is the efficiency of scale and the ease with which abundant proteins or multicopy genomic fragments can be detected, identified and quantitatively analyzed.

Topics: Cell Differentiation; Cell Fractionation; Cloning, Molecular; DNA, Neoplasm; Electrophoresis, Gel, Two-Dimensional; G1 Phase; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Genome; Heat-Shock Proteins; Microtubule Proteins; Monomeric GTP-Binding Proteins; Neoplasm Proteins; Neoplasm Staging; Neuroblastoma; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phosphoproteins; Proliferating Cell Nuclear Antigen; Stathmin; Transcription Factors; Tretinoin; Tumor Cells, Cultured

1. The electrophysiological properties of voltage-dependent sodium currents were studied in the human neuroblastoma cell line SH-SY5Y before and after in vitro differentiation with retinoic acid, with the use of the whole cell variant of the patch-clamp technique. 2. Voltage steps from a holding level of -90 mV to depolarizing potentials elicited, in both undifferentiated and differentiated cells, fast inward sodium currents that were full inactivating and tetrodotoxin sensitive. 3. In undifferentiated cells the current peaked at -10 mV, the half-activation potential was -35 mV, and the half-inactivation potential was -81 mV. In differentiated cells the current peaked at + 10 mV, the half-activation potential was -28 mV, and the half-inactivation potential was -56 mV. Moreover, the peak current amplitude was about a factor of 2 larger and inactivation kinetics was about a factor of 2 slower than in undifferentiated cells. 4. This diversity in sodium channel properties was related to differences in cell excitability. Under current-clamp conditions, intracellular injection of rectangular depolarizing current stimuli from a hyperpolarized membrane potential of about -100 mV elicited graded and weak regenerative responses in undifferentiated cells, whereas overshooting action potentials with faster rising phases could be elicited in differentiated cells.

Topics: Cell Differentiation; Electric Conductivity; Humans; Membrane Potentials; Neuroblastoma; Neurons; Patch-Clamp Techniques; Regression Analysis; Sodium Channels; Tetrodotoxin; Tretinoin; Tumor Cells, Cultured

Neuronal nicotinic acetylcholine receptors are expressed on a variety of cells in the nervous system where they play key roles in synaptic transmission and information transfer. Little is known, however, about the molecular mechanisms that control their expression, distribution, and function during nervous system development. We have investigated the control of expression during differentiation of one class of acetylcholine receptors that bind alpha-bungarotoxin of human neuroblastoma cells. We report that induction of differentiation of SH-SY5Y, SK-n-SH or IMR-32 cells by the phorbol ester 12-O-tetradecanoyl phorbol 13-myristate (10 nM, TPA) or by retinoic acid resulted in as much as a 70% decline in alpha-bungarotoxin receptors on the cells. The response to the phorbol ester was blocked by the protein kinase C inhibitors staurosporine and bisindolylmaleimide. The decrease in receptors induced by 10 microM retinoic acid was not affected by either agent. However, responses to lower (10 nM) concentrations of retinoic acid were blocked by staurosporine but not bisindolylmaleimide, suggesting a dual mechanism of action for retinoic acid in regulating acetylcholine receptors. It appears that acetylcholine receptors on neuroblastoma cells are regulated during differentiation by both protein kinase C-dependent and -independent mechanisms.

Topics: Binding Sites; Bungarotoxins; Cell Differentiation; Cyclic AMP; Down-Regulation; Enzyme Activation; Humans; Iodine Radioisotopes; Kinetics; Neuroblastoma; Neurons; Protein Kinase C; Receptors, Nicotinic; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) decreased growth and increased morphologic differentiation of human neuroblastoma LA-N-1 cells. These phenomena correlated with a specific enhancement of PHA-E lectin binding to a glycoprotein of MW 67 kDa (gp67). Gp67 was found susceptible to N-glycanase and displayed BSA binding by affinity chromatography analysis. The chemotherapeutic agent methotrexate (MTX) also reduced growth and induced differentiation of LA-N-1 cells. In addition, the cells responded to MTX as well as to doxorubicin by a marked increase in PHA-E binding to gp67. We conclude that reduced growth and induction of morphological differentiation of LA-N-1 cells correlates with increased binding of PHA-E to gp67.

Topics: Amidohydrolases; Antibodies; Cell Differentiation; Cell Division; Chromatography, Affinity; Humans; Lectins; Membrane Glycoproteins; Methotrexate; Neoplasm Proteins; Neuroblastoma; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Phytohemagglutinins; Protein Binding; Sensitivity and Specificity; Sepharose; Serum Albumin, Bovine; Staining and Labeling; Tretinoin; Tumor Cells, Cultured

The relationship of genes associated with contact inhibition of cell growth and the commitment for differentiation was studied in the human neuroblastoma cell line SH5Y. These cells could be induced to differentiate in vitro into neuronal-like cells upon incubation with retinoic acid, an event that was accompanied by an enhancement in levels of neuron-specific acetylcholinesterase. The kinetics of differentiation, based on morphology and acetylcholinesterase levels, showed that proliferation arrest always preceded differentiation and may be a prerequisite for differentiation. To determine if this growth arrest is mediated by the same pathway underlying contact inhibition of proliferation, the expression of a gene associated with the induction of contact inhibition, protein disulfide isomerase (PDI), was quantified by Northern blot analysis and enzymatic activity after retinoic acid treatment. Retinoic acid caused a significant elevation of PDI-mRNA within 24 hrs. after treatment with a corresponding increase in enzyme activity which immediately preceded proliferation arrest and differentiation. Bacitracin, a specific inhibitor of PDI, abrogated the ability of retinoic acid to induce differentiation. However, treatment with interferon also increased PDI activity and caused proliferation arrest and SH5Y differentiation but into a fibroblastoid cell without neurite outgrowth. These results suggest that the commitment for differentiation of SH5Y cells involves a form of proliferation arrest in which activation of PDI activity is a required and early event but one that does not determine the final differentiation pathway.

Topics: Cell Differentiation; Cell Division; Enzyme Induction; Humans; Isomerases; Neuroblastoma; Protein Disulfide-Isomerases; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Recent evidence suggests that resistance to antineoplastic therapy may result from mutations in genes mediating the apoptotic response to DNA damage. To determine the effects of epigenetic changes on tumor responsiveness to cytotoxic agents inducing DNA damage, we examined the chemosensitivity of neuroblastoma (NB) after differentiation by retinoic acid (RA). Differentiation of the cell lines SH-SY5Y and SMS-KCNR by RA abolished the cytotoxic effects of adriamycin (Adr) and cisplatin. Chemoresistance was not the result of decreased proliferation induced by RA because: (a) growth arrest by nutrient deprivation did not affect sensitivity; (b) growth arrested NB cell lines, which did not differentiate, remained chemosensitive; and (c) RA concentrations which promoted differentiation without affecting growth, induced resistance. Apoptosis characterized NB cells responding to Adr, although differentiated SH-SY5Y did not apoptose and were resistant to Adr and cisplatin. Marked induction of bcl-2 in NB cells followed RA-induced differentiation, whereas in cell lines failing to differentiate, bcl-2 was not detected. Our data indicate that NB differentiation induces drug resistance after a loss of the apoptotic response to antineoplastic drugs and suggest that bcl-2 overexpression is an important mechanism of resistance in differentiated tumor cells.

Topics: Apoptosis; Cell Differentiation; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; In Vitro Techniques; Neuroblastoma; Neurons; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

Topics: Alprostadil; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Brain; Calcium Channels; Calcium Channels, L-Type; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; Enhancer Elements, Genetic; Exons; Gene Expression Regulation, Neoplastic; Genomic Library; Glioma; Hybrid Cells; Introns; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Insertional; Nerve Tissue Proteins; Neuroblastoma; Neurons; Oligodeoxyribonucleotides; Rats; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; RNA, Messenger; Sequence Deletion; Transcription, Genetic; Transfection; Tretinoin

Trk receptors are a family of genes implicated in the survival, differentiation, and growth of certain neurons and tumors of the nervous system. A better understanding of the regulation of Trk receptors is relevant for developmental and oncological studies. Human neuroblastoma (NB) cell lines constitutively express low levels of TrkA mRNA, while TrkB mRNA is not readily detectable. Differentiation of NB cells is accompanied by a differential modulation of Trk expression in human NB cells. Nanomolar concentrations of RA induce a stable increase of TrkB mRNA. A transient induction of TrkA mRNA levels requires micromolar concentrations of RA. Induction of both TrkA and TrkB mRNA does not require new protein synthesis. However, RA-induced TrkB mRNA expression is transcriptionally regulated, while the transient RA-induced increase of TrkA mRNA is a consequence of extended mRNA stability. Interferon gamma (IFN-gamma) selectively increases TrkA mRNA without affecting TrkB mRNA levels. Similar to RA, IFN-gamma does not modify the transcriptional rate of TrkA mRNA, but rather increases TrkA mRNA stability. Thus, RA and IFN-gamma differentially regulate TrkA or TrkB expression in the same cell type by predominantly transcriptional (TrkB) or post transcriptional (TrkA) mechanisms. Such experiments indicate the complexity of Trk mRNA regulation and also indicate compounds that may affect neurotrophin responsiveness in developing neural cells.

Topics: Gene Expression Regulation; Humans; Interferon-gamma; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptor, trkB; Receptors, Nerve Growth Factor; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

We report the first evidence that differential transcriptional regulation of human chromogranin A (CHGA) gene expression occurs during in vitro treatment of tumorigenic neuroblastoma (NB) cells with retinoic acid (5 microM) and/or dibutyryl-cAMP (1 mM). The CHGA gene encodes a tissue specific protein restricted to cells of the diffuse neuroendocrine system, but also widely expressed among NB tumours. We previously reported that CHGA as well as other neuroendocrine markers are modulated during NB differentiation in vitro. To investigate, at the molecular level, the mechanisms leading to NB tumour cell differentiation during the treatment with biologically active compounds, we sequenced and functionally characterised 2169 bp of a genomic DNA clone encompassing the 5' flanking region of the human CHGA gene. Computer-assisted analysis of the sequence revealed the presence of a cAMP responsive element at positions -56 to -49, and Sp1 binding sites at positions -181 to -176 and -216 to -210. Two novel 9 bp motifs, located at position -462 to -454 and -91 to -83 of the CHGA promoter were identified in the regulatory regions of two other neuroendocrine genes encoding for tyrosine hydroxylase and neuropeptide Y. In addition, in the first 1000 bp of the untranslated 5' region, we found the presence of several putative DNA binding sites of bHLH molecules, a protein family regulating tissue specific differentiation. Transient transfection experiments of chloramphenicol acetyltransferase (CAT) deletion constructs, showed the presence of an active promoter within the first 455 bp upstream from the start site. This region conferred tissue specific expression to a CAT reporter gene. In addition, the transcriptional activity of this fragment was modulated during the induction of differentiation of NB cells treated by retinoic acid and/or dibutyryl-cAMP. These observations provide preliminary data regarding CHGA transcriptional regulation in NB cells, and indicate that retinoic acid and cAMP activate distinct, apparently competitive, transcriptional pathways during NB cell differentiation. The molecular characterisation of the mechanisms regulating CHGA expression in tumour and normal neuroendocrine tissue could lead to the identification of novel molecules potentially relevant for future gene therapy of NB tumours.

Topics: Base Sequence; Bucladesine; Cell Differentiation; Chromogranin A; Chromogranins; Humans; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; Sequence Analysis, DNA; Transfection; Tretinoin; Tumor Cells, Cultured

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. This protein is important in the regulation of cell growth and differentiation in a number of different cell types. Therefore, the aim of the present study was to examine the role of p21ras and regulators of its activity in the differentiation of neuroblastoma cells induced by retinoic acid (RA). Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and neurofibromin. RA-induced differentiation of the two neuroblastoma cell lines SK-N-SH and IMR-32 was closely related to growth inhibition. Differentiation induced by RA resulted in an increase in both type I GAP120 and neurofibromin mRNAs. This increase was accompanied by a decrease in the activation of p21ras. These results suggest that, in neuroblastoma, activation of p21ras is not associated with RA-induced differentiation. However, the GTPase activating proteins type I GAP120 and neurofibromin may have effector functions in RA-induced differentiation of neuroblastoma.

Topics: Base Sequence; Blotting, Western; Cell Differentiation; Cell Division; GTPase-Activating Proteins; Humans; Keratolytic Agents; Molecular Sequence Data; Neuroblastoma; Neurofibromin 1; Polymerase Chain Reaction; Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Tretinoin; Tumor Cells, Cultured

Using monoclonal antibodies against the RAR-alpha and RAR-beta retinoic receptors, we demonstrated that these receptors were present together in C6 glioma cells as two isoforms of 50 and 55 kDa. For RAR-beta, the 50 kDa isoform predominated (60 to 80% of the total of the two isoforms). After a treatment for 48 h with retinoic acid 10 microM, the 55 kDa form was enhanced, while no effect was observed either on RAR-alpha isoforms from C6 cells and on both RAR-alpha and RAR-beta forms from neuroblastoma SKN SH SY5Y used as a control. Using purified neuronal and glial rat brain nuclei, we showed that the 55 kDa isoform from RAR-beta predominated in glial cells. These results suggest that retinoic acid treatment of C6 cells led to a partial differentiation, the enhancement of the heavy form of RAR-beta being a marker of this phenomenon.

Topics: Animals; Antibodies, Monoclonal; Biomarkers; Cell Nucleus; Glioma; Molecular Weight; Neuroblastoma; Neuroglia; Neurons; Rats; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

1,25-Dihydroxycholecalciferol (D3) plays an important role in embryonic development and cell differentiation. It has previously been reported to decrease c-myc expression by HL-60 cells and downregulate c-myc expression by breast and ovarian cancer cells. We report the results of our investigations into the differentiating effects of D3 on LA-N-5 human neuroblastoma cells.. LA-N-5 human neuroblastoma cell line was treated with D3, retinoic acid (RA), D3 and RA, or solvent control. Growth inhibitory effects, neurite extension, acetylcholinesterase activity, invasiveness, motility, and N-myc protein expression were examined following treatment.. Growth inhibition was observed at concentrations of > 24 nM. D3 stimulated the differentiation of LA-N-5 cells as demonstrated by increased neurite outgrowth, increased acetylcholinesterase activity, and decreased invasiveness. A decrease in N-myc expression was observed in immunostained cells treated with either agent alone, with a more profound effect appreciated with the combination.. Vitamin D3 decreases N-myc expression in LA-N-5 human neuroblastoma cells, with extended treatment causing growth inhibition and differentiation. When used in combination with RA, these effects are more profound than with either agent alone. The therapeutic use of differentiating agent combinations such as D3 and RA may provide a relatively nontoxic means of treating susceptible tumor types.

Topics: Calcitriol; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (ATRA) has been shown to be one of the most potent chemical inducers of human neuroblastoma differentiation. The recent discovery that the stereoisomer of ATRA, 9-cis-retinoic acid (9-cis-RA), binds to both the retinoic acid and retinoid X series of receptors prompted us to evaluate the ability of this compound to promote differentiation of this cell type. Using the LA-N-5 cell line, we have now determined that 9-cis-RA can induce the differentiation of human neuroblastoma cells as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and reduction of N-myc mRNA expression. In comparing the effects of 9-cis-RA to ATRA, we found that while both compounds induced qualitatively similar cholinergic (versus adrenergic) features in LA-N-5 cells, 9-cis-RA was 5-to-10-fold more potent than ATRA in its antiproliferative and differentiation activity. These results were supported by transient transfection experiments utilizing chloramphenicol acetyltransferase (CAT) plasmid constructs containing a retinoic acid responsive regulatory element which showed a 2-to-3-fold increase in reporter gene activity induced with 9-cis-RA over that seen with ATRA at pharmacologically relevant retinoid concentrations (> 10(-8) M). Furthermore, we have determined that 9-cis-RA can significantly enhance mRNA levels of the nuclear retinoic acid receptors alpha and beta in LA-N-5 cells. Taken together, these findings have established the ability of 9-cis-RA to induce neuroblastoma differentiation and suggest that this retinoic acid isomer may have better therapeutic characteristics than ATRA.

Topics: Acetylcholinesterase; Animals; Cattle; Cell Differentiation; Cell Division; Chloramphenicol O-Acetyltransferase; Dose-Response Relationship, Drug; Gene Expression; Genes, myc; Humans; Neuroblastoma; Neurons; Plasmids; Receptors, Retinoic Acid; RNA, Messenger; Stereoisomerism; Transfection; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, beta-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

Topics: Animals; Cell Differentiation; Cell Division; Ceramides; Mice; Models, Neurological; Neuroblastoma; Neurons; Sphingolipids; Sphingosine; Tretinoin; Tumor Cells, Cultured

Immature neurons, including fetal and tumoral cells, are used for investigating neuronal differentiation in vitro. The human neuroblastoma cell line NB69 could be induced to differentiate to dopamine or acetylcholine neurons by different compounds, including neurotrophins and activators of the protein kinases. In these NB69 cells dibutyryl cyclic AMP (dbcAMP) at 2 mM reduced the division rate and increased the levels of catecholamines, tyrosine hydroxylase (TH) activity, and monoamine oxidase activity. The dbcAMP also increased cell size, dendritic arborization, density of the sites for high-affinity dopamine uptake, and activity of choline acetyltransferase. In fetal rat midbrain neurons treatment with dbcAMP increased the levels of dopamine and the number of TH-immunoreactive neurons in the culture. When embryonic day 14 fetal midbrain neurons, previously exposed to 1 microM retinoic acid (a compound that severely reduces the number of fetal midbrain dopamine neurons), were treated with dbcAMP, the levels of dopamine and the number of TH-immunoreactive cells returned to normal levels. This suggests that dbcAMP induces the differentiation to dopamine neurons of quiescent progenitor or facilitates expression of the dopamine phenotype in immature neurons. Therefore, dbcAMP not only differentiates uncommitted immature dopamine neurons, but also reverses the antidopaminergic effects of retinoic acid. These properties of dbcAMP could be of therapeutic value in Parkinson's disease.

Topics: Alprostadil; Animals; Bucladesine; Cell Death; Cell Differentiation; Dopamine; Fetus; Humans; Mesencephalon; Neuroblastoma; Neurons; Rats; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.

Topics: Blood Physiological Phenomena; Bucladesine; Cell Differentiation; Cell Division; Humans; Neuroblastoma; Receptors, Tumor Necrosis Factor; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma (NB) tumor cell lines treated in vitro with the retinoid, all-trans-retinoic acid (aRA), form neurites and undergo growth arrest. Retinoids exert their diverse morphologic effects through a signalling pathway which involves the nuclear retinoid receptors. Defective retinoic acid receptor (RAR) function contributes to the malignant phenotype of several human and experimental tumors. Considerable evidence from gene disruption studies now suggests that one of the RARs, RAR gamma, may directly mediate some retinoid effects on embryonic and malignant cells. We, firstly, examined primary NB tumor tissue for a correlation between endogenous RAR gamma expression and clinical stage of the tumor and secondly, the effects of exogenous over-expression of the RAR gamma gene on a human NB tumor cell line. RAR gamma mRNA expression in 32 primary NB tumor tissue samples were significantly higher in clinically localised tumors compared with advanced or disseminated tumors. The human NB tumor cell line, BE(2)-C, was stably transfected with a mammalian expression vector (pREP4) over-expressing the human RAR gamma cDNA. Two selected clones over-expressing RAR gamma (BE/G1 and 2) exhibited a reduced growth rate compared to control cells. Tumorigenicity was inhibited for BE/G1 cells and there was a delayed onset to tumor formation for BE/G2 cells. aRA caused growth inhibition but not neuritic differentiation of the BE/G clones, while 9-cis-retinoic acid caused both growth arrest and neuritic differentiation. Taken together these results suggest that reduced endogenous RAR gamma expression may contribute to the malignant phenotype of human NB. In NB cells the retinoid signalling pathway for neuritic differentiation may be distinct from that causing growth inhibition.

Topics: Adrenal Glands; Base Sequence; Cell Differentiation; Cell Division; Cells, Cultured; DNA Primers; Humans; In Vitro Techniques; Molecular Sequence Data; Neurites; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transfection; Tretinoin

All-trans-retinoic acid (RA) and retinoids induce synthesis of tissue-type plasminogen activator (t-PA) in endothelial and neuroblastoma cells in vitro and in rats in vivo. In HT1080 fibrosarcoma cells, induction of t-PA-related antigen secretion and t-PA mRNA steady state levels by RA were found to depend on de novo protein and mRNA synthesis. Fragments derived from the 5'-flanking region of the t-PA gene (+197 to -9578 base pairs (bp)) were linked to the chloramphenicol acetyltransferase gene. Transfection studies demonstrated that the region spanning bp -7145 to -9578 mediated induction by RA. A functional retinoic acid response element (RARE), consisting of a direct repeat of the GGGTCA motif spaced by 5 nucleotides (t-PA/DR5), was localized at -7.3 kilobases. The t-PA/DR5 element interacted with the heterodimer composed of retinoic acid receptor alpha and retinoid X receptor alpha in vitro, whereas its mutation abolished induction by RA in transient expression. In human EA.hy926 hybrid endothelial and in SK-N-SH neuroblastoma cells, the activity of t-PA/DR5 was found to be independent of the intervening sequence (-632 to -7144 bp) and of its distance from the transcription initiation site. Staurosporine, an inhibitor of protein kinase activity, inhibited induction by RA, suggesting that it required protein phosphorylation.

Topics: Alkaloids; Animals; Base Sequence; Cell Line; Chloramphenicol O-Acetyltransferase; DNA; Dose-Response Relationship, Drug; Endothelium; Enzyme Induction; Fibrosarcoma; Gene Expression; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Mutagenesis; Neuroblastoma; Oligodeoxyribonucleotides; Protein Kinase C; Protein Multimerization; Rats; Receptors, Retinoic Acid; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Sequence Deletion; Staurosporine; Tissue Plasminogen Activator; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

p36 is a calcium/lipid-binding phosphoprotein that is expressed at high levels in proliferating and transformed cells, and at low levels in terminally differentiated cells, such as CNS neurons. The calcium-dependent binding to membrane phospholipids, and its capacity to interact with intermediate filament proteins suggest that p36 may be involved in the transduction of extracellular signals. The present work examines p36 gene expression in the mature CNS, primary primitive neuroectodermal tumors (PNETs), and transformed PNET cell lines. p36 immunoreactivity was not observed in normal adult human brain, but low levels of the protein were detected by Western blot analysis. Following acute anoxic cerebral injury, the mean levels of p36 protein were elevated two-fold, and injured neurons exhibited increased p36 immunoreactivity. This phenomenon was likely to have been mediated by post-transcriptional mechanisms since there was no corresponding change in the level p36 mRNA. p36 immunoreactivity was detected in 8 of 9 primary PNETs, and in 3 of 3 neurofilament-expressing PNET cell lines. The levels of p36 protein in PNET cell lines were 5-fold higher than in adult human brain tissue. Although p36 gene expression was generally high in proliferating PNET cells, the levels of p36 mRNA and protein were not strictly correlated with DNA synthesis. Instead, p36 gene expression was modulated in both proliferating and non-proliferating PNET cell cultures by treatment with 50 mIU/ml of insulin, 100 mM ethanol, or 5 microM retinoic acid. The frequent discordances observed experimentally and in vivo between p36 mRNA and p36 protein expression suggest that the steady-state levels of p36 protein in neuronal cells may be regulated primarily by post-transcriptional mechanisms.

Topics: Adult; Animals; Annexin A2; Astrocytoma; Brain; Brain Ischemia; Brain Neoplasms; Cell Differentiation; Ethanol; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Hypoxia, Brain; Insulin; Membrane Lipids; Neoplasm Proteins; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neuroectodermal Tumors, Primitive; Neurons; Rats; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The effects of retinoic acid on components of the cAMP-dependent signalling system were examined in two related human neuroblastoma cell lines SK-N-SH-F (SHF) and SK-N-SH-N (SHN). Retinoid treatment for a week significantly increased the concentration of intracellular cAMP and the levels of activity of protein kinase A and adenylate cyclase in both cell lines. Retinoic acid treatment also caused a very marked translocation of nucleoside diphosphate kinase from the cytosol to the membrane fraction. The increases in cyclic nucleotide and protein kinase A activity were observed to occur as early as within 1 and 2 days respectively and preceded or were concurrent with the onset of observable morphological differentiation. Results also indicated that agents which elevated intracellular cAMP caused neuronal differentiation and blunted retinoic acid-induced melanocytic differentiation in SHF cells. However, increases in cAMP brought about by treatment of SHF cells with retinoic acid alone were several-fold smaller and thus insufficient to induce neuritogenesis in these cells. The results as a whole indicate that one overall effect of retinoic acid treatment is to upgrade the activity of components of the cAMP-dependent signalling system in both neuroblastoma cell lines. However, retinoic acid causes the SH-F and SH-N cell lines to differentiate along different routes which means that the upgrading responses may be related to more general aspects of differentiation rather than to specific phenotype expression.

Topics: Adenylyl Cyclases; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Humans; Neuroblastoma; Nucleoside-Diphosphate Kinase; Signal Transduction; Time Factors; Tretinoin

Topics: Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 3; Female; Fetal Diseases; Humans; Neuroblastoma; Pregnancy; Retinal Diseases; Retinol-Binding Proteins; Translocation, Genetic; Tretinoin

Recent work on a variety of normal and malignant cell lines has shown that induction and secretion of biologically active TGF-beta may occur after exposure to all-trans-retinoic acid (RA), coincident with decreased growth rate and/or differentiation. This study evaluates the expression and regulation of transforming growth factor beta (TGF-beta) and its receptors during RA-induced cell growth arrest and induction of differentiation in the RA-sensitive human neuroblastoma cell line SMS-KCNR and the RA-resistant neuroblastoma cell line SK-N-AS. RA treatment of SMS-KCNR cells results in a 40-fold increase in TGF-beta 1 mRNA after 4 days of RA, a dose-dependent increase in TGF-beta 1 secretion, an increase in types I (TBRI) and III (TBRIII) TGF-beta receptor proteins, and an increase in type II TGF-beta receptor (TBRII) mRNA coincident with RA-responsiveness of the cells. However, in the RA-resistant line SK-N-AS, TGF-beta 1 is constitutively secreted at levels that are unchanged after RA treatment, and although TBRI and TBRIII mRNA is expressed in untreated SK-N-AS cells, levels of TBRI and TBRIII protein and TBRII mRNA decrease after RA treatment. Thus, in RA-sensitive neuroblastoma cells, RA treatment may result in the induction of a negative autocrine TGF-beta 1 growth regulatory loop. These results suggest the hypothesis that: (a) induction of a TGF-beta 1 negative autocrine growth loop may be a necessary component for RA-responsiveness of neuroblastoma cells in vivo; and (b) the inability to induce or maintain this TGF-beta 1 negative autocrine growth loop may be a mechanism of RA resistance in neuroblastoma.

Topics: Base Sequence; Cell Differentiation; Cell Division; Evaluation Studies as Topic; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neuroblastoma; Protein Processing, Post-Translational; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Two families of nuclear retinoid receptors, retinoic acid receptor and retinoid X receptor (RAR and RXR respectively), and a family of cellular retinoic acid-binding proteins (CRABPI and II) participate in the retinoic acid (RA) signaling pathway. The presence and function of many of these receptors and cellular binding proteins have not been fully explored in RA-responsive human neuroblastoma cells. We have previously shown that RAR transcripts and protein are present in human neuroblastoma cells, and that all-trans RA induces the expression of the RAR beta mRNA. In this paper, we demonstrate that human neuroblastoma cells express mRNA for RXR alpha and beta. The mRNA for CRABPI is present in untreated human neuroblastoma cells, whereas the mRNA for CRABPII is induced in cells treated with either all-trans RA or 9-cis RA. Furthermore, 9-cis RA, a ligand that binds to both the RAR and the RXR families, selectively activates the CRABPII gene. In contrast, all-trans RA and 9-cis RA are equally effective in the induction of RAR beta transcript and inhibition of cell proliferation. Since both retinoids inhibit human neuroblastoma cell proliferation, it appears that induction of RAR beta rather than of CRABPII is more likely linked to the regulation of human neuroblastoma cell growth.

Topics: Base Sequence; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

In addition to the mu- and delta-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of kappa 3 receptors, accounting for 40% of total opioid binding, as measured with [3H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in kappa 3 binding. Fully 75% of the increased binding corresponds to kappa 3 sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the mu antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). Naloxone benzoylhydrazone, a kappa 3 agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional kappa 3 receptors.

Topics: Binding Sites; Binding, Competitive; Cell Differentiation; Cyclic AMP; Diprenorphine; Humans; Morphine; Naloxone; Neuroblastoma; Receptors, Opioid, kappa; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma I-type cells isolated from cell lines in vitro are morphologically intermediate between neuroblastic (N) cells, with properties of embryonic sympathoblasts, and substrate-adherent (S) cells having properties of embryonic Schwann/glial/melanocytic cells of the neural crest. I cells have biochemical features of both N and S cells. We propose that the I-type cell represents a malignant neural crest stem cell. The strongest evidence in support of this hypothesis is that: (a) I cells can generate progeny that have neuronal properties, i.e., are committed neuroblasts, or properties of nonneuronal, embryonic neural crest-derived cells; and (b) I-type cells can generate multipotent I-type progeny, indicating their capacity for self-renewal, a feature of stem cells. We report here that I-type cells, derived from four different human neuroblastoma cell lines and experimentally induced to differentiate, give rise to cells with distinct N or S cell phenotypes, indicative of I cell multipotentiality. Experiments with a large panel of I-type subclones, isolated from clonal I-type BE(2)-C cells and exposed to retinoic acid to induce neuronal differentiation or 5-bromo-2'-deoxyuridine to obtain S-type cells, demonstrated that differentiation occurs via induction and selection and not by selection of spontaneously arising variants. The differentiation phenotype was stable. We conclude that human neuroblastoma I-type cells are multipotent embryonic precursor cells of the peripheral nervous system, capable of either neuronal or nonneuronal neural crest cell differentiation.

Topics: Bromodeoxyuridine; Cell Differentiation; Cell Transformation, Neoplastic; Clone Cells; Humans; Neural Crest; Neuroblastoma; Stem Cells; Tretinoin

Atomic force microscopy (AFM) is gaining ever-increasing attention in biology as it allows us to achieve very high resolution both in air and in liquid. Recently, this technique has been employed for the observation of dynamic phenomena of cells in their culture medium. We employed this technology for comparing different morphologies, neuronal and substrate-adherent type, of cell lines of human neuroblastoma, a tumor derived from neuroectodermal tissue. The AFM image allows to confirm and enrich the information given by optical microscopes adding further details especially on the neural protrusions. Furthermore, we took advantage of the possibility to perform the observation of the cells in their culture medium for studying the neuroblastoma cell differentiation. For the first time, we detected the very early events of the outgrowth of neurite-like structures induced by retinoic acid. A time-course experiment has showed that the acid induces changes in the cellular membrane and dramatic modifications in the cytoskeleton already within 2 h.

Topics: Cell Differentiation; Cell Membrane; Culture Media; Cytoskeleton; Humans; Microscopy, Atomic Force; Neurites; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Phenylacetate, a natural metabolite of phenylalanine which was originally described as a plant growth hormone, has recently gained attention as a possible differentiation inducer for a variety of human tumor cell types. This interest prompted us to assess the ability of sodium phenylacetate (NaPA) to promote the differentiation of human neuroblastoma cells, both alone and in combination with retinoic acid (RA), a known inducer of neuroblastoma differentiation and maturation. Using the LA-N-5 cell line, we have determined that NaPA can stimulate the differentiation of neuroblastoma cells, as evidenced by dose-dependent inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity and reduction of N-myc expression. Furthermore, NaPA and RA synergized in inducing differentiation, in that combination treatment resulted in cessation of cell growth along with morphologic and biochemical changes indicative of the loss of malignant properties. We have determined that NaPA can markedly enhance mRNA levels of the nuclear RA receptor-beta (RAR beta) in LA-N-5 cells prior to morphologic or other phenotypic changes induced by this compound. This effect appeared to be distinct from the ability of NaPA to alter tumor cell lipid metabolism via inhibition of protein isoprenylation. Thus among its varied effects on LA-N-5 cells, NaPA appears to interact with the RA pathway at the nuclear level by up-regulating RAR beta expression.

Topics: Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Genes, myc; Glutamine; Humans; Immunoenzyme Techniques; Neurites; Neuroblastoma; Phenylacetates; Protein Prenylation; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramaticall

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Division; Cytoskeletal Proteins; DNA Damage; DNA, Neoplasm; Fenretinide; Fluorescent Antibody Technique; Humans; Neuroblastoma; Neurons; Tretinoin

RET proto-oncogene products are involved in neural crest development, and constitutional RET mutations are associated with syndromes characterized by tumors of neural crest origin. To study the regulation of RET transcription during neuronal differentiation we analyzed RET expression in neuroblastoma cell lines treated with various differentiating agents. A marked increase in RET mRNA levels was observed in all the cell lines examined shortly after retinoic acid (RA) treatment and before the onset of detectable morphological changes. Upregulation of RET expression was also found in SK-N-BE cells induced to differentiate by 12-O-tetradecanoylphorbol-13-acetate, glial cell-conditioned medium, alpha or gamma interferon, and in SH-SY-5Y cells exposed to nerve growth factor. Induction of RET expression by RA occurred in the absence of de novo protein synthesis. On the other hand, cycloheximide treatment by itself caused upregulation of RET transcripts. These results indicate that the positive transcriptional regulation of RET is closely associated with early neuronal differentiation and suggest that a negative regulatory factor/s controls RET transcription in neuroblastoma cells. Finally, anti-Ret antibodies immunoprecipitated four bands with apparent molecular weights of 150, 155, 170, and 175 kDa in RA-induced SK-N-BE cells. These bands likely represent differently glycosylated forms of the two RET primary products (117 and 122 kDa) detected in tunicamycin-treated cells.

Topics: Cell Differentiation; Cycloheximide; Drosophila Proteins; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Neurons; Protein Synthesis Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Expression of the RET proto-oncogene, a cell surface receptor for an as yet unknown ligand, is associated with tumors, tissues, and cell lines of neural crest origin. Accumulating evidence suggests that RET activity is involved in the process of neuronal differentiation. Moreover, induction of phenotypic differentiation of neuroblastoma cell lines is associated with the rapid accumulation of RET transcripts. To verify the role of RET in neuronal differentiation, we introduced into the human neuroblastoma cell line SK-N-BE four versions of the RET oncogene, activated by different mechanisms: RET/PTC1 and RET/PTC3, which are activated by rearrangement with heterologous genes; and two activated RET mutants, which carry the single amino acid substitution found associated to the inheritance of the multiple endocrine neoplasia type 2A (retMEN2A allele) and type2B (retMEN2B allele), respectively. We demonstrate that, after transfection with the RET oncogenes, SK-N-BE cells display a reduced growth rate and acquire a neurite-bearing phenotype accompanied by enhanced expression of the axonal growth-associated protein, GAP-43, and the high molecular weight neurofilament, NF200. These results indicate that, when activated, RET is able to cause growth inhibition and to promote neuronal differentiation of neuroblastoma cells.

Topics: Blotting, Northern; Cell Differentiation; Cell Division; Drosophila Proteins; Fluorescent Antibody Technique; Humans; Mutation; Neuroblastoma; Oncogenes; Phenotype; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

It is a well established fact that the calcium-binding protein, calbindin-D28k, is influenced by vitamin D in intestine and kidney. However, very little is known concerning the regulation of calbindin-D28k in brain. Although few genes that are regulated by retinoic acid (RA) have been identified in the nervous system, we now report that the human medulloblastoma cell line D283 (which is derived from cerebellum and has a distinctly neuronal phenotype) contains calbindin-D28k endogenously and that calbindin protein and mRNA can be induced 10- to 15-fold in these cells by 10(-7) M RA. These findings are the first evidence of RA-mediated regulation of calbindin. The time course of response, as determined by Northern blot analysis, indicated that the first significant increase in calbindin-D28k mRNA is at 12 h with a plateau of calbindin mRNA induction at 72 h after RA treatment. The induction of calbindin mRNA by RA was preceded by an induction of retinoic acid receptor-alpha mRNA and was accompanied by an induction of retinoid X receptor-alpha mRNA. Calbindin-D28k mRNA levels in D283 medulloblastoma cells as well as the induction of calbindin mRNA by RA were not significantly affected by 1,25-dihydroxyvitamin D3 treatment. Deletion mutant analysis of the native calbindin-D28k promoter and cotransfection of CV-1 or D283 medulloblastoma cells in the presence of retinoic acid receptor-alpha and/or retinoid X receptor-alpha expression vectors as well as results of nuclear transcription assays did not indicate transcriptional regulation of calbindin-D28k by RA. Studies of calbindin-D28k mRNA in control and RA-pretreated D283 medulloblastoma cells at various times (3-24 h) after treatment with 4 micrograms/ml actinomycin D indicated that the half-life of calbindin-D28k mRNA was significantly increased in the presence of RA, suggesting regulation of calbindin-D28k mRNA stability by RA. Thus, calbindin-D28k is one of the few known targets of RA action in cells that express a neuronal phenotype. In addition, our findings present further evidence of an interrelationship between the actions of 1,25-dihydroxyvitamin D3 and the active metabolites of vitamin A.

Topics: Animals; Calbindin 1; Calbindins; Chlorocebus aethiops; Gene Expression Regulation; Humans; Medulloblastoma; Mice; Neuroblastoma; Promoter Regions, Genetic; Rats; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA, Messenger; S100 Calcium Binding Protein G; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage-sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid-treated cells. Both high K(+)- and maitotoxin-induced [Ca2+]i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd3+, an inorganic Ca(2+)-entry blocker, and by the snail toxin omega-conotoxin GVIA, which interacts with the N subtype of voltage-sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel subtype, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K(+)-induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K(+)-induced increase of [Ca2+]i was still present.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Calcium; Cell Differentiation; Cytophotometry; Humans; Marine Toxins; Membrane Potentials; Neuroblastoma; Nimodipine; omega-Conotoxin GVIA; Oxocins; Peptides; Potassium; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factors (IGFs) regulate the autocrine/paracrine growth of neuroblastomas. The IGFs bind to specific binding proteins (IGFBPs) which modulate their biological activity. We investigated, by Western ligand blotting (WLB), the presence of IGFBPs and their possible modulation by retinoic acid (RA), IGF-I, IGF-II and truncated Des(1-3)IGF-I in conditioned medium (CM) of the human neuroblastoma SK-N-BE(2) cell line. We demonstrated the presence of two IGFBPs, with MW 37 kDa and 25 kDa. Following immunoprecipitation, they turned out to be IGFBP-2 and -4, respectively. The RA-induced differentiation in SK-N-BE(2) cells was accompanied by a marked reduction of the intensity of both IGFBP bands after 48 h (32% and 24% of control, respectively) and 72 h (2% and 0% of control, respectively) incubation. The addition of exogenous IGFs, which did not induce cell differentiation, did not change the IGFBP pattern significantly, except for the truncated form of IGF-I, which induced a marked decrease in both the 37 kDa and 25 kDa bands after 72 h incubation (45% and 18% of control, respectively). These findings suggest that IGFBPs have a role in RA-induced differentiation in human neuroblastoma cells.

Topics: Blotting, Western; Carrier Proteins; Cell Differentiation; Down-Regulation; Humans; Immunosorbent Techniques; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Proteins; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

G21.3, a monoclonal antibody previously shown to block central nervous system (CNS) myelination, is now demonstrated to recognize an extracellular matrix (ECM) component. The antigen is present on the surface of neurons but not oligodendrocytes and is highly abundant in the white matter of the adult rat brain; however, it is not found in isolated myelin. Double immunostaining studies indicate a neuronal and ependymal cell source of the G21.3 antigen and a developmental expression pattern distinct from known markers of CNS myelination. The antigen is found at low levels in non-neuronal tissue and is mainly localized to basement membranes. G21.3 immunoreactive proteins are upregulated by retinoic acid-induced differentiation of SK-N-SHF neuroblastoma cells. These results suggest that the G21.3 antigen is an axolemma-associated ECM component with roles in postnatal CNS development and cell-matrix interactions during morphological differentiation of neuroblastoma cells.

Topics: Antibodies, Monoclonal; Brain Neoplasms; Cell Differentiation; Central Nervous System; Extracellular Matrix; Humans; Immunohistochemistry; Myelin Sheath; Nerve Tissue Proteins; Neuroblastoma; Peripheral Nervous System; Tretinoin; Tumor Cells, Cultured

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. Like other guanine nucleotide-binding proteins p21ras is active when GTP bound and inactive when GDP bound. Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and NF1-GRD. In this study we have identified type I GAP120 and two NF1-GRD mRNAs in three neuroblastoma cell lines, IMR-32, SK-N-SH and SK-N-MC. NF1-GRD mRNA was expressed in all cell lines at a similar level but type I GAP120 mRNA was more abundant in the IMR-32 cell line. Retinoic acid induced differentiation of all three cell lines, this effect was most marked in the SK-N-SH line. This differentiation was accompanied by an increase in both type I GAP120 and NF1-GRD mRNAs. Retinoic acid induced differentiation had no effect on the ratio of type I to type II NF1-GRD mRNA. In seven patient tumour samples examined type I GAP120 and NF1-GRD were coexpressed, type I GAP120 at a higher level than NF1-GRD in all tumour stages. Type I was the predominant NF1-GRD mRNA. The expression of type I GAP120 was similar in all tumour stages but the total level of NF1-GRD was higher in stage 2 and 3 tumours than in stage 4 tumours. In summary, these results suggest increased type I GAP120 and NF1-GRD mRNA are associated with differentiation in neuroblastoma cells.

Topics: Base Sequence; Cell Differentiation; GTPase-Activating Proteins; Humans; Infant; Molecular Probes; Molecular Sequence Data; Neoplasm Staging; Neuroblastoma; Polymerase Chain Reaction; Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (dopa) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of dopa. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of dopa and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-69, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and enzyme expressions. The RA treatment modulated the activities of tyrosine hydroxylase and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.

Topics: Cell Differentiation; Dihydroxyphenylalanine; Dopa Decarboxylase; Dopamine; Humans; Neuroblastoma; Tretinoin; Tritium; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

The observation that neuroblastoma cells differentiate in response to retinoic acid (RA) in vitro has led to clinical trials using either 13-cis or all-trans RA. Since 9-cis RA may also have important biological functions, we have compared the potential of RA isomers to induce differentiation and inhibit cell proliferation of SH SY 5Y neuroblastoma cells. 9-cis RA at high concentrations is better at inducing morphological differentiation than either all-trans or 13-cis RA and as effective at inhibiting proliferation. Hence, 9-cis RA or stable analogues may have important therapeutic potential in the treatment of neuroblastoma.

Topics: Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Neuroblastoma; Osmolar Concentration; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

Retinoids are chemical compounds which play important roles in ontogenetic development and cranio-caudal differentiation in animals, but their effect on phenotypic expression of neurotransmitters are unknown. We studied the pharmacological and morphological effects of retinoic acid (RA) on two types of immature vertebrate neurons, the human derived neuroblastoma cells, NB69, and fetal rat mid brain neurons in culture. The pharmacological effects of RA on the cultures and their relation to catecholamine and acetylcholine neurotransmission were evaluated according the levels of catecholamines, tyrosine hydroxylase (TH) activity, TH immunostaining, and choline acetyltransferase (CAT) activity, respectively. RA reduces catecholamine levels and TH activity in NB69 cells and the number of dopamine neurons in cultures derived from rat fetal mid brain. The detrimental effect of RA on mid brain neurons is dose- dependent; limited to TH+ cells at low concentrations (100 to 500 nM) and toxic for all types of cells at high concentrations (1 to 2 microM). RA increases CAT activity in NB 69 cells and produces phenotypic differentiation of these to a more mature neuronal phenotype with more prolonged neurite extensions. Therefore, RA may play a trophic positive role in the differentiation of immature cells to cholinergic neurons; this contrasts with the detrimental effects of RA on catecholamine neurons.

Topics: Animals; Catecholamines; Dopamine; Dose-Response Relationship, Drug; Humans; Mesencephalon; Neuroblastoma; Neurons; Rats; Synaptic Transmission; Tretinoin; Tumor Cells, Cultured

The promoter region of genes involved in cell growth and differentiation is bound by specific transcription factors which regulate its expression. Our previous study showed that the calcyclin gene, which belongs to the large family of Ca2+-binding proteins, is differently expressed in SK-N-BE(2)C and LA-N-5 neuroblastoma cell lines. We analysed the region upstream the transcription initiation site of the gene before and during retinoic acid (RA)-induced differentiation. Gel-shift analysis showed that the -161,-135 untranslated region is bound by an AP-1-like protein both in SK-N-BE(2)C and LA-N-5 cells. Competition assay demonstrated that AP-2,AP-3 and NF1 transcription factors did not bind in the same region. Calcyclin mRNA is induced in RA-treated LA-N-5 cells and reaches maximal expression at 96 h, suggesting that its gene is involved in cell differentiation. Gel-shift analysis shows a strong signal of binding after 96 h of RA treatment. Our results indicate that RA induces an increase in the binding protein or improves its affinity for the AP-1-like region during neuronal differentiation. These preliminary data suggest that the calcyclin gene is involved in neuronal pathway differentiation and that AP-1-like binding sequence could be one of the gene regions that is under transcriptional factor control during cell differentiation.

Topics: Base Sequence; Binding, Competitive; Calcium-Binding Proteins; Cell Differentiation; Molecular Sequence Data; Neuroblastoma; Oligonucleotide Probes; Promoter Regions, Genetic; S100 Proteins; Transcription Factor AP-1; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The identification and molecular characterization of Brn-3.2 has revealed a family of Brn-3-related mammalian POU proteins that share homology with the C. elegans developmental regulator Unc-86 and extended similarity with the Drosophila neurodevelopmental gene I-POU, which defines a novel POU-IV box. Brn-3.2 exhibits DNA binding properties similar to those of Brn-3.0, but its expression is uniquely regulated by retinoic acid in teratocarcinoma and neuroblastoma cells. In the developing PNS and retina, the expression pattern of Brn-3.2 is similar to that of Brn-3.0. In the caudal CNS (spinal cord, hindbrain, and midbrain) Brn-3.2 and Brn-3.0 are initially coexpressed, but diverge later in development. Rostral to the midbrain, Brn-3.2 and Brn-3.0 exhibit nonoverlapping patterns of expression, suggesting divergence of gene function in more recently evolved structures. Our analysis suggests that in the CNS Brn-3.2 is selectively expressed in postmitotic neurons, implying a role in specifying terminally differentiated neuronal phenotypes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain; Cell Differentiation; Cell Line; DNA-Binding Proteins; DNA, Complementary; Drosophila; Embryo, Mammalian; Embryo, Nonmammalian; Gene Expression; Gene Expression Regulation; Gene Library; Genes, Homeobox; Homeodomain Proteins; In Situ Hybridization; Mesencephalon; Mice; Mitosis; Molecular Sequence Data; Neuroblastoma; Neurons; Organ Specificity; Rhombencephalon; Sequence Homology, Amino Acid; Spinal Cord; Teratoma; Transcription Factor Brn-3; Transcription Factor Brn-3A; Transcription Factor Brn-3B; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Intercellular adhesion molecule-1 (ICAM-1) is an important cell surface adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells, including cancer cells, is regulated by various proinflammatory cytokines. In the present study, we investigated the role of calcium (Ca2+) and calmodulin (CaM) in the retinoic acid and gamma-interferon (IFN-gamma) signaling in the human neuroblastoma cell line SK-N-SH for up-regulating ICAM-1 expression. A 24-h incubation in the presence of Ca(2+)-mobilizing agents (A23187 and thapsigargin) resulted in the induction of ICAM-1 expression. Both Ca(2+)-mobilizing agents stimulated ICAM-1 expression additively to IFN-gamma but not to retinoic acid, suggesting that IFN-gamma does not use Ca2+ to stimulate ICAM-1, whereas retinoic acid might use it in part. As a second messenger, Ca2+ can be coupled with calmodulin. Using calmodulin inhibitors (W7 and calmidazolium), we found that retinoic acid-stimulated, A23187-stimulated, and thapsigargin-stimulated but not FIN-gamma-stimulated ICAM-1 were inhibited. Calmodulin signaling elicited by retinoic acid was an early event occurring within the first h of retinoic acid treatment, providing evidence that they may both be coupled to regulate gene expression. Using a novel CaM kinase II inhibitor, KN-62, we demonstrated that retinoic acid stimulated ICAM-1 expression in a CaM kinase II-dependent fashion. The mechanisms whereby CaM kinase II mediates retinoic acid activity on ICAM-1 expression remain to be elucidated.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Adhesion Molecules; Enzyme Activation; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Sulfonamides; Terpenes; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The MRP gene (Cole et al., Science (Washington DC), 258: 1650-1654, 1992) encodes a membrane-bound glycoprotein the expression of which correlates with non-P-glycoprotein-mediated multidrug resistance in a variety of cultured human cell lines. Using an RNA-polymerase chain reaction assay, expression of this gene was examined in the highly chemoresistant pediatric malignancy, neuroblastoma. MRP expression was observed in 5 human neuroblastoma cell lines and in all 25 primary neuroblastoma tumors of stage I through IVS. Tumors with amplification of the N-myc oncogene were found to have significantly higher MRP expression that those with no amplification (P = 0.0016). Expression of the MRP gene in the tumor specimens was highly correlated with expression of the N-myc gene (P = 0.0009), while expression of the MDR1 gene, encoding P-glycoprotein, was not related to expression of either the N-myc or MRP genes. Decreased expression of the N-myc oncogene in neuroblastoma cell lines SH-SY5Y and BE(2)-C, following treatment with retinoic acid, was paralleled by down-regulation of MRP gene expression, contrasting with increased expression of the MDR1 gene. Expression of the MRP gene is thus common in both primary neuroblastoma tumors and cultured cell lines, and correlates with amplification and overexpression of the N-myc oncogene, which is central to the malignant phenotype of this disease.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Base Sequence; Cell Differentiation; Drug Resistance; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Molecular Sequence Data; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.

Topics: Aspartic Acid Endopeptidases; Blotting, Northern; Cycloheximide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Proprotein Convertases; Protein Precursors; Radioimmunoassay; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

Topics: Apoptosis; Cell Adhesion Molecules, Neuronal; Cloning, Molecular; Gene Expression; Genetic Variation; Humans; Neural Crest; Neuroblastoma; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; RNA, Antisense; Selection, Genetic; Transcription, Genetic; Transfection; Transglutaminases; Tretinoin; Tumor Cells, Cultured

To determine whether over-expression of the MDR1 gene may result from activation of its promoter by differentiating agents, the activity of the human MDR1 proximal promoter (MDR1 pp) transfected to 2 neuroblastoma lines treated with retinoic acid and forskolin was first measured using transient expression assays while the MDR1 mRNA levels were measured by Northern blots. The results indicate that retinoic acid and forskolin were able to activate the human MDR1 pp in a dose dependent manner after transfection of the MDR1pp- CAT constructs in the 2 cell models tested, i.e., SK-N-SH and IGR-N-91, a new human neuroblastoma cell line. A significant increase in MDR1 gene transcript levels was observed upon treatment with differentiation inducers in SK-N-SH but not in IGR-N-91 neuroblasts. These results suggest that the induction of the MDR1 gene promoter is necessary but not sufficient to lead to an increase in MDR1 gene transcript levels, according to the neuroblast cell line considered.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Base Sequence; Cell Differentiation; Child; Colforsin; Dose-Response Relationship, Drug; Gene Expression; Humans; Male; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Topics: Cell Death; Gene Expression; Humans; Immunoblotting; Neuroblastoma; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Topics: Apoptosis; Cell Differentiation; Humans; Neuroblastoma; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Four children with advanced or relapsed neuroblastoma were treated with oral 13-cis-retinoic acid 0.75 mg/kg/day. Clinical response to retinoic acid was noted only in the two children with tumors coexpressing trk protooncogene mRNA, encoding an essential part of the nerve growth factor (NGF) high affinity receptor, and low affinity NGF receptor gene (LNGFR) mRNA. Clinical stage or age, plasma neuropeptide Y, tumor DNA ploidy and N-myc amplification did not as accurately predict response to retinoic acid as NGF receptor mRNAs. In vitro data have shown that retinoic acid up regulates LNGFR expression and NGF sensitivity via interaction with specific regulatory elements in the LNGFR gene promoter. We hypothesize that part of the therapeutic effect of retinoic acid in neuroblastoma in vivo may be exerted via increased NGF receptor expression and NGF sensitivity. Analysis of trk and LNGFR mRNA may be useful to predict clinical response to retinoic acid in these children.

Topics: Administration, Oral; Child; Child, Preschool; DNA, Neoplasm; Female; Gene Expression; Genes, myc; Humans; Male; Neuroblastoma; Neuropeptide Y; Ploidies; Proto-Oncogene Proteins; Receptors, Nerve Growth Factor; RNA, Messenger; Treatment Outcome; Tretinoin

Topics: Blotting, Northern; Brain-Derived Neurotrophic Factor; Cell Differentiation; Humans; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptor, trkB; Receptors, Nerve Growth Factor; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Blotting, Northern; Cell Differentiation; Gene Amplification; Gene Expression; Genes, myc; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Blotting, Northern; Cell Differentiation; Gene Expression; Genes, Homeobox; Homeodomain Proteins; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Topics: Base Sequence; Blotting, Northern; DNA Primers; Gene Expression; Humans; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Topics: Antigens, Surface; Biomarkers; Blotting, Northern; Bromodeoxyuridine; Cell Differentiation; Cell Line, Transformed; Humans; Neural Crest; Neuroblastoma; Phenotype; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Cells, Cultured

The BE (2)-M17 human neuroblastoma has previously been shown to express corticotropin-releasing factor (CRF) mRNA following retinoic acid treatment. It is demonstrated in this report that both cell extracts and cell incubation medium of retinoic acid-treated BE (2)-M17 cells were shown to contain CRF-like immunoreactivity (CRF-LI) by RIA. CRF-LI secretion and content were also dose-dependently increased by forskolin. In addition, cell extracts were applied to a C18 Vydac column and peak CRF-LI from the collected fractions was shown to coincide in time of elution with peak immunoreactivity seen with oxidized synthetic CRF standard. Thus, in containing the CRF peptide, the BE (2)-M17 cells are useful models for further study of CRF cellular and genetic regulation.

Topics: Brain Neoplasms; Chromatography, High Pressure Liquid; Colforsin; Corticotropin-Releasing Hormone; Humans; Neuroblastoma; Radioimmunoassay; Tretinoin; Tumor Cells, Cultured

Dopamine D2 receptor (D2-receptor) expression and its coupling to Gi sensitive adenylate cyclase was investigated in human neuroblastoma SHSY-5Y cells. Incubation of SHSY-5Y cells in the presence of 100 nM retinoic acid (RA) for 24 hours resulted in phenotypic differentiation accompanied by a 47% increase in D2-receptor mRNA and a significant increase in the specific binding of a D2-receptor antagonist, [3H]YM09151-2. Stimulation of D2-receptors in differentiated cells by LY171-555, a D2-agonist, attenuated cellular cAMP levels by 30%. The effect of LY171-555 on cAMP levels was blocked by the D2-antagonist, (-)sulpride. Application of these drugs to control undifferentiated cells or differentiated cells incubated with vehicle only had no effect on cellular cAMP levels. These studies suggest that differentiated SHSY-5Y cells express functional D2-receptors and will provide a useful model for future studies on the regulation of expression and function of D2-receptors in cellular differentiation of neuronal cells.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Base Sequence; Benzamides; Cell Differentiation; Cyclic AMP; Dopamine Agonists; Dopamine Antagonists; Ergolines; Humans; Molecular Sequence Data; Neuroblastoma; Neurons; Quinpirole; Receptors, Dopamine D2; RNA, Messenger; Second Messenger Systems; Sulpiride; Tretinoin; Tumor Cells, Cultured

The objective of this study was to compare the properties of 9-cis and all-trans retinoic acid with respect to the induction of expression of retinoic acid receptor beta (RAR-beta) and cellular retinoic acid-binding protein (CRABP) II in human neuroblastoma SH SY 5Y cells. RAR-beta and CRABP II mRNA was induced by both all-trans and 9-cis retinoic acid in SH SY 5Y cells. Induction was rapid, detectable within 2-4 h, and inhibited by actinomycin D. Time-courses of induction for RAR-beta and CRABP II differed: RAR-beta mRNA levels reached a maximum 4-6 h after adding all-trans or 9-cis retinoic acid, whereas CRABP II mRNA levels increased over at least 18 h. These differences were attributed to the longer half-life of CRABP II mRNA (20 h) compared with RAR-beta mRNA (3.9 h). The dose-response characteristics of all-trans and 9-cis retinoic acid were different: all-trans was effective at nanomolar concentrations, whereas 10-fold higher levels of 9-cis retinoic acid were required to achieve comparable induction of RAR-beta and CRABP II. Conversely, at high concentrations, 9-cis retinoic acid gave a greater induction of RAR-beta and CRABP II than all-trans. The induction of RAR-beta and CRABP II by all-trans retinoic acid was maintained in the subsequent absence of all-trans retinoic acid, whereas induction by 9-cis retinoic acid was dependent on its continued presence in the culture medium. These results suggest that, at high concentrations, 9-cis retinoic acid may produce its transcriptional effects via retinoid X receptor (RXR) homodimers. This has implications for the cellular functions of 9-cis retinoic acid and its use as a biological response modifier.

Topics: Dactinomycin; Dose-Response Relationship, Drug; Humans; Neuroblastoma; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Human NB cell lines express features of one or more of three recognizable phenotypes that include N-type (neuroblastic), S-type (Schwannian), and I-type (intermediate phenotype) cells. The I-type cell, which shares properties of both N- and S-type cells, is thought to represent the progenitor cell from which the other two cell types are derived. The MYCN amplified NB cell line NUB-7, established in our laboratory, is now shown to be composed principally of I-type cells. The observed phenotype was stable in culture and was representative of the original surgically resected tumor. The I-type cell designation was established based on morphological characteristics, the coexpression of various N-type (neurofilaments, peripherin, GAP-43, NCAM, MYCN) and S-type cell (vimentin, laminin, fibronectin) markers, and the relatively high level of expression of these markers in comparison to five predominantly N-type, one S-type, and one N/S mixed NB cell lines. Dibutyryl cyclic AMP and retinoic acid induced enhanced expression of N- and S-type phenotypes, respectively, in NUB-7 as supported by specific morphological changes, reduced growth, and changes in the levels of expression of both N- and S-type markers. Our studies with the NUB-7 cell line have now provided convincing evidence for the existence of a bipotential progenitor of N- and S-type cells in NB. As well, the NUB-7 cell line may also represent the tumor counterpart of a sympathetic ganglion progenitor cell.

Topics: Bucladesine; Cell Differentiation; Cell Division; Humans; Karyotyping; Neuroblastoma; Neurons; Phenotype; Schwann Cells; Stem Cells; Tretinoin; Tumor Cells, Cultured

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.

Topics: Amino Acid Sequence; Animals; Base Sequence; Breast Neoplasms; Cells, Cultured; Chlorocebus aethiops; Choline O-Acetyltransferase; Choriocarcinoma; Enzyme Induction; Female; Humans; Molecular Sequence Data; Neoplasms, Hormone-Dependent; Neuroblastoma; Neurons; Organ Specificity; Proto-Oncogene Proteins c-jun; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Steroids; Transcriptional Activation; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Uterine Neoplasms

SH-SY5Y Neuroblastoma cells were used to study the effect of retinoic acid (RA)-induced differentiation on the expression of gangliosides and neuronal markers. In the presence of 10 microM RA, more than 70% of the cells differentiate to a neuronal phenotype within 8 days. They extend long neuritic processes and show an enhanced immuno-expression of neurone-specific enolase (NSE), neurofilament protein (NF-M), and polysialic acid (PSA). SH-SY5Y cells were found to express at least 12 different gangliosides. RA-induced neuronal differentiation led to a decrease in the content of GM2, GD3, and GD2 and to a 3-7 fold increased concentration of the ganglio-tetraosyl gangliosides GM1, GD1a, GT1a, GD1b, and GT1b. Thus, RA-induced neuronal differentiation of SH-SY5Y cells is accompanied by ganglioside changes similar to those observed during embryonic neuronal differentiation.

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Line; Gangliosides; Humans; Nervous System Neoplasms; Neuroblastoma; Neurons; Neuropeptides; Tretinoin; Tumor Cells, Cultured

The sequential methylation of ethanolamine (Etn) or phosphorylethanolamine to the corresponding choline (Cho) derivatives was studied in both undifferentiated and retinoic acid (RA) differentiated human neuroblastoma clones LA-N-1 and LA-N-2. Conversion of Etn derivatives to the respective Cho metabolites was low in both cell types. However, after treatment of the cultures with ethanol or RA, the methylation of phosphoryl-Etn was stimulated while that of phosphatidyl-Etn was severely reduced in both cholinergic LA-N-2 and catecholaminergic LA-N-1 cells.

Topics: Biological Transport; Cell Differentiation; Choline; Clone Cells; Ethanol; Ethanolamine; Ethanolamines; Humans; Kinetics; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Drebrins are developmentally regulated actin-binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SH-SY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F-actin was more stable against cytochalasin D than F-actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F-actin.

Topics: Actins; Brain Neoplasms; Cell Differentiation; Cytochalasin D; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Immunohistochemistry; Neuroblastoma; Neurons; Neuropeptides; Subcellular Fractions; Tretinoin; Tumor Cells, Cultured

We have studied human immunodeficiency virus type 1 (HIV-1) infection in human SH-SY5Y neuroblastoma cells at various stages of morphological differentiation. Two days' treatment of the cells with retinoic acid (RA) or dibutyryl cAMP (db-cAMP) resulted in the appearance of elongated neurites and enhanced production of 160K to 200K neurofilament proteins as shown by indirect immunofluorescence. DNA synthesis was reduced only in RA-treated cells as detected by 5-bromo-2'-deoxyuridine incorporation. The cells were infected with two T-lymphotropic virus strains (IIIB and NDK) and two fresh isolates (39001 and 46001) from bronchoalveolar lavage samples of AIDS patients. The latter two isolates were unable to form syncytia in infected CD4-positive T-lymphoblastoid C8166 cells which was in contrast to our T-lymphotropic virus strains. Interphase in situ hybridization showed that 14 to 16% of SH-SY5Y cells become positive for HIV-1 DNA. Regardless of the virus strain, morphological differentiation of the cells with RA or db-cAMP inhibited infection by 50% at a single cell in situ resolution. Nested PCR confirmed the presence of proviral DNA in the infected cells. These results show that human neuroblastoma cells, tumour cells of neuroectodermal origin, can be infected by different HIV-1 isolates and that the infection is inhibited by neurotypic cell differentiation.

Topics: Base Sequence; Bucladesine; Cell Differentiation; HIV-1; Humans; Molecular Sequence Data; Nerve Growth Factors; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) nuclear receptors (RARs) are thought to mediate the cellular and molecular effects of RA on a wide variety of tissues. In most cell types, RAR alpha expression remains relatively constant following exposure to RA, while that of RAR beta is rapidly induced. In this study, we show that in human neuroblastoma, a cell type exceptionally sensitive to RA-induced differentiation, RAR alpha as well as RAR beta is markedly up-regulated by RA treatment. This effect was consistent in all 5 neuroblastoma cell lines tested and was reflected in a 2- to 5-fold increase in receptor mRNA levels as assessed by Northern-blot analysis. Using LA-N-5 human neuroblastoma cells, we found that receptor up-regulation occurred in a time- and dose-dependent fashion with increases in both RAR alpha and beta mRNA detectable 1-2 hr after the addition of RA. These inductions were not abrogated by cycloheximide, indicating that protein synthesis was not required for the RA responses. Nuclear run-off experiments combined with Northern-blot analysis of RAR alpha stability directly demonstrated that the up-regulation of RAR alpha mRNA levels reflected an increased rate of transcription without changes in message half-life. These findings, showing direct activation by RA of RAR alpha gene transcription in human neuroblastoma cells, suggest differences in the overall regulation of this receptor from that found in most other RA-inducible tissue.

Topics: Cell Differentiation; Cell Nucleus; Cycloheximide; Dose-Response Relationship, Drug; Humans; Interferon-gamma; Neuroblastoma; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH-SY5Y cells: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased. This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-imido)triphosphate [Gpp(NH)p; 10 nmol/L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prostaglandin E1 (PGE1) receptors and Gs alpha, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1-stimulated adenylate cyclase activity, but significantly reduced amounts of Gs alpha were found. This down-regulation is paralleled by a decrease in the stimulatory activity of Gs alpha as assessed in S49 cyc- reconstitution assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Alprostadil; Blotting, Western; Cell Differentiation; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Kinetics; Neuroblastoma; Receptors, Opioid, mu; Tretinoin; Tumor Cells, Cultured

Neurotrophins are responsible for the differentiation and survival of neurons in the developing and in the adult nervous system. They bind to specific membrane receptors with tyrosine kinase activity whose prototype is the product of the trkA proto-oncogene. TrkB, a member of this family, is the receptor for the neurotrophins brain derived growth factor (BDNF) and neurotrophins-3, -4/5. In this study, we show that stable expression of the c-erbA proto-oncogene, which encodes the alpha 1-isoform of the nuclear receptor for thyroid hormone (Tr alpha 1) induces the expression of trkB mRNA with a concomitant decrease to undetectable levels of trkA and trkC mRNAs in the mouse neuroblastoma N2a cell line. trkB induction by c-erbA is ligand independent, since addition of T3 had no effect. The induced trkB transcript encodes a functional gp145trkB protein, which is phosphorylated on tyrosine in response to BDNF. Furthermore, induction of trkB mRNA is also caused by transient expression of either TR alpha 1 or beta 1 isoforms. Our results are compatible with the idea that there are certain pathways which are under control of unliganded thyroid hormone receptor, and that one of these pathways results in regulation of trk expression.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Differentiation; Colforsin; Gene Expression Regulation, Neoplastic; Mice; Nerve Tissue Proteins; Neuroblastoma; Phosphorylation; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptor, trkB; Receptor, trkC; Receptors, Growth Factor; Receptors, Nerve Growth Factor; Receptors, Thyroid Hormone; RNA, Messenger; Thyroid Hormones; Transfection; Tretinoin; Tumor Cells, Cultured

In retinoic acid (RA)-differentiated SH-SY5Y cell cultures, colchicine could induce cell death, accompanied by the typical ladder pattern of DNA fragmentation found in apoptotic cells. This effect could be attenuated by the protein synthesis inhibitor cycloheximide. The protein kinase inhibitor H-89 efficiently reduced colchicine-induced cell death. These results suggest that the mechanism for colchicine-induced cell death may act, at least in part, through the activation of apoptosis in differentiating SH-SY5Y cells.

Topics: Apoptosis; Cell Differentiation; Cell Line; Colchicine; Dose-Response Relationship, Drug; Humans; Isoquinolines; Kinetics; L-Lactate Dehydrogenase; Neuroblastoma; Neurotoxins; Protein Kinase Inhibitors; Sulfonamides; Tretinoin; Tumor Cells, Cultured

The nuclear factor kappa B (NF-kappa B) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF-kappa B translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor alpha (TNF alpha), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells NF-kappa B was present in the cytosol and translocated into nuclei as a result of TNF alpha treatment. The TNF alpha-activated NF-kappa B was transcriptionally functional. NF-kappa B activation by TNF alpha was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated NF-kappa B, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated NF-kappa B, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF-kappa B activation. We found, moreover, that in SK-N-Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF-kappa B was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF-kappa B activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNF alpha proved that NF-kappa B activation was not sufficient for induction of neuroblastoma differentiation.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Chloramphenicol O-Acetyltransferase; Humans; Molecular Sequence Data; Nerve Growth Factors; Neuroblastoma; NF-kappa B; Nuclear Proteins; Oligodeoxyribonucleotides; PC12 Cells; Protein Kinase C; Recombinant Proteins; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Membrane; Cloning, Molecular; DNA; Electric Stimulation; Electrophoresis, Polyacrylamide Gel; Embryonal Carcinoma Stem Cells; Enzyme Activation; Mice; Molecular Sequence Data; Neoplastic Stem Cells; Neuroblastoma; Neurons; Phosphorylation; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins pp60(c-src); Transfection; Tretinoin; Tumor Cells, Cultured

Two human neuroblastoma cell lines, LAN-5 and GI-CA-N, have been analyzed for their capability to adhere to different extracellular matrix (ECM) components. The GI-CA-N cells adhered to all the tested substrates: laminin (LN), type I and type IV collagen (Coll I, Coll IV), vitronectin (VN), and fibronectin (FN). Conversely LAN-5 cells weakly attached to FN and VN, whilst adhesion on LN and Coll I and IV was strong and induced a rapid elongation of cell processes. By means of RT-PCR and immunoprecipitation we showed that the integrin pattern of these two lines was different and could explain their diversity in adhesion capability. Both cell lines express a large amount of the beta 1 integrin subunit, associated with different alpha chains, probably responsible for their adhesion to some ECM proteins. After treatment of LAN-5 cells with biological differentiating agents, such as gamma-interferon, alone or in combination with tumour necrosis factor-alpha (TNF-alpha), or retinoic acid, the levels of alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 integrin expression were enhanced, while the amount of alpha v remained constant. In contrast, treatment of LAN-5 cells with TNF-alpha, that did not induce any maturation, or starvation in 2% foetal calf serum, that inhibited cell proliferation without affecting neural differentiation, did not induce any change in the integrin assessment. Messenger-RNAs for the two alpha 6 isoforms, A and B, were present in both cell lines. However, in LAN-5 cells, the protein product was neither detectable nor inducible by differentiation. Our results confirm the specific modulation of the alpha 1 beta 1 integrin expression in human neuronal development, and show, for the first time, the involvement of alpha 2 beta 1 and alpha 3 beta 1 heterodimers in this maturational process.

Topics: Cell Adhesion; Cell Differentiation; Collagen; Fibronectins; Gene Expression; Glycoproteins; Humans; Integrins; Interferon-gamma; Laminin; Macromolecular Substances; Neuroblastoma; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitronectin

Human neuroblastoma cells (SH-SY5Y) differentiated by retinoic acid exhibited high threshold-activated N-type Ca2+ currents, which were largely inhibited by the two enkephalins, DAGO and DPDPE, as well as by dopamine and somatostatin. The inhibitory effects were fully abolished after pretreatment of cells with pertussis toxin. After washing out the toxin, the inhibitory effects re-established with a time constant of about 16 h. The recovery of Ca2+ current inhibition was similar for all tested agonists. Unexpectedly, we observed a neurotransmitter-induced stimulation of Ca2+ currents in approximately 10% of all investigated cells during the recovery phase. Such a stimulatory effect by otherwise inhibitory receptors was never seen in control cells. It did also not occur when exogenous purified G-proteins of the Gi family were reconstituted via the patch pipette, suggesting that additional mechanisms may play a role in the appearance of stimulatory effects during the recovery phase after pertussis toxin pretreatment.

Topics: Calcium; Cell Differentiation; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Membrane Potentials; Neuroblastoma; Pertussis Toxin; Somatostatin; Tretinoin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Intercellular adhesion molecule 1 (ICAM-1) is a major adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells including cancer cells regulated by various proinflammatory cytokines. Incubation of the human glioma cell line HS 683 and the neuroblastoma cell line SK-N-SH with 12-phorbol 13-myristic acid (PMA), retinoic acid, or gamma-interferon (IFN-gamma) strongly stimulates ICAM-1 expression. In the present study, we investigated the role of the protein kinase C (PKC)-mediated signal transduction pathway in this process. We found that IFN-gamma, but not retinoic acid, was able to induce activation and translocation of PKC after 60 min in a dose-dependent fashion, contrasting with the very rapid activation and translocation induced by PMA which occurred at 15 min. The PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride and staurosporine, as well as depletion of PKC by a 24-h treatment with 100 nM PMA, decreased the PMA-mediated stimulation but not the retinoic acid- or the IFN-gamma-mediated stimulation of ICAM-1 expression. On the contrary, they rather stimulated ICAM-1 expression. Furthermore, this stimulation was additive with retinoic acid and IFN-gamma. A 24-h incubation in the presence of retinoic acid or IFN-gamma strongly inhibited activation and translocation of PKC by PMA. These results suggest that although PMA-induced ICAM-1 expression is PKC dependent on HS 683 and SK-N-SH cells, the stimulation of ICAM-1 expression by retinoic acid and by IFN-gamma may be due to PKC inactivation at longer time points (24 h), as mimicked by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, staurosporine, or PKC depletion by high doses of PMA.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Cell Adhesion Molecules; Enzyme Activation; Glioma; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

We report the induction of apoptosis in a human neuroblastoma cell line SK-N-BE(2) by cisplatin or retinoic acid, and its relation to cell cycle. Apoptosis was monitored by counting apoptotic bodies and evaluating the activity of 'tissue' transglutaminase (EC 2.3.2.13), one of the genes specifically expressed in apoptotic cells. Data indicate that both agents enhance apoptosis, even though cells arrest at different cell cycle phases. In fact, retinoic acid causes accumulation in G1, whilst cisplatin induces accumulation of cells in the G2/M phase. This evidence suggests the presence of multiple start points for the apoptotic death programme within the cell cycle of human neuroblastoma cells.

Topics: Apoptosis; Caffeine; Cell Cycle; Cisplatin; Ethers, Cyclic; Humans; Interphase; Mitosis; Neuroblastoma; Okadaic Acid; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Tissue transglutaminase (tTG) activity was used to test the potent regulatory role of vasoactive intestinal peptide (VIP) on Retinoic Acid-induced effect in human neuroblastoma cell line. The comparison between both differentiation and cell death related to tissue transglutaminase was discussed in this model. VIP alone was a potent differentiating agent in SK-N-SH cells but in the presence of retinoic acid (RA), this peptide rather potentiates RA-induced tTG activity which is now considered as an apoptosis marker in neuroblastoma cell line. This paper demonstrated an additional neuromodulator role for VIP.

Topics: Apoptosis; Biomarkers; Drug Synergism; Enzyme Induction; Humans; Neoplasm Proteins; Neuroblastoma; Transglutaminases; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The human neuroblastoma cell line SH-SY5Y was used to demonstrate morphine-induced down-regulation and naloxone-induced up-regulation of opiate receptors in a mu receptor containing neuronally derived preparation capable of desensitization to morphine. Chronic exposure to morphine decreased the number but not the affinity of mu opiate receptors in SH-SY5Y cells. Differentiation of the cells with retinoic acid or with the phorbol agent TPA (12-O-tetradecanoyl-phorbol-13-acetate) increased the number of mu receptors. Morphine-induced down-regulation, however, was observed in the absence of differentiation as well as after differentiation with retinoic acid or TPA. The decrease in the number of receptors was related to time of exposure, with a half-maximum disappearance time (T1/2) of about 3 hr during the initial phase. The receptor decrease was near maximum at 24 hr with no further significant change up to 72 hr. The loss of [3H] DAMGO ([3H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) binding was also dose-dependent, with reductions occurring at 0.3, 1 and 10 microM. The loss of receptors was dependent on temperature, with reductions at 37 but not 23 degrees C. The down-regulation was blocked by naloxone and the mu-selective antagonist CTOP (D-Phe-Cys-Tyr-D(-Trp-)Orn-Thr-Pen-Thr-NH2), but not by the delta antagonist ICI 174864 ([N,N-diallyl-Tyr1,Aib2,3]Leu-enkephalin). Cholinergic ([3H]quinclidinyl benzilate) binding was not affected by the morphine treatment, indicating that the down-regulation was homologous for opiate receptors. In SH-SY5Y cells, unlike other cell models, the opiate antagonist naloxone upregulated mu receptors by more than 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Cell Differentiation; Dose-Response Relationship, Drug; Down-Regulation; Humans; Kinetics; Molecular Sequence Data; Morphine; MSH Release-Inhibiting Hormone; Naloxone; Neuroblastoma; Pentazocine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Receptors, Opioid, mu; Temperature; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Up-Regulation

When treated in vitro with retinoic acid, many neuroblastoma cell lines undergo neuronal differentiation and show a significant increase in human multi-drug resistance gene (MDRI) transcript levels. To determine whether over-expression of the gene may result from activation of its promoter by differentiating agents, the activity of the human MDRI proximal promoter (MDRI pp) transfected to 2 neuroblastoma lines treated with retinoic acid and forskolin was first measured using transient expression assays. The MDRI gene transcript levels of these 2 lines were then measured by Northern blots. The results indicate that retinoic acid and forskolin were able to activate the human MDRI pp in a dose-dependent manner after transfection of the MDRI pp-CAT constructs in the 2 cell models tested, i.e., SK-N-SH and IGR-N-91, a new human neuroblastoma cell line. A significant increase in MDRI gene transcript levels was observed upon treatment with differentiation inducers in SK-N-SH but not in IGR-N-91 neuroblasts. These results suggest that the induction of the MDRI gene promoter is necessary but not sufficient to lead to an increase in MDRI gene transcript levels, according to the neuroblast cell line considered.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Base Sequence; Blotting, Northern; Carrier Proteins; Cell Differentiation; Colforsin; Cyclic AMP; DNA Probes; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Molecular Sequence Data; Neuroblastoma; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, a malignancy of early childhood arises in the embryonal neural crest. Neuroblastoma cells are in a state of arrested differentiation; however, they can be induced to differentiate in vitro by retinoic acid. As a first step toward understanding the molecular mechanisms of neuroblastoma differentiation we analyzed the expression pattern of the developmentally important Homeobox genes in cells treated with retinoic acid. The strategy employed involved rapid screening of a cDNA library prepared from retinoic acid treated human LA-N-5 neuroblastoma cells for Homeobox genes by the polymerase chain reaction. Multiple Homeobox genes were amplified from recombinant phage DNA using degenerate primers directed against the conserved homeobox. To date 6 Homeobox genes (HoxC6, HoxC8, HoxD1, HoxD4, HoxD8, and HoxD9) have been identified in the cDNA library prepared from LA-N-5 cells treated with retinoic acid. HoxD1 and HoxC8 are being reported for the first time to be expressed in neuroblastoma cells. Preliminary studies indicate that there is an induction of Homeobox gene expression in differentiating LA-N-5 neuroblastoma cells.

Topics: Amino Acid Sequence; Base Sequence; Cell Differentiation; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Neoplastic; Gene Library; Genes, Homeobox; Humans; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Sequence Alignment; Tretinoin

Neuroblastoma cells in response to retinoic acid (RA) exhibit differentiation. RA, which can promote tumor cell differentiation, has also been shown to regulate tumor-infiltrating leukocytes. In an attempt to explore the relationship between RA-induced neuroblastoma cell differentiation and leukocyte chemotaxis, we investigated expression of IL-1 beta, IL-8, granulocyte-macrophage colony stimulating factor, and tumor necrosis factor-alpha in the undifferentiated and RA-induced differentiated neuroblastoma cells. Using SK-N-SH neuroblastoma cells, we found that RA induced differentiation of SK-N-SH cells as demonstrated by down-regulation of N-myc gene expression, cell-cycle arrest in G1 phase, and phenotypic change. Neither RA-treated nor untreated neuroblastoma cells expressed IL-1 beta, granulocyte-macrophage colony stimulating factor, or tumor necrosis factor-alpha mRNA. RA-treated but not untreated SK-N-SH cells expressed IL-8 mRNA in a time- and dose-dependent fashion. As determined by ELISA, IL-8 levels were detectable in the culture supernatants from RA-treated, but not untreated, neuroblastoma cells (2.65 +/- 0.43 versus 0.05 +/- 0.04 ng/mL). Using neutrophil and lymphocyte chemotactic assays, we found that RA-treated but not untreated culture supernatants of neuroblastoma cells promoted neutrophil and lymphocyte chemotaxis. The RA enhancement of neuroblastoma cell-mediated leukocyte chemotaxis was significantly blocked by anti-IL-8 neutralizing antibodies. These results suggest that RA-induced neuroblastoma cell differentiation is associated with production of functional IL-8, which may be involved in the leukocyte infiltration and activation resulting in tumor regression.

Topics: Cell Differentiation; Chemotaxis, Leukocyte; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Lymphocyte Activation; Lymphocytes; Lymphocytes, Tumor-Infiltrating; Neuroblastoma; Neutrophils; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

A neuronal cell model endogenously expressing the 5-HT1A receptor, in which to study the function and regulation of this gene, has yet to be identified. We examined murine SN-48 cells, a septum x neuroblastoma fusion cell line that proliferates in a nondifferentiated state but can be induced to differentiate into neurofilament-positive cells following 24-96 hr treatment with 10 microM retinoic acid in low serum. Northern blot analysis demonstrated the presence of a single 10.9 kilobase (kb) 5-HT1A receptor RNA species in differentiated SN-48 cells, which was not detected in undifferentiated SN-48 cells. The presence of receptor RNA in differentiated SN-48 cells correlated with the appearance of functional responses (i.e., pertussis toxin-sensitive inhibition of cAMP accumulation) to 5-HT1A agonists in differentiated but not in undifferentiated cells. In order to verify that the large 10.9 kb RNA species in SN-48 cells truly corresponded to the mouse 5-HT1A receptor RNA, a cDNA fragment from differentiated SN-48 cells was used to clone the corresponding mouse brain cDNA. The 2.4 kb cDNA contained a single open reading frame that displayed high (> 85% predicted amino acid identity) homology to the human and rat 5-HT1A receptor genes. When transfected into receptor-negative Ltk- cells, this cDNA was found to direct expression of a murine 5-HT1A receptor. Thus, we conclude that upon differentiation SN-48 cells express RNA encoding functional 5-HT1A receptors. The SN-48 septal cells will provide a useful cellular model system for investigating aspects of neuronal differentiation leading to the development of sensitivity to serotonergic input.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Animals; Base Sequence; Cell Differentiation; Cloning, Molecular; Gene Expression; Hippocampus; Mice; Molecular Sequence Data; Neuroblastoma; Neurons; Receptors, Serotonin; RNA, Messenger; Septal Nuclei; Tretinoin; Tumor Cells, Cultured

Newcastle disease virus (NDV), an avian pathogen, selectively replicates in and kills neuroblastoma (NB) cells, but not normal fibroblasts in vitro and in vivo in nude mice. NDV cytotoxicity towards NB cells is enhanced by N-myc oncogene amplification. To further define the antineoplastic effects of NDV, we examined NDV's interaction with NB cells following short-term exposure to the differentiating agent, all-trans retinoic acid (RA), and to neuraminidase. The human NB cell line IMR-32, after treatment with 50 mumol/L RA, became eight times more sensitive to NDV in a cytotoxicity assay. A time course study to determine the optimal incubation period of IMR-32 cells with RA indicated that a fourfold increase in sensitivity towards NDV killing occurred after only 8 hours of RA incubation prior to addition of virus. Maximal sensitivity was achieved at 24 hours of RA incubation and remained constant for longer incubation periods (up to 72 hours). The sensitization of IMR-32 NB cells to NDV was constant for RA doses between 3 mumol/L and 50 mumol/L. Plaque formation, which indicates replication, virus spread and cytotoxicity by a single infectious virus particle, was also enhanced by RA. This effect does not appear to require N-myc amplification in the target NB cells since RA had similar effects upon the high N-myc (IMR-32) and the low N-myc expressing cells (SK-N-SH). Enhanced sialylation has been shown by others to mediate the growth inhibitory effects of RA on a variety of tumor lines. Removal of sialic acid from the IMR-32 NB cell surface using Clostridium neuraminidase (2.7 mg/mL) inhibited 75% of NDV plaque formation. These results demonstrate that NDV killing of two NB cell lines is enhanced using clinically achievable levels of RA and that sialylation of the NB cell surface is important for virus binding and cytotoxicity.

Topics: Cytopathogenic Effect, Viral; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neuraminidase; Neuroblastoma; Newcastle disease virus; Time Factors; Tretinoin; Tumor Cells, Cultured; Viral Plaque Assay

The ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied by incubating the cells in the presence of labeled testosterone (T) or progesterone (P) to measure, respectively, the formation of dihydrotestosterone (DHT) or dihydroprogesterone (DHP) (5 alpha-reductase activity). The 3 alpha-hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol). The results show that undifferentiated neuroblastoma cells possess a significant 5 alpha-reductase activity, as shown by the considerable conversion of T into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester TPA, but not after differentiation induced by retinoic acid (RA). The 5 alpha-reductase(s) present in SH-SY5Y cells is also able to convert P into DHP. In undifferentiated cells, this conversion was about 8 times higher than that of T into DHT. Under the influences of TPA and RA, the formation of DHP followed the same pattern observed for the formation of DHT. SH-SY5Y cells also appear to possess the enzyme 3 alpha-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3 alpha-diol. This enzymatic activity is not altered following TPA-induced differentiation and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful "in vitro" model for the study of the mechanisms involved in the control of androgen and P metabolism in nervous cells.

Topics: 20-alpha-Dihydroprogesterone; 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); 3-Hydroxysteroid Dehydrogenases; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Biotransformation; Cell Differentiation; Cell Line; Dihydrotestosterone; Humans; Kinetics; Neuroblastoma; Progesterone; Testosterone; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured

Neuroblastoma cell lines have been reported to contain two proopiomelanocortin (POMC) mRNA transcripts. We have now shown by immunocytochemistry and radioimmunoassay (RIA) that a number of neuroectodermally derived cell lines contain immunoreactive beta-endorphin although cell concentrations were not characteristic of any tumour type. To explore further the functional significance of beta-endorphin expression, we analysed neuroblastoma cell lines having intermediate (I), substrate adherent (S) and neuronal (N) phenotypes. No differences in cell beta-endorphin content were detected. However, the expression of POMC mRNA and of immunoreactive beta-endorphin was reduced within a few hours of treatment of these cell lines with retinoic acid. Culture of the cell lines in the presence of beta-endorphin resulted in small but significant increases in growth. Although the POMC gene is in the same chromosomal segment as N-myc, which is normally amplified in neuroblastoma, no corresponding amplification of POMC could be demonstrated. The data suggest that POMC gene products may contribute to the autocrine/paracrine growth of neuroectodermal tumours.

Topics: beta-Endorphin; Cell Line; Clone Cells; Ectoderm; Gene Expression; Glioblastoma; Humans; Immunohistochemistry; Melanoma; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Pro-Opiomelanocortin; Radioimmunoassay; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, the most common malignant solid cancer of children, has an ability to differentiate in vitro and in vivo. This biological property has a significant influence upon the prognosis of patients with neuroblastomas. Neuronal cells express three alternatively spliced forms of c-src mRNA (nonneuronal c-src, neuronal c-srcN1, and neuronal c-srcN2), which are found at different levels in adult and fetal human brain tissue. In this study, the transcriptional levels of the three c-src mRNAs were examined in relation to the neural differentiation in eight human neuroblastoma cell lines and two clonal sublines and in seven primary neuroblastoma tissues by S1 nuclease protection assays. Neuronal c-srcN1 mRNA was expressed at high levels in neuroblastoma cell lines with the ability to differentiate but not in the cell lines lacking the capacity to mature in response to chemical inducers irrespective of N-myc gene amplification and overexpression. In terminally differentiated neuroblastoma cells, the expression of neuronal c-srcN2 mRNA, which was barely detectable at a steady-state level in the uninduced cells, increased to significant levels. Infantile neuroblastomas identified by mass screening tests expressed both neuronal c-srcN1 and c-srcN2 mRNAs at levels almost identical to that found in human brain tissue, but terminally differentiated neuroblastoma cells, neuroblastomas from older children identified based on clinical symptoms, did not. These results suggest that neuronal c-src expression and the ability of neuroblastomas to differentiate in vitro and in vivo may be correlated.

Topics: Base Sequence; Brain Chemistry; Cell Differentiation; DNA; DNA, Neoplasm; Gene Expression; Humans; Molecular Sequence Data; Neuroblastoma; Neurons; Polymerase Chain Reaction; RNA Splicing; RNA, Messenger; Tretinoin

Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.

Topics: Cell Differentiation; Cell Division; Cell Survival; Culture Media; DNA, Neoplasm; Humans; Neuroblastoma; Phenylacetates; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factor II (IGF-II) is implicated in the development of the vertebrate neural circuitry, and increases neurite growth in vitro and in vivo. We examined the relationship of IGF-II expression to the in vitro differentiation induced by retinoic acid (RA). We find that RA stimulates an increase in IGF-II messenger RNA (mRNA) in the SK-N-SH (SH) neuroblastoma cell line. An increase of IGF-II mRNA is detected within 12 h of treatment and precedes morphological differentiation. A RA dose response test indicates that an increase in IGF-II mRNA occurs within 2 days in SH cells treated with doses of RA from 1 x 10(-8) to 1 x 10(-5) M. We suggest that IGF-II expression may be regulated either directly or indirectly by RA in vitro and may lead to neuroblastoma differentiation.

Topics: Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Insulin-Like Growth Factor II; Neurites; Neuroblastoma; RNA, Messenger; Time Factors; Tretinoin

Buprenorphine is an opiate drug with a mixed agonist-antagonist profile and has therapeutic efficacy in attenuating drug craving and addiction. Because the adenylyl cyclase system has been implicated in the biochemical basis of opiate withdrawal phenomena, we have compared the acute and chronic effects of buprenorphine with the full opiate agonist etorphine on cyclic AMP (cAMP) synthesis in the human neuroblastoma cell SK-N-SH. Both drugs acutely inhibited prostaglandin (PG)E1-stimulated cAMP accumulation; the inhibition caused by either drug was prevented by pretreatment with the opiate antagonist naltrexone or with pertussis toxin. Chronic treatment of the cells with etorphine induced an increase in PGE1-stimulated cAMP synthesis which was observed after withdrawal of the inhibitory drug. Chronic treatment with buprenorphine appeared to have the opposite effect, resulting in an attenuated PGE1 stimulation; additionally, buprenorphine prevented the etorphine-induced enhancement in cAMP synthesis, whether administered before or after prolonged incubation of the cells with etorphine. The attenuating effect of buprenorphine occurred within 5 min and was prevented by a prior application of naltrexone, but could not be reversed by a subsequent treatment with antagonist. These findings suggest that buprenorphine was binding (pseudo)irreversibly to the opiate receptor, resulting in a persistent inhibition of cAMP synthesis which masks the etorphine-induced enhancement of adenylyl cyclase activity. This hypothesis was confirmed by experiments demonstrating that treatment of the cells with buprenorphine significantly reduced available opiate receptor binding sites despite extensive washing of the cells to remove unbound buprenorphine. These pharmacodynamic actions of buprenorphine may be relevant to its therapeutic efficacy in treating drug abuse and addiction.

Topics: Adenylyl Cyclases; Alprostadil; Buprenorphine; Cell Differentiation; Cyclic AMP; Drug Interactions; Etorphine; Humans; Neuroblastoma; Receptors, Opioid, mu; Sensitivity and Specificity; Stimulation, Chemical; Time Factors; Tretinoin; Tumor Cells, Cultured

AHNAK is a newly identified human gene notable for the exceptional size (c.a. 700 kD) and structure of its product, and for the repression of its expression in human neuroblastoma cells. Here we report the identification and partial characterization of the protein encoded by AHNAK. The protein is located principally (but not exclusively) in the nucleus and is phosphorylated on both serine and threonine. The abundance of the protein increases appreciably when cells withdraw from the division cycle, in response to either withdrawal of serum (fibroblasts) or differentiation (neuroblastoma cells). By contrast, the amount of phosphorylation appears to diminish in those settings. The considerable abundance and conjectured fibrous structure of AHNAK protein suggest a role in cytoarchitecture, but no function can yet be discerned.

Topics: Amino Acid Sequence; Blotting, Western; Carcinoma, Small Cell; Cell Differentiation; Cell Fractionation; Cell Nucleus; Fluorescent Antibody Technique; Humans; Immune Sera; Lung Neoplasms; Melanoma; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins; Neuroblastoma; Peptides; Phosphoproteins; Tretinoin; Tumor Cells, Cultured

A series of human neuroblastoma (NB) cell lines was analyzed for expression of peripherin, a class-III intermediate filament protein expressed at high levels in ganglia of the peripheral nervous system. By Western blotting, peripherin protein was detected in all human NB cell lines examined. The highest level of peripherin was found in the NUB-7 cell line, previously characterized as homogeneously neuroblastic. By immunofluorescence labeling, peripherin was shown to be organized in a perinuclear filamentous pattern and, exemplified by IMR32 cells, was also shown to be localized to spontaneously formed neurites. Peripherin was expressed in neuroblastic but not substrate-adherent cells, and was found at low levels in I-type cells. There was a pronounced redistribution of peripherin to neurites formed in response to dibutyryl cyclic adenosine monophosphate (dbcAMP) and all-trans-retinoic acid (RA). In NUB-7 cells, which do not extend neurites in response to nerve growth factor, there was no change in the level of peripherin protein following treatment with this agent. Both dbcAMP and RA induced a redistribution of peripherin to neurite extensions, but only treatment with RA increased the level of the protein as demonstrated with NUB-6A4 and NUB-6C4 subclones. Peripherin was also variably expressed in peripheral neuroepithelioma (NE) cell lines tested, but was organized into a more basket-like filamentous pattern in these cells. The heterogeneous expression and distribution of peripherin in NB and NE cell lines indicate that this protein is associated with maturation of the neuronal phenotype and hence serves as a differentiation marker for tumors derived from the neural crest.

Topics: Blotting, Western; Bucladesine; Cell Differentiation; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Intermediate Filament Proteins; Membrane Glycoproteins; Nerve Growth Factors; Nerve Tissue Proteins; Neurites; Neuroblastoma; Peripherins; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces the differentiation of tumor cells of neural origin and may do so by binding to one or more nuclear receptor proteins. We have identified transcripts and nuclear RA receptor (RAR) protein in a clonal line of human neuroblastoma cells that differentiate in response to RA. Prior to any exposure to RA, LA1-15n cells express two transcripts for RAR alpha (approximately 3.6 and approximately 2.7 kb) as well as low levels of transcript for RAR beta (approximately 3.4) and RAR gamma (approximately 2.8 kb). Exposure of LA1-15n cells to RA leads to the induction of a approximately 2.9-kb RAR beta mRNA, whereas the expression of transcripts for RARs alpha and gamma does not change appreciably. The 2.9-kb RAR beta transcript is increased by 4 h (8-fold) and continues to increase for 24-48 h (40- to 60-fold). The RA-associated increase in RAR beta mRNA in LA1-15n cells is not diminished by the addition of the protein synthesis inhibitor, cycloheximide, but is abolished by the addition of the RNA synthesis inhibitor, actinomycin D. In addition to RAR transcripts, LA1-15n cells contain a nuclear protein with the requisite characteristics of a RAR. The nuclear protein binds all-trans-[3H]RA with high affinity (Kd approximately 0.2 nM). The nuclear protein sediments at approximately 4S, which is consistent with the molecular mass deduced from RAR cDNAs (approximately 50,000 Da). The nuclear protein is clearly distinguishable from a all-trans-[3H]RA-binding protein found in the cytosolic fraction of LA1-15n cells. The cytosolic protein sediments at approximately 2S on sucrose density gradients, consistent with the expected molecular mass of the cellular retinoic acid-binding protein (approximately 16,000 Da). The nuclear [3H]RA-binding protein binds to DNA-cellulose and to the RAR beta response element. These results support the hypothesis that RARs are present in human neuroblastoma cells and may be involved in human neuroblastoma cell differentiation. They also demonstrate that RA markedly influences the expression of steady-state levels of mRNA for one of its own receptors, the RAR beta.

Topics: Blotting, Northern; Carrier Proteins; Cell Nucleus; Chromatography, Affinity; Cycloheximide; Cytosol; Dactinomycin; Humans; Kinetics; Neuroblastoma; Receptors, Retinoic Acid; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Neuroectodermal tumours express hormones which are post-translationally processed and inactivated by the action of specific proteases and peptidases. The data reported here show the presence of a novel thermolysin-like metallo-endopeptidase activity in several human cell lines. The soluble fractions of neuroblastoma, melanoma and a glioblastoma tumour cell lines are able, with different degrees, to cleave the Ser12-Phe13 bond of a DVDERDVRGFAS decreases FLNH2 substrate. The inhibition pattern suggests a metallo-endopeptidase thermolysin-like character, with the involvement of thiol group(s), clearly distinct from neutral endopeptidase (NEP; EC 3.4.24.11). This metallo-endopeptidase activity is down regulated during retinoic acid(RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells but not in the RA-resistant BE(2)-M17 cells, suggesting that the down regulation is related to neuronal differentiation and not a direct effect of RA on the enzymatic activity.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Glioma; Humans; Kinetics; Melanoma; Metalloendopeptidases; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neprilysin; Neuroblastoma; Oligopeptides; Recombinant Proteins; Skin; Substrate Specificity; Thermolysin; Tretinoin; Tumor Cells, Cultured; Xenopus laevis

The effects of the thyroid hormone triiodothyronine (T3), nerve growth factor (NGF) and stress (exposure to heat or aluminum sulfate) on growth, development and ageing of human neuroblastoma cells were studied in vitro. Differentiation of cells using retinoic acid and NGF inhibits cell growth and proliferation; simultaneously, it promotes acquisition of neuronal phenotype, down-regulation of T3 receptors, and an increase in catecholaminergic tyrosine hydroxylase activity and microtubule assembly. The actions of T3 on neuronal differentiation resemble those of NGF and suggest the existence of NGF-T3 interactions. Exposure to stress inhibits cell growth and proliferation, increases immunoreactivity to the microtubule-assembling protein tau (which occurs in paired filaments of neurofibrillary tangles in the aged human brain), and facilitates formation of tau-ubiquitin complexes (which also occur in the aged brain). Stress does not prevent the inhibition of cell proliferation by high doses of T3; however, T3 doses that are equivalent to physiological levels reduce stress-induced inhibition of growth. Previous studies have shown that stress may also induce in these cells facsimile lesions of normal and abnormal ageing, such as accumulation of lipofuscin pigments, formation of paired helical filaments and increased immunoreactivity to tau, beta-amyloid proteins, and ubiquitin. These lesions may represent cellular and molecular manifestations of increased vulnerability and susceptibility to genetic and extrinsic factors (e.g. hormones and environmental influences) with ageing. It is proposed that neuroblastoma cells may serve as a model to study mechanisms of neuronal ageing and to identify agents and conditions capable of preventing, delaying or reducing metabolic abnormalities leading to age-associated disorders.

Topics: Aging; Alum Compounds; Brain; Cell Differentiation; Cell Division; Cellular Senescence; DNA, Neoplasm; Hot Temperature; Humans; Models, Neurological; Nerve Growth Factors; Neuroblastoma; Receptors, Thyroid Hormone; tau Proteins; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Ubiquitins

1. The mouse neuroblastoma cell line N-115 was used as a model system to study neuronal differentiation induced by treatment of cells with different agents. 2. The extent of morphological differentiation obtained with dibutyryl cyclic AMP (dbc-AMP), dimethyl sulfoxide (DMSO), retinoic acid (RA), and serum-free medium was correlated to the expression of the mRNA for the gamma isoform of the glycolytic enzyme enolase, a recognized neuron-specific marker. 3. A 4-day treatment of the cells with any of the differentiation inducing agents used in this study resulted in the extension of long neurites, though differences in cell body shape were observed depending on the agent used. 4. Northern blot analysis revealed that changes in the level of gamma enolase-specific mRNA correlate with the extent of morphological differentiation, with a 5- to 20-fold increase depending on the differentiation inducing agent used. 5. Finally, we found that a high cell density causes a significative increase in the level of the gamma enolase-specific message in cells maintained in growing conditions.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Contact Inhibition; Culture Media, Serum-Free; Dimethyl Sulfoxide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Isoenzymes; Mice; Neoplasm Proteins; Neuroblastoma; Neurons; Phosphopyruvate Hydratase; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces the neuronal differentiation of many human neuroblastoma cell lines. In this study, we show that RA treatment of neuroblastoma cells induces the expression of TrkB, the receptor for the neurotrophins BDNF, NT-3, and NT-4/5. BDNF addition to RA-treated SH-SY5Y neuroblastoma cells stimulated the tyrosine phosphorylation of TrkB and neuronal differentiation. RA treatment of KCNR neuroblastoma cells, which constitutively express BDNF mRNA, resulted in the expression of TrkB and differentiation in the absence of added BDNF. Finally, in 15N neuroblastoma cells, which express BDNF mRNA but do not differentiate in response to RA, RA induced only a truncated form of TrkB. 15N cells transfected with full-length TrkB differentiated in the absence of RA. These results indicate that RA induces the neuronal differentiation of neuroblastoma cells by modulating the expression of neurotrophin receptors.

Topics: Cell Differentiation; Gene Expression Regulation; Humans; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurons; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor, trkA; Receptors, Cell Surface; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tyrosine

When established in culture, human neuroblastoma cell lines typically are comprised of heterogeneous cellular subpopulations, including neuroblastic (N-type), substrate-adherent (S-type), and intermediate (I-type) cells that can be distinguished by their characteristic morphologies and expression of differentiation-associated antigens. Here we examined the relative levels of the Bcl-2 oncoprotein in 15 clones derived from four different neuroblastoma cell lines. Among six clones isolated from the SK-N-SH line, levels of p26-Bcl-2 correlated with morphology and differentiation markers with the hierarchy of bcl-2 expression being: N-type cells > N/I-type > I-type > S-type. Furthermore, stimulation of one of the N-type clones, SH-SY5Y, with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induced differentiation toward a more neuronal-like phenotype and resulted in a 5- to 10-fold elevation in the relative levels of Bcl-2 protein. High relative amounts of p26-Bcl-2 protein were also found in an N-type clone derived from the SMS-KCN line. In two N-type clones derived from the LA-N-1 line, however, levels of Bcl-2 protein were only moderately elevated, and in one N-type clone from the SK-N-BE(2) line the levels of Bcl-2 protein were low. Thus, high relative levels of Bcl-2 oncoprotein are not a universal feature of N-type cells (three of six clones tested). In contrast, all 5 of the S-type clones evaluated contained relatively low levels of Bcl-2 protein, suggesting that these cells (which may represent embryonic precursors of Schwann, glial, and melanocytic cells) do not typically express the bcl-2 gene at high levels. Consistent with this inverse correlation between Bcl-2 protein levels and S-type characteristics, stimulation of an I-type clone derived from the SK-N-BE(2) line with 5-bromodeoxyuridine was accompanied by an accumulation of S-type cells in these cultures, decreased Bcl-2 protein, diminutions in the neuronal markers neurofilament-M and neuron-specific enolase, and an increase in the relative levels of the S-type marker proteins vimentin and beta-2-microglobulin. Conversely, stimulation of this I-type clone with retinoic acid resulted in an accumulation of N-type cells (which are thought to represent embryonic precursors of sympathetic neurons), decreased vimentin and beta-2-microglobulin, increased neurofilament-M, and a marked elevation in p26-Bcl-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Antineoplastic Agents; Carcinogens; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Clone Cells; Drug Resistance; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Neuroblastoma; Oncogenes; Phorbol Esters; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Muscarinic receptor density increased by approximately 36% after differentiation induced by retinoic acid (Bmax, control = 126 +/- 13 fmol/mg protein; Bmax, retinoic acid-treated = 170 +/- 17 fmol/mg protein; P < 0.05), corresponding to a 170% increase in receptor content per cell. The affinity of [3H]NMS for the receptors was somewhat lower in the retinoic acid-treated cells (Kd, control = 0.14 +/- 0.04 nM; Kd, retinoic acid-treated = 0.25 +/- 0.04 nM; P < 0.05). Reverse transcriptase/polymerase chain reaction analysis using subtype-specific primers revealed that undifferentiated Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by retinoic acid treatment. [3H]NMS displacement curves with subtype selective receptor ligands (pirenzepine, m1; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m3 and m1 receptor subtypes, and differentiation did not affect the pharmacological profile of the expressed receptor populations. The present results indicate that differentiation induces selective changes in the expression and activity of muscarinic receptors in a neuronal cell line.

Topics: Base Sequence; Cell Differentiation; Humans; Molecular Sequence Data; N-Methylscopolamine; Neuroblastoma; Receptors, Muscarinic; RNA, Messenger; Scopolamine Derivatives; Tretinoin; Tumor Cells, Cultured

Insulin-like growth factor 2 (IGF-2) is the major autocrine growth factor for neuroblastoma. IGF-2 mRNA can just be detected in SK-N-BE(2) cell line; higher levels are present in two clones derived from it [BE(2)-C; BE(2)-M17]. IGF-2 mRNA is increased by retinoic acid (RA) only in the clones. IGF-2 expression/induction is more marked in BE(2)-M17, which shows more RA-resistance (evaluated as growth inhibition, neurite outgrowth and induction of programmed cell death). Under RA exposure, the parental line shows a more pronounced growth inhibition, neurite outgrowth and programmed cell death, as compared to its clones. BE(2)-C cells also express type 1 IGF receptor mRNA, though with a different time course than for expression of IGF-2. The data suggest that IGF-2 expression is correlated with growth, and may counteract the growth retardation, neurite outgrowth and programmed cell death effects of retinoic acid. Therefore the autocrine pattern of IGF-2 production by neuroblastoma cells may promote RA-resistance.

Topics: Brain Neoplasms; Cell Differentiation; Cell Movement; Growth Hormone; Humans; Indicators and Reagents; Insulin-Like Growth Factor II; Neurites; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Topics: Administration, Oral; Animals; Cell Differentiation; Cell Division; Humans; Intermediate Filaments; Mice; Mice, Nude; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Monoclonal antibodies that recognize the human, mouse, and rat retinoic acid receptor alpha (RAR alpha) protein have been generated using synthetic peptides. Less well-characterized monoclonal antibodies were also generated against the RAR beta and RAR gamma proteins. Monoclonal antibodies of the IgG1 (R alpha 10) and IgG2a (R alpha 13) isotypes effectively and specifically recognize both the human and mouse RAR alpha protein. Preincubation of the antibodies with the synthetic RAR alpha peptide, but not with the RAR beta or RAR gamma peptides, blocked recognition of the approximately 55 kDa RAR alpha protein on western blots. These monoclonal antibodies also detected differing levels of RAR alpha in various rat tissues. These monoclonal antibodies will serve as powerful reagents to study the structure and regulation of the retinoic acid receptor protein.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blotting, Western; Carrier Proteins; Cell Line; Humans; Immunoglobulin G; Immunoglobulin Isotypes; Macromolecular Substances; Mice; Molecular Sequence Data; Neuroblastoma; Rats; Rats, Inbred Strains; Receptors, Retinoic Acid; RNA, Messenger; Sequence Homology, Nucleic Acid; Tretinoin

Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

Topics: Bucladesine; Carbachol; Humans; Neuroblastoma; Peptide Biosynthesis; Radioimmunoassay; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In this report we provide evidence for the activation of distinct differentiation pathways during treatment of the neuroblastoma cell line SMS-KCNR with 1 mM dibutyryl cyclic AMP (dbcAMP) and/or 5 microM retinoic acid (RA). Our results show that the adrenal gland specific gene pG2 is induced only during dbcAMP treatment, while RA induces a neuronal phenotype and expression of all neural related genes while decreasing the expression of many chromaffin related genes. Furthermore dbcAMP does not affect the DNA content distribution of SMS-KCNR [G1 = 61.8 +/- 4.1% (SD); S = 20.3 +/- 6.3%; G2-M = 18 +/- 5.4%] despite morphological and molecular signs of cellular differentiation. Conversely, RA arrests cell growth causing a decrease in cells in the growth fraction (S + G2 + M = 15.6 +/- 6.1%) and an increase in cells in G1 (G1 = 84.3 +/- 5%). Using cyclic AMP and RA in combination, we found that RA inhibited expression of adrenal gland specific gene pG2 and induced a neuronal phenotype. Since dbcAMP does not cause a significant G1 block in SMS-KCNR cells we propose that this agent may be able to induce SMS-KCNR only to an intermediate stage of chromaffin differentiation in which cells retain their proliferative potential.

Topics: Bucladesine; Cell Differentiation; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Genetic Markers; Humans; Neuroblastoma; Phenotype; RNA, Messenger; RNA, Neoplasm; S Phase; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

Topics: Cell Transformation, Neoplastic; Humans; Neuroblastoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In large part, malignancy is the end result of aberrant cell growth and differentiation. Control of these processes is anticipated to result in a suppression of oncogenicity. Retinoic acid (RA), a derivative of vitamin A, has been shown to inhibit proliferation, induce cell differentiation and reverse the malignant phenotype of a variety of tumor cell types. In order to further characterize the antitumor potential of RA, this study examined the in vitro and in vivo effects of this retinoid on cell lines derived from human neuroblastoma (NB). The in vitro phase of this study tested the ability of various compounds to raise intracellular cyclic adenosine 3':5'-monophosphate (cAMP) levels and either alone or in combination with RA, to promote differentiation of two relatively RA-resistant cell lines. Direct activation of the synthetic enzyme adenylate cyclase by forskolin or cholera toxin increased intracellular cAMP levels over 10-fold after 1 hour of treatment, declining over the next 16 to 24 hours. After 5 days of continuous growth in the presence of these agents, cAMP levels remained elevated 2- to 7-fold above control values and were accompanied by a decrease in cell proliferation and an increase in cell differentiation. All these effects were exaggerated in the presence of phosphodiesterase inhibitors. Isoproterenol and epinephrine did not alter cAMP levels and had no discernible biological effects. RA promoted differentiation with little effect on cAMP levels. Combination treatment of cells with RA plus agents that raised cAMP levels resulted in greater degrees of differentiation than seen with single-agent treatment. From these data, it was concluded that: 1. the cAMP synthetic and degradative pathways are functional in the NB cell lines studied; 2. elevation of cAMP is a sufficient but not necessary condition for inhibiting proliferation and promoting differentiation in these cells; 3. elevation of intracellular cAMP potentiates the differentiation-inducing activity of RA; and 4. overcoming retinoid resistance in some tumor cell lines may be feasible by alterations in the cAMP system. This would be of particular value in treating tumors that have lost retinoid responsiveness. The in vivo phase of this study examined the effects of single-agent treatment using RA on the development and growth in nude mice of tumors derived from a NB cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cyclic AMP; Drug Screening Assays, Antitumor; Female; In Vitro Techniques; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

A variety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. In the current study, we have assessed the ability of S-allyl cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was observed in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 micrograms/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.

Topics: Acetylcholinesterase; Antineoplastic Agents; Cell Differentiation; Cell Division; Cyclic AMP; Cysteine; Garlic; Gene Expression; Genes, myc; Humans; In Vitro Techniques; Neuroblastoma; Plants, Medicinal; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

1. The expression of the gene codifying for CD4, the most important human immunodeficiency virus type 1 (HIV-1) receptor molecule, was analyzed in 11 fetal brains at various gestational ages and in 9 human neuroblastoma (NB) cell lines. CD4 gene expression in fetal and malignant neural cells was then compared with that observed in a hematopoietic cell line and adult hippocampus. 2. In addition, CD4 mRNA was evaluated in two NB cell lines induced to differentiate in vitro with retinoic acid (RA) or 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H7), a protein kinase C inhibitor. 3. All fetal brains and NB cell lines express a 1.8-kb signal when hybridized with pT4BcDNA probe, while a 3.0-kb signal such as observed in hematopoietic human cells was found in 1 of 11 fetal brains and in 0 of 9 NB cell lines. The 1.8-kb signal was lost in all analyzed poly(A)+ mRNA samples. 4. Moreover, CD4 gene expression was not induced in either RA- or H7-treated NB cells at any tested time and dose. The analysis of NB cells by polymerase chain reaction failed to demonstrate CD4 expression in either poly(A)+ or poly(A)- RNA. 5. In conclusion, the results show that the 1.8-kb signal observed in RNA extracted from fetal or transformed human neural cells is probably due to an aspecific hybridization. However, the gene codifying for CD4 can rarely be expressed by fetal brain cells early during gestation, in still unclear circumstances.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Base Sequence; Brain; CD4 Antigens; Cell Differentiation; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Gestational Age; Humans; Isoquinolines; Molecular Sequence Data; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Piperazines; Poly A; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

A new human cell line, termed Muraoka, has been established from the recurrent tumor of a case of congenital primitive neuroectodermal tumor (PNET) arising at the temporofacial region of a male infant. The microscopic findings of this cell line were epithelioid, and the xenografted tumor in a nude mouse consisted of the malignant epithelioid cells. Immunohistochemically, the cells were positive for neuron-specific enolase, S-100 protein, carcinoembryonic antigen, cytokeratin, epithelial membrane antigen, and glial fibrillary acidic protein. These findings were quite similar to those of the epithelioid cells in the original tumor and of the xenografted tumor cells. Neither chromosomal abnormalities nor N-myc amplification were observed. Morphological differentiation after treatment with N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2-cAMP), all-trans-retinoic acid (RA), prostaglandin E1 (PGE1), and 5-bromo-2'-deoxyuridine (BrdU) showed two different results. Bt2-cAMP and PGE1 induced neuronal differentiation with the extension of neurites, whereas RA and BrdU predominantly induced Schwannian differentiation (flat cells). In these respects, the cell line Muraoka seems to be useful for studying characteristics of PNET as well as for developing the new treatments against such tumors.

Topics: Animals; Bromodeoxyuridine; Bucladesine; Carcinoembryonic Antigen; Cell Line; Facial Neoplasms; Glial Fibrillary Acidic Protein; Humans; Infant, Newborn; Karyotyping; Male; Mice; Mice, Nude; Neurites; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Phosphopyruvate Hydratase; S100 Proteins; Tretinoin

1. The effects of retinoic acid, gamma-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction. 2. Cell morphology was strongly affected by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and gamma-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects. 3. Acetylcholinesterase activity was significantly stimulated by retinoic acid and by gamma-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold. 4. The cellular catecholamine and related metabolite content was lowered by retinoic acid and gamma-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation. 5. Finally, the cell-associated vasointestinal peptide level was strikingly increased by gamma-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. 6. It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.

Topics: Biomarkers, Tumor; Catecholamines; Cell Differentiation; Cytarabine; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type. The two uninduced neuroblastoma cell lines show a similar pattern of expression for a number of HOX2 genes although the levels of expression are different for individual cell lines. The embryonal carcinoma cell line F9 expresses low levels of several HOX2 genes which is restricted to the 5' region of the HOX2 cluster. The glioblastoma cell line, 251-MG expresses almost all of the genes of the HOX2 locus. Differentiation of these cells modulates the expression of the HOX2 genes in a manner that is dependent upon the cell type as well as the differentiation factor. Differentiation affects both the level of HOX2 gene expression and the distribution of transcript sizes. In conclusion, our analysis reveals a complex pattern of expression for the genes of the HOX2 locus in neuronal and glial cells and suggests that the cell-specific expression of these genes may be correlated with the phenotypic differences that are observed between different neuronal and glial cell populations within the nervous system.

Topics: Animals; Blotting, Northern; Bucladesine; Cell Differentiation; Gene Expression; Genes, Homeobox; Glioma; Humans; Mice; Nerve Growth Factors; Neuroblastoma; Neurons; RNA, Neoplasm; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Cadmium; Cadmium Chloride; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Cell Line; Cell Membrane; Chlorides; Evoked Potentials; Fura-2; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Nicotinic Acids; Nifedipine; omega-Conotoxins; Oxadiazoles; Peptides, Cyclic; Potassium; Tretinoin

Human neuroblastoma (NB) cell lines have been suggested to represent a model of neural crest differentiation. The expression of several Schwann-cell-associated antigens was examined by flow cytometry and Northern blot analysis. Variable reactivity of the human NB cell lines was found in both the level and pattern of reactivity. Retinoic acid treatment of cell line SMS-KAN resulted in a neuron-like morphological differentiation and a decrease in several of the glial markers under study. Similarly, Northern blot analysis illustrated myelin-associated glycoprotein expression, and decreased expression of this message with retinoic acid treatment was consistent with the neuron-like morphological changes. Overall, human NB in vitro was found to be multipotential, but we have shown that it is capable of expressing several Schwann cell markers which are modulated during induced differentiation.

Topics: Biomarkers, Tumor; Blotting, Northern; Cell Line; Flow Cytometry; Gene Expression; Humans; Myelin Basic Protein; Myelin Proteins; Myelin-Associated Glycoprotein; Neuroblastoma; RNA; Schwann Cells; Tretinoin

Insulin-like growth factors (IGF-I and IGF-II) are mitogenic polypeptides that play an important role in normal growth and development. IGF-II has been shown to stimulate the growth of neuroblastoma tumors in an autocrine and paracrine fashion. Critical in determining the role of IGF-II in tumorigenesis is the necessity to delineate factors affecting the transcription of IGF-II in normal and tumor tissues. To date such factors are poorly characterized. In this study we find that retinoic acid (RA), a naturally occurring morphogen, that has been shown to be indispensable in the development of the chick limb bud, stimulates an increase in IGF-II messenger RNA (mRNA) in the Lan-1-15N neuroblastoma cell line. This increase in IGF-II is coincident with RA mediated inhibition of DNA synthesis. An increase in the steady state levels of IGF-II mRNA is detectable within 2 h of RA treatment and maximal by 24 h. In RA-treated Lan-1-15N cells, IGF-II mRNA levels are regulated at the level of new gene transcription and result in an increase in IGF-II protein in the culture supernatant. These studies suggest one mechanism affecting the production of IGF-II in vivo may be mediated by RA and detail a model system by which transcriptional regulation of IGF-II mRNA can be analyzed.

Topics: Cell Division; Cell Line; Cell Nucleus; DNA Replication; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor II; Kinetics; Neuroblastoma; Poly A; RNA; RNA, Messenger; Thymidine; Transcription, Genetic; Tretinoin

Neuroblastomas in culture are characterized by the presence of 2 morphologically and biochemically distinct phenotypes (i.e., neural "N-type" and flat substrate-adherent "S-type") which undergo transdifferentiation. Human neuroblastoma SK-N-BE(2) cells differentiate toward a neural phenotype upon retinoic acid (RA) treatment. However, we recently showed that, during the RA treatment, a subset of SK-N-BE(2) cells undergo apoptosis; these cells specifically express a high "tissue" transglutaminase (tTG) level. This study was undertaken to investigate the cellular and molecular basis of the action of retinoic acid on apoptosis in human neuroblastoma cells. As a biochemical marker of the phenomenon we studied the tTG gene expression in the parental line SK-N-BE(2) and in 2 clones which stably express neuroblastic [BE(2)-M17] and substrate-adherent [BE(2)-C] features, respectively. Data showed a differential phenotype-specific regulation of tTG gene expression. In fact, RA treatment enhanced tTG expression and apoptotic index in the flat substrate-adherent variant, whereas, in cells expressing the neural phenotype, very low tTG expression and apoptosis were found. Northern-blotting analysis revealed that the substrate-adherent cells had a basal 3-fold higher level of tTG mRNA. An increase in tTG mRNA major transcript levels (3.7 kb) occurred within a few hours of exposure to RA in both the phenotypic variants. By contrast, tTG protein level was very low in the cell expressing the neuronal phenotype, even after prolonged exposure to RA. Immunohistochemical analysis indicated that tTG protein, in addition to mature apoptotic cells, was specifically localized in the flat substrate-adherent variant both in the wild-type and in the BE(2)-C clone. These findings suggest that the ability to undergo apoptosis in the neuroblastoma cells is associated with the expression of a non-neuronal neuroectodermal (substrate-adherent cells) immature phenotype.

Topics: Cell Death; Cell Differentiation; Cell Division; Humans; Neuroblastoma; Phenotype; RNA, Messenger; RNA, Neoplasm; Staining and Labeling; Transglutaminases; Tretinoin; Tumor Cells, Cultured

In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.

Topics: Cell Membrane; Fibroblast Growth Factor 2; Gene Amplification; Genes, myc; Humans; Neuroblastoma; Oxidation-Reduction; Transfection; Tretinoin; Tumor Cells, Cultured

Two cell clones [BE(2)-C and BE(2)-M17] derived from the human neuroblastoma cell line SK-N-BE(2) express corticotrophin-releasing hormone as well as interleukin-6 mRNA. Both genes are overexpressed, although with a different time course, following exposure to 5 microM retinoic acid, in parallel to the induction of neuroblastic differentiation. On the contrary, we are unable to detect interleukin-1 beta mRNA in these cell lines. Both cytokines are known to increase hypothalamic CRH mRNA. The production of cytokines and neuropeptides by neuroblastoma cells indicate a complex dialogue between tumour cells and anti-tumour immunity.

Topics: Blotting, Northern; Cell Differentiation; Corticotropin-Releasing Hormone; Gene Expression; Humans; Interleukin-1; Interleukin-6; Kinetics; Neuroblastoma; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.

Topics: Biomarkers, Tumor; Cell Differentiation; Chromogranin A; Chromogranin B; Chromogranins; Golgi Apparatus; Humans; Immunohistochemistry; Neurites; Neuroblastoma; Protein Biosynthesis; Proteins; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, a malignant neoplasm that arises in the adrenal medulla or sympathetic ganglion, is one of the most common solid tumors of childhood. Reports that neuroblastomas spontaneously mature to form benign ganglioneuromas have prompted investigations into the efficacy of using agents that induce neuronal differentiation in the treatment of this malignancy. Retinoic acid is one agent in particular that has been shown to induce growth inhibition and terminal differentiation of neuroblastoma cell lines in vitro. Using the human neuroblastoma cell line SMH-KCNR, we have investigated the role of the extracellular matrix protein thrombospondin in retinoic acid induced neuroblastoma differentiation. Treatment with retinoic acid results in a rapid induction (within 4 h) of thrombospondin (TSP) message which is independent of intervening protein synthesis and superinducible in the presence of cycloheximide. This suggests that TSP functions as a retinoic acid inducible immediate early response gene. A concomitant increase in both cell associated and soluble forms of TSP protein can be detected within 24 h of retinoic acid treatment. A functional role for TSP in SMH-KCNR differentiation was established in experiments which showed that exposure to anti-TSP monoclonal antibodies delay retinoic acid differentiation for 48 h. At the time the cells overcome the effects of TSP inhibition, laminin production becomes maximal. Treatment of the cells with a combination of anti-TSP and antilaminin antibodies results in complete inhibition of differentiation.

Topics: Cell Differentiation; Humans; Laminin; Neuroblastoma; Oligopeptides; Platelet Membrane Glycoproteins; Thrombospondins; Tretinoin; Tumor Cells, Cultured

Cellular differentiation is often associated with striking changes in ganglioside metabolism. Because retinoic acid causes cellular differentiation in vitro, we have characterized its effect on ganglioside synthesis and shedding by LAN-5 human neuroblastoma cells. Three major observations were made: (a) 20 microM retinoic acid caused a marked (twofold) increase in cellular ganglioside content, with a slight relative enhancement in GD1a and GT1b synthesis, (b) ganglioside shedding increased in parallel with increased cellular ganglioside content, and also, unexpectedly, (c) retinoic acid caused a quantitatively similar increase in content of cell membrane phospholipids, which are also shed. We conclude that enhanced ganglioside synthesis and shedding by retinoic acid are part of a previously undescribed generalized stimulatory effect on membrane lipid metabolism.

Topics: Chromatography, Thin Layer; DNA; Gangliosides; Humans; Neuroblastoma; Phospholipids; Thymidine; Tretinoin; Tritium; Tumor Cells, Cultured

The differentiation pattern of two related human neuroblastoma cell lines, SK-N-SHF and SK-N-SHN, induced by retinoic acid and staurosporine was studied. Immunohistochemical and electron microscopic examination of the cells indicated that the SHF variant could undergo differentiation along a melanocytic route when treated with retinoic acid and to neuronal cells when treated with retionic acid and staurosporine together. Treatment of SHN cells with either or both these agents caused neuronal differentiation. The melanocytic pathway was characterized in part by the flattening of the cells, the appearance of melanocytic antigens and various forms of melanosomes, an increase in tyrosinase activity, and the absence of neuronal marker proteins. The neuronal route was typified by the development of long neuritic processes containing microtubules and numerous neurosecretory granules as well as by immunohistochemical reactions for neural cell adhesion molecule, synaptophysin, and neurofilament proteins. The significance of these results is discussed in terms of the differentiation responses of neuroblastoma cells to chemical agents as well as some of the factors involved in the regulation of phenotype expressions of these cells.

Topics: Alkaloids; Cell Differentiation; Cell Line; Humans; Melanocytes; Microscopy, Electron; Monophenol Monooxygenase; Neurites; Neuroblastoma; Neurons; Protein Kinase C; Staurosporine; Tretinoin

Expression of the N-myc oncogene is an important determinant of tumor behavior in human neuroblastoma. To study the regulation of N-myc, we have subcloned fragments of the 5' flanking region of the human N-myc gene upstream of the chloramphenicol acetyl transferase (CAT) reporter gene, and assayed for promoter activity in transient transfections into neuroblastoma and other cell lines. Upstream sequences were found to possess promoter activity to within 121 bp of the major cap site (-121). Negative regulatory elements were identified in regions approximately 2 kb and 500 bp upstream from the major cap site, as well as 150-1000 bp downstream. Promoter constructs containing downstream elements from bp +150 to +1000 were active in N-myc-expressing neuroblastoma cell lines, but not in non-expressing Epstein-Barr virus (EBV)-transformed 729-6 B-cell or HeLa cell lines, while those lacking this element were active in all cell types tested. All tested constructs retaining promoter activity showed decreased activity in parallel with the down-regulation of endogenous N-myc in response to treatment of transfected cells with retinoic acid. These studies suggest that N-myc regulation may be controlled at different levels, and provide a basis for further characterization of N-myc regulation in neuroblastoma.

Topics: DNA Mutational Analysis; Gene Expression Regulation; Genes, myc; Humans; In Vitro Techniques; Neuroblastoma; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Restriction Mapping; Tretinoin; Tumor Cells, Cultured

We have isolated a human neuroblastoma (NB) cell line, HTLA230, from the bone-marrow aspirate of a patient with stage-IV disease. Subcutaneous tumors after inoculation of HTLA230 cells into nude mice were composed of primitive neuroblasts which rarely contained neuro-secretory granules. Cytogenetic studies of the cell line demonstrated 2 distinct populations of cells with common chromosomal markers. Stable sub-clones with a differentiated or undifferentiated cell morphology were isolated, demonstrating phenotypical heterogeneity of the HTLA230 parental cell line. Treatment with retinoic acid (RA) induced extensive neurite outgrowth in the parental cell line and in phenotypically differentiated sub-clones, but rarely in undifferentiated ones. Long-term treatment with RA was not associated with down-modulation of mycN-gene expression, which could be achieved only in cultures treated additionally with aphidicolin, a DNA-synthesis inhibitor, thus eliminating growing NB cells. A RA resistant subclone (CI-5) was isolated from parental HTLA230 cells grown at clonal cell density. Cells originally showed a homogeneously differentiated morphology; however, flat cells (F-cells) appeared with time and were subsequently separately propagated. Transdifferentiation of isolated F-cells into cells with neuron-like (N-cell) morphology was observed. Immunohistochemical analysis demonstrated that F-cells had lost the expression of neuronal markers, including HNK-I and A2B5, and expressed the intermediate filament, vimentin. Furthermore, F-cells showed high incorporation of [methyl-3H] thymidine (3H-TdR) by autoradiography but no mycN protein could be detected, although present in the parental cell line. These results then suggest that the isolated NB cell line and the RA-resistant variant line represent an excellent in vitro model with which the bi-modal differentiation pathway of NB can be analyzed on a molecular biological level.

Topics: Animals; Cell Differentiation; Drug Resistance; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Infant; Male; Mice; Mice, Nude; Neuroblastoma; Nucleic Acid Hybridization; Phenotype; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Immunocytochemical, immunoblotting and in situ hybridization studies were used to map the distribution of SNAP-25 protein and mRNA in the rodent nervous system. These experiments demonstrated that subsets of neurons expressed SNAP-25, and that several patterns of expression emerged: SNAP-25 expression in caudate nucleus was initially concentrated in axons, which subsequently was localized in presynaptic regions of these axons. Other regions, typified by neocortex, showed developmental increases and persistent adult neuronal immunoreactivity for SNAP-25. Finally, olfactory bulb contained neurons which initially expressed SNAP-25, but lost expression during maturation. Additional studies in cultured human and rat cell lines derived from neural crest suggested that SNAP-25 is expressed in such lines, but not in glial or fibroblast lines. Differentiation of rat PC-12 cells with nerve growth factor failed to alter steady-state levels of SNAP-25 protein; similar responses were seen in human SMS-KCNR neuroblastoma cells differentiated using retinoic acid. The presence of SNAP-25 in presynaptic regions of numerous neuronal subsets and in neural crest cell lines suggests that this protein subserves an important function in neuronal tissues.

Topics: Animals; Blood Proteins; Brain; Evans Blue; Female; Humans; Immunohistochemistry; Membrane Proteins; Nerve Tissue Proteins; Neuroblastoma; Nucleic Acid Hybridization; PC12 Cells; Pregnancy; Rats; Rats, Inbred Strains; Skin; Synaptosomal-Associated Protein 25; Tretinoin; Tumor Cells, Cultured

A comparable pattern of morphological neuronal differentiation was induced in the human neuroblastoma cell line SMS-KCNR by treatment with either retinoic acid (RA) or exogenous laminin (LM). LM expression and synthesis by SMS-KCNR was increased upon RA treatment which involved the cell bound, rather than the secreted protein. These data suggest an involvement of LM in the neuroblastoma differentiation process manifested both as an ability of LM to induce a morphological neuronal differentiation and as a selective control on LM metabolism during RA induced neuronal differentiation.

Topics: Cell Differentiation; Collagen; Fibronectins; Fluorescent Antibody Technique; Humans; Laminin; Microscopy, Phase-Contrast; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The effect of a complex in vitro synthesized extracellular matrix (ECM) and its components on growth and phenotypical differentiation of a human neuroblastoma (NB) cell line (HTLA230) was investigated. Rat smooth-muscle-cell (R22CIF)-derived ECM composed of collagen, glycoproteins, and glycosaminoglycans (GAGs) promoted spontaneous neurite outgrowth of HTLA230 cells but did not alter their growth kinetic or cloning efficiency as compared with cells seeded onto gelatin-coated dishes. The matrix significantly enhanced, quantitatively and qualitatively, the responsiveness of HTLA230 cells to retinoic acid (RA), and a substantially reduced growth rate was observed in the presence of RA with cells grown on the ECM. Biochemical modification of the composition of the R22CIF-matrix by trypsin digestion and/or high-salt extraction (4 M guanidinium) demonstrated that the ratio of chondroitin sulfate to hyaluronic acid (HA) present in the ECM determines the capacity of the matrix to promote NB differentiation. A human fibroblast (T-1)-derived ECM, which has a biochemical composition of the GAG component similar to that of the trypsinized R22CIF-matrix, but which has a high amount of glycoproteins, confirmed these results. Nerve-growth-factor (NGF)-induced differentiation in a variant HTLA 230 cell line was inhibited when cells were grown on an ECM with a low ratio of chondroitin sulfate/HA. The composition of the ECM thus modulates the responsiveness to various differentiation-inducing agents and alters the phenotype of NB cells.

Topics: Animals; Cell Differentiation; Clone Cells; Collagen; Extracellular Matrix; Glycoproteins; Glycosaminoglycans; Humans; Neurites; Neuroblastoma; Phenotype; Rats; Tretinoin; Trypsin; Tumor Cells, Cultured

We have investigated the effects of retinoic acid (RA), human recombinant gamma interferon (gamma-IFN), and the association of both agents on the growth of human neuroblastoma (NB) cells in [CD1(nu/nu)] nude mice. Two human NB cell lines, namely LAN-5 and GI-LI-N, were previously adapted to grow in syngeneic animals for 7 consecutive passages. At the eighth passage, only animals which developed 10-mm diameter tumors within 40 days from xenograft were admitted to the study. RA and/or gamma-IFN were administered subcutaneously 3-5 days per week for 3 consecutive weeks. The number of days necessary for each tumor mass to grow up to 20 mm diameter (in vivo doubling time, ivDT) was then evaluated. Tumor growth was significantly inhibited in gamma-IFN (P less than 0.005) and RA (P less than 0.05) treated mice grafted with GI-LI-N. The combination of the two agents did not further enhance ivDT. The tumor growth inhibition was not statistically significant in LAN-5 bearing mice treated with RA or gamma-IFN alone, while a synergistic effect between the two drugs was observed (P less than 0.05). We conclude that parenteral combined administration of RA and gamma-IFN may prove to be useful in inhibiting the growth of tumors derived from human NB cells resistant to single inducers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Drug Administration Schedule; Drug Synergism; Female; Humans; Infant; Injections, Intralesional; Injections, Subcutaneous; Interferon-gamma; Mice; Mice, Inbred Strains; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Overexpression of the nuclear phosphoprotein p53 has been detected in many different transformed human cell lines and primary adult tumors. Elevated steady-state levels of p53 appear to be the result of an increase in the stability of the protein and, in adult cancers, high levels of the protein are associated with mutation of the p53 gene. In this study, overexpression of p53 was detected in 4 out of 5 human neuroblastoma-derived cell lines. The protein expressed by each of these four lines had a significantly prolonged half-life relative to the p53 protein in immortalized rodent fibroblasts and normal bovine adrenal medullary cells. However, no mutations were detected in the highly conserved regions of the p53 gene in these four neuroblastoma lines and the protein being expressed was not recognized by the mutant-specific anti-p53 monoclonal antibody, PAb 240. Upon retinoic acid-induced differentiation of the LA-N-5 neuroblastoma cell line, the level of p53 protein declined, as did the level of p53 mRNA, but the half-life of the protein remained unchanged. The high level of protein observed in the undifferentiated cell lines appears to result from expression of a stable wild-type p53 protein and increased transcription. In contrast, p53 protein was undetectable in two neuroepithelioma-derived cell lines; the p53 gene in one of these lines contained a nonsense mutation, while the other transcribed truncated p53 mRNA.

Topics: Base Sequence; Cell Differentiation; Genes, p53; Half-Life; Humans; Molecular Sequence Data; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.

Topics: Cell Differentiation; Electrophysiology; Humans; Membrane Potentials; Neuroblastoma; Sodium Channels; Tetrodotoxin; Tretinoin; Tumor Cells, Cultured

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Animals; Axons; Bucladesine; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Cyclic AMP; DNA Replication; Neuroblastoma; Tetradecanoylphorbol Acetate; Tretinoin

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.

Topics: Bucladesine; Cell Differentiation; Cell Division; Gene Expression Regulation; Heat-Shock Proteins; Humans; Neuroblastoma; Nuclear Proteins; Nucleolin; Phosphoproteins; Proto-Oncogene Proteins c-myc; RNA-Binding Proteins; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.

Topics: Calcium; Calcium Channels; Carbachol; Cell Line; Cell Membrane; Cell Membrane Permeability; Colforsin; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Kinetics; Neuroblastoma; Phorbol 12,13-Dibutyrate; Plant Extracts; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Terpenes; Thapsigargin; Tretinoin

We have recently demonstrated that acute and chronic treatments with estradiol and progesterone induce changes in the responsiveness of endogenous opioid systems to painful stimulation. In the present study the neuroblastoma SH-SY5Y subclone known to contain predominantly mu opioid receptors was used as a model to characterize the gonadal steroid effect on this opioid receptor system. The function of opioid receptors was assessed by measuring prostaglandin E1 (PGE1)-induced cyclic AMP accumulation after various treatments with estradiol and progesterone. Differentiated SH-SY5Y cells respond to PGE1 with a dramatic increase in cAMP level. Morphine (MOR) inhibits by about 75% the stimulatory effect of PGE1 on cAMP. Pretreatment with 5 nM of estradiol for 6 days resulted in a significant increase of PGE1-stimulated cAMP accumulation. Exposure of cells for 48 h to estradiol in doses of 5 nM or 50 nM did not affect cell sensitivity to the PGE1 effect on cAMP. Moreover, neither dose of estradiol changed the inhibitory effect of morphine on PGE1-induced cAMP response. There was a significant increase in PGE1-stimulated cAMP accumulation after treatment with 100 nM progesterone for 1 h or 15 min and a marked elevation of cAMP levels was also measured after 15 min treatment with 10 nM progesterone. Exposure to either dose of progesterone for 8 h, 48 h or 6 days did not affect basal or PGE1-induced cAMP in neuroblastoma cells. Progesterone-treated groups responded to MOR with 56-67% inhibition of PGE1-stimulated cAMP accumulation. The potency of MOR-induced inhibition was comparable to the MOR effect in cells not treated with the steroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alprostadil; Animals; Cyclic AMP; Estradiol; Glioma; Humans; Morphine; Neuroblastoma; Progesterone; Radioimmunoassay; Receptors, Opioid; Receptors, Opioid, mu; Tretinoin; Tumor Cells, Cultured

Differentiation-promoting effects of interferon-gamma (IFN-gamma), both alone and in combination with retinoic acid (RA), were studied on the human neuroblastoma cell line, LA-N-5. The results show that IFN-gamma inhibited the growth and induced morphological differentiation in a dose- and time-dependent manner with measurable effects appearing at 20-40 IU/ml after 3 to 4 days of treatment in vitro. Acetylcholinesterase activity, used as a biochemical index of neuroblastoma differentiation, increased up to 2.5-fold in the presence of IFN-gamma with a half maximal concentration of approximately 100 IU/ml. Concomitantly, modest IFN-induced increases (less than or equal to 2-fold) in choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) activities were seen. Combination treatment of cells with IFN-gamma and RA resulted in synergistic effects on morphological differentiation, growth inhibition and induction of ChAT. Reversal of IFN-gamma's ability to influence neuroblastoma cell growth as well as potentiate the anti-tumor effects of RA was obtained in the presence of an antibody against the IFN-gamma receptor, implying receptor-mediated physiological events. Taken together, these data confirm the differentiating effects of IFN-gamma on human neuroblastoma cells and suggest that combination therapy with RA may be beneficial in the treatment of this disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholinesterase; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Choline O-Acetyltransferase; Cyclic AMP; Drug Synergism; Humans; Interferon-gamma; Kinetics; Neuroblastoma; Receptors, Immunologic; Receptors, Interferon; Recombinant Proteins; Tretinoin; Tyrosine 3-Monooxygenase

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Differentiation; Cell Division; Cell Line; Cytoskeletal Proteins; Drug Resistance; Genes, myc; Humans; Kinetics; Membrane Glycoproteins; Models, Biological; Neuroblastoma; Proto-Oncogene Proteins c-myc; Tretinoin

Topics: Bromodeoxyuridine; Bucladesine; Cell Differentiation; Cell Line; Colforsin; Cytoskeletal Proteins; Humans; Neuroblastoma; Neurotransmitter Agents; Tretinoin

Human neuroblastoma cell lines are induced to differentiate and display neuronal phenotypes when treated with interferon (IFN)-alpha 2, retinoic acid (RA), or dibutyryl cyclic AMP (dbcAMP). We investigated the effects of combinations of these agents in induction-differentiation in the neuroblastoma cell line, NUB-6. The inductive effect of IFN-alpha 2 was markedly enhanced when used in combination with RA or dbcAMP. In parallel, RA or dbcAMP also enhanced the level of 2'-5'-oligoadenylate (2-5A) synthetase, and enzyme induced by IFNs and implicated in their biological action. The levels of another IFN-inducible enzyme, p68 kinase, were not enhanced by the combination treatments. The enhancement effects appeared to be exerted largely at the posttranscriptional level as both RA and dbcAMP stabilized IFN-induced 2-5A synthetase mRNA, resulting in increased enzyme activity. Thus, the 2-5A synthetase system is likely involved in mediating the IFN-alpha 2-induced differentiation of neuroblastoma cells and may also mediate the enhancement effects of RA and dbcAMP on IFN activity in these cells. These results also provide a rational basis for establishing a combination therapeutic approach for the treatment of neuroblastoma.

Topics: 2',5'-Oligoadenylate Synthetase; Base Sequence; Bucladesine; Cell Differentiation; Cell Division; eIF-2 Kinase; Enzyme Induction; Gene Expression; Humans; Interferon Type I; Molecular Sequence Data; Neuroblastoma; Protein Kinases; Protein Processing, Post-Translational; Recombinant Proteins; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Survival; Eflornithine; HeLa Cells; Humans; Immunohistochemistry; Neuroblastoma; Polyamines; Transglutaminases; Tretinoin; Tumor Cells, Cultured

The direct effects of the neurotransmitter serotonin on the catecholaminergic enzyme, tyrosine hydroxylase and the microtubule-associated tau protein were studied in a human neuroblastoma cell line. Undifferentiated LAN-5 cells, cultured in serum supplemented basal medium, or cells induced to differentiate by 6 day exposure to 10 uM retinoic acid were treated for 48 hr with 50 nM and 50 uM serotonin. In undifferentiated cells, serotonin treatment (50 uM) decreased both tyrosine hydroxylase activity and a 50 kD cytoplasmic fraction tau protein while 50 nM serotonin treatment caused this 50 kD protein to increase in the cytoplasmic fraction but decrease in the membrane fraction. While basal tyrosine hydroxylase activity increased in differentiated vs. undifferentiated cells, serotonin treatment had no effect on the enzyme or tau in differentiated LAN-5. This study shows serotonin to have direct effects on the biochemistry and cytoskeleton of undifferentiated cultured human neuroblastoma.

Topics: Cell Differentiation; Cell Division; Cell Line; Humans; Kinetics; Neuroblastoma; Serotonin; tau Proteins; Tretinoin; Tyrosine 3-Monooxygenase

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Line; Gene Expression; Genes, myc; Genes, Retinoblastoma; Genes, src; Humans; Kinetics; Neuroblastoma; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins pp60(c-src); Retinoblastoma Protein; Tretinoin

Topics: Blotting, Northern; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Cell Line; Cytokines; Flow Cytometry; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Intercellular Adhesion Molecule-1; Neuroblastoma; Poly A; Radioimmunoassay; Recombinant Proteins; RNA; RNA, Messenger; Transcription, Genetic; Tretinoin

Gangliosides of 11 different neuroblastoma cell lines, grown to confluence, were extracted and quantified with respect to: (a) total lipid-bound sialic acid, (b) total gangliotetraose family, and (c) GM1 content. The cultured cells were induced to grow neurites in 3 ways: (a) serum reduction, (b) exogenous ganglioside, and (c) retinoic acid. Neurite outgrowth was quantified in terms of % of cells bearing neurites and average number of neurites per cell. No correlation was observed between neurite outgrowth and total ganglioside concentration, but a reasonably good correlation was observed with respect to neuritogenesis and gangliotetraose content. When exogenous ganglioside was the stimulant the best correlation was with GM1, whereas retinoic acid-stimulated outgrowth was approximately proportional to GD1a content. The 'neurite minus' N1A-103 line, which had the lowest level of GM1, GD1a, and total gangliotetraose gangliosides, showed little if any response to any of the stimuli.

Topics: Animals; Brain; Cattle; Cell Differentiation; Cholera Toxin; Chromatography, Thin Layer; G(M1) Ganglioside; Horseradish Peroxidase; Humans; Mice; Neurites; Neuroblastoma; Rats; Staining and Labeling; Tretinoin; Tumor Cells, Cultured

1. The effects of gamma-interferon (gamma-IFN), retinoic acid (RA), and cytosine arabinoside (ARA-C) on the growth, morphology, and phenotype of the human neuroblastoma (NB) cell lines, LAN-1 and GI-ME-N, have been extensively tested. 2. RA, gamma-IFN, and ARA-C induced a dose-dependent morphological differentiation and growth inhibition, without affecting cell viability. Cells exposed to 10(-6) M RA or 1000 U/ml gamma-IFN significantly decreased their growth rate within the first 24 and 48 hr of culture, respectively. Cells became smaller and polygonal and sprouted long cellular processes with varicosities along their courses. In contrast, ARA-C-differentiated cells were larger and flattened, with few elongated dendritic processes. 3. Analysis of membrane and cytoskeletal markers by immunofluorescence and Western blot showed several changes in NB-specific antigen expression after 5 days of treatment with all inducing agents. Analysis of labeled phosphatidylinositol metabolites from prelabeled cells showed, within 1 min of treatment with RA, a rapid decrease in inositol 1,4,5-trisphosphate and of 1,2-diacylglycerol levels. No changes in inositol phospholipid metabolism were observed in gamma-IFN- or ARA-C-treated cells. 4. We conclude that RA-induced decrease in phosphatidylinositol (PI) hydrolysis is not likely to be a consequence of the acquisition of a different phenotype, as its changes precede the acquisition of neuronal markers. In addition, gamma-IFN and ARA-C, both inducing a mature phenotype, did not affect PI hydrolysis. 5. Decreased PI hydrolysis seems to be sufficient, although not necessary, to commit NB cells to neuronal differentiation. Analysis of molecular mechanisms associated with NB cell differentiation may be helpful to clarify the potential of various biological agents in affecting the development of the neural cell.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cytarabine; Cytoskeletal Proteins; DNA Polymerase I; Dose-Response Relationship, Drug; Humans; Inositol Phosphates; Interferon-gamma; Neoplasm Proteins; Neuroblastoma; Stimulation, Chemical; Tretinoin; Tumor Cells, Cultured

Topics: Cell Differentiation; Cell Division; Cell Line; DNA Replication; Drug Synergism; Glucagon; Growth Hormone-Releasing Hormone; Humans; Kinetics; Neuroblastoma; Peptide Fragments; Secretin; Sermorelin; Thymidine; Tretinoin; Vasoactive Intestinal Peptide

Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.

Topics: Cell Differentiation; Humans; Integrins; Laminin; Neurites; Neuroblastoma; Neurons; Receptors, Immunologic; Receptors, Laminin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

A small acidic polypeptide, termed thymosin beta 10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin beta 10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin beta 10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin beta 10, as well as its homologue gene product, thymosin beta 4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin beta 10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time- and dose-dependent increase in the content of thymosin beta 10, but not that of thymosin beta 4. The increase in thymosin beta 10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin beta 4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on beta-thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin beta 10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin beta 10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.

Topics: Animals; Bucladesine; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Kinetics; Neuroblastoma; Nucleic Acid Hybridization; Rats; RNA, Messenger; Thymosin; Tretinoin; Tumor Cells, Cultured

We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Bromodeoxyuridine; Cell Differentiation; Cyclic AMP; Flow Cytometry; Humans; Immunohistochemistry; Melanoma; Mice; Neuroblastoma; Precipitin Tests; Tretinoin

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.

Topics: 4-Aminopyridine; Animals; Cell Line; Dimethyl Sulfoxide; Gene Expression; Membranes; Mice; Nervous System Neoplasms; Neuroblastoma; Potassium Channels; Solutions; Tetraethylammonium Compounds; Tretinoin

The conversion of choline in cultures of the human neuroblastoma cell lines, LA-N-1 and LA-N-2 cells, was investigated in order to identify potential precursors in acetylcholine (AcCho) synthesis. LA-N-1, a catecholaminergic and LA-N-2, a cholinergic, cell line were incubated with [3H-methyl]choline (Cho) for varying periods of time up to 72 h. The radioactivity present in lipids and water-soluble metabolites increased linearly up to 24 h in both cell lines. Approximately 20% of the radioactivity associated with the water-soluble metabolites in both control (untreated) and retinoic acid-induced differentiated (RA-treated cells) LA-N-2 cells was present as Cho and AcCho. There was no detectable AcCho in the catecholaminergic cell line, LA-N-1. The untreated and RA-treated LA-N-1 and LA-N-2 cells were labeled for 24 h with [3H-methyl]Cho, followed by a chase in growth medium containing 100 microM unlabeled choline. The distribution of radioactivity in the LA-N-2 cells was 6-10% of AcCho, 84-89% as phosphocholine (PCho), 1-3% as glycerophosphocholine (GroPCho), and 2-4% as Cho. The distribution of radioactivity in the LA-N-1 cells was similar except for the absence of AcCho. The distribution of radioactivity in the culture medium of LA-N-1 cells was 70-80% as Cho, 20-30% as PCho, and 1-3% as GroPCho. In contrast, the radioactivity was equally distributed between Cho (50%) and PCho (50%), with only 1-3% as GroPCho in the medium of LA-N-2 cells.

Topics: Acetylcholine; Cell Differentiation; Choline; Chromatography, Thin Layer; Humans; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The maturation of embryonal neural crest cells is thought to be regulated in part by the milieu into which these cells migrate. Neuroblastoma (NB) is a tumor of very early childhood that is thought to arise in association with the arrested differentiation of embryonal neural crest cells. In culture, neuroblastoma tumor cells differentiate in the presence of retinoic acid, which is also known to influence extracellular matrix protein synthesis. We have cultured neuroblastoma cells on laminin (LN) and fibronectin (FN) substrata to examine the role of extracellular matrix in retinoic acid (RA)-induced differentiation of these tumor cells. These proteins caused morphologic changes in NB cells indistinguishable from those caused by RA. Antiserum to each of these proteins blocked the effects induced by the corresponding protein, but neither antiserum affected the action of RA. Despite the induction of a neuronal morphologic change, matrix proteins did not alter the proliferation of NB cells. These results indicate that LN and FN modulate the differentiation of NB cells without inducing growth arrest and that RA-induced differentiation does not require these matrix proteins.

Topics: Blotting, Northern; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Extracellular Matrix Proteins; Fibronectins; Humans; Laminin; Neural Crest; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Neuroblastomas are malignant childhood neoplasms that arise from derivatives of the neural crest. We report the characterization of a new neuroblastoma cell line, designated NBL-W, derived from the primary tumor of a patient with stage IVS disease (S. L. Cohn, C. V. Herst, H. S. Maurer, and S. T. Rosen, J. Clin. Oncol., 5: 1441-1444, 1987) according to the criteria of Evans [A. E. Evans, G. J. D'Angio, and J. Randolf, Cancer (Phila.), 27: 374-378, 1971]. Neurite-bearing (N) and substrate-adherent (S) cell lines have been subcloned from the parent line. N and S cells can interconvert, and both cell types label with the neural crest cell surface marker antibody, HNK-1. Cells in the subcloned lines and in the parent line have been shown by Southern blot analysis to contain approximately 100 copies of the N-myc gene. Cytogenetic analysis shows a homogeneously staining region present on chromosome 19. Although these subclones are of identical genotype, the S cells express lower amounts of N-myc mRNA and protein as compared to the N cells. N cells express several neuronal proteins including the neurotransmitter-processing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, the neuronal intermediate filament proteins peripherin and NF66/alpha-internexin, and the neural cell adhesion molecule. S cells generally lack neuronal markers but express the mesenchymal intermediate filament protein vimentin, and a small subset of the S cells express glial fibrillary acidic protein. Some S cells were labeled weakly with neural cell adhesion molecule antibody; others were negative. S cells did not express the glial marker S-100 or a melanocyte marker, tyrosinase. Thus, S cells express the neural crest marker HNK-1 but do not express a set of antigens characteristic of any known cell type derived from the neural crest. These results are consistent with the suggestion that differential N-myc expression may be involved in the interconversion of N and S cells but indicate that the S cell phenotype need not represent a highly differentiated neural crest derivative.

Topics: Cell Differentiation; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Intermediate Filaments; Karyotyping; Nerve Growth Factors; Neural Crest; Neuroblastoma; Phenotype; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured; Vimentin

Calcyclin gene expression was evaluated in different neuroblastoma cell lines and during neuronal differentiation induced by retinoic acid. Calcyclin gene expression was more frequently detected in epithelial-type or Schwann-like cells rather than in neuroblastic cells. This result indicates an increase of G1 cell fraction, which may explain the limited growth potential usually observed for these cells. LAN-5 cell (neuronal type) differentiation experiments showed that calcyclin gene is detectable after 4 days of retinoic acid treatment, which induces G1 phase accumulation (as detected by cytofluorometric analysis), and cell growth arrest. Otherwise, neither block of cell proliferation by 0.5% fetal calf serum medium nor addition of 15% fresh fetal calf serum after cell arrest induce calcyclin expression. The increase of calcyclin mRNA levels during cell differentiation shows that calcyclin gene expression is associated with neuronal differentiation. This bivalent role of the calcyclin gene, which is normally expressed in the G1 phase of the cell cycle but also expressed during retinoic acid-induced neuroblastoma cell differentiation, suggests that (at least in neuroblastoma cells) the gene is subject to a complex transcriptional regulation.

Topics: Calcium-Binding Proteins; Cell Cycle Proteins; Cell Differentiation; Culture Media; Gene Expression Regulation; Humans; Neuroblastoma; S100 Calcium Binding Protein A6; S100 Proteins; Schwann Cells; Sequence Homology, Nucleic Acid; Tretinoin; Tumor Cells, Cultured

It is known that PKC is differently expressed in brain and the peripheral nervous system and is involved in cellular differentiation. We have analyzed 9 human neuronal-derived crest-cell lines for PKC-alpha mRNA. Seven out of nine expressed 9.0 kb and 4.0 kb PKC-alpha mRNAs, but three had high level of 9.0 kb transcription. The different expression of the two messenger RNAs may result from alternative splicing and a different degree of cell maturation. The same cell lines were studied for MYCN gene expression. A possible relation between the two genes is discussed. One cell line expressing high levels of both PKC-alpha mRNA was treated with 10(-5) M retinoic acid (RA). The expression of both messenger RNAs was suppressed when the cells achieved a morphological differentiation and showed neurite-like processes. A decrease of PKC-alpha gene expression was associated to down regulation of MYCN mRNA. These preliminary results suggest that PKC suppression of PKC-alpha mRNA is associated with reversion of the malignant phenotype.

Topics: Cell Transformation, Neoplastic; Gene Expression Regulation, Enzymologic; Humans; Neuroblastoma; Oncogenes; Protein Kinase C; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

A cloned thymosin beta-10 cDNA was used to study modulation of thymosin beta-10 mRNA levels in the rat B104 neuroblastoma cell line in response to retinoic acid. Northern blot analysis revealed the presence of a single greater than 600-nucleotide thymosin beta-10 mRNA species that was constitutively expressed in proliferating neuroblastoma cells. Addition of retinoic acid to the culture medium induced a dose- and time-dependent increase in thymosin beta-10 mRNA abundance. Additional studies showed that although thymosin beta-4 and beta-10 are coexpressed in this cell line, the stimulatory action of retinoic acid is specific for the thymosin beta-10 gene. Serum was found to augment the stimulatory action of retinoic acid. Blockade of protein synthesis with cycloheximide abrogated the stimulatory action of retinoic acid upon thymosin beta-10 mRNA accumulation; this observation suggests that activation of the thymosin beta-10 gene in this cell line by retinoic acid is dependent upon the de novo synthesis of a labile protein. Collectively, these findings demonstrate that the developmentally regulated thymosin beta-10 gene is a target for morphogenic retinoids in cells derived from the neural crest.

Topics: Animals; Blood; Blotting, Northern; Cattle; Chromatography, High Pressure Liquid; Culture Media; Cycloheximide; DNA Probes; Gene Expression Regulation, Neoplastic; Kinetics; Neuroblastoma; RNA, Messenger; RNA, Neoplasm; Thymosin; Tretinoin

Topics: Blotting, Northern; Cell Division; Cell Line; DNA Replication; Drug Resistance; Humans; Insulin-Like Growth Factor II; Kinetics; Neuroblastoma; RNA, Neoplasm; Tretinoin

Topics: Cell Differentiation; Cell Line; Child; Dactinomycin; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Kinetics; Neuroblastoma; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Proto-Oncogenes; Tretinoin

Several human neuroblastoma cell lines express transcripts of the proopiomelanocortin (POMC) gene of 0.8 kb and 9.5 kb, thus different from the characteristic pituitary 1.2 kb POMC mRNA. The expression of POMC mRNA decreases when the cell lines are differentiated with retinoic acid or alpha-difluoromethylornithine. Since both POMC and MYCN genes are present on the same chromosome segment (2p23), it is possible that the 2 genes are coamplified and coexpressed. Alternatively the expression of opiate peptides by neuroblastoma cells might act as an autocrine factor or it may modulate the action of tumour infiltrating lymphocytes.

Topics: Cell Differentiation; Cell Line; Chromosomes, Human, Pair 2; Eflornithine; Gene Expression; Humans; Neuroblastoma; Nucleic Acid Hybridization; Pro-Opiomelanocortin; RNA, Messenger; Tretinoin

Topics: Adolescent; Bone Marrow; Cell Division; Cell Line; Child; Child, Preschool; Genes, myc; Humans; Immunoenzyme Techniques; Neoplasm Metastasis; Neuroblastoma; Proto-Oncogene Proteins c-myc; Tretinoin

Human neuroblastoma cells SK-N-BE(2) can be induced to differentiate towards a neuronal phenotype by retinoic acid (RA) or a schwannian/glial phenotype by alpha-difluoromethylornithine (DFMO), producing differential binding of 14 antibodies (MAbs). RA induced the expression of the neural cell adhesion molecule, NCAM (also confirmed by northern blot); whereas DFMO enhanced the binding of MAbs UJ181.4, UJ127.11 which recognise an identical protein doublet of 220-240 kDa, thought to be the L1 protein(s). The data presented demonstrate that neuroblastoma cells differentiate toward separate phenotypes associated with a specific induction of two different adhesion molecules, NCAM on neuronal cells and L1 on schwannian/glial cells.

Topics: Antibodies, Monoclonal; Blotting, Northern; Blotting, Western; Cell Adhesion Molecules, Neuronal; Cell Differentiation; Cell Line; Eflornithine; Fluorescent Antibody Technique; Gene Expression; Humans; Models, Neurological; Molecular Weight; Neuroblastoma; Neurons; Phenotype; Tretinoin

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR-32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage-independent growth. By Southern blot analysis, BIM cells had both human and rat types of N-myc genes. The human N-myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR-32. The rat N-myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N-myc genes may be involved in the properties of partial reverse-transformation in BIM cells. When treated with various drugs such as db-cAMP, forskolin, and cAMP with isobutyl-methylxanthine, BIM cells expressed a nerve-like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve-like cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Axons; Bucladesine; Calcimycin; Cell Cycle; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Fibroblasts; Humans; Hybrid Cells; Methylcellulose; Neuroblastoma; Neurons; Phenotype; Rats; Thymidine; Tretinoin; Tumor Cells, Cultured

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (-30 to -20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from -90 to +10 mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10-300 microM) and gadolinium (10-30 microM) as well as by high concentrations of nickel and cobalt. omega Conus toxin (1 microM) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3-10 microM) and antagonists, nifedipine (3-10 microM) and nimodipine (10 microM) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.

Topics: Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Cyclic AMP; GTP-Binding Proteins; Humans; Kinetics; Mollusk Venoms; Neuroblastoma; omega-Conotoxin GVIA; Phosphorylation; Tretinoin; Tumor Cells, Cultured

The cytolytic activity of lymphokine-activated killer (LAK) cells against human neuroblastoma (NB) cells was investigated using the continuous NB cell lines, IMR-32, Kelly, and two subclones of SK-N-SH, SH-SY5Y (neuroblastic phenotype), and SH-EP (non-neuronal phenotype). NB cells were found to be sensitive targets of LAK. Of the SK-N-SH subclones, the neuroblasts, SH-SY5Y, were more susceptible to LAK killing than were the non-neuronal cells, SH-EP. Pretreatment of the targets SH-SY5Y and SH-EP with the differentiating agents, retinoic acid (RA, 10 microM), herbimycin A (236 nM), or nerve growth factor (10 ng/ml), did not substantially alter LAK killing. Furthermore, these differentiating agents did not measurably affect LAK activity during the cytolysis assay or with 1-h preincubation of the LAK effectors. However, co-incubation of the LAK cultures over the 3-day activation period with RA (1 microM) or PGE2 (1 microM) inhibited cytolysis by 80%, suggesting that these agents interfere with an early activation step of LAK. These results support the potential use of LAK treatment for neuroblastoma, in combination with differentiation agents that do not affect neuroblastoma sensitivities toward LAK cells. However, some differentiation agents, (e.g., RA) and endogenous prostaglandins (e.g., PGE2) may interfere with LAK activation.

Topics: Cell Differentiation; Cytotoxicity, Immunologic; Dinoprostone; Humans; Killer Cells, Lymphokine-Activated; Neuroblastoma; Neurotransmitter Agents; Phenotype; Tretinoin; Tumor Cells, Cultured

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.

Topics: Amino Acid Sequence; Base Sequence; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Molecular Sequence Data; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Studies on the involvement of protein kinase C in retinoic acid-induced differentiation of human neuroblastoma were carried out with two variants of the SK-N-SH cell line namely the SH-F subline, which differentiates to give a fibroblast-like phenotype, and the SH-N subline, which develops into the typical neuronal phenotype. In SH-F, a substantial increase in protein kinase C activity accompanied morphological differentiation. Accordingly, after 7 days of retinoic acid treatment, EDTA-extracted, cytosolic protein kinase C activity increased by slightly more than 2-fold over vehicle-treated controls. Again, detergent-extracted activity, representing membrane-bound or total protein kinase C, showed a similar 2.6- to 5.1-fold increase in treated cells. A time-course study revealed an earliest increase in total activity after two days of retinoic acid treatment which continued linearly for the first 6 to 8 days, and then levelled off. A study of the effect of retinoic acid on the protein kinase C in vitro with SH-F cell extracts showed only a slight increase in activity (of 25%) at the relatively high concentration of 10(-4) M; however, no significant differences were observed at lower concentrations. In contrast, the SH-N cell line responded to retinoic acid by a 45% decrease in EDTA-extractable, and a 63% decrease in detergent-extractable protein kinase C activity. Added to SH-F cell cultures, 15 nM staurosporine was found to inhibit protein kinase C in vivo and to a lesser extent, the protein kinase A. Present together with retinoic acid, staurosporine not only prevented the augmentation but caused a marked decrease of protein kinase C activity in this cell line. Morphological studies indicated that when SH-N cells are treated with staurosporine, or staurosporine and retinoic acid together, a neuronal phenotype similar to that produced by retinoic acid alone is observed. In contrast, when the SH-F cell line is treated with staurosporine or staurosporine and retinoic acid together, the flattened fibroblast-like cell type normally induced by retinoic acids is not observed. Instead, these cells display much smaller cell bodies and elaborate extensions resembling the neuronal phenotype produced by retinoic acid induced differentiation of the SH-N variant. These results suggest that changes in the protein kinase C activity may be involved in regulating the expression of the phenotype during cell differentiation.

Topics: Alkaloids; Cell Differentiation; Cytosol; Edetic Acid; Fibroblasts; Humans; Neuroblastoma; Neurons; Protein Kinase C; Staurosporine; Tretinoin; Tumor Cells, Cultured

1. Neuroblastoma (NB) is an unusual neuroectodermal tumor showing a high degree of spontaneous regression. NB cells can be induced to differentiate in vitro by various agents. Cell differentiation results in morphological changes characteristic of the mature neuronal phenotype, including outgrowth of neurite-like structures with several interconnections. 2. Recent experiments indicate that morphological differentiation of NB cells is associated with changes in expression of N-myc, c-myc, and c-myb oncogenes and synthesis of neurofilament proteins. However, little is known about the transcription of neurofilament genes during differentiation. 3. We have analyzed the expression of both the N-myc oncogene and mid-size neurofilament (NF) genes in the LAN-1 human NB cell line, cultured in the presence of retinoic acid (RA). Continuous treatment with RA induced morphological differentiation within 5-6 days. The transcription of N-myc was down-modulated within 24 hr of the initial exposure to RA. The mid-size NF mRNA was increased at this time. The expression of N-myc was not modified in serum-deprived LAN-1 cells, indicating that N-myc transcription is unaffected by the arrest of the cells in the G1 phase. 4. We conclude that new synthesis of mid-size NF mRNA and a decrease in N-myc transcription precede de novo formation of neurite-like processes and morphological cell differentiation of neuroblastoma cells.

Topics: Cell Differentiation; Gene Expression Regulation, Neoplastic; Humans; Intermediate Filament Proteins; Neoplasm Proteins; Neuroblastoma; Neurofilament Proteins; Proto-Oncogene Proteins c-myc; RNA, Messenger; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

We have tested the ability of various compounds to raise intracellular cyclic AMP (cAMP) levels and, either alone or in combination with retinoic acid (RA), to promote differentiation of two "RA-resistant" sublines of LA-N-5 human neuroblastoma cells, designated LA-N-5HP and LA-N-5R9. Direct activation of adenylate cyclase by forskolin and cholera toxin increased intracellular cAMP levels over 10-fold in both cell lines after 1 h of treatment, after which the levels slowly declined for the next 16 to 24 h. After 5 days of continuous treatment, cAMP levels still remained 2- to 7-fold elevated above controls and were accompanied by a decrease in cell proliferation and an increase in neurite outgrowth. All these effects were exaggerated when the agents were combined with phosphodiesterase enzyme inhibitors. Increasing cAMP levels (up to 24-fold) with N6,O2'-dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cAMP also resulted in decreased proliferation and an increase in morphological differentiation. Isoproterenol and epinephrine did not alter cAMP levels and had no discernible biological effects. Of the agents that raised cAMP levels, only dbcAMP caused an increase in acetylcholinesterase activity. This effect was duplicated with sodium butyrate and prostaglandin E1 in the absence of an increase in cAMP. RA promoted differentiation but also had little effect on cAMP levels. Combination treatment of cells with RA plus agents that raised cAMP levels resulted in greater degrees of differentiation than seen with single agent treatments. We conclude that: (a) the cAMP synthetic and degradative pathways are functional in LA-N-5HP and LA-N-5R9 cells; (b) elevation of cAMP is sufficient for inhibiting proliferation and promoting neurite outgrowth from these cells, but is not a necessary condition for inducing differentiation; and (c) elevation of intracellular cAMP potentiates the differentiation-inducing activity of RA.

Topics: Acetylcholinesterase; Alprostadil; Cell Differentiation; Cell Division; Cholera Toxin; Colforsin; Cyclic AMP; Humans; In Vitro Techniques; Neuroblastoma; Norepinephrine; Theophylline; Tretinoin; Tumor Cells, Cultured

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line that can be induced to differentiate along the neuronal pathway by retinoic acid (RA). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H]inositol or [1(3)-3H]glycerol. No changes were observed in both [3H]inositol and [3H]glycerol uptake within 24 h of RA treatment. Decreased incorporation of the metabolic precursor into PI 4-monophosphate and PI 4,5-bisphosphate occurred within 1 h of RA treatment. No changes were seen in the specific radioactivity of the precursor pools up to 1 h of treatment with RA. Analysis of labeled PI metabolites from prelabeled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol content within 1 min of induction of LAN-1 cell differentiation. These findings constitute the earliest reported events in neuroblastoma cell differentiation.

Topics: Cell Differentiation; Deoxyglucose; Glycerol; Humans; Inositol; Inositol Phosphates; Neuroblastoma; Phosphatidylinositols; Tretinoin; Tumor Cells, Cultured

Human fetal brain expresses high levels of a polypeptide identified by protein biochemistry and molecular cloning as thymosin beta 10. Within the first 18 months after birth, the thymosin beta 10 content of human brain falls to undetectable levels. In order to establish the molecular basis of this process we screened a number of human tumor cell lines derived from the nervous system for the presence of thymosin beta 10. All of the cell line expressed authentic thymosin beta 10. However, in the HTB-10 neuroblastoma, retinoic acid caused a reduction in the level of thymosin beta 10. This effect of the retinoid was conditional upon its continual presence in the tissue culture medium and was not evident in the other cell lines examined. These results suggest that the thymosin beta 10 gene may be a target for retinoids in the developing nervous system.

Topics: Amino Acid Sequence; Base Sequence; Brain; Brain Chemistry; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes; Humans; Infant; Infant, Newborn; Molecular Sequence Data; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Thymosin; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induced differentiation of SH-SY5Y neuroblastoma cells is associated with more than a tenfold induction of total Alzheimer's disease beta A4 amyloid protein precursor (APP) mRNA as analyzed by Northern blot hybridisation. S1 nuclease protection experiments reveal that the splicing pattern of these differentiated cells is altered in favor of APP695 mRNA, coding for the shortest amyloidogenic beta A4 amyloid precursor protein. Induction of differentiation of SH-SY5Y cells with NGF leads to a fivefold increase of total APP mRNA without change in the splicing pattern. This suggests that RA but not NGF induces factor(s) which are responsible for an APP hnRNA splicing favoring APP695 mRNA.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Blotting, Northern; Cell Differentiation; Cell Line; Deoxyribonuclease EcoRI; Genes; Humans; Nerve Tissue Proteins; Neuroblastoma; Nucleic Acid Hybridization; Restriction Mapping; RNA Splicing; RNA, Neoplasm; Tretinoin

We have investigated the effects of retinoic acid (RA) on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5. When cells were treated with 4 x 10(-6) M RA in vitro there was a marked reduction in the number of mice developing tumors when compared to solvent-treated controls. In vivo treatment with RA reduced tumor formation when the retinoid was given for 5 days before tumor injection and continued for 14 days thereafter. In established tumors, RA inhibited progressive tumor growth. There was no demonstrable effect of RA in vivo on the morphologic phenotype of the tumor cells when these regimens were used. We conclude that oral retinoid administration may prove useful in inhibiting or arresting the growth of neuroblastoma, particularly when there is a small initial tumor burden.

Topics: Animals; Cell Division; Drug Screening Assays, Antitumor; Female; Humans; Infant; Male; Mice; Mice, Nude; Neuroblastoma; Tretinoin

Activin/EDG, a stimulator of the secretion of follicle stimulating hormone (FSH) from pituitary gland and an inducer of erythroid differentiation for Friend leukemia cells, has since been implicated in a variety of biological roles. Here, we show some novel effects of activin on murine embryonal carcinoma cells (EC cells). First, activin acts as a growth factor on undifferentiated P19 cells, a well characterized EC cell line for the study of mammalian development. Second, activin inhibits the retinoic acid (RA) induced differentiation of P19 cells to neurons and glial cells. The inhibitory effect of activin on neural differentiation, which has yet to be proved in other physiological peptides, is confirmed also on the differentiation of various neuroblastoma cell lines. Our results suggest a possible role of activin as a negative regulator of neural differentiation in mammalian development.

Topics: Activins; Animals; Cell Differentiation; Cell Division; Cell Line; Inhibins; Kinetics; Neuroblastoma; Neurons; Teratoma; Tretinoin

A potential role of the protein kinase C (PKC) system in differentiation of human neuroblastoma cell line LA-N-5 was investigated. It was found that neurite outgrowth induced by 12-O-tetradecanoylphorbol 13-acetate (TPA, 81 nM) was associated with a down-regulation of PKC as determined independently by immunocytochemistry, immunoblot, and enzyme activity assay. Down-regulation of PKC in cells induced to differentiate by retinoic acid (1 microM) was less pronounced, whereas it was undetected in cells induced to differentiate by nerve growth factor (100 ng/ml). The in vitro phosphorylation of an 80-kilodalton protein present in control LA-N-5 cells or in cells treated with TPA, retinoic acid, or nerve growth factor for 1 day decreased to various extents at days 4 or 7 concomitant with neuritogenesis. Pretreatment of LA-N-5 cells with a high concentration (1 microM) of TPA to deplete cellular PKC rendered the cells unresponsive to the differentiating effect of the agents. It was observed that CHP-100 cells, another human neuroblastoma line shown to be resistant to differentiation induced by the agents, had a reduced PKC level and the amount of in vitro phosphorylation of the 80-kilodalton protein was greatly reduced in control cells and remained relatively unchanged when the cells were treated with the agents for up to 7 days. The present studies suggested that PKC and its 80-kilodalton substrate protein were likely involved in initiation and/or progression of LA-N-5 cell differentiation induced by TPA and that separate PKC-independent pathways might also be involved in the differentiating effect of retinoic acid or nerve growth factor.

Topics: Axons; Calcium; Cell Line; Humans; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Phosphorylation; Protein Kinase C; Substrate Specificity; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The effects of cell density and retinoic acid-induced differentiation on the class and molecular species composition of mouse neuroblastoma NB2a cell glycosphingolipids were examined under conditions where the period of culture was controlled. The total amount of neutral glycosphingolipids per cell decreased both with differentiation and as the cells became confluent. The relative amount of the neutral glycosphingolipid classes was not affected by differentiation, whereas there were small but significant changes in the relative amount of the neutral glycosphingolipid classes as the cells became confluent. The total amount of the gangliosides was unaffected by either differentiation or cell density, but there were significant changes in the ganglioside class composition as a result of both cell density and differentiation, and the effects were additive. The molecular species of all the major neutral glycosphingolipid and ganglioside classes were essentially identical, and were altered only slightly by either differentiation or cell density.

Topics: Animals; Cell Count; Cell Differentiation; Chemical Phenomena; Chemistry; Glycosphingolipids; Mice; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The presence of 10(-5) M retinoic acid (RA) in the culture medium of LA-N-1, a catecholaminergic cell line, and LA-N-2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydroxylase (TH) activity of the LA-N-1 cells was increased in the RA-treated cells compared with control cultures at day 4 and remained elevated. Choline acetyltransferase (ChAT) activity in the LA-N-2 cells gradually increased until 8 days in vitro (DIV) both in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradually to a constant level of activity. The ChAT activity at 8 DIV of RA-treated LA-N-2 cells was increased 2.1-fold (P less than 0.001) as compared to the control cultures. This increase in ChAT activity was accompanied by a 73% decrease of acetylcholinesterase (AChE) activity in LA-N-2 cells by 8 DIV. AChE activity of LA-N-1 cells was unchanged during the time course of the experiment. Phospholipase-A2 (PL-A2) activity in RA-treated LA-N-2 cells was increased at day 4 as compared with the control cultures. There were no differences observed in phospholipase-D (PL-D), choline kinase and GPC-phosphodiesterases activities in RA-treated and -untreated LA-N-1 and LA-N-2 cells.

Topics: Acetylcholinesterase; Adrenergic Fibers; Cell Differentiation; Cell Line; Cells, Cultured; Choline O-Acetyltransferase; Cholinergic Fibers; Humans; Neuroblastoma; Phospholipases; Tretinoin

The aim of this study was to analyze by flow cytometry the effect of cis-diamminedichloroplatinum II (CDDP) and retinoic acid (RA) on the cell cycle of a neuroblastoma cell line (SK-N-BE (2)C NB) and to correlate the kinetic data with cell morphology. CDDP at 1 microgram/ml induced a dramatic G2 + M cell cycle phases block (nearly 200% increase with respect to control) 2 days after treatment. The G2 + M block was spontaneously reversed starting from the 4th day. The cells treated with 10 microM RA were, instead, induced to irreversibly enter the G0 + G1 phase of the cell cycle (nearly 20% increase with respect to control) 48 h after treatment. Neurite-like structures were observed for both CDDP and RA treated cells. These data suggest different cell cycle dependent molecular mechanisms and different degrees of differentiation during CDDP or RA treatment of NB cells.

Topics: Cell Cycle; Cell Differentiation; Cisplatin; DNA, Neoplasm; Flow Cytometry; Humans; In Vitro Techniques; Neuroblastoma; Neurons; Tretinoin; Tumor Cells, Cultured

Four human neuroblastoma cell lines exhibited differences in their ability to differentiate into neuron-like cells in response to three different treatments, serum deprivation, or additions of dibutyryl cyclic-AMP or retinoic acid. Expression of N-myc gene product was reduced in neuroblastoma cell line SK-N-DZ differentiated by retinoic acid as compared with untreated cells. On the contrary, expression of c-src gene product, pp60c-src, was considerably enhanced in differentiated SK-N-DZ cells. Tyrosine phosphorylation of several cellular proteins was found to be enhanced in differentiated cells. Alteration in expression of these proto-oncogene products might be important in the differentiation of neuroblastoma cells into neuron-like cells.

Topics: Bucladesine; Cell Differentiation; Cell Line; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neuroblastoma; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins pp60(c-src); Tretinoin; Tumor Cells, Cultured

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN-1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and gamma-interferon (gamma-IFN). Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol within 1 min of induction of LAN-1 cell differentiation by RA, while no changes were observed in gamma-IFN-treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA-treated LAN-1 cells and suggest that phosphoinositide-derived metabolites may not constitute general regulators of cellular differentiation.

Topics: Cell Differentiation; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Humans; Interferon-gamma; Neuroblastoma; Phosphatidylinositols; Tretinoin; Tumor Cells, Cultured

The fibrinolytic enzyme profile of SMS-KAN human neuroblastoma cells was found to vary dramatically during the differentiation process. Five maturational agents--retinoic acid, dibutyryl cAMP, 5-bromodeoxyuridine, sodium butyrate and phorbol myristate acetate were tested for their effects on cellular morphology, DNA synthesis, plasminogen activator (PA) and PA inhibitor (PAI) activity. SMS-KAN cells secrete urokinase (UK) and tissue PA (tPA) as well as a possibly unique PAI. Treatment of cells with 1 microM RA resulted in an inhibition of proliferation, extension of neurite-like processes indicative of differentiation, as well as a switch from secretion of UK to tPA and a reduction in PAI secretion. Other agents which caused neural process formation and decreased cell proliferation also induced alterations in PA/PAI while agents which had no detectable effect on cell growth induced little change in the fibrinolytic enzyme profile.

Topics: Bromodeoxyuridine; Bucladesine; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; DNA Replication; Fibrinolysis; Glycoproteins; Humans; Neuroblastoma; Plasminogen Activators; Plasminogen Inactivators; Tetradecanoylphorbol Acetate; Tretinoin

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.

Topics: Avidin; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Calmodulin-Binding Proteins; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Neuroblastoma; Phosphoprotein Phosphatases; Phosphoric Diester Hydrolases; Phosphorylation; Protein Kinases; Synaptosomes; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.

Topics: Cell Differentiation; Cyclic AMP; Humans; Neuroblastoma; Radioimmunoassay; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We established the subline RT-BMV-C6 from the parent human neuroblastoma cell line RT-BM by a process that required repeated subculture of cells, which were prone to disaggregation. RT-BMV-C6 and the parent cloned line, RT-BM-1, had an identical marker chromosome, confirming that both lines were derived from a common progenitor. In the analysis of surface antigen expression, RT-BMV-C6 did not react with UJ-127-11, Leu7 or KP-NAC2 MAbs to which RT-BM-1 showed positive binding. The levels of both N-myc amplification and expression in RT-BMV-C6 were twice as high as the level obtained in RT-BM-1. Colony-forming efficiency in soft agar was 2.0 +/- 0.8% for RT-BMV-C6 and 3 times greater than that for RT-BM-1 (0.6 +/- 0.1%). When 100 x 10(6) cells of RT-BM-1 and RT-BMV-C6 were inoculated into nude mice, tumor incidence was significantly higher for RT-BMV-C6 (6/6; 100%) than for RT-BM-1 (0/6; 0%). Our data show that N-myc is closely related to tumorigenicity in NB. When RT-BM-1 and RT-BMV-C6 were co-cultured with a new synthetic retinoid, polyprenoic acid (E5166), and dibutyryl cyclic AMP, RT-BM-1 was induced to neuronal differentiation, defined by the formation of neuronal processes and expression of neurofilaments, whereas RT-BMV-C6 was not. However, when exposed to E5166, N-myc expression of RT-BMV-C6 was more strongly reduced than that of RT-BM-1, and colony formation of RT-BMV-C6 was significantly inhibited as compared to RT-BM-1. These findings suggest that the reduction of N-myc expression might closely correlate with growth inhibition accompanying neuronal differentiation of neuroblastoma cells.

Topics: Animals; Antigens, Surface; Bucladesine; Cell Differentiation; Gene Amplification; Humans; Mice; Mice, Inbred BALB C; Neuroblastoma; Oncogenes; Tretinoin; Tumor Cells, Cultured

Human SMS-KCNR cells differentiated in response to either retinoic acid or phorbol esters; differentiated cells extended numerous, complex neurites and showed reduced proliferation. Tyrosine hydroxylase (TH) immunoreactivity was measured in this cell line following treatment with retinoic acid (1-10 microM), 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 16-160 nM), or combinations of these agents. After 21 days of treatment with either TPA or retinoic acid (RA), TH immunoreactivity was measured in this using densitometric scans of Western blots, was doubled relative to untreated or serum-deprived SMS-KCNR cultures. Increases in TH immunoreactivity could be detected after 6 days of treatment. Treatment with RA for 3 days followed by phorbol esters for an additional 3 days resulted in a 3-fold increase in TH immunoreactivity at day 6; reversing the order of drug treatment did not have this effect. Treatment of cultures with the divalent cationophore A23187 caused treated cells to retract neurites; expression of TH immunoreactivity was decreased relative to drug-treated and control cultures. These results suggest that retinoic acid treatment may 'prime' SMS-KCNR cells for the subsequent effects of phorbol esters, and indicate that the patterns of biochemical differentiation induced by TPA or RA are different.

Topics: Cell Differentiation; Cell Line; Cells, Cultured; Humans; Neuroblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.

Topics: ADP Ribose Transferases; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bacterial Toxins; Blotting, Western; Butyrates; Butyric Acid; Cell Differentiation; Cell Membrane; Clone Cells; Drug Resistance; Exotoxins; Gene Expression; Humans; Immunotoxins; Membrane Glycoproteins; Neuroblastoma; Pseudomonas; Pseudomonas aeruginosa Exotoxin A; RNA, Messenger; RNA, Neoplasm; Time Factors; Tretinoin; Tumor Cells, Cultured; Virulence Factors

The relationship between plasminogen activator (PA)/plasminogen activator inhibitor (PAI) activity and morphological differentiation was investigated in human neuroblastoma (NB) cells treated with retinoic acid (RA). Conditioned medium from nine NB cell lines and one closely related neuroepithelioma line was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. All NB cell lines were shown to secrete urokinase (UK)-type PA (mol. wt., 52 kDa), and all except two produced tissue PA (mol. wt., 65 kDa). Identification of the PAs was made based on molecular weight and sensitivity to inhibition by anti-UK and anti-tPA antibodies. Several cell lines expressed PA inhibitory molecules; two molecular-weight forms were observed (35 and 40 kDa) in different cell lines. Complex formation with [125]I-labelled proteases revealed specific binding with UK and trypsin but not thrombin, plasmin, or kallikrein. After treatment for 6 days with 1 microM RA, six of the cell lines exhibited an increase in cell-associated and/or secreted tPA activity, corresponding to morphological differentiation of the cells as manifested by extensive neurite outgrowth. A decrease in UK and UK-complex secretion was observed in several of these cell lines. Three cell lines exhibiting no detectable morphological alterations with RA treatment also showed no dramatic changes in PA/PAI activity. These results suggest that morphological differentiation of NB cells may be associated with alterations in the regulation of PA activity.

Topics: Cell Line; Cell Transformation, Neoplastic; DNA, Neoplasm; Drug Screening Assays, Antitumor; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Neuroblastoma; Plasminogen Activators; Plasminogen Inactivators; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

In 1986 we reported on the capacity of cis-diaminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 micrograms/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.

Topics: Antigens, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Cisplatin; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Neuroblastoma is one of the most common solid tumors of childhood and is notable for its ability to spontaneously regress and, in some instances, to differentiate to less malignant ganglioneuromas. Since immune mechanisms may account for these phenomena, identification of in vivo immune responses to tumor cell surface antigens may be important to the progression of the disease. As determined by analysis on the fluorescence-activated cell sorter, sera from 10 of 18 neuroblastomas patients were found to contain antibodies to a cell surface antigen present on subpopulations of cells from human neuroblastoma cell lines maintained in vitro. Eight human neuroblastoma cell lines were examined and found to vary in reactivity with sera. Induction of differentiation of cell lines with retinoic acid (RA) in vitro resulted in most cell lines bearing higher percentages of positive cells but with a decreased mean cell fluorescence. Preliminary Western blot analysis of lysates of the human cell lines NMB/N7, SMS-KAN, and SK-N-MC showed two principal antigen bands on reducing gels. Comparison of sera from different individuals on lysates of cell lines showed reactivity principally with bands of 105-110 kD and 65-70 kD and an additional minor band of slightly lower molecular weight with the higher titer sera. The ability of different sera to recognize a common antigen pattern suggests that this represents an immunodominant cell surface antigen. Examination of reactivity of other cell lines in this system showed that positive sera reacted with all neuroblastoma lines examined, one neuroepithelioma (SK-N-MC), two melanoma lines (MeWo, G361), and one adrenal-derived adenocarcinoma (SW-13).

Topics: Antibody Formation; Antigens, Neoplasm; Antigens, Surface; Blotting, Western; Cell Differentiation; Fluorescent Antibody Technique; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H] inositol or [1,(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.

Topics: Cell Differentiation; Cell Line; Diglycerides; Drug Resistance; Humans; Neuroblastoma; Phosphatidylinositols; Tretinoin

A prerequisite for the study of biological characteristics in neuroblastoma is the establishment of cell lines from tumors of patients. For our study a neuroblastoma cell line, IGR-N-835, was established from a primary tumor. During in vitro establishment of this line morphological changes were observed. IGR-N-835 exhibited both anchored cells and floating clusters. When cultured on bovine endothelial corneal cellular matrix, all tumor cells anchored to the matrix and proliferated, giving a continuous cell line. IGR-N-835 was studied in vitro and in vivo. Treatment with retinoic acid resulted in cell growth arrest and morphological differentiation. IGR-N-835 was highly tumorigenic in nude mice, exhibited a doubling time of 65 hr and was able to form colonies in methyl-cellulose with a cloning efficiency of 0.46%. Immunocytochemical studies showed reactivity of this line and its primary passages with CE7 monoclonal antibody (MAb). Cytogenetic analyses revealed stable mode and markers resulting from structural changes in chromosomes 10, 11 and 21 with no homogeneously staining regions or double minute chromosomes. Genomic amplification and overexpression of the N-myc oncogene exhibited by the patient's tumor remained unchanged in nude mouse xenografts and the IGR-N-835 cell line. These phenotypic and genotypic traits were unchanged during establishment of this neuroblastoma line. IGR-N-835 could therefore constitute a suitable material for the study of the biology or therapeutics of human neuroblastoma.

Topics: Animals; Cell Differentiation; Child, Preschool; Chromosome Aberrations; Female; Gene Amplification; Genotype; Humans; Mice; Mice, Nude; Neuroblastoma; Phenotype; Proto-Oncogenes; Tretinoin; Tumor Cells, Cultured

Choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, abbreviated ChAT), the biosynthetic enzyme for acetylcholine and acetylcholinesterase (EC 3.1.1.7, abbreviated AChE) are expressed in a human cholinergic neuroblastoma cell line, MC-IXC. We have shown that ChAT activity can be regulated in culture by retinoic acid, an active metabolite of vitamin A, and by sodium butyrate, an organic fatty acid. Optimal concentrations of these agents produce 4.3-fold and 1.6-fold increases in ChAT activity, respectively. The effects of retinoic acid are statistically significant after 24 h, whereas for sodium butyrate significant differences are seen only after 48 h. Since retinoic acid stimulation of ChAT activity was reversed only by trypsin treatment and not by removal of retinoic acid from the medium, this suggests that this agent may be acting at the level of the cell surface. Other differentiating conditions, such as culture in serum-free medium or addition of 1-2% dimethylsulfoxide did not increase ChAT activity. Acetylcholinesterase activity was shown to increase only in the presence of sodium butyrate, suggesting that retinoic acid and sodium butyrate may be acting via different pathways. Retinoic acid and sodium butyrate both seem to be permissive rather than instructive in regulating ChAT activity in that they are unable to induce ChAT expression de novo in cell lines which do not already express ChAT activity.

Topics: Butyrates; Butyric Acid; Cell Line; Choline O-Acetyltransferase; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

In our previous report we have shown that the enzyme choline acetyltransferase (ChAT), responsible for the synthesis of the neurotransmitter acetylcholine, can be regulated in response to treatment by either retinoic acid or sodium butyrate. These responses were dose and time dependent, but the mechanism by which these agents were acting was not understood. We now report the results of studies aimed at elucidating the level at which both sodium butyrate and retinoic acid are able to increase ChAT activity. The effects of these agents on macromolecular synthesis appeared to be limited to small but statistically significant increases in the rate of RNA synthesis. However, inhibition of DNA, RNA and protein synthesis in these cells had no effect on the stimulation of ChAT activity by either sodium butyrate or retinoic acid. Several experiments appeared to rule out a role for cyclic AMP or protein kinase C in the regulation of ChAT activity, even though retinoic acid treatment could increase endogenous levels of cyclic AMP 3- to 4-fold over the time course of ChAT activity stimulation. Experiments performed to determine kinetic parameters of this enzyme demonstrated changes only in the Vmax, but not the Km of ChAT, suggesting that the affinity of enzyme for either of its substrates was not responsible for the increase in specific activity. Taken together, this evidence suggests that the activation of choline acetyltransferase in this human neuroblastoma cell line occurs at the post-translational level.

Topics: Butyrates; Butyric Acid; Cell Line; Choline O-Acetyltransferase; Cycloheximide; Humans; Kinetics; Neuroblastoma; Nucleotides, Cyclic; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Upon differentiation with retinoic acid of the human neuroblastoma cells SH-SY5Y into mature neurons, opioid drugs become highly effective in suppressing prostaglandin E1 (50% inhibition)- and forskolin (70% inhibition)-stimulated adenylate cyclase activity, which was assessed by measuring cyclic AMP accumulation in intact cells. Whereas the SH-SY5Y cells carry both mu and delta receptors in a ratio of mu/delta approximately equal to 5/1, the response is predominantly mediated by the mu receptor. Morphine acts as a strong agonist with an EC50 of 50 to 100 nM which falls into the therapeutic range expected for narcotic analgesic effects mediated by the mu receptor. Narcotic analgesic drugs with only partial agonism fail to evoke full response, which suggests that this cell model could provide a rapid screening assay for narcotic analgesic efficacy. Continued exposure of the cells to morphine resulted in partial tolerance within 12 hr with a 4-fold shift of morphine's EC50 to higher concentrations, whereas longer morphine exposure did not cause any further shift. Thus, the differentiated SH-SY5Y cells provide a suitable system for studying the molecular mechanisms of the narcotic analgesics.

Topics: Adenylyl Cyclases; Algorithms; Alprostadil; Analgesics; Cell Differentiation; Cell Line; Colforsin; Humans; Morphine; Narcotics; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tretinoin

Topics: Bucladesine; Catecholamines; Cell Differentiation; Cell Line; Cyclic AMP; Humans; Microscopy, Electron, Scanning; Neuroblastoma; Phosphopyruvate Hydratase; Tretinoin; Tumor Cells, Cultured

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.

Topics: Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Drug Combinations; Humans; Lactams, Macrocyclic; Neuroblastoma; Neurons; Quinones; Receptors, Opioid; Rifabutin; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

Neuroblastoma cells in culture contain low levels of cyclic AMP, a second messenger which plays a major role in neuronal maturation. In this study, human neuroblastoma cells, SK-N-SH-SY5Y, were induced to differentiate by treatment with either nerve growth factor (50 ng/ml), retinoic acid (10 microM), dibutyryl cyclic AMP (1 mM), or 12-O-tetradecanoylphorbol-13-acetate (0.1 microM), and the ability of several neurotransmitters or hormones to stimulate adenylyl cyclase was tested. Although all four differentiation factors caused morphological changes towards a neuronal phenotype, only retinoic acid dramatically enhanced cyclic AMP accumulation, specifically upon stimulation with prostaglandin E1 (PGE1). PGE2 was also active, but less potent, than PGE1, whereas the other cyclic AMP-stimulating agents tested were largely unaffected. Further, the rapid desensitization of the PGE1-cyclic AMP response observed in control cells after 20 min of PGE1 exposure did not occur in retinoic acid-treated cells, and the EC50 values for PGE1 were reduced from approximately 240 to 14 nM after retinoic acid treatment. The increased sensitivity to PGE was associated with an increase of high-affinity PGE1 binding sites, whereas the Gs coupling proteins and adenylyl cyclase were not measurably affected. A similar enhancement of the PGE1-cyclic AMP response by retinoic acid was also observed in two additional human neuroblastoma cell lines tested, Kelly and IMR-32, suggesting that up-regulation of the prostaglandin response by retinoic acid is common among neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Alprostadil; Bucladesine; Cell Differentiation; Cyclic AMP; Dinoprostone; Drug Synergism; Humans; Indomethacin; Nerve Growth Factors; Neuroblastoma; Prostaglandins E; Receptors, Prostaglandin; Receptors, Prostaglandin E; Tachyphylaxis; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.

Topics: Cell Differentiation; Cell Line; Eflornithine; Humans; Neuroblastoma; Polyamines; Transglutaminases; Tretinoin

The influence of 13-cis-retinoic acid on the light-caused injury of hematoporphyrin-pretreated nontransformed 10 T1/2 fibroblasts and N2a neuroblastoma cells was investigated. Retinoid reduced the percentage of damaged cells as assessed by the plating efficiency assay. N2a neuroblastoma cells were found to be better protected than 10 T1/2 cells by 13-cis-retinoic acid on hematoporphyrin light-induced injury.

Topics: Animals; Cell Division; Cell Line; Cells, Cultured; Hematoporphyrin Derivative; Hematoporphyrins; Isomerism; Isotretinoin; Mice; Neuroblastoma; Radiation-Sensitizing Agents; Tretinoin

Human neuroblastoma (NB) is a highly malignant tumor arising in cells that originate in the embryonal neural crest. Several lines of investigation suggest that both NB and other tumors of developing tissues are blocked in their ability to differentiate and achieve growth arrest. Since in vivo differentiation of NB has been frequently observed and may be of clinical importance (Fox et al., 1959; Evans et al., 1976), we have utilized the in vitro induction of NB differentiation by retinoic acid (RA) to study the molecular events associated with NB differentiation. We have focused our studies on changes that occur in the expression of various proto-oncogenes during NB tumors cell differentiation because proto-oncogenes are likely to be of central importance in mediating processes critical for cellular growth and maturation. In these studies, we have found that the expression of no fewer than five proto-oncogenes including c-Ha-ras, c-ets-1, and c-fos change during the differentiation of NB cells, while the expression of c-erb-B changes in association with the arrest of growth that occurs during NB differentiation. In some cases the altered expression of a proto-oncogene was transcriptionally regulated, while in others post-transcriptional mechanisms were important.

Topics: Cell Differentiation; Gene Expression Regulation; Humans; Neuroblastoma; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogenes; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Alpha tocopheryl succinate treatment (6-8 micrograms/ml), which inhibited the growth of murine neuroblastoma (NBP2) cells (46 +/- 3%), reduced basal and prostaglandin (PG)E1- and PGA2-stimulated adenylate cyclase (AC) activity in vitro. It also inhibited sodium fluoride (NaF)- and forskolin-stimulated AC activity, suggesting that the effect of vitamin E succinate on AC activity is mediated via stimulatory GTP-binding protein (Gs) and catalytic subunit. Vitamin E succinate-induced reduction of AC activity is not strictly related to inhibition of cell growth. This is substantiated by the finding that, although retinoic acid and butylated hydroxyanisole reduced the growth by over 50%, they did not inhibit AC activity. On the other hand, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (R020-1724, 200 micrograms/ml), which inhibited growth (73 +/- 3%) and induced differentiation in NB cells, increased basal and PGE1-stimulated AC activity. Vitamin E succinate treatment also reduced PGE1- and PGA2-AC activity in murine fibroblasts (L-cells) without inhibiting growth.

Topics: Adenylyl Cyclases; Animals; Cell Division; Colforsin; Mice; Neuroblastoma; Prostaglandins A; Prostaglandins E; Sodium Fluoride; Tocopherols; Tretinoin; Tumor Cells, Cultured; Vitamin E

Topics: Cell Differentiation; Glutathione Peroxidase; Humans; Neuroblastoma; Superoxide Dismutase; Tretinoin; Tumor Cells, Cultured

Inositol phospholipid turnover is part of a signal transduction mechanism which mobilize intracellular calcium and activate a calcium- and phospholipid-dependent protein kinase, protein kinase C. Phosphatidylinositol turnover has recently been implicated in the regulation of cell proliferation and transformation. Its role in differentiation has now been investigated using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by RA. Treatment of LAN-1 cells with RA was followed by a rapid decrease of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H] inositol or [1(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol (1,4,5)trisphosphate and (1,2)diacylglycerol within 1 min. of induction of LAN-1 cell differentiation. These findings suggest that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain RA signals are transduced, playing a key role in control of neuroblastoma cell differentiation.

Topics: Cell Differentiation; Cell Line, Transformed; Diglycerides; Humans; Neuroblastoma; Phosphatidylinositols; Time Factors; Tretinoin

LA-N-5 human neuroblastoma cells were found to express high levels of an Mr 53,000 cellular tumor antigen (p53), a protein that has been implicated as playing a fundamental role in the control of cell division and differentiation processes in a variety of tumor systems. When LA-N-5 cells are treated with retinoic acid, they undergo growth and morphological, biochemical, and electrophysiological changes that are characteristic of neuronal maturation and a reduction of the malignant phenotype. We find that these retinoic acid-induced changes are accompanied by a marked decrease in the levels of p53 and p53 mRNA. In our study, p53 levels decreased in concert with both morphological differentiation and with inhibition of cellular proliferation in vitro. These results suggest that p53 levels are intimately related to an undifferentiated phenotype in neuroblastoma cells and support studies which relate p53 levels to the malignant phenotype in other tumor systems.

Topics: Cell Differentiation; Humans; Neoplasm Proteins; Neuroblastoma; Nuclear Proteins; Phosphoproteins; RNA, Messenger; Thymidine; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

Topics: Cell Differentiation; Cell Line; Gene Expression Regulation; Humans; Neuroblastoma; Proto-Oncogene Mas; Proto-Oncogenes; RNA, Messenger; Transcription, Genetic; Tretinoin

Topics: Cell Line; Genes; Humans; Neuroblastoma; Nucleic Acid Hybridization; RNA, Messenger; Tretinoin

The prognosis of patients with advanced neuroblastoma remains poor despite recent progress in chemo/radiotherapy. Therapeutic trials on the induction of differentiation of neuroblastoma by chemical and biological agents have been attempted to improve patients' prognosis. Recently a new synthetic polyprenoic acid, E5166, having retinoic acid properties, has been described. In this study two human neuroblastoma cell lines, KP-N-RT(LN) and SK-N-DZ, were treated in vitro by E5166. Morphological differentiation of KP-N-RT(LN) and SK-N-DZ cells could be induced by E5166 in the presence of 1.7 X 10(-5) M E5166 for 10 days in culture. Levels of catecholamines (dopamine, adrenaline, and noradrenaline) were not elevated in the E5166-differentiated cells. E5166-induced differentiation may not be cyclic AMP dependent, since levels of cyclic AMP did not increase after exposure of cells to this agent. No significant increase in neuron-specific enolase levels could be demonstrated in E5166-treated neuroblastoma as compared to control untreated cells. E5166 treatment of KP-N-RT(LN) and SK-N-DZ cells was found to inhibit colony formation in soft agar in a dose-dependent manner. Colonies of KP-N-RT(LN) cells in the presence of E5166 showed morphological differentiation as defined by the expression of long neurite processes. E5166 is a less toxic reagent than the retinoic acids used for the induction of differentiation, it can be administered to patients p.o., and the concentration of E5166 which induces the morphological differentiation in vitro can be achievable in vivo. Therefore our study suggests that E5166 could be a useful therapeutic agent in advanced neuroblastoma to differentiate residual anaplastic tumor cells to a benign form (ganglioneuroma) after surgery and chemotherapy.

Topics: Cell Differentiation; Cell Line; Cyclic AMP; Dopamine; Epinephrine; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Neuroblastoma; Norepinephrine; Prognosis; Tretinoin

Differentiation of human neuroblastoma (NB) was studied in vitro with five NB cell lines treated with dibutyryl cyclic adenosinemonophosphate and retinoic acid. Although the above agents induced different responses in the various cell lines, three overall morphologic phenotypes emerged: a neuronal, characterized by cell processes and neurosecretory granules, a flat cell without pigment, which displayed basal lamina pertinent to Schwann cells, and a flat pigmented cell which exhibited melanosomes, similarly to melanocytes. The activity of the Schwann cell enzyme cyclic nucleotidyl phosphohydrolase increased considerably in one condition, after induction of a predominantly flat cell phenotype. All studied NB cell lines were capable of synthesizing and expressing the extracellular matrix proteins laminin (LM), fibronectin (FN), and Type IV collagen; but a specific pattern of expression emerged after differentiation, which was proportional to normal tissue equivalents: neuronal--none; melanocytic--FN only; and Schwann cell--large amounts of FN, LM, and Type IV collagen.

Topics: 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; 2',3'-Cyclic-Nucleotide Phosphodiesterases; Bucladesine; Cell Differentiation; Cell Line; Choline O-Acetyltransferase; Collagen; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Fibronectins; Humans; Laminin; Melanocytes; Microscopy, Electron; Neural Crest; Neuroblastoma; Neurons; Neurotransmitter Agents; Phosphoric Diester Hydrolases; Protein Biosynthesis; S100 Proteins; Schwann Cells; Tretinoin

Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of 125I-NGF to solubilized receptors. Analysis of 125I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.

Topics: Cell Line; Cell Membrane; Humans; Nerve Growth Factors; Neuroblastoma; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Tretinoin

Testing with a panel of 26 monoclonal antibodies (MAbs) showed the antigenic profile of 13 human neuroblastoma cell lines to be characterized by a generally poor antigenic expression; therefore, Interferon-gamma (IFN-gamma), dibutyryl cyclic-AMP and retinoic acid were used to analyse the modulation of surface antigenic expression during differentiation. Treatment of neuroblastoma cell lines with IFN-gamma resulted mainly in induction or increase of class-I MHC antigenic expression. Induction of class-II MHC antigens was obtained on only one neuroblastoma cell line out of 13, thus representing an exceptional event. An increase in some other antigens expressed by neuroblastoma cell lines was also observed. In contrast, and in addition to morphological maturation, treatment of these cell lines with the differentiation inducer dibutyryl-cyclic-AMP (dbc-AMP), resulted in general down-modulation of antigenic expression, particularly of neuroblastoma-associated 5A7 or Leu7 antigens. Retinoic acid treatment had no significant effect on MHC antigens, but it decreased expression of 5A7 and Leu7 antigens, and markedly increased the expression of the melanoma-associated antigen Me14-D12. The similarity between the antigenic profile of in vitro differentiated neuroblastoma cells and that of mature ganglioneuroma cells suggests that compounds like cyclic-AMP or retinoic acid are excellent tools for further investigations of the mechanisms of neuroblastoma differentiation and might have important clinical applications.

Topics: Antigens, Neoplasm; Antigens, Surface; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Dose-Response Relationship, Drug; HLA Antigens; HLA-DR Antigens; Humans; Interferon-gamma; Neuroblastoma; Phenotype; Tretinoin

Dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) and beta-all-trans retinoic acid (RA) have been shown separately, and in some cases in combination, to modulate the growth, differentiation, and cAMP-dependent protein kinase (PK-A) activity of various tumor cells. The effects of Bt2cAMP and RA on a cholinergic clone (S20) of C1300 mouse neuroblastoma cells were explored in the present study. Treatment of these cells with 1 mM Bt2cAMP for 3 or more days resulted in 93% inhibition of cell proliferation in monolayer cultures and in 98% inhibition of colony formation in semisolid medium (0.5% agarose). In contrast, treatment of the cells with 1 or 10 microM RA had no inhibitory effects on cell proliferation in monolayer cultures but enhanced colony formation in agarose by up to 130%. The growth of cells treated with a combination of Bt2cAMP and RA was inhibited, although less so than with Bt2cAMP alone. Cells treated with Bt2cAMP alone or Bt2cAMP and RA extended long, neurite-like, cellular processes indicative of differentiation, whereas only a few untreated or RA-treated cells produced such extensions. The amount of [3H]cAMP-binding protein increased gradually up to 2-fold during a 3-day treatment with Bt2cAMP; in contrast it decreased by nearly 2-fold during RA treatment. These changes occurred in the level of the type I regulatory subunit (RI) of PK-A as determined by photoaffinity labeling with 8-azidoadenosine cyclic 3':5'-[32P]monophosphate. The increase in RI following Bt2cAMP treatment was corroborated by DEAE-cellulose chromatography. This analysis also demonstrated that type I PK-A is the predominant kinase in the untreated S20 cells and that RI exists as a free subunit in Bt2cAMP-treated cells. The activity of PK-A decreased by about 20% following treatment with either Bt2cAMP or RA and by 45% following treatment with a combination of both agents. These results suggest that the distinct effects of Bt2cAMP and RA on the anchorage-independent growth of S20 cells may be related to their opposite effects on the level of RI.

Topics: Animals; Bucladesine; Carrier Proteins; Cells, Cultured; Cyclic AMP Receptor Protein; Mice; Microscopy, Phase-Contrast; Neuroblastoma; Protein Kinases; Tretinoin

The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane.

Topics: Cell Differentiation; Cell Membrane; Clone Cells; GTP-Binding Proteins; Humans; Neoplasm Proteins; Nerve Growth Factors; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The mRNA expression of c-myc and N-myc in the human neuroblastoma cell line SH-SY5Y was found not to change appreciably during the cell cycle and was also unaffected by proliferative inhibition induced by serum starvation or polyamine depletion. However, an early (0.5-8.0 h post-induction) transient reduction of c- and N-myc transcripts were observed in these cells upon induction to differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of these neuroblastoma cells with TPA for longer periods (1-8 days), which induces morphological and functional differentiation and growth arrest, was followed by decreased expression of both myc genes. However, the rate of disappearance differed considerably. The N-myc mRNA level was slightly decreased after 4 days and was still detectable 8 days after induction, whereas the c-myc transcript was down-regulated much faster. In contrast, when the cells were exposed to retinoic acid, which results in a maturation along an alternative pathway, the inhibition of N-myc and c-myc expression was similar. The c-fos mRNA expression increased in TPA-treated SH-SY5Y cells and remained high during extended exposure to the drug. The highest c-fos transcript level in induced cells coincided in time with the transient reduction of N-myc and c-myc. Thus, the TPA-induced neuronal differentiation of SH-SY5Y cells was compatible with high c-fos and a substantial N-myc mRNA expression.

Topics: Cell Cycle; Cell Differentiation; Gene Expression Regulation; Neuroblastoma; Proto-Oncogene Proteins; Proto-Oncogenes; Tetradecanoylphorbol Acetate; Tretinoin

The aim of the study was to investigate the influence of the substances modifying cell phenotype (phorbol ester and 13-cis retinoic acid) on the growth of mouse neuroblastoma N2a cells in the mixed culture system. The results were compared with monoculture system and with tumor multicellular spheroids system where neoplastic cells are cultivated in the close contact from all sides. The suppression of the pleyotypic effect of phorbol ester was observed when N2a cells were cocultured with 3T3 fibroblasts. On the contrary, the close contact of neoplastic cells to each other (spheroids) do not change the stimulatory effect of phorbol ester on tumor cells proliferation. 13-cis retinoic acid suppressed neoplastic cells growth in all cell culture systems we used.

Topics: Animals; Cell Communication; Cell Differentiation; Cell Division; Cell Line; Fibroblasts; Glioma; Neuroblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The specific binding of the muscarinic ligand [3H](-)quinuclidinyl benzilate [( 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a Kd = 0.28 +/- 0.06 nM and a Bmax of 337 +/- 47 pmol/g protein. Pirenzepine inhibited [3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, KH = 10 nM) and a low affinity site (66%, KL = 1 microM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M1/M2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to non-treated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.

Topics: Acetylcholinesterase; Atropine; Binding, Competitive; Cell Differentiation; Cell Line; Choline O-Acetyltransferase; Humans; Neuroblastoma; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Tetradecanoylphorbol Acetate; Tretinoin

We have studied the regulation of lysosomal glycosidases during morphological differentiation of NB2a neuroblastoma cells. Cells treated with dibutyryl cAMP induced axon-like neuritis and showed a 2-4 fold increase in the activity of 6 lysosomal glycosidases, reaching their highest level after 5 days of treatment. Cells treated with retinoic acid, which induced dendrite-like neurites, did not show significant changes in the glycosidases activity although cell proliferation was also inhibited. There was no change in the pattern of the enzyme secretion during the dibutyryl cAMP treatment and morphological analysis using electron microscopy and cytochemical staining with acid phosphatase indicated the presence of lysosomes in the induced neurites.

Topics: Animals; Bucladesine; Cell Line; Cell Transformation, Neoplastic; Glucuronidase; Glycoside Hydrolases; Lysosomes; Mice; Microscopy, Electron; Neuroblastoma; Staining and Labeling; Tretinoin

The activity of neurotoxin-responsive Na+ channels in mouse neuroblastoma cells, N-18, was examined after treating the cells with compounds that are reported to perturb intracellular traffic. The compounds used have been shown to either alter glycoprotein synthesis and processing, (swainsonine, castanospermine, monensin, and retinoic acid) or receptor mediated endocytosis (mevinolin, 7-ketocholesterol, and chloroquine), or both. All of these compounds inhibited the activity of the neurotoxin-responsive Na+ channel with the exception of retinoic acid which increased the activity. Na+ channel activity was measured by two methods: (a) In vivo, the efflux of 86Rb was measured by use of the cells in monolayer culture, and (b) in vitro, the flux of 86Rb was measured from artificial phospholipid vesicles containing the partially purified Na+ channel. In both cases, 86Rb flux responded to stimulating neurotoxins, veratridine and scorpion venom, and was inhibited by tetrodotoxin as characteristic of excitable membranes. One of the perturbing compounds, swainsonine, was examined in detail. Treatment of N-18 cells with 10 microM swainsonine for 24 h markedly reduced the activity of the neurotoxin-responsive Na+ channel, as shown by the neurotoxin-stimulated efflux of 86Rb in vivo. In addition, after reconstitution into phospholipid vesicles of the partially purified Na+ channel from swainsonine-treated cells, reduced 86Rb flux was observed when compared with that of nontreated cells. Furthermore, the activity was not recovered in other less purified fractions. A comparison of the glycopeptides from the treated and nontreated cells by size, charge, and lectin-binding affinities was consistent with the formation of hybrid oligosaccharides after swainsonine treatment. It is concluded that the oligosaccharide residues of the Na+ channel glycoprotein must be processed to the mature complex-type for full activity. The stimulation of channel activity by treatment with retinoic acid supported this conclusion.

Topics: Animals; Cell Line; Glycoproteins; Ion Channels; Kinetics; Mice; Neuroblastoma; Neurotoxins; Protein Processing, Post-Translational; Rubidium; Scorpion Venoms; Sodium; Tetrodotoxin; Tretinoin; Tritium; Veratridine

SY5Y neuroblastoma cells were treated with retinoic acid to induce differentiation of neuroblast-like cells and with aphidicolin (an inhibitor of DNA polymerase) to eliminate the flat cells present in the long-term cultures and masking some of the biochemical developmental changes. Catecholaminergic enzyme (tyrosine hydroxylase and monoamine oxidase-A) activity was consistently increased with development and the increase was significantly greater after aphidicolin-induced elimination of dividing, non-neuronal cells.

Topics: Animals; Aphidicolin; Cell Differentiation; Cell Line; Clone Cells; Diterpenes; Isoenzymes; Models, Neurological; Monoamine Oxidase; Neuroblastoma; Neurons; Tretinoin; Tyrosine 3-Monooxygenase

Retinoic acid (RA) has been shown to induce the differentiation of human neuroblastoma cells in vitro. In this study, we describe two variants of the SK-N-SH human neuroblastoma cell line that have dramatically different responses to RA. RA induces neuronal-like differentiation characterized by extensive neurite outgrowth, thick neurite bundles, and large cellular aggregates of SK-N-SH-N (SH-N) cells. In contrast, RA treatment of SK-N-SH-F (SH-F) cultures transforms the small neuroblast cells into large flattened, fibroblastic or epithelial-like cells. Karyotype analysis verified that the SH-N and SH-F cultures were derived from a common precursor cell. Confirmation of their markedly different responses to RA was obtained by metabolic labelling of glycoproteins and SDS-PAGE analysis. While both sublines showed very similar Coomassie-labelled protein bands and glycoprotein profiles in control cultures, dramatic differences between the lines were revealed following RA treatment. In contrast to their similar protein profiles, untreated SH-N and SH-F cells had quite different patterns of ganglioside biosynthesis in that GM3 was detected in SH-F cells but not in SH-N, while GM1 was only detected in SH-N. Cellular RA binding protein (CRABP) was detected in both SH-F and SH-N cells and their RA-transformed derivatives. These results demonstrate heterogeneity in the response to RA of neuroblastoma cells derived from a common origin that cannot be accounted for by differences in CRABP content. The SH-N and SH-F neuroblastoma sublines should provide a useful system for further studies of the molecular processes through which RA exerts its differentiation-inducing activity on this type of tumor.

Topics: Carrier Proteins; Cell Differentiation; Cells, Cultured; Chromosome Aberrations; Gangliosides; Glycoproteins; Humans; Isoenzymes; Karyotyping; Molecular Weight; Neuroblastoma; Receptors, Retinoic Acid; Tretinoin

Topics: Antigens, Neoplasm; Antigens, Surface; Axons; Bromodeoxyuridine; Bucladesine; Cell Differentiation; Cell Line; Humans; Neuroblastoma; Neuroglia; Neurons; Schwann Cells; Tretinoin

Topics: Bucladesine; Cell Differentiation; Cells, Cultured; Collagen; Extracellular Matrix; Glycoproteins; Humans; Melanocytes; Neuroblastoma; Neurons; Schwann Cells; Tretinoin

To a limited extent, we corroborated a previous report of human neuroblastoma sensitivity to 13-cis-retinoic acid. Seven cultured human neuroblastoma, two primitive neuroectodermal tumor, and one melanoma cell line were exposed to 0.001 to 10.0 microM 13-cis-retinoic acid for six to fourteen days. The neuroblastoma cell line, SK-N-DZ, was the only cell line lysed by all concentrations of 13-cis-retinoic acid. The other cell lines were refractory to concentrations as high as 10 microM. Increased cell process formation was observed in three neuroblastoma, SK-N-SH, SK-N-BE, SK-N-LE, and one melanoma cell line. We conclude that sensitivity to 13-cis-retinoic acid is unevenly distributed among histogenetically similar tumors from different patients.

Topics: Cell Line; Cell Survival; Humans; Melanoma; Neoplasms, Germ Cell and Embryonal; Neuroblastoma; Tretinoin; Tumor Stem Cell Assay

We have characterized the effects of retinoic acid (RA) on the growth, morphology and biosynthesis of cytoskeletal proteins in NB2a mouse neuroblastoma cells. In addition, the morphological and biochemical changes were compared to those induced by dibutyryl cyclic AMP (db cAMP). Growth inhibition by RA was concentration-dependent and was first detected 24 h after addition of RA. The proliferation of RA-treated NB2a was more dependent on serum than was the proliferation of untreated cultures and RA decreased the saturation density of NB2a cells grown in serum. Morphological changes induced by RA include the formation of an elaborate network of branching neurites in NB2a cells. In contrast, neurites induced by db cAMP or serum deprivation were bipolar and unbranching. Ultrastructural observations of neurites induced by RA revealed dendritic characteristics such as polysomes, spines and absence of intermediate filaments, while neurites induced by db cAMP had axonal characteristics such as filament bundles, absence of ribosomes, and the formation of membrane densities when neurite endings contacted another cell body. These morphological differences were also reflected in a number of changes in the biosynthesis of cytoskeletal proteins. These results suggest that NB2a cells treated with RA and db cAMP are a model system for the study of distinct stages of differentiation.

Topics: Animals; Blood Physiological Phenomena; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Growth Inhibitors; Mice; Neuroblastoma; Protein Biosynthesis; Time Factors; Tretinoin

We investigated the effects of retinoic acid (RA) on natural killer (NK) cell activity and on the susceptibility of various tumor target cells to NK cell lysis. These studies were undertaken to assess the overall effects of RA treatment on the natural killing of tumor cells in humans. We also evaluated how RA affects the generation of NK-like activity in mixed leucocyte culture, to determine whether or not this compound can influence the regulation of natural cytotoxicity during the induction phase of NK cell development. Our results indicate that pharmacological levels of RA have little, if any, influence on the development, cytotoxic potential, or activity of NK cells in humans. In contrast, RA can significantly reduce the sensitivity of some tumor target cells to natural killing. This effect appears to be directly related to the differentiation-promoting properties of RA, since reduced NK susceptibility was observed with target cells that were "differentiated" by RA treatment, but not with cells that were either unaffected by RA or were only growth inhibited without concomitant differentiation. These findings indicate that the immunomodulating properties of RA do not extend to human NK cell activity. However, a possible decreased susceptibility of tumor cells to natural killing should be a consideration when planning therapeutic applications of RA in certain cancers.

Topics: Adult; Cell Line; Cells, Cultured; Cytotoxicity, Immunologic; Humans; Immunity, Innate; Killer Cells, Natural; Lymphocyte Culture Test, Mixed; Neoplasms, Experimental; Neuroblastoma; Tretinoin

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.

Topics: Cell Differentiation; Cell Line; Gene Expression Regulation; Humans; Neuroblastoma; Oncogenes; RNA, Messenger; RNA, Neoplasm; Tretinoin

This report examines the morphological changes that occur in a line of human neuroblastoma cells (LA-N-5) following treatment with retinoic acid, in vitro. The results demonstrate that retinoic acid induces pronounced differentiation of these cells. Perikarya aggregate into tight clusters and extend long processes that are frequently fasciculated. Growth cones appear at the ends of these processes. Transmission electron microscopy reveals that after 10 days of treatment these long neurites give rise to varicosities which contain clusters of large dense-core vesicles and smaller clear vesicles. After 18 days of treatment the cultures cease to differentiate further. The pattern of neurite outgrowth is very complex by this point and the frequency of growth cones and vesicle-containing varicosities is greatly increased compared with shorter treatments. Most of these varicosities contain a mix of large dense-core vesicles and smaller clear vesicles and in some profiles the clear vesicles are round while in others they are pleomorphic. Despite this increase in the number of vesicle-containing profiles no membrane specializations were seen that resemble mature synapses. The present results demonstrate that retinoic acid can produce morphological changes in these cells in culture, and that these changes closely mimic those of normal differentiating neurons in culture. Considered with previous studies, these findings suggest that this cell line might provide a useful model system for studying neural differentiation.

Topics: Cell Differentiation; Cell Line; Humans; Microscopy, Electron; Models, Neurological; Neuroblastoma; Neurons; Time Factors; Tretinoin

Topics: Acetylcholinesterase; Axons; Cell Aggregation; Cell Differentiation; Cell Line; Cytoskeleton; Electric Conductivity; Enzyme Activation; Humans; Neuroblastoma; Tretinoin

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.

Topics: Acetylcholinesterase; Cell Differentiation; Cell Line; Fluorescent Antibody Technique; Humans; Neuroblastoma; Tretinoin

Cultured human SH-SY5Y neuroblastoma cells differentiated in the presence of retinoic acid (RA) or 12-0-tetradecanoyl-phorbol-13-acetate (TPA). In both cases, the cells acquired long cell processes and the cell growth was partially inhibited. Treatment with RA or TPA resulted in an increased neuron-specific enolase activity, relative to the total cellular enolase activity. At the optimal concentration, TPA induced a 200-fold increase in the concentration of noradrenalin, whereas in RA-treated cells the corresponding increase was only fourfold. Cells treated with a combination of RA and TPA were morphologically differentiated and growth inhibited and had a high relative activity of neuron-specific enolase. The increase in the concentration of noradrenalin induced by TPA was inhibited by RA in a concentration-dependent fashion. However, despite this result there seemed to be no general antagonistic effect of RA on the TPA-induced differentiation. The phenotypes of the cells treated by RA, TPA, or the combination of RA and TPA, did, on the other hand, differ from each other. Our results suggest that RA and TPA induce the SH-SY5Y cells to differentiate along different pathways.

Topics: Cell Differentiation; Cell Division; Cell Line; Humans; Neuroblastoma; Neurons; Norepinephrine; Phenotype; Phorbols; Phosphopyruvate Hydratase; Tetradecanoylphorbol Acetate; Tretinoin

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.

Topics: Cell Differentiation; Cell Division; Cell Line; Enzyme Induction; Humans; Neuroblastoma; Ornithine Decarboxylase; Tetradecanoylphorbol Acetate; Tretinoin

The antineoplastic effects of a novel retinoid derivative E5166 on cultured human neuroblastoma, GOTO cells, were investigated. E5166, as well as retinoic acid and retinol, suppressed the proliferation of GOTO cells and the synthesis of DNA, RNA and protein; the inhibitory potency of E5166 was not stronger than that of the natural vitamin A derivatives. E5166 also inhibited amino acid transport, but was found to stimulate sugar transport in the neuroblastoma cells.

Topics: Amino Acids; Biological Transport, Active; Carbohydrate Metabolism; Cell Division; Cell Line; Diterpenes; DNA, Neoplasm; Humans; Neoplasm Proteins; Neuroblastoma; RNA, Neoplasm; Tretinoin

Neuroblastoma cells are a good model for neuronal development because of their ability to extend neurites in response to various stimuli, including retinoic acid. In the present experiments, we have examined five human neuroblastoma cell lines (LA-N-1, IMR-32, LA-N-5, SK-N-MC, and CHP-100) for the presence of cellular retinoic acid binding protein (CRABP), a receptor-like protein implicated in the molecular functioning of vitamin A. CRABP is identified and quantitated by sucrose gradient centrifugation, selective inhibition by the mercurial reagent p-chloromercuribenzene sulfonic acid (PCMBS), and saturation analysis. All five lines contain significant levels of cytosolic CRABP (2.5-7.5 pmol/mg of protein), which display typical properties of specific high affinity retinoic acid binding, a sedimentation coefficient of 2 S, and inhibition by PCMBS. Three of the lines (LA-N-1, IMR-32, and LA-N-5) are strongly growth inhibited by 1 microM retinoic acid in monolayer culture, whereas two (LA-N-1 and LA-N-5) undergo marked differentiation to a stellate, fusiform morphology with characteristic neurite outgrowths. The SK-N-MC and CHP-100 lines are relatively resistant to the antiproliferative effects of retinoic acid under these conditions. Nevertheless, all five lines are effectively inhibited by retinoic acid in their ability to form anchorage-independent colonies in soft agar. Thus, although CRABP is not necessarily correlated with growth inhibition in monolayer culture, it is associated with retinoic acid's ability to inhibit neuroblastoma colony formation in soft agar. More experiments will be required to determine if this effect on growth in soft agar reflects the putative ability of retinoic acid to convert tumorigenic neuroblastoma cell lines into the normal differentiated phenotype.

Topics: Carrier Proteins; Cell Division; Cell Line; Centrifugation, Density Gradient; Humans; Neuroblastoma; Receptors, Retinoic Acid; Tretinoin

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alternations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.

Topics: Axons; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Humans; Neuroblastoma; Neurons; Receptors, Retinoic Acid; Thymidine; Tretinoin; Vacuoles

Retinoic acid (RA) induced concentration-dependent morphologic differentiation and growth inhibition in the LA-N-1 human neuroblastoma cell line. Time course studies demonstrated a significant increase in the formation of long neurites in LA-N-1 cultures within 48 hours of RA addition; maximum expression of differentiation occurred at approximately 4 days. This differentiation profile corresponded to a detectable decrease in [3H]thymidine incorporation at 48 hours and complete inhibition of cell growth after 3-4 days. The RA-induced morphologic differentiation and growth inhibition persisted despite removal of the drug. A soft agar assay system showed that RA also inhibited the ability of LA-N-1 cells to form anchorage-independent colonies and induced morphologic differentiation in colonies that did develop. These findings suggest that RA promoted the differentiation of LA-N-1 neuroblastoma cells, resulting in an altered expression of the malignant phenotype.

Topics: Cell Differentiation; Cell Division; Cell Line; DNA; Humans; Microscopy, Phase-Contrast; Neuroblastoma; Time Factors; Tretinoin

Topics: Animals; Antineoplastic Agents; Carboxy-Lyases; Cell Division; Cell Line; Drug Evaluation, Preclinical; Eflornithine; Glioma; Mice; Neuroblastoma; Ornithine; Ornithine Decarboxylase Inhibitors; Rats; Retinaldehyde; Tretinoin; Vitamin A