tretinoin and Carcinoma

Differentiation is defined as the ability of a cell to acquire full functional behavior. For instance, the function of bladder urothelium is to act as a barrier to the diffusion of solutes into or out of the urine after excretion by the kidney. The urothelium also serves to protect the detrusor muscle from toxins present in stored urine. A major event in the initiation and progression of bladder cancer is loss of urothelial differentiation. This is important because less differentiated urothelial tumors (higher histologic tumor grade) are typically associated with increased biologic and clinical aggressiveness. The differentiation status of urothelial carcinomas can be assessed by histopathologic examination and is reflected in the assignment of a histologic grade (low-grade or high-grade). Although typically limited to morphologic evaluation in most routine diagnostic practices, tumor grade can also be assessed using biochemical markers. Indeed, current pathological analysis of tumor specimens is increasingly reliant on molecular phenotyping. Thus, high priorities for bladder cancer research include identification of (1) biomarkers that will enable the identification of high grade T1 tumors that pose the most threat and require the most aggressive treatment; (2) biomarkers that predict the likelihood that a low grade, American Joint Committee on Cancer stage pTa bladder tumor will progress into an invasive carcinoma with metastatic potential; (3) biomarkers that indicate which pTa tumors are most likely to recur, thus enabling clinicians to prospectively identify patients who require aggressive treatment; and (4) how these markers might contribute to biological processes that underlie tumor progression and metastasis, potentially through loss of terminal differentiation. This review will discuss the proteins associated with urothelial cell differentiation, with a focus on those implicated in bladder cancer, and other proteins that may be involved in neoplastic progression. It is hoped that ongoing discoveries associated with the study of these differentiation-promoting proteins can be translated into the clinic to positively impact patient care.

Topics: Biomarkers, Tumor; Carcinoma; Cell Differentiation; Disease Progression; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 3-alpha; Humans; Muscles; Neoplasm Metastasis; Neoplasm Staging; PPAR gamma; Signal Transduction; Transcription Factors; Tretinoin; Urinary Bladder Neoplasms; Urothelium

Prevention of epithelial cancers is needed, as the treatment of such cancers in the advanced state has failed. Vitamin A and its structural analogs called retinoids will prevent cancers of the lung, breast, bladder, stomach, and cervix in animals exposed to carcinogens. Synthetic retinoids, offer less toxicity than natural retinoids and may be acceptable to human trials. Identification of a suitable group at risk for an epithelial cancer which might be reasonably expected to benefit from retinoids should precede planning of a clinical trial.

Topics: Animals; Carcinogens; Carcinoma; Cricetinae; Epithelium; Female; Forecasting; Humans; Male; Neoplasms, Experimental; Rats; Research Design; Soft Tissue Neoplasms; Tretinoin; Vitamin A

A review of recent investigations in the retinoid field is presented. Retinoic acid exerts a prophylactic and a therapeutic effect on chemically induced benign and malignant epithelial tumors in mice. In clinical studies positive therapeutic results have been obtained in patients with preneoplastic and neoplastic epithelial lesions. However, treatment with retinoic acid is limited by serious side effects (hypervitaminosis A syndrome). Therefore, the synthesis of analogs of retinoic acid (retinoids) possessing a more favorable therapeutic ratio has been initiated. Among a large series of synthesized compounds, certain aromatic analogs proved to have a particularly favorable therapeutic ratio. The structure-activity relationship of the most active retinoids is discussed including some biological data concerning prophylaxis and therapy of epithelial tumors. The total synthesis of retinoids according to various building schemes is discussed in detail. Methods for the synthesis of the cyclic end group, of the polyene chain component, and of the full retinoid skeleton are described. Metabolic studies of retinoic acid and of the most active retinoid, as well as the synthesis of some isolated metabolites are outlined. Suggestions concerning the mechanism of action of retinoids are made. Some clinical results on the treatment of acne, psoriasis and precancerous conditions are reported.

Topics: Animals; Carcinoma; Humans; Neoplasms, Experimental; Papilloma; Retinaldehyde; Skin Diseases; Skin Neoplasms; Structure-Activity Relationship; Tretinoin; Vitamin A

Treatment options are limited for recurrent/metastatic adenoid cystic carcinoma of the head and neck (R/M ACCHN). We aimed to evaluate the preliminary results of the efficacy and safety of all-trans retinoic acid (ATRA) combined with low-dose apatinib in patients with R/M ACCHN according to a secondary analysis of a phase II study.. A total of 16 patients were included with nine (56.3%) males and aged 35-69 years old. All recruited patients previously received anti-angiogenic therapy then withdrew due to toxicities or progression occurred. The objective response rate (ORR) and disease control rate (DCR) were 18.8% and 100%, respectively. During a median follow-up of 23.9 months (range:17.8-31.7 months), 11 (68.8%) patients developed PD and one of them died in 20.9 months. The median of progression-free survival (PFS) was 16.3 months (95% CI: 7.2-25.4 months), and the 6-month, 12-month, and 24-month PFS rates were 100%, 81.3%, and 33.3%, respectively. The grade 3 adverse events were albuminuria (n = 2, 12.5%) and hand-foot syndrome (n = 1, 6.25%).. All-trans retinoic acid combined with low-dose apatinib might be a potential efficacy therapeutic option for patients with R/M ACCHN. This finding will be further confirmed by our registered ongoing trial, the APLUS study (NCT04433169).

Topics: Adult; Aged; Antineoplastic Agents; Carcinoma; Carcinoma, Adenoid Cystic; Female; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Middle Aged; Tretinoin

Topical tretinoin is commonly prescribed, but its frequent adverse effects are barriers to use. Predictors of resistance or susceptibility to retinoid irritation are not known.. To identify baseline patient characteristics associated with adverse effects of topical tretinoin.. This cohort study used data collected from 324 participants in the Veterans Affairs Topical Tretinoin Chemoprevention trial who were randomized to apply tretinoin cream on the face and ears. Univariate and multivariate logistic regression models were used to examine the associations between baseline characteristics and local adverse effects.. One hundred and ninety-seven patients (61% of those randomized to tretinoin) reported local adverse effects within 6 months. Clinical signs of severe photodamage at baseline [odds ratio (OR) 0·15, 95% confidence interval (CI) 0·04-0·54] and history of acne (OR 0·46, 95% CI 0·27-0·77) were associated with a decreased risk of adverse effects to tretinoin. The use of other topical medications at enrolment (OR 1·88, 95% CI 1·15-3·08) predicted an increase in adverse effects.. In this study population, the common indications of topical tretinoin treatment were associated with lower risks of adverse effects. The concurrent use of other topical medications may worsen irritation caused by tretinoin.

Topics: Acne Vulgaris; Aged; Anticarcinogenic Agents; Carcinoma; Drug Eruptions; Female; Humans; Keratinocytes; Male; Ointments; Photosensitivity Disorders; Risk Factors; Skin Neoplasms; Tretinoin

Observational studies have reported significant negative associations between sporadic non-steroidal anti-inflammatory drug (NSAID) use and keratinocyte carcinoma (KC) while reporting null results for regular use. This pattern may be partially explained by the operational expression of NSAID exposure and analytic model assumptions. Our goals were to quantify the association between NSAIDs and KC and to explore the impact of exposure metrics and modeling assumptions on observed associations.. We conducted a prospective cohort study by linking data from the Veterans Affairs Topical Tretinoin Chemoprevention Trial and the VA Pharmacy Benefits Management database. NSAID use was categorized according to cyclooxygenase selectivity, timing of initiation, and frequency of use. Data were analyzed using time-varying and time-fixed multivariable-adjusted Cox proportional hazard models [Correction made here after initial online publication]. Simulated null data were generated and analyzed to explore potential biases introduced by the models and the exposure metrics.. During a median follow-up time of 2 years for basal cell carcinoma and 2.5 years for squamous cell carcinoma, 472 occurrences of BCC and 309 occurrences of SCC were observed. Time-fixed analyses of NSAID exposure metrics produced significant negative associations, whereas time-varying analyses produced null results. Analysis of simulated null data revealed the potential for strong bias in the time-fixed analyses.. This study did not identify a negative association between NSAIDs and KC. The disparity between the time-fixed and the time-varying analyses highlights the extent to which operational definitions of drug exposures and reliance on time-fixed methods may introduce bias.

Topics: Administration, Topical; Aged; Aged, 80 and over; Anti-Inflammatory Agents, Non-Steroidal; Carcinoma; Cohort Studies; Double-Blind Method; Female; Humans; Keratinocytes; Male; Middle Aged; Skin Neoplasms; Tretinoin; United States; United States Department of Veterans Affairs

In addition to suppressing breast cancer cell growth, retinoids potentiate growth inhibition in human breast cancer when tested in vitro and in vivo with tamoxifen and/or interferon. The purpose of this study was to ascertain the biologic effects of all-trans-retinoic acid (ATRA) administered alone and with tamoxifen +/- interferon and to identify the relationship between ATRA plasma concentrations and optimal biological dose (the lowest dose that produces a biological response). Three consecutive groups of 15 patients with locally advanced operable breast cancer were treated, in accordance with good clinical practice (GCP) requirements, with ATRA at 3 dose levels alone or with tamoxifen +/- alpha-interferon 2a at flat doses. After 3 weeks, the tumors were surgically removed. Biological parameters measured at the beginning (in biopsy tissue) and end (in surgical tissue) of the study were compared. The optimal biological dose for ATRA was 15 mg/m2/day. Treatments influenced tumor grade but not cell cycle kinetics (G0-G1 phase) or proliferation (Ki67 levels). ATRA induced progesterone receptors independent of dose level and co-administered drugs, but did not induce estrogen receptors when administered alone. Retinoic acid receptor (RAR)-alpha was not affected by treatment and RAR-alpha was moderately influenced whereas RAR-beta (concomitantly with transforming growth factor-beta) was induced in 33% of patients by ATRA alone. ATRA pharmacokinetics were dose- and time-dependent. Neither the ATRA + tamoxifen nor the ATRA + tamoxifen + interferon combinations potentiated the ATRA-induced biological changes. Future studies evaluating the role of RAR-beta as a biological marker of retinoid activity are warranted.

Topics: Aged; Aneuploidy; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Bone Marrow Diseases; Breast Neoplasms; Carcinoma; Drug Administration Schedule; Drug Interactions; Female; Follow-Up Studies; Headache; Humans; Hypercholesterolemia; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Mastectomy; Middle Aged; Neoplasm Proteins; Receptors, Retinoic Acid; Receptors, Steroid; Recombinant Proteins; Safety; Tamoxifen; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome; Tretinoin

A 69-year-old man was referred to the hematology department for the evaluation of pancytopenia. He had been treated with radiation and epirubicin for hepatocellular carcinoma, and with docetaxel and pembrolizumab for lung adenocarcinoma. Bone marrow smears exhibited markedly increased promyelocytes, and polymerase chain reaction (PCR) study demonstrated chimeric fusion genes of

Topics: Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Carcinoma, Hepatocellular; Docetaxel; Epirubicin; Humans; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Lung Neoplasms; Male; Neoplasms, Multiple Primary; Treatment Outcome; Tretinoin

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.

Topics: Alu Elements; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Nanog Homeobox Protein; Octamer Transcription Factor-3; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; RNA Polymerase III; Teratocarcinoma; Teratoma; Transcription, Genetic; Tretinoin

Interactions between cancer cells and microenvironment are emerging issue in tumor progression. Aldehyde dehydrogenase 1 (ALDH1) is a recognized cancer stem cell marker but little is known about its role in intratumoral stroma. Therefore, we focused on ALDH1 expression in tumor-associated stroma of breast carcinomas (BrCa). Stromal and tumoral ALDH1 expression was evaluated immunohistochemically in BrCa and their lymph node metastases (LNMs), and related to clinico-pathological characteristics, patients' outcome, presence of CD68, HLADR, retinoic acid (RA) in stroma, and selected proteins in tumor cells. ALDH1(+) stromal cells were detected in 53% of 374 BrCa and 61% of 102 LNMs. ALDH1(+) stroma in primary tumor correlated to longer disease-free (p = 0.030), metastasis-free (p = 0.024), and overall survival (p = 0.043) having an independent prognostic impact on DFS (multivariate analysis, p = 0.047). It was associated with concomitant presence of HLA-DR(+) stromal cells and RA in tumor cells (both p < 0.001), and inversely associated with vimentin expression in tumor cells (p = 0.036). ALDH1(+) stroma in LNMs correlated inversely to presence of disseminated tumor cells in patients' bone marrow (p = 0.014) and was independent prognosticator of shorter DFS and MFS (multivariate analysis, p = 0.004 and p = 0.002, respectively). In conclusion, ALDH1 expression in tumor-associated stromal cells indicates reduced BrCa progression, possibly via RA secretion.

Topics: Aged; Aldehyde Dehydrogenase 1 Family; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Breast Neoplasms; Carcinoma; Cohort Studies; Disease Progression; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Germany; HLA-DR Antigens; Humans; Immunohistochemistry; Isoenzymes; Kaplan-Meier Estimate; Lymphatic Metastasis; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplastic Stem Cells; Poland; Retinal Dehydrogenase; Stromal Cells; Tissue Array Analysis; Tretinoin

Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis-related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin-induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII-tubulin in 8-day-differentiated cells. However, the neurites of dendrite marker microtubule-associated protein-2 (MAP-2)-positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP-2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII-tubulin and MAP-2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression.

Topics: Animals; Antimetabolites; Carcinoma; Cell Differentiation; Cell Line, Tumor; Dendrites; Deoxyglucose; Doublecortin Domain Proteins; Endoplasmic Reticulum Stress; Glial Fibrillary Acidic Protein; Heat-Shock Proteins; Mice; Microtubule-Associated Proteins; Neurons; Neuropeptides; Receptors, Peptide; RNA, Messenger; Transcription Factor CHOP; Tretinoin; Tunicamycin; Ubiquitin-Protein Ligases

Human SPLUNC1 can suppress nasopharyngeal carcinoma (NPC) tumor formation; however, the correlation between SPLUNC1expression and NPC patient prognosis has not been reported. In the present study, we used a large-scale sample of 1015 tissue cores to detect SPLUNC1 expression and its association with patient prognosis. SPLUNC1 expression was reduced in NPC samples compared to nontumor nasopharyngeal epithelium tissues. Positive expression of SPLUNC1 in NPC predicted a better prognosis (disease-free survival, P = 0.034; overall survival, P = 0.048). Cox's proportional hazards model revealed that SPLUNC1 could be a significant prognostic factor affecting disease-free survival (P = 0.027). A cDNA micro-array analyzed by significant analysis of micro-array (SAM) and ingenuity pathway analysis (IPA) revealed that an indirect interaction existed between SPLUNC1 and retinoic acid (RA) in the cancer regulatory network. To further investigate the molecular mechanisms involved, we utilized several bioinformatics tools and identified 12 retinoid X receptors heterodimer binding sites in the promoter region of the SPLUNC1 gene. The transcriptional activity of the SPLUNC1 promoter was up-regulated significantly by all-trans-retinoic acid (ATRA). SPLUNC1 and retinoic acid receptor expression were induced significantly by ATRA, and removal of ATRA led to a progressive loss of SPLUNC1 and retinoic acid receptor expression. ATRA inhibited proliferation and induced the differentiation of NPC cells. Interestingly, over-expression of SPLUNC1 sensitized NPC cells to ATRA, whereas knockdown of SPLUNC1 in HNE1 cells increased cell viability. Under SPLUNC1 knockdown conditions, differentiation was reversed by ATRA treatment. We concluded that SPLUNC1 could potentially predict prognosis for NPC patients and play an important role in ATRA-induced growth inhibition and differentiation in NPC cells.

Topics: 5' Untranslated Regions; Adolescent; Adult; Aged; Binding Sites; Carcinoma; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Lymphatic Metastasis; Male; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Proteins; Phosphoproteins; Prognosis; Promoter Regions, Genetic; Recombinant Fusion Proteins; Retinoid X Receptors; RNA Interference; RNA, Small Interfering; Tretinoin; Young Adult

Retinoids have shown potential for the inhibition of tumour growth and progression. The objective of this study was to investigate retinoic acid nuclear receptor subtypes RAR/RXR and iodothyronine 5'-deiodinase, type I expression pattern in papillary thyroid tumour tissue of 26 patients in order to compare with those of the non-neoplastic thyroid tissue of the corresponding patients. The expression of selected parameters mRNA was examined by semi-quantitative RT-PCR. Papillary thyroid carcinoma (PTC) expressed RXRγ, when compared to non-neoplastic thyroid tissues of the corresponding patients that were lacking expression of RXRγ or its expression was very low. Moreover, we found significantly increased expression of RARα and RARγ in the overall group of PTC. This increase was detected in cases with positive lymph node metastasis (LNM), but not in cases with negative LNM. RARβ was significantly reduced in the subgroup of classic variant (CV). We also detected absence or significantly lower expression of hDIO1 mRNA in tumour tissue when compared to non-neoplastic tissue in both overall PTC cases and in the CV subgroup. However, the significantly decreased levels of hDIO1 mRNA were detected in cases with negative LNM but not in cases with positive LNM when compared to corresponding non-tumour tissue in both overall PTC cases and in the CV subgroup. Differences in RAR and RXR subtype mRNA expression patterns in various PTCs may contribute to the immunochemistry data available, and may thus find exploitation in clinical oncology, particularly in the differential diagnosis of thyroid neoplasms.

Topics: Adult; Aged; Carcinoma; Carcinoma, Papillary; Diagnosis, Differential; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Lymph Nodes; Male; Middle Aged; Neoplasm Metastasis; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor gamma; Retinoid X Receptors; RNA, Messenger; Thyroid Cancer, Papillary; Thyroid Neoplasms; Tretinoin

The purpose of this study was to determine the effect of all-trans retinoid acid (ATRA) on two nasopharyngeal cancer (NPC) cell lines and to evaluate the synergistic effect of ATRA used in combination with cisplatin, a standard chemotherapeutic agent for NPC. Two NPC cell lines (NPC-TW01 and NPC-TW04) were used, and the MTT assay was used to analyze cell growth inhibition under various concentrations of ATRA and cisplatin. Under low concentrations of ATRA, the growth of both cell lines was significantly inhibited. When ATRA was used in combination with cisplatin, the resulting synergistic effects were robust and significant. Only a low concentration of ATRA was required to produce a synergistic effect when combined with cisplatin, indicating the potential usefulness of a combination therapy regimen. Therefore, the combination of ATRA and cisplatin is a viable treatment option for NPC and should be further investigated.

Topics: Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Cisplatin; Drug Synergism; Drug Therapy, Combination; Growth Inhibitors; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Tretinoin; Tumor Cells, Cultured

VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed β-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Density; Carcinoma; Cell Size; Drug Screening Assays, Antitumor; Estradiol; Female; Imidazoles; Lipid Metabolism; Mammary Neoplasms, Experimental; Methylnitrosourea; Neoplasms, Hormone-Dependent; Organ Size; Ovariectomy; Rats; Rats, Sprague-Dawley; Tretinoin; Tumor Burden; Uterus

Since all-trans retinoic acid (ATRA) has shown promising results in differentiation therapy, the present study was designed to investigate the effects of ATRA on thyroid carcinoma and to evaluate the effectiveness of ATRA in redifferentiation induction of thyroid carcinoma. Therefore, we investigated cell growth rate, morphological and nuclear: cytoplasmic ratio, adherent-dependent growth, response to chemotherapy drug following differentiation, T3 and T4 measurement, and critical genes expression pattern. Papillary cell line showed more growth inhibition by ATRA, in addition, mesenchymal and spindle-shape of 8305C cells changed to polygonal. Additionally, high nuclear: cytoplasmic ratio of anaplastic decreased significantly. Redifferentiation significantly suppressed the anchorage-dependent growth in the both cell lines in a dose-dependent manner, potentiated the arsenic trioxide (ATO) effects in anaplastic and papillary cell lines. Furthermore, reduction in the expression of stemness, and invasion related genes was observed in the both cell lines. Altogether, ATRA treatment could hold the aggressive behavior of thyroid carcinoma in restraint and/or potentiate the effect of chemotherapy drug ATO.

Topics: Antigens, Differentiation; Antineoplastic Agents; Carcinoma; Cell Dedifferentiation; Cell Differentiation; Cell Line, Tumor; Cell Nucleus Size; Cell Proliferation; Cell Shape; Cell Size; Cell Survival; Gene Expression; Humans; Thyroid Neoplasms; Thyroxine; Tretinoin; Triiodothyronine

CYP46A1 is a neuron-specific cytochrome P450 that plays a pivotal role in maintaining cholesterol homeostasis in the CNS. However, the molecular mechanisms underlying human CYP46A1 expression are still poorly understood, partly because of the lack of a cellular model that expresses high levels of CYP46A1. Our previous studies demonstrated that specificity protein (Sp) transcription factors control CYP46A1 expression, and are probably responsible for cell-type specificity. Herein, we have differentiated Ntera2/cloneD1 cells into post-mitotic neurons and identified for the first time a human cell model that expresses high levels of CYP46A1 mRNA. Our results show a decrease in Sp1 protein levels, concomitant with the increase in CYP46A1 mRNA levels. This decrease was correlated with changes in the ratio of Sp proteins associated to the CYP46A1 proximal promoter. To examine if the increase in (Sp3+Sp4)/Sp1 ratio was observed in other Sp-regulated promoters, we have selected four genes--reelin, glutamate receptor subunit zeta-1, glutamate receptor subunit epsilon-1 and μ-opioid receptor--known to be expressed in the human brain and analyzed the Sp proteins binding pattern to the promoter of these genes, in undifferentiated and differentiated Ntera2/cloneD1. Our data indicate that the dissociation of Sp1 from promoter regions is a common feature amongst Sp-regulated genes that are up-regulated after neuronal differentiation.

Topics: Carcinoma; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cholesterol 24-Hydroxylase; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Nerve Tissue Proteins; Neurons; Promoter Regions, Genetic; Receptors, N-Methyl-D-Aspartate; Reelin Protein; RNA, Messenger; Sp Transcription Factors; Statistics, Nonparametric; Steroid Hydroxylases; Transcriptional Activation; Tretinoin

Pluripotent cells maintain a unique gene expression pattern and specific chromatin signature. In this study, we explored the effect of the methyltransferase inhibitor adenosine dialdehyde (AdOx) on pluripotency maintenance and gene expression in P19 embryonal carcinoma cells.. After AdOx treatment, the pluripotency-related gene network became disordered, and the early developmental genes were released from the repression. Remarkably, AdOx caused contrasting effects on the expression of two key pluripotency genes, nanog and oct3/4, with the reduction of the repressive histone marks H3K27me3, H3K9me3 and H3K9me2 only in the nanog gene.. Key pluripotency genes were controlled by different mechanisms, including the differential enrichment of repressive histone methylation marks. These data provided novel clues regarding the critical role of histone methylation in the maintenance of pluripotency and the determination of cell fate in P19 pluripotent cells.

Topics: Adenosine; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histones; Homeodomain Proteins; Mice; Nanog Homeobox Protein; Nerve Tissue Proteins; Octamer Transcription Factor-3; Time Factors; Tretinoin

The receptor for advanced glycation end products (RAGE) is a multiligand receptor protein thought to play an important role in neuronal differentiation. RAGE can bind a number of ligands and activate a variety of signalling pathways that lead to diverse downstream effects. Amphoterin and S100B are endogenous ligands, the interaction of which with RAGE is known to be involved in defined physiological processes. The present study investigated the spatiotemporal pattern of the expression for RAGE and its ligands, amphoterin and S100B, during neuronal differentiation of NT2/D1 cells. In this study, all three proteins were shown to increase with progression of neuronal differentiation as determined by Western blotting, raising the possibility that both amphoterin and S100B may interact with RAGE and have important functions during the process of cell differentiation. Moreover, blocking the activation of RAGE with neutralizing antibody in the presence of retinoic acid disrupted the progression of normal neuronal differentiation. Immunocytochemistry (ICC) studies showed that amphoterin partially colocalized with RAGE within differentiating NT2 cells, whereas S100B showed a high degree of colocalization. This result suggests that S100B is more likely to be the principal ligand for RAGE during the differentiation process and that RAGE and amphoterin might have both independent and combined roles. Moreover, RAGE was expressed only in cells that were committed to a neuronal phenotype, suggesting direct involvement of RAGE in mediating cellular changes within differentiating neuronal cells. Further detailed studies are now required to characterize fully the role of RAGE during the neuronal differentiation period.

Topics: Antibodies; Antineoplastic Agents; Carcinoma; Cell Cycle; Cell Differentiation; Cell Line, Tumor; HMGB1 Protein; Humans; Nerve Growth Factors; Neurons; Receptor for Advanced Glycation End Products; Receptors, Immunologic; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Tretinoin; Up-Regulation

Previous research in our laboratory showed that retinol inhibited all-trans retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism in vitro. The objective of the current study was to determine if dietary vitamin A supplementation inhibited metastasis of ATRA-resistant colon cancer cells in a nude mouse xenograft model. Female nude mice (BALB/cAnNCr-nu/nu, n = 14 per group) consumed a control diet (2,400 IU retinyl palmitate/kg diet) or a vitamin A supplemented diet (200,000 IU retinyl palmitate/kg diet) for 1 mo prior to tumor cell injection to preload the liver with vitamin A. HCT-116, ATRA-resistant, human colon cancer cells were intrasplenically injected. Mice continued to consume their respective diets for 5 wk following surgery. Consumption of supplemental vitamin A decreased hepatic metastatic multiplicity to 17% of control. Hepatic and splenic retinol and retinyl ester concentrations were significantly higher in the mice supplemented with vitamin A when compared to mice consuming the control diet. Supplemental vitamin A did not decrease body weight, feed intake, or cause toxicity. Thus, supplemental dietary vitamin A may decrease the overall number of hepatic metastasis resulting from colon cancer.

Topics: Animals; Antineoplastic Agents; Biotransformation; Carcinoma; Colorectal Neoplasms; Dietary Supplements; Diterpenes; Drug Resistance, Neoplasm; Female; HCT116 Cells; Humans; Liver; Liver Neoplasms, Experimental; Mice; Mice, Nude; Random Allocation; Retinyl Esters; Spleen; Tissue Distribution; Tretinoin; Vitamin A; Xenograft Model Antitumor Assays

Nasopharyngeal carcinoma is closely associated with infection with Epstein-Barr Virus (EBV); however, the mechanism is still unclear. Here, we report that the EBV oncoprotein, latent membrane protein 1 (LMP1), suppresses apoptotic cell death provoked by all-trans retinoic acid (ATRA) in NPC cells. For this purpose, LMP1 downregulated levels of Bak whilst it upregulated levels of Bcl2, lowering the ratio of Bak to Bcl2. In addition, LMP1 suppressed ATRA-mediated activation of Caspase 9, Caspase 3, and PARP but not Caspase 8 in Ad-AH cells, suggesting that LMP1 acts by blocking the activation of intrinsic apoptosis pathway by ATRA. These effects were almost completely abolished when levels of retinoic acid receptor-β(2) (RAR-β(2)) in the LMP1-expressing cells were recovered by either exogenous gene expression or treatment with a universal DNMT inhibitor, 5-Aza-2'dC, indicating that LMP1 executes its antiapoptotic effects by downregulating levels of RAR-β(2) via DNA methylation.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Carcinoma; Caspases; Cell Line, Tumor; DNA Methylation; Down-Regulation; Humans; Nasopharyngeal Neoplasms; Poly(ADP-ribose) Polymerases; Receptors, Retinoic Acid; Tretinoin; Viral Matrix Proteins

The aim of this study was to assess the clinical outcome of redifferentiation therapy using retinoic acid (RA) in combination with 131I therapy, and to identify biological parameters that predict therapeutic response in Korean patients with radioiodine-refractory papillary thyroid carcinoma (PTC).. A total of 47 patients (13 men, 34 women; age 54.2±13.6 years) with radioiodine-refractory PTC underwent therapy consisting of consecutive treatment with 131I and RA. Each 131I/RA treatment cycle involved the administration of oral isotretinoin for 6 weeks at 1-1.5 mg/kg daily followed by a single oral dose of 131I (range 5.5-16.7 GBq). Therapeutic responses were determined using serum thyroglobulin (Tg) levels and the change in tumour size 6 months after completing the 131I/RA therapy. Biological parameters and pathological parameters before and after combined therapy were compared.. After completing 131I/RA therapy, 1 patient showed a complete response, 9 partial response, 9 stable disease, and 28 progressive disease, representing an overall response rate of 21.3%. Univariate analysis revealed that an age of <45 years and a persistently high serum Tg level were related to a good response. No clinical response was achieved when metastases showing no iodine uptake were present. Multivariate regression analysis showed that an age of <45 years was significantly associated with a good response. Of the 24 patients with well-differentiated carcinoma, 5 (20.8%) responded to 131I/RA therapy, whereas all 6 patients with poorly differentiated carcinoma failed to respond.. 131I/RA therapy was found to elicit a response rate of 21.3% among patients with radioiodine-refractory PTC, and an age of <45 years was found to be significantly associated with a good response.

Topics: Carcinoma; Carcinoma, Papillary; Combined Modality Therapy; Female; Humans; Iodine Radioisotopes; Male; Middle Aged; Republic of Korea; Retrospective Studies; Thyroid Cancer, Papillary; Thyroid Neoplasms; Treatment Failure; Tretinoin

FERM domain containing protein 7 (FRMD7) mutations are associated with X-linked idiopathic congenital nystagmus (ICN). The purpose of this study is to identify a novel splice variant of FRMD7 (FRMD7-S) in both humans and mice with a shortened exon 4 relative to the original form of FRMD7 (FRMD7-FL),and to detect the role of FRMD7-FL and FRMD7-S in the process of neuronal development.. The splice variant of FRMD7 was identified by PCR. Expression levels of hFRMD7-FL and hFRMD7-S transcripts in developing human fetal brain were tested by RT-PCR, and expression levels in the human pluripotent embryonic carcinoma NTera 2/cl.D1 (NTERA-2; NT2) cell line with all-trans retinoic acid (ATRA) or bone morphogenetic protein-2 (BMP-2) treatment were tested by real-time qPCR. hemaglutinin (HA)-tagged recombinant plasmids DNA encoding hFRMD7-FL and Myc-tagged recombinant plasmids DNA encoding hFRMD7-S were used to transiently transfect the human NT2 cells. Further, immunofluorescence experiments were performed to determine the co-localization of the two fusion proteins. Finally, using co-immunoprecipitation analyses, we demonstrated that FRMD7-FL and FRMD7-S interacted with each other.. A novel splice variant of FRMD7 (FRMD7-S) with a shortened exon 4 relative to the original form of FRMD7 (FRMD7-FL) was identified from the cDNA of the human NT2 cell line and mouse fetal brain. The FRMD7 transcripts showed similar tissue distributions and were upregulated following all trans retinoic acid (ATRA)-induced differentiation of NT2 cells. FRMD7-FL and FRMD7-S co-localized and co-immunoprecipitated with each other. Further, overexpression of FRMD7-FL in NT2 cells resulted in altered neurite development and upregulation of FRMD7-S.. Although the significance of the 45 bp deletion remains unknown, our observations suggest that the FRMD7 isoforms may play a significant role during neuronal differentiation and development.

Topics: Alternative Splicing; Animals; Bone Morphogenetic Protein 2; Brain; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; Exons; Fetus; Gene Expression Regulation, Developmental; Humans; Membrane Proteins; Mice; Neoplasms, Germ Cell and Embryonal; Neurons; Nystagmus, Congenital; Plasmids; Protein Isoforms; Real-Time Polymerase Chain Reaction; Recombinant Fusion Proteins; Transfection; Tretinoin

NeuroD, a basic helix-loop-helix transcription factor, is capable of converting embryonic epidermal cells into neuronal cells. However, whether histone deacetylases (HDACs) are involved in the autoregulation of neuroD or not is unclear. In this study, neuroD expression was found to be significantly increased in the all-trans retinoid acid-treated P19 cells. Meanwhile, neuroD could itself enhance its promoter activity and mRNA expression. By using specific inhibitors to histone modification enzymes, HDAC3 was identified to specifically augment the autoactivation of neuroD in P19 cells. The data suggest that the elevation of HDAC3 and neuroD in all-trans retinoid acid-treated cells exponentially increases the neuroD expression and mediates an early commitment of P19 cells for neuronal differentiation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma; Cell Differentiation; Cell Line; Chromatin Immunoprecipitation; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylases; Homeostasis; Humans; Luciferases; Mice; Nerve Tissue Proteins; RNA, Messenger; Transfection; Tretinoin

The effectiveness rate of all-trans-retinoic acid (RA) is only about 30% in the clinical application of inducing thyroid carcinoma differentiation. In addition, there are severe toxic side effects, which limit its clinical application. Phase I-III clinical studies have been conducted on the combined application of two or more kinds of inductors in tumors. Nevertheless, the combination of RA with histone deacetylase inhibitors is rarely reported. This article studied the effects of differentiation for papillary thyroid carcinoma and follicular thyroid carcinoma cell lines induced by RA combined with trichostatin A (TSA), enhancing the effect of induction, while reducing the toxic side effects of a single drug, to provide a theoretical basis for preclinical trials.. After incubation with RA combined with TSA, K1 and FTC-133 were grouped into Group 1 (RA 10(-4) mol/L plus TSA 1.65 x 10(-7) mol/L), Group 2 (RA 1 x 10(-4) mol/L plus TSA 3.31 x 10(-7) mol/L), Group 3 (RA 10(-5) mol/L plus TSA 1.65 x 10(-7) mol/L), Group 4 (RA 1 x10(-5) mol/L plus TSA 3.31 x 10(-7) mol/L) by four varied concentrations and three time points (12 h, 24 h, and 48 h). The cell proliferation, conformation, toxic effect, and induced differentiation on K1 and FTC-133 cell lines were studied microscopically with hematoxylin-eosin (HE) to observe cell quantity and morphology, methyl-thiazolyl-tetrazolium (MTT) to calculate cell survival rates, and electrochemiluminescence analysis measuring in vitro thyroglobulin (Tg) levels.. The research showed that K1 and FTC-133 cells had cell spacing increases, with an outer edge of smooth, nuclear chromatin condensation after RA combined TSA. Survival rate were assessed by an analysis of variance (ANOVA) by concentration and time point, F values of K1 and FTC-133 were 23.52 and 170.14, and 57.09 and 224.35, respectively. There were significant differences for both cells (P < 0.01). The SNK analysis indicated that survival rates were in the order of Group 2 < Group 1 < Group 4 < Group 3. Tg was also assessed by ANOVA, F values of K1 were 69.63 and 101.07, and F values of FTC-133 were 79.77 and 81.72 (P < 0.01). Group 1 was compared with Group 3 of K1 and FTC-133 by the least significant difference (LSD) method, and there was no statistical difference between the two group (P = 0.06, 0.2, respectively; P > 0.05), yet a significant difference was seen between the other Groups.. Lower concentrations of RA combined with lower concentrations of TSA have both inhibited cell proliferation, decreased toxicity of the drugs, and increased the effect of K1 and FTC-133 cell differentiation. The mechanism of action may be that TSA has pretranscription DNA regulation and that RA has posttranscriptional signal regulation to enhance the effects of inhibited proliferation and differentiation of cells by transcription systems.

Topics: Adenocarcinoma, Follicular; Antineoplastic Agents; Apoptosis; Carcinoma; Carcinoma, Papillary; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Thyroglobulin; Thyroid Cancer, Papillary; Thyroid Neoplasms; Tretinoin

Our previous studies demonstrated that retinoic acid (RA)-induced reduction of both, the key glycolytic enzyme ENO1 and proliferation-promoting c-Myc, resulted in decreased vitality and invasiveness of the follicular thyroid carcinoma cell lines FTC-133 and FTC-238. By employing two-dimensional electrophoresis and mass spectrometry, we identified proteins affected by RA treatment. In addition to previously reported decrease in ENO1 expression, we found that RA led to significantly reduced levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase isoenzymes M1/M2 (PKM1/M2), peptidyl-prolyl cis-trans isomerase A (PPIA), transketolase (TKT), annexin A2 (ANXA2), glutathione S-transferase P (GSTP1) and peroxiredoxin 2 (PRDX2) as compared to untreated control. The same proteins investigated on thyroid tissues were found to be significantly up-regulated in follicular, papillary and undifferentiated thyroid carcinomas when compared with goiter and adenoma tissues. These findings identify new target proteins for RA-mediated anti-tumor and re-differentiation therapies and provide novel insights into treatments for thyroid carcinoma.

Topics: Adolescent; Adult; Aged; Biomarkers, Pharmacological; Biomarkers, Tumor; Carcinoma; Cell Line, Tumor; Female; Humans; Male; Metabolome; Middle Aged; Proteomics; Thyroid Neoplasms; Tretinoin; Young Adult

Retinoic acid presently is the most advanced agent able to improve the efficacy of radioiodine therapy in differentiated thyroid carcinoma. In order to identify compounds with higher efficacy a panel of pharmacologically well-characterized compounds with antitumour action in solid cancer cell lines was screened.. The effects of the compounds on iodide uptake, cell number, proliferation and apoptosis were evaluated.. In general, compounds were more effective in cell lines derived from more aggressive tumours. The effectiveness in terms of number of responsive cell lines and maximal increase in iodide uptake achieved decreased in the order: APHA > valproic acid approximately sirolimus approximately arsenic trioxide > retinoic acid approximately lovastatin > apicidine approximately azacytidine approximately retinol approximately rosiglitazone approximately bortezomib.. We hypothesize that testing of cells from primary tumours or metastases in patients may be a way to identify compounds with optimum therapeutic efficacy for individualized treatment.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Boronic Acids; Bortezomib; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Iodides; Lovastatin; Neoplasm Metastasis; Oxides; Pyrazines; Sirolimus; Thymus Gland; Thyroid Neoplasms; Tretinoin

The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) beta is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RAR beta could act as a tumor suppressor. However, the status of RAR beta in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RAR alpha, RAR beta, and RAR gamma using AM580 (a RAR-specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RAR beta and RAR gamma mRNA was significantly induced by AM580, compared with vehicle controls, whereas RAR alpha mRNA was significantly attenuated by AM580, compared with vehicle. RAR beta was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RAR beta mRNA expression in the Ishikawa cell line, and the expression level of RAR beta in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma.

Topics: Antineoplastic Agents; Benzoates; Carcinoma; Cell Proliferation; Endometrial Hyperplasia; Endometrial Neoplasms; Endometrium; Female; Gene Expression; Humans; Receptors, Retinoic Acid; RNA, Messenger; Tetrahydronaphthalenes; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) displays potent anticarcinogenic activities that are mediated by the nuclear retinoic acid receptors (RARs). However, use of RA in oncology is limited by RA resistance acquired during carcinogenesis. Moreover, in some cancers, RA facilitates rather than inhibits growth. A clue to this paradoxical behavior was recently suggested by the findings that RA also activates PPARbeta/delta, a receptor involved in mitogenic and anti-apoptotic activities. The observations that partitioning of RA between its two receptors is regulated by two intracellular lipid-binding proteins-CRABP-II, which targets RA to RAR, and FABP5, which delivers it to PPARbeta/delta-further suggest that RA resistance may stem from the deregulation of the binding proteins, resulting in activation of PPARbeta/delta rather than RAR. Here, we show that, in the RA-resistant mouse model of breast cancer MMTV-neu, RA indeed activates the nonclassical RA receptor PPARbeta/delta. This behavior was traced to an aberrantly high intratumor FABP5/CRABP-II ratio. Decreasing this ratio in mammary tissue diverted RA from PPARbeta/delta to RAR and suppressed tumor growth. The data demonstrate the existence of a mechanism that underlies RA resistance in tumors, indicate that CRABP-II functions as a tumor suppressor, and suggest that the inhibition of FABP5 may comprise a therapeutic strategy for overcoming RA resistance in some tumors.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Fatty Acid-Binding Proteins; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Neoplasm Proteins; PPAR delta; PPAR-beta; Receptors, Retinoic Acid; Transcription, Genetic; Tretinoin

Nurr1 has been implicated as a transcription factor mediating the endogenous neuroprotective mechanism against stroke. We examined the in vivo and in vitro properties of a new human embryonic carcinoma Ntera-2 cell line carrying the human Nurr1 gene (NT2N.Nurr1). Adult Sprague-Dawley rats underwent experimental stroke initially and 14 days later were assigned randomly to receive stereotaxic transplantation of NT2N.Nurr1 cells or infusion of vehicle into their ischemic striatum. Transplantation of NT2N.Nurr1 cells promoted significant attenuation of behavioral impairments over a 56-day period after stroke, characterized by decreased hyperactivity, biased swing activity, and neurologic deficits, as well as significant reduction in ischemic striatal cell loss compared to vehicle-infused stroke animals. Transplanted NT2N.Nurr1 cells survived and expressed neuronal phenotypic markers in the ischemic striatum. In vitro results showed that cultured NT2.Nurr1 cells were already negative for nestin even before retinoic acid treatment, despite strong nestin immunoreactivity in NT2 cells. This indicates Nurr1 triggered a rapid commitment of NT2 cells into a neuronal lineage. Indeed, NT2.Nurr1 cells, at 4 weeks into RA treatment, displayed more abundant tyrosine hydroxylase positive cells than NT2 cells. Parallel ELISA studies showed further that cultured NT2N.Nurr1, but not NT2N cells, secreted glial cell derived neurotrophic factor. The present study shows efficacy of NT2N.Nurr1 cell grafts in ischemic stroke, with in vitro evidence suggesting the cells' excellent neuronal differentiation capability and ability to secrete GDNF as likely mechanisms mediating the observed therapeutic benefits.

Topics: Animals; Behavior, Animal; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Transplantation; Corpus Striatum; Disease Models, Animal; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Glial Cell Line-Derived Neurotrophic Factor; Humans; Male; Motor Activity; Multivariate Analysis; Nerve Tissue Proteins; Nuclear Receptor Subfamily 4, Group A, Member 2; Rats; Rats, Sprague-Dawley; Stroke; Time Factors; Transcription Factors; Tretinoin

Studies were performed to determine the regulation of DMT1 (divalent metal transporter 1) during RA (retinoic acid)-induced differentiation of P19 embryonic carcinoma cells. Protein and mRNA expression for the +/-IRE (iron response element) forms of DMT1, but not the 1A isoform, were down-regulated within the first few hours upon removal of RA, at which time the cells began to differentiate. The turnover of the +/-IRE isoforms of DMT1 protein during this period was found to be dependent on both the proteasomal and lysosomal pathways. Changes in mRNA levels were shown to be regulated by nitric oxide produced by the induction of neuronal nitric oxide synthase after removal of RA. Nitric oxide functions by inhibiting NF-kappaB (nuclear factor kappaB) nuclear translocation and the subsequent binding to the putative NF-kappaB response element (at -19 to -23) within the 1B promoter. Gel-shift analysis and chromatin immunoprecipitation assay indicated that nuclear NF-kappaB is capable of binding to this response element and that binding decreases during early stages of differentiation. Luciferase reporter gene assay demonstrated that a mutation in this binding domain leads to decreased activity. These results demonstrate that during neuronal differentiation of P19 cells, there is a decrease in specific isoforms of DMT1 via both post-translational and transcriptional mechanisms.

Topics: Carcinoma; Cation Transport Proteins; Cell Differentiation; Cell Line; Humans; Immunoglobulins; Iron-Binding Proteins; NF-kappa B; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Protein Processing, Post-Translational; RNA, Messenger; Time Factors; Transcription, Genetic; Tretinoin

Although primary neuronal cell cultures, usually obtained from embryonic or early postnatal rodents, have been used in vitro to study the neural cannabinoid signalling system, development of cell lines with neural properties exhibiting native expression of cannabinoid receptors is desirable. This study was undertaken to investigate the expression of CB1 and CB2 cannabinoid receptors in neurons that develop from retinoic acid (RA)-primed mouse P19 embryonal carcinoma cells. Both undifferentiated P19 cells and RA-treated P19 neurons were positive, by using reverse transcription-polymerase chain reaction (RT-PCR), for CB1 (but not CB2) mRNA. Neuronal differentiation increased the CB1 mRNA expression, and Western blotting with a CB1 receptor antibody showed a strong immunoreactive band at approximately 62 kDa in membranes from P19-derived neurons. The cannabinoid receptor agonists CP 55,940 and HU-210 produced concentration-dependent inhibition of forskolin-induced (3 microM) cyclic AMP production in the P19-derived neurons (29% at 1 microM CP 55,940 and 34% at 1 microM HU-210), which could be blocked by the CB1-selective receptor antagonist AM251, but not by the CB2-selective antagonist AM630. Furthermore, glutamate (100 microM) induced a sustained increase in [Ca2+]i in P19-derived neurons that could be concentration-dependently blocked by the cannabinoid receptor agonists WIN 55,212-2. Thus, the protocol used provides an in vitro model system expressing CB1 cannabinoid receptors at the level of mRNA, protein, and AM251-sensitive agonist-induced inhibition of intracellular cyclic AMP accumulation, which may be useful to investigate the developmental regulation, expression and function of neuronal cannabinoid receptors.

Topics: Analysis of Variance; Animals; Antineoplastic Agents; Benzoxazines; Blotting, Western; Calcium; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; gamma-Aminobutyric Acid; Gene Expression; Mice; Morpholines; Naphthalenes; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Temperature; Tretinoin; Tritium

Although it is uncommon, pancreatic cancer is known to have a poor prognosis. The aim of the present study was to determine the inhibitory effects of all-trans-retinoic acid (ATRA) on cell growth, cell cycle and alkaline phosphatase (ALP) activity in the human pancreatic cancer cell line PANC-1 in vitro.. Human pancreatic cancer PANC-1 cells were treated by various concentrations of ATRA, and then the cell growth was determined by MTT viability assay. Cell cycle distribution and ALP activity were analyzed by flow cytometry and chemical analyzer, respectively.. ATRA inhibited the growth of PANC-1 cells grown in culture; a dose-dependent inhibitory influence was found. ATRA arrested PANC-1 cells at G2/M phase. The ALP activity of PANC-1 cells was significantly increased by 1-50 micromol/L ATRA.. The antitumor effects of ATRA on human pancreatic cancer cells are associated with G2/M phase arrest and increased ALP activity.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma; Cell Cycle; Cell Line, Tumor; DNA Fragmentation; Flow Cytometry; Humans; In Vitro Techniques; Pancreatic Neoplasms; Treatment Outcome; Tretinoin

The plasma membrane glycoprotein sodium/iodide symporter (NIS) is crucial for thyroid hormone biosynthesis and mediates the iodide uptake of thyrocytes. It has been shown that retinoic acid (RA) alters NIS gene expression in thyroid carcinoma lines and stimulates their iodide uptake. Here, we generated an ARO human thyroidal cancer cell line that expresses the NIS gene (ARO-NIS) and found that its baseline 125I uptake was threefold higher than that of its parental ARO cells. However, a 1-microM all-trans retinoic acid (tRA) treatment significantly increased this 125I uptake up to approximately approximately 6.5-fold on Day 3. tRA also elevated NIS mRNA expression in ARO-NIS cells, with peaks of expression being observed on Day 3. To investigate the underlying genomic mechanisms involved in these tRA-induced phenotypic changes, we subjected tRA-treated and untreated ARO-NIS cells to cDNA microarray analysis. Of 1152, genes spotted onto the microarray membrane, 18 were up-regulated (z ratio>2.0) and 33 were down-regulated (z ratio<-2.0) in ARO-NIS cells after 3 days of tRA treatment. More specifically, tRA increased the expression of BCL3, CSRP3, v-fos, and CDK5 genes and decreased the expression of the FGF12 and IGFBP6 genes. Thus, tRA treatment of human anaplastic thyroid carcinoma cells stably expressing the NIS gene significantly elevates their NIS-mediated radioiodine uptake and alters the expression of many genes involved in cell growth and cellular differentiation. Therefore, tRA treatment and NIS gene transfection are potential tools for the diagnosis and treatment of thyroid cancer.

Topics: Carcinoma; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Metabolic Clearance Rate; Neoplasm Proteins; Radiopharmaceuticals; Symporters; Thyroid Neoplasms; Tretinoin

The distribution of the cancer-associated protein p62 in human gastric carcinoma (BGC-823) cells was examined by confocal laser scanning microscopy and electron microscope immunocytochemistry. In control cells p62 was cytoplasmic in location and concentrated in the cytoplasmic matrix, but when cell growth was inhibited by treatment with 50 microM all-trans-retinoic acid (ATRA) for 5 days, p62 expression decreased and the protein was translocated from cytoplasm to nucleus. Ultrastructural localization using gold particles showed that p62 was bond mainly to a linear structure in nucleus. The speculation that p62 binds Insulin-like growth factor (IGF)-II mRNA indicates its probable involvement in the posttranscriptional IGF-II mRNA processing and p62 could play a role in tumorgenesis by regulating the expression of IGF-II. Further studies will be needed to confirm this view.

Topics: Carcinoma; Cell Line, Tumor; Cell Nucleus; Humans; Microscopy, Confocal; RNA-Binding Proteins; Stomach Neoplasms; Tretinoin

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in P19 embryonic stem cells but is first suppressed and reactivated during retinoic acid (RA)-induced neuronal differentiation. However, no RA response element (RARE) can be found in this gene regulatory region. The suppression and reactivation of the KOR gene in this neuronal differentiation model suggested chromatin remodeling occurred on this gene promoter triggered by RA induction. This study asks whether RA induces alteration in the nucleosomal structure of this gene promoter that has no apparent RARE and, if so, how RA remodels chromatin of this promoter. The results revealed two loose nucleosomes, N1 at -44 (3' boundary) from the transcription initiation site and N2 spanning the transcription initiation site, that are relevant to active transcription. RA formed a repressive chromatin configuration of this promoter by compacting nucleosome N1, followed by nucleosome N2 condensation. Chromatin immunoprecipitation assay demonstrated RA induced replacement of the c-Myc/Max complex with the Max/Mad1 complex on the E box located within nucleosome N1, coinciding with reduced Sp1 binding to GC boxes located within nucleosome N2 and recruitment of chromatin remodeling factor Brahma-related gene 1 (BRG-1) to this promoter. Consistently, histone deacetylation, Lys9 methylation, and hypophosphorylation of RNA polymerase II C-terminal domain were detected on this promoter after RA treatment. It is concluded that RA induces KOR gene suppression, as early neuronal differentiation marker, by inducing substitution of c-Myc/Max with Max/Mad on the E box and by BRG-1 involved nucleosome recruitment and chromatin condensation, thereby abolishing Sp1 binding.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Southern; Blotting, Western; Carcinoma; Cell Line, Tumor; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; Chromosome Mapping; DNA Helicases; Gene Expression Regulation; Histone Deacetylase 1; Histone Deacetylases; Histones; Mice; Nuclear Proteins; Nucleosomes; Receptors, Opioid, kappa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription Factors; Tretinoin

We investigated the preventive effects of a synthetic acyclic retinoid, NIK-333, on the early and late events of hepatocarcinogenesis in male F344 rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). NIK-333 was administered once a day on consecutive days at a dose of 10, 40, or 80 mg/kg body weight along with the supplementation with 3'-MeDAB-containing diet for 16 wk. Animals from each group were sacrificed at 4 and 16 wk after the commencement of the experiment to determine the effect of NIK-333 on the early and late stages of carcinogenesis, respectively. NIK-333 suppressed the emergence of both oval-like cells expressing transforming growth factor (TGF)-alpha, putative progenitors of hepatocellular carcinoma (HCC), and activated hepatic stellate cells, major matrix-producing cells of the liver, in the early stage and inhibited the incidence of HCC in the late phase. These results suggest that NIK-333 is a promising drug for the chemoprevention of HCC by uniquely suppressing the early events of hepatocarcinogenesis, that is, development of both oval-like cells and fibrogenesis.

Topics: Actins; Adenoma; Animals; Antineoplastic Agents; Carcinoma; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Fibrosis; Liver; Liver Neoplasms, Experimental; Male; Methyldimethylaminoazobenzene; Rats; Rats, Inbred F344; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor alpha; Tretinoin

In previous studies we have shown that all-trans retinoic acid (atRA)-treatment of the atRA-sensitive ovarian carcinoma cell line CA-OV3 repressed AP-1 activity by about 50%, while a similar effect was not observed in the atRA-resistant ovarian carcinoma cell line, SK-OV3. These results suggested that the repression of AP-1 activity may be one of the mechanisms by which atRA inhibits the growth of atRA-sensitive CA-OV3 cells. In the present studies, we investigated further the molecular mechanism by which AP-1 activity is repressed by atRA. We show that the repression of AP-1 activity correlates with an increase in JunB protein expression and a decrease in N-terminal phosphorylation of c-Jun. The decrease in N-terminal phosphorylation of c-Jun does not appear to be modulated by JNK or ERK, since their protein expression patterns and kinase activity do not correlate with the repression of AP-1 activity following treatment with atRA. However, the activity of the protein phosphatase PP2A was found to increase 24 h following atRA treatment in CA-OV3 cells. Moreover, the catalytic subunit of PP2A was found to associate with c-Jun in vivo following atRA treatment. Since the inhibition of AP-1 activity following atRA treatment of CA-OV3 cells was abolished in the presence of specific PP2A inhibitors, it is likely that PP2A plays an important role in the atRA-induced repression of AP-1.

Topics: Carcinoma; Cell Line, Tumor; Dimerization; Down-Regulation; Female; Humans; JNK Mitogen-Activated Protein Kinases; Ovarian Neoplasms; Phosphoprotein Phosphatases; Phosphorylation; Serine; Transcription Factor AP-1; Tretinoin

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.

Topics: Abnormalities, Drug-Induced; Aging; Aldehyde Oxidoreductases; Animals; Animals, Newborn; Body Patterning; Bromodeoxyuridine; Carcinoma; Cell Death; Cell Differentiation; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Embryo, Mammalian; Embryonic Induction; Extremities; Female; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Galactosides; Gene Expression Regulation, Developmental; High Mobility Group Proteins; Homeodomain Proteins; In Situ Hybridization; In Situ Nick-End Labeling; Indoles; Limb Buds; Male; Mesoderm; Mice; Mice, Knockout; Models, Biological; Pregnancy; Retinoic Acid 4-Hydroxylase; SOX9 Transcription Factor; Time Factors; Trans-Activators; Transcription Factors; Transfection; Tretinoin

Retinoids, which include vitamin A (retinol; ROL) and its derivatives, have been investigated in the treatment of bladder cancer. We have shown that expression of the enzyme lecithin:ROL acyltransferase (LRAT), which converts ROL to retinyl esters, is reduced in several human cancers. Here we evaluated expression of LRAT protein and mRNA in normal and malignant bladder tissue specimens from human patients. We also examined the effect of retinoids on LRAT expression in bladder cancer cell lines.. We evaluated 49 bladder cancer specimens for LRAT protein expression using immunohistochemistry with affinity-purified antibodies to human LRAT. LRAT mRNA expression was assessed using reverse transcription-PCR in bladder specimens from an additional 16 patients. We examined the effect of retinoic acid and ROL on LRAT mRNA expression in five human bladder cancer cell lines.. LRAT protein was detected throughout the nonneoplastic bladder epithelium in all of the specimens. In bladder tumors, LRAT protein expression was reduced compared with the nonneoplastic epithelium or was completely absent in 7 of 32 (21.9%) superficial tumors versus 16 of 17 (94.1%) invasive tumors (P < 0.001). All of the non-neoplastic bladder specimens tested (11 of 11) showed LRAT mRNA expression, compared with 5 of 8 (62%) superficial tumors and 0 of 5 (0%) invasive tumors (P = 0.001). Three of five human bladder cancer cell lines expressed LRAT mRNA independent of retinoid exposure, whereas in two cell lines LRAT mRNA expression was induced by retinoid treatment.. We report a significant reduction in LRAT expression in bladder cancer. Moreover, we demonstrate an inverse correlation of LRAT mRNA and protein expression with increasing tumor stage. These data suggest that loss of LRAT expression is associated with invasive bladder cancer.

Topics: Acyltransferases; Adult; Aged; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Disease Progression; Esters; Extracellular Matrix; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Prognosis; Proportional Hazards Models; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; Risk; Risk Factors; RNA, Messenger; Tretinoin; Urinary Bladder Neoplasms

Urinary bladder epithelial cells play an important role in the host defense against urinary tract infections. Interferon-gamma (IFN-gamma) is a potent cytokine that regulates immune responses by inducing multiple genes in many types of cells including urinary bladder epithelial cells. Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH-box family, which is involved in various reactions related to RNA metabolism, and is induced in leukemic cells by retinoic acid or in endothelial cells by lipopolysaccharide. We have studied the expression of RIG-I in T24 cells, a cell line derived from human urinary bladder epithelial carcinoma cells. IFN-gamma stimulated T24 cells to express RIG-I mRNA and protein in concentration- and time-dependent manners. Immunohistochemical analysis revealed the expression of RIG-I in the urinary bladder epithelium from a patient with chronic urinary tract infection and in a bladder epithelial carcinoma. We conclude that RIG-I may play some role in inflammatory reactions in the urinary tract epithelium.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma; Cell Line, Tumor; DEAD Box Protein 58; DEAD-box RNA Helicases; Dose-Response Relationship, Drug; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interferon-gamma; Interleukin-4; Kinetics; Receptors, Immunologic; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Helicases; RNA, Messenger; Tretinoin; Up-Regulation; Urinary Bladder Neoplasms

During early rodent development, the parietal endoderm appears from an inner cell mass and produces large amounts of basement membrane components, such as laminin-1 and collagen IV. To elucidate the regulatory network for gene expression during these procedures, we constructed a series of short interfering RNA expression vectors targeted to various transcription factors, transfected them into F9 embryonal carcinoma cells, and evaluated the effects of the gene silencing on the induction of parietal endoderm differentiation and basement membrane component production by treating F9 cells with all trans-retinoic acid and dibutyryl cyclic AMP. Among the transcription factors tested, silencing of Sox7 or combined silencing of Gata-4 and Gata-6 resulted in suppression of cell shape changes and laminin-1 production, which are the hallmarks of parietal endoderm differentiation. In cells silenced for Sox7, induction of Gata-4 and Gata-6 by retinoic acid and cyclic AMP treatment was inhibited, while induction of Sox7 was not affected in cells silenced for Gata-4 and Gata-6, indicating that Sox7 is an upstream regulatory factor for these Gata factors. Nevertheless, silencing of Sox7 did not totally cancel the action of retinoic acid, since upregulation of coup-tf2, keratin 19, and retinoic acid receptor beta2 was not abolished in Sox7-silenced F9 cells. Although overexpression of Sox7 alone was insufficient to induce parietal endoderm differentiation, overexpression of Gata-4 or Gata-6 in Sox7-silenced F9 cells restored the differentiation into parietal endoderm. Sox7 is therefore required for the induction of Gata-4 and Gata-6, and the interplay among these transcription factors plays a crucial role in parietal endoderm differentiation.

Topics: Animals; Basement Membrane; Blotting, Western; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Collagen Type IV; Cyclic AMP; DNA-Binding Proteins; DNA, Complementary; Endoderm; GATA4 Transcription Factor; GATA6 Transcription Factor; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Vectors; High Mobility Group Proteins; Laminin; Mice; Models, Biological; Plasmids; Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; SOXF Transcription Factors; Time Factors; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Up-Regulation

The prognosis of advanced colorectal carcinoma (CC) is poor. Established chemotherapy shows only limited efficacy but significant side effects. We investigated how far a combination of tamoxifen (TAM), 9-cis-retinoic acid (CRA) and the fluoroquinolone ciprofloxacin (CIP) synergize to inhibit proliferation and promote apoptosis of CC cells in vitro. The CC cell lines LOVO, CC-531 and SW-403 were incubated with TAM, CRA and CIP (10(minus;4)-10(minus;6) M) as single agents and in combination. Cell proliferation was assessed by bromodeoxyuridin incorporation. Apoptosis was quantified immunohistochemically and by FACS analysis after staining with propidium iodide. Changes in the expression of caspase 3, bax, bcl-2 and p21cip/waf were assessed by quantitative Western blotting. CRA and TAM monotherapy was moderately effective. Their combination enhanced apoptosis from 60% to more than 80% in all cell types. Apoptosis was paralleled by inhibition of proliferation and further potentiated by addition of CIP. The combination effectively up-regulated caspase 3 and bax and down-regulated bcl-2 and p21cip/waf. Combinations of biomodulaters act synergistically to inhibit proliferation and promote apoptosis in CC cells. Due to their known safety profile, this justifies clinical trials for colorectal cancer using combinations of these biological response modifiers.

Topics: Alitretinoin; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bromodeoxyuridine; Carcinoma; Caspase 3; Caspases; Cell Division; Cell Line, Tumor; Cell Separation; Ciprofloxacin; Colonic Neoplasms; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunohistochemistry; Indicators and Reagents; Kinetics; Prognosis; Propidium; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Time Factors; Tretinoin

Retinoic acid (RA) receptor (RAR) beta2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RAR beta2 promoter and 5'-UTR is the underlying cause. Here we show that RAR alpha2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR alpha2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR alpha2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR alpha2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR alpha2 and RAR beta2. On the basis of a clonogenic assay, RAR alpha2 displayed ligand-dependent growth-suppressive activity similar to that of RARb eta2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR alpha2-transfected cells compared with 75 and 77%, respectively, in RAR beta2-transfected cells. We conclude that the silencing of the RAR alpha2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.

Topics: Breast Neoplasms; Carcinoma; Cell Division; DNA Methylation; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and its derivatives inhibit the proliferation of normal human mammary epithelial cells (HMEC) and some breast carcinoma lines by mechanisms which are not fully understood. To identify genes that mediate RA-induced cell growth arrest, an HMEC cDNA library was synthesized and subtractive screening was performed. We identified the interleukin-1beta (IL-1beta) gene as an RA induced gene in HMEC. Northern blot analyses showed that the IL-1beta gene was up-regulated as early as 2 h after RA treatment. Results from the treatment of HMEC with cycloheximide and actinomycin D indicated that the regulation of the IL-1beta gene by RA occurred at the transcriptional level and that the IL-1beta gene is a direct, downstream target gene of RA. To evaluate the effects of IL-1beta on cell proliferation, the proliferation of HMEC was measured in the presence of RA or IL-1beta, or both. Either RA or IL-1beta could significantly inhibit the proliferation of HMEC. However, the addition of soluble IL-1 receptor antagonist (sIL-1ra) to the cell culture medium did not block RA-induced HMEC growth inhibition, whereas sIL-1ra did block the growth inhibition of HMEC by IL-1beta. IL-1beta expression was not observed in the three carcinoma cell lines, MCF-7, MDA-MB-231, and MDA-MB-468, as compared to the HMEC. Growth curves of the breast carcinoma cell lines showed strong inhibitory effects of RA and IL-1beta on the growth of the estrogen receptor (ER) positive MCF-7 cell line, but only a small effect on the ER negative MDA-MB-231 cells. The expression of the IL-1beta gene was also transcriptionally activated by RA in normal epithelial cells of prostate and oral cavity. Our results suggest that: (a) the IL-1beta gene is a primary target of RA receptors in HMEC; (b) the enhanced expression of the IL-1beta gene does not mediate the RA-induced growth arrest of HMEC; and (c) the expression of the IL-1beta gene is low or absent in all three human breast carcinoma cell lines examined, but the defect in the IL-1beta signaling pathway may be different in ER positive versus ER negative carcinoma cells.

Topics: Breast; Breast Neoplasms; Carcinoma; Cell Division; Cycloheximide; Dactinomycin; Drug Synergism; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Kinetics; Male; Prostate; Protein Synthesis Inhibitors; Receptors, Estrogen; Receptors, Retinoic Acid; Recombinant Fusion Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The effects of E2 are dependent on ERs and local E2 concentration in target cells. Modulation of intracellular E2 concentration involves the action of 17beta-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme converting E2 to estrone. In the present study, the influence of RAs on the growth of endometrial cancer cell line RL 95-2 as well as the expression of ERs and 17HSD type 2 have been investigated. It was found that RAs repress the growth of RL 95-2 cells, which express all subtypes of RXR and RAR, as examined by RT-PCR. Also, quantitative RT-PCR analysis showed that both ERalpha and ERbeta are present in RL 95-2 cells, and Western blot assay further revealed that ERalpha expression was decreased by all trans-RA treatment. In contrast, RAs induced 17HSD type 2 mRNA expression in a dose- and time-dependent fashion. This stimulatory effect was also detected at the level of in vivo oxidative 17HSD activity in cultured cells. On the other hand, the abundance of 17HSD type 2 mRNA was not altered by RAs in cultured normal epithelial cells isolated from human early- and late-secretory endometrium. The data indicate that RAs have an inhibitory effect on the growth of RL 95-2 cells and a cross-talk with the estrogen pathway in estrogen-responsive endometrial cancer cells.

Topics: 17-Hydroxysteroid Dehydrogenases; Carcinoma; Cell Division; Cells, Cultured; Endometrial Neoplasms; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Isoenzymes; Oxidation-Reduction; Receptors, Estrogen; Receptors, Retinoic Acid; Reference Values; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin

Peroxisome proliferator-activated receptor gamma (PPARgamma) forms a heterodimeric DNA-binding complex with the retinoid X receptor (RXR) and regulates the transcription of its target genes. Activation of PPARgamma has been shown to induce G1 arrest and to inhibit cell growth of human pancreatic carcinoma cell lines. The purpose of the present study was to examine the effect of ligand activation of PPARgamma and RXR on cell growth and on the expression of G1 cyclins in a pancreatic cancer cell line PANC-1, which expresses PPARgamma at high levels. Troglitazone, a specific ligand for PPARgamma, was found to cause a reduction in the growth rate and induced G1 cell cycle arrest and this effect was additive with that of 9-cis retinoic acid (9-cis RA), a ligand for RXR. Of the G1 cyclins tested, troglitazone specifically reduced the expression of cyclin D1 mRNA and the corresponding protein and this effect was also additive with 9-cis RA. These results suggest that the activation of PPARgamma together with RXR may be useful for the suppression of pancreatic cancer cell growth through the reduction in cyclin D1 levels.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinoma; Cell Division; Chromans; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; G1 Phase; Humans; Pancreatic Neoplasms; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Transfection; Tretinoin; Troglitazone; Tumor Cells, Cultured

The active form of vitamin D(3), 1,25(OH)(2)D(3), inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D(3) analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3) (Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)(2)D(3) in inhibiting 50% clonal growth (ED(50)) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10(-8) M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)(2)D(3) (10(-8) M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)(2)D(3) induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10(-8) M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)(2)D(3) at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G(1)/G(0) phase and expression level of p27(kip1). Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphoric Monoester Hydrolases; Prostatic Neoplasms; PTEN Phosphohydrolase; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

The clearance of apoptotic cells is crucial to avoid chronic inflammation and autoimmunity. Little is known about the factors that regulate it in vivo. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF) administration to carcinoma patients confers to their leukocytes a significantly higher ability to phagocytose apoptotic cells than before (P < 0.005). GM-CSF increased the concentration of monocytes and polymorphonuclear leukocytes in the peripheral blood and activated circulating polymorphonuclear leukocytes. Both effects abated early after treatment, whereas phagocytosis of apoptotic cells was still significantly higher after 18 days compared with basal values (P < 0.005 and P < 0.025 for monocytes and polymorphonuclear leukocytes, respectively). On in vitro phagocytosis of apoptotic cells monocytes, but not polymorphonuclear leukocytes, up-regulated MHC class II membrane expression. These findings are consistent with the possibility that GM-CSF endows both scavenger and antigen-presenting leukocytes with the ability to internalize apoptotic tumor cells.

Topics: Adult; Aged; Antineoplastic Agents; Apoptosis; Carcinoma; Carcinoma, Renal Cell; Colonic Neoplasms; Combined Modality Therapy; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Factors; Interferon-alpha; Jurkat Cells; Kidney Neoplasms; Leukocyte Count; Male; Middle Aged; Monocytes; Neutrophils; Pancreatic Neoplasms; Phagocytosis; Tretinoin

Retinoic acid (RA)-resistant cell lines are highly malignant. To inhibit the growth of the RA-resistant cells we used 4-HPR, a synthetic retinoid, which may act through alternative signal transduction pathways. 4-HPR induced cell growth inhibition and apoptosis in all RA-sensitive as well as -resistant cells, demonstrating a wider spectrum of potency over RA. 4-HPR induced tissue TGase activity. A tight correlation between the induction of tissue TGase, the inhibition of cell growth, and apoptosis was evident in all eight RA-sensitive cell lines. However, basal TGase differed in the different cells, suggesting inducibility rather than basal levels as the relevant parameter. In sharp contrast to the RA-sensitive cells, RA-resistant cells showed sporadic response to 4-HPR for tissue TGase. The wider spectrum of activity of 4-HPR in inhibiting cell growth and inducing apoptosis makes it a good candidate for the treatment of RA-resistant cancer cells.

Topics: 3T3 Cells; Animals; Apoptosis; Carcinoma; Cell Division; Fenretinide; Humans; Mice; Transglutaminases; Tretinoin; Tumor Cells, Cultured

CD97 is a dimeric glycoprotein belonging to the secretin receptor superfamily and is abundantly expressed in cells of hematopoietic origin. The aim of this study was to analyze the expression of the CD97 protein in thyroid carcinomas and the role of all-trans-retinoic acid (RA) in the regulation of CD97 protein in monolayer culture of the human follicular thyroid carcinoma cell line FTC-133. In normal thyroid tissue, no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas, CD97 expression was either lacking or low. Undifferentiated anaplastic thyroid carcinomas revealed high CD97 expression. The expression of CD97 protein seems to be correlated to the postoperative histopathological classification staging. Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in the number of CD97-positive cells after TSH, forskolin, and insulin treatment compared to control values. Epidermal growth factor treatment led to an increase in CD97 immunostaining (up to 90%), whereas phorbol 12-myristate 13-acetate slightly decreased the immunoreactivity of CD97 (from 50% to 30%). Under basal conditions, RA treatment for 72 h led to a decrease in total cell number by 33% and in CD97-positive cells from 50% to 30%. TSH, forskolin, phorbol 12-myristate 13-acetate, and insulin showed no effect after 72-h pretreatment with RA, whereas epidermal growth factor treatment led to a slight increase in the number of the CD97-positive cells (from 30% to 40%) compared to the control value. These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors, and RA might interfere with this process in thyroid carcinoma by suppressing the dedifferentiation marker CD97.

Topics: Adult; Aged; Antigens, CD; Carcinoma; Cell Count; Cell Division; Child; Female; Humans; Male; Membrane Glycoproteins; Middle Aged; Receptors, G-Protein-Coupled; Stimulation, Chemical; Thyroid Neoplasms; Tretinoin; Tumor Cells, Cultured

Tumor cells expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene become sensitive to ganciclovir (GCV), and the phenomenon by which tumor cells surrounding the HSV-tk expressing cells also become sensitive to GCV is known as the "bystander effect." The purpose of this study was to investigate the bystander effect in human lung-cancer cell lines, and the role of gap-junctional intercellular communication as the mechanism responsible for it. Gap-junctional intercellular communication was measured both with a dye-transfer assay involving single-cell microinjection of Lucifer Yellow and with a PKH26/calcein-AM double-dye-transfer assay. Significant bystander tumoricidal effect was observed in lung-cancer cell lines when cultured cells contained only 10% HSV-tk expressing cells. This was also observed to occur with cell lines of different origin or from different species. Although gap-junctional intercellular communication characterized by rapid transfer of Lucifer Yellow was not observed, we did detect gap-junctional communication marked by the slow transfer of calcein-AM in lung-cancer cell lines. However, neither an inhibitor (1-octanol) nor an enhancer (all trans-retinoic acid [ATRA]) of gap-junctional communication affected the extent of the bystander effect. These findings suggest that low levels of gap-junctional communication may be efficient for producing the bystander effect in lung-cancer cells, or that other mechanisms may underlie this effect. Although gap-junctional communication may play an important role in generating the bystander effect in tumor cells expressing the HSV-tk gene, further knowledge of the mechanism of this effect may help improve the treatment of lung cancer with an HSV-tk system.

Topics: 1-Octanol; Animals; Antimetabolites, Antineoplastic; Carcinoma; Cell Communication; Cell Division; Cell Transformation, Viral; Coculture Techniques; Ganciclovir; Gap Junctions; Genetic Vectors; Humans; Lung Neoplasms; Mice; Moloney murine leukemia virus; Simplexvirus; Thymidine Kinase; Tretinoin; Tumor Cells, Cultured

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.

Topics: Carcinoma; Feasibility Studies; Humans; Neoplasm Proteins; Polymerase Chain Reaction; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; RNA, Messenger; Thyroid Neoplasms; Tretinoin; Tumor Cells, Cultured

Retinoids inhibit growth and induce differentiation in a variety of pancreatic carcinoma cells. The goal of this study was to examine the molecular mechanisms responsible for retinoid sensitivity.. Anchorage-independent growth was examined in AR42J, DSL-6A/C1, and Capan-2 cells using a human tumor clonogenic assay. Retinoid receptors were characterized by a reverse-transcription polymerase chain reaction. Retinoic acid receptor gamma1 (RARgamma1) was stably transfected into AR42J cells using lipofectamin and into DSL-6A/C1 using ballistomagnetic gene transfer. Receptor expression was verified using Southern and Northern blotting as well as electrophoretic mobility shift assays.. Retinoid treatment resulted in a dose-dependent growth inhibition of Capan-2 cells, whereas growth was not affected in AR42J and DSL-6A/C1 cells. A selective loss of RARgamma1 expression was observed in both retinoid-resistant cell lines, whereas all other retinoid receptor subtypes showed an identical expression pattern. Retinoid treatment of three independent RARgamma1-expressing cell clones of AR42J and DSL-6A/C1 cells resulted in pronounced growth inhibition compared with wild-type control cells.. RARgamma1 expression determines sensitivity of pancreatic carcinoma cells to retinoid-mediated growth inhibition and might therefore serve as a valuable predictive marker for retinoid treatment of pancreatic cancer.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Division; Cell Line; Dose-Response Relationship, Drug; Drug Resistance; Humans; Pancreas; Pancreatic Neoplasms; Rats; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transfection; Tretinoin

The initial step in tumor invasion and metastasis is determined by adhesion of tumor cells to basement membranes. To evaluate their potential therapeutic use in controlling local growth and metastasis, the effects of retinoids on the adhesive properties in the human pancreatic carcinoma cell line DAN-G were examined.. The effects of retinoids on cellular adhesion were assessed by adhesion assays in vitro. The expression of laminin-binding proteins was characterized by Northern blotting, radioimmunoprecipitation, and flow-cytometric analysis.. Treatment with retinoids results in a time- and dose-dependent inhibition of DAN-G cell adhesion to fibronection and laminin but not to collagens I, IV, and VI. The adhesion of DAN-G cells to laminin could be blocked completely by anti-alpha 6 and anti-beta 1 antibodies but not by the synthetic peptide YIGSR. Flow-cytometric analysis of DAN-G cells showed no quantitative difference for alpha 6-integrin expression in retinoid-treated and -untreated DAN-G cells. Furthermore, radioimmunoprecipitation showed no difference in the appearance of alpha 6 beta 1-integrin expression after retinoid incubation.. Retinoids decrease pancreatic carcinoma cell adhesion to laminin via an as yet unidentified mechanism involving alteration of the alpha 6 beta 1-integrin receptor function and thereby open interesting perspectives for the modulation of infiltrative growth and metastasis in pancreatic cancer.

Topics: Animals; Carcinoma; Cell Adhesion; Integrin alpha6beta1; Integrins; Laminin; Pancreatic Neoplasms; Rats; Retinoids; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Apoptosis; Carcinoma; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Female; Gene Expression; Humans; Retinoids; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Human stromelysin-3 and interstitial collagenase are matrix metalloproteinases whose expression by stromal cells in several types of carcinomas has been associated with cancer progression. We compared here the regulation of the expression of both proteinases by retinoids in human fibroblasts. Physiological concentrations of retinoic acid were found to simultaneously induce stromelysin-3 and repress interstitial collagenase. In both cases, the involvement of a transcriptional mechanism was supported by run-on assays. Furthermore, in transient transfection experiments, the activity of the stromelysin-3 promoter was induced by retinoic acid through endogenous receptors acting on a DR1 retinoic acid-responsive element. The ligand-dependent activation of the receptors was also investigated by using selective synthetic retinoids, and we demonstrated that retinoic acid-retinoid X receptor heterodimers were the most potent functional units controlling both stromelysin-3 induction and interstitial collagenase repression. However, specific retinoids dissociating the transactivation and the AP-1-mediated transrepression functions of the receptors were found to repress interstitial collagenase without inducing stromelysin-3. These findings indicate that such retinoids may represent efficient inhibitors of matrix metalloproteinase expression in the treatment of human carcinomas.

Topics: Carcinoma; Collagenases; Drug Synergism; Enzyme Repression; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 11; Metalloendopeptidases; Promoter Regions, Genetic; Receptors, Retinoic Acid; Rhabdomyosarcoma; Transcription Factor AP-1; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The ability of all-trans retinoic acid (atRA), interferon-alpha2a (IFN-alpha2a) or a combination thereof to modulate the growth of three human cervical carcinoma cell lines (ME180, MS751 and CaSki) and the relationship between responsiveness and the expression of cytosolic retinoid-binding proteins (CRBP and CRABP II), nuclear RA receptors (RAR-alpha, -beta and -gamma) and retinoid X receptor (RXR alpha) were investigated. atRA induced an antiproliferative effect on two of the cell lines (ME180 > MS751), whereas CaSki was much less responsive. An additive growth inhibition on the latter two cell lines was achieved with the combined treatment of atRA and IFN-alpha2a. Receptor expression appeared to be unrelated to growth inhibition in these cell lines in so far as atRA exerted minimal effect on the growth of CaSki, although these cells expressed four of these nuclear receptors. However, mRNA for CRABP II was not demonstrable in CaSki, in contrast to the other two atRA responsive cell lines, as evaluated with RT-PCR and ethidium bromide staining. Treatment with atRA or IFN-alpha2a did not induce any change in mRNA for the nuclear retinoid receptors or cellular retinoid binding proteins after 3 or 6 days of treatment.

Topics: Antineoplastic Agents; Carcinoma; Cell Division; Cell Line; Drug Synergism; Female; Humans; Interferon alpha-2; Interferon-alpha; Oligonucleotide Probes; Polymerase Chain Reaction; Receptors, Retinoic Acid; Recombinant Proteins; Tretinoin; Uterine Cervical Neoplasms

Cell cycle analysis indicates that retinoic acid (RA) inhibition of MCF-7 cell growth occurs through induction of G1 arrest with a concomitant reduction in the proportion of cells in S and G2 + M phases. RA did not affect cyclins D1, A, and E and cyclin-dependent kinase 2 (CDK2) expression, but significantly reduced cyclin D3 and CDK4 expression after 24 h. RA also inhibited cyclin B1 and CDC2 expression, possibly responsible for the reduction of the proportion of cells in G2 + M and S phases. RA did not induce p16 and p27 expression, but obviously reduced p21 level in MCF-7 cells. The retinoid markedly reduced pRB protein level and abrogated pRB phosphorylation after 48 h; it also reduced transcription factor E2F1 expression at both the mRNA and protein levels. E2F1 promoter activity was reduced by 60%, which is probably responsible, at least in part, for the reduction of E2F1 expression in RA-treated MCF-7 cells. These observations demonstrate a marked effect of RA on some of the key cell cycle regulatory proteins in MCF-7 cells. Cyclin D3 and CDK4 are likely the early targets of RA, followed by reduced pRB expression and phosphorylation, as well as by the inhibition of the E2F1 transcription factor which controls progression from G1 to S phase. Most of these events precede the observed reduction in MCF-7 cell growth, which begins at Day 3 of RA treatment.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Phosphorylation; Promoter Regions, Genetic; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; Transcription Factor DP1; Transcription Factors; Tretinoin

We report the cDNA cloning of Stra13, a novel retinoic acid (RA)-inducible gene from P19 embryonal carcinoma cells that encodes a basic helix-loop-helix (bHLH) protein that shows the highest sequence similarities to the Drosophila Hairy and Enhancer of split and mouse Hes proteins. Stra13 does not bind to the known consensus motifs (E-box and N-box) for bHLH proteins, but can repress activated transcription (through an alpha-helix rich domain) in part by interaction with general factors of the basal transcription machinery. During mouse embryogenesis, Stra13 RNA is expressed in the neuroectoderm, and also in a number of mesodermal and endodermal derivatives. Remarkably, overexpression of Stra13 in P19 cells results in neuronal differentiation in monolayer culture, under conditions where wild-type P19 cells only undergo mesodermal/endodermal differentiation. This neuronal differentiation is accompanied by an altered expression of mesodermal and neuronal markers, indicating that Stra13 could be one of the earliest RA target genes whose expression is required for repression of mesodermal/endodermal differentiation and/or induction of neuronal differentiation when P19 cell aggregates are exposed to RA. Our results raise the possibility that Stra13 could be involved as a repressor in a number of decision events occurring during differentiation of various cell lineages.

Topics: Amino Acid Sequence; Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Carcinoma; Cell Differentiation; Cloning, Molecular; COS Cells; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Helix-Loop-Helix Motifs; Homeodomain Proteins; Mesoderm; Mice; Molecular Sequence Data; Neurons; Tissue Distribution; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The retinoic acid (RA) signaling pathway was investigated by transient transfection of a chloramphenicol acetyltransferase (CAT) reporter gene construct containing the RA response element (RARE) of the murine (m) RARbeta2 gene into murine primary epidermal keratinocytes (PEK), papilloma-derived SP1 cells, and carcinoma-derived 3P2 cells. Murine PEK transfected in a low-Ca2+ medium (0.05 mM Ca2+) exhibited a strong transactivation of the CATgene after exposure of the cells to 0.1 microM RA. Transactivation of the CATgene could, however, also be achieved by shifting RAREbeta2-transfected low-Ca2+ PEK to high-Ca2+ conditions (0.15-1.2 mM Ca2+). Concomitantly, the Ca2+ raise also led to the induction of both cellular retinol (ROL)-binding protein I (CRBPI) and cellular RA-binding protein II (CRABPII), whereas expression of cellular RA-binding protein I (CRABPI) was not observed. Moreover, induction of in vitro differentiation also activated the ROL-->RA converting enzyme system in PEK. These findings suggest the following sequence of events involved in the high Ca2+-mediated activation of RAREbeta2. First, high Ca2+ induces the synthesis of mCRBPI, which binds ROL released from retinyl ester stores and makes it accessible to the ROL-RA converting enzyme system. Enzymatically generated RA is taken over by mCRABPII and transported to the nucleus, where it acts as ligand for nuclear receptors, which complex with RAREbeta2 to activate the reporter gene. This hypothetical cascade of RA signaling was supported by our findings that inhibition of the ROL-->RA converting enzyme system by citral abolished the Ca2+-mediated transactivation of the CAT gene in a nontoxic manner. Studies in transformed murine cell lines revealed that Ca2+-induced activation of RAREbeta2 was essentially maintained in papilloma-derived SP1 cells, although all parameters of the Ca2+-dependent RAREbeta2 activation cascade were induced to a much lower extent. In contrast, strong RAREbeta2 activity was already observed in low-Ca2+ carcinoma-derived 3P2 cells. Low-Ca2+ 3P2 cells also expressed high levels of both mCRBPI and mCRABPII and possessed a highly active ROL-->RA converting enzyme system. Again, inhibition of the enzyme by citral abolished RAREbeta2 activity in low-Ca2+ 3P2 cells. Our data show that Ca2+-induced differentiation in cultured murine PEK entails a series of events that ultimately lead to the activation of RARE-containing genes. These properties are maintained in transformed e

Topics: Animals; Calcium; Carcinoma; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Genes, Reporter; Keratinocytes; Mice; Mice, Inbred Strains; Papilloma; Receptors, Retinoic Acid; Signal Transduction; Skin; Skin Neoplasms; Skin Physiological Phenomena; Transcriptional Activation; Transfection; Tretinoin; Vitamin A

Retinoids have been demonstrated to inhibit epithelial cell growth and differentiation. We examined the anti-proliferative effects of retinoic acid (RA) in an HPV positive and negative cervical carcinoma cell line. Our findings indicate that HPV-negative C33A cervical carcinoma cells are more sensitive to the growth inhibitory activity of retinoic acid (RA) than are HPV-positive CaSki cervical carcinoma cells. However, conditioned medium from RA-treated C33A cells displayed strong growth inhibitory activity in both C33A and CaSki cells. Since RA has been shown to modulate the expression of insulin-like growth factor binding proteins (IGFBPs) in many cells, we examined RA regulated expression of IGFBPs in medium isolated from RA treated C33A cells. IGFBP-5 was detectable in medium from C33A cells exposed to RA, and addition of purified exogenous IGFBP-5 resulted in growth inhibition of C33A cells. These results indicate that RA exerts it's anti-neoplastic effect in HPV negative cervical carcinoma cells via the overproduction of IGFBP-5.

Topics: Antineoplastic Agents; Carcinoma; Cell Division; Female; Growth Inhibitors; Humans; Insulin-Like Growth Factor Binding Protein 5; Papillomaviridae; Papillomavirus Infections; Tretinoin; Tumor Cells, Cultured; Tumor Virus Infections; Uterine Cervical Neoplasms

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Topics: Bone Marrow; Brain Neoplasms; Carcinoma; Cell Division; Culture Media; Dihydrotestosterone; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We have investigated the role of protein kinase C (PKC) isoenzymes in the differential growth regulation of human pancreatic carcinoma cell lines by all-trans retinoic acid (RA). RA treatment results in dose-dependent stimulation of anchorage-independent growth in AsPc1 cells and growth inhibition in Capan 2 cells. Both cell lines express an identical pattern of nuclear RA and retinoid X receptors as determined by RT-PCR. Western blotting using monospecific antibodies revealed that both cell lines express PKC isoenzymes alpha and zeta, whereas beta, gamma, delta, and epsilon were not detected. Incubation with RA in the growth-stimulated AsPc1 cell line resulted in induction of PKC alpha expression, whereas PKC alpha expression was decreased by RA in the growth-inhibited Capan 2 cell line. In contrast, PKC zeta expression was not affected by RA in either cell line. Incubation of AsPc1 cells with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate resulted in a time- and dose-dependent selective down-regulation of PKC alpha but not zeta. The dose-dependent decrease of intracellular PKC alpha concentration correlated well with the anchorage-independent growth rate of AsPc1 cells. Furthermore, selective down-regulation of PKC alpha blocks subsequent growth stimulation by RA in AsPc1 cells. When PKC alpha concentration was decreased by stably transfecting AsPc1 cells with a PKC alpha complementary DNA antisense construct, RA-stimulated growth could also be partially blocked. These data, therefore, suggest that differential regulation of PKC alpha expression plays a central role in determining the bidirectional effects of RA on growth in pancreatic carcinoma cells.

Topics: Base Sequence; Carcinoma; Cell Division; Humans; Isoenzymes; Molecular Probes; Molecular Sequence Data; Pancreatic Neoplasms; Protein Kinase C; Protein Kinase C-alpha; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Evidence exists to suggest that human cytomegalovirus (hCMV) may opportunistically use retinoic acid (RA) to advance its own replication, in which transcriptional activation of the viral major immediate-early promoter is a crucial control point. We demonstrate that the enhancer of the viral promoter contains three RA-response-elements that cooperate in mediating RA activation. These elements are direct repeats of two sequence motifs separated by 2 bp (DR2 site, REa) and 5 bp (DR5 sites, REb and c). DNA-binding experiments revealed that each of these elements bind RA receptor (RAR)-retinoid X receptor (RXR) heterodimers more efficiently than either homodimer. Apparent equilibrium dissociation constants of RAR-RXR heterodimers for sites REa, REb, and REc were estimated to be 5 nm, 10 nm, and 20 nm, respectively. The level of contribution of each of these elements to RA inducibility correlated with the strength of binding by RAR-RXR heterodimers to each site. These experiments demonstrate that RAR and RXR are necessary for RA responsiveness of the viral promoter. Using synthetic RA analogs, which selectively activate RARs and RXRs, the RAR partner within the heterodimeric complex appeared to be sufficient while the RXR partner was insufficient to independently activate transcription. However, joint activation of RARs and RXRs indicated that RXRs (in the presence of a transcriptionally active RAR) could contribute to transactivation. This restricted co-dependent ligand activation of RXR varied depending on the particular response element and the cell context. These studies further indicate that signaling of retinoid receptors (in particular RAR) by RA plays an important role in modulating hCMV infection.

Topics: Carcinoma; Cytomegalovirus; Embryo, Mammalian; Enhancer Elements, Genetic; Humans; Prohibitins; Protein Conformation; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated P19 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19-derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.

Topics: Animals; Astrocytes; Brain Tissue Transplantation; Carcinoma; Cell Differentiation; Cell Line; Cell Transplantation; Cells, Cultured; Mice; Mice, Inbred Strains; Microscopy, Electron; Oligodendroglia; Tretinoin

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01-0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene.

Topics: Alkyl and Aryl Transferases; Animals; Carcinoma; Cell Compartmentation; Cell Membrane; Cell Transformation, Neoplastic; Cytosol; Diterpenes; Enzyme Inhibitors; Mice; Papilloma; Proto-Oncogene Proteins p21(ras); Skin; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Transferases; Tretinoin

Liarozole inhibits cytochrome P-450-dependent enzymes that play a key role in all-trans-retinoic acid (ATRA) catabolism. In MCF-7 cells, liarozole potentiates the antiproliferative effects of ATRA. The present study demonstrates this synergistic effect on cell differentiation of MCF-7 cell cultures as measured by immunocytochemistry for cytokeratins 8, 18, and 19, actin, E-cadherin, desmoglein and desmoplakins I & II. ATRA concentration-dependently (10(-8) M-10(-6) M) induced changes in actin stress fibers and cytokeratin intermediate filaments. These changes were accompanied by a more obvious interaction of these filaments with junctional complexes. Surface area and volume of the MCF-7 cells increased markedly after ATRA exposure, with extensive filopodia formation. Liarozole (10(-6) M) alone had no effect on cell morphology, cytokeratin or actin organization, or on cellular junctions. In combination with ATRA (10(-9) M and 10(-8) M), liarozole potentiated the ATRA-induced effects. The MCF-7 cell cultures used showed morphological heterogeneity, consisting of at least two cellular subpopulations. This was reflected in the staining for E-cadherin, desmoglein and desmoplakins I & II. ATRA increased E-cadherin staining at cell-cell contact sites, but had no influence on the staining patterns of desmoglein and desmoplakins I & II. Similar to what has been observed for the cytoskeletal differentiation parameters, liarozole alone had no influence on E-cadherin, desmoglein or desmoplakins I & II expression, but in combination with ATRA again intensified the effects on E-cadherin distribution. These effects on MCF-7 cells agree with previously obtained observations concerning the inhibition of ATRA catabolism by liarozole. Furthermore, our data support the hypothesis that the antiproliferative properties of the drug are accompanied by induction of differentiation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Adhesion Molecules; Cell Division; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Imidazoles; Neoplasm Proteins; Tretinoin

The P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum-containing medium are capable of differentiating into neuron-like cells in N2 medium. Dividing fibroblast-like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron-like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron-like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron-like cells.

Topics: Animals; Carcinoma; Cell Differentiation; Cell Line; Cells, Cultured; Culture Media; Neurons; Tretinoin

Retinoic acid (RA) induces P19 embryonal carcinoma cells to differentiate into neurons with the extension of neuritic processes. We used the P19 cell as a model system to elucidate the regulation of neurofilament (NF) expression. Four mammalian NF proteins, NF-66 (alpha-internexin), peripherin, NF-L and NF-M, and the neural-specific, growth-associated gene, GAP-43, were studied during the RA treatment of P19 cells in vitro. As controls, untreated P19 cells were maintained in parallel. Indirect immunofluorescent staining showed that in RA-treated, morphologically differentiated P19 cells NF-66 was expressed in neuron-like cells characterized by phase bright cell bodies and long neuritic processes. At various times P19 cells were harvested for protein analysis by immunoblotting with antibodies to individual NF proteins or for total RNA extraction and Northern blotting with cDNA probes for NF-66, -L, -M, peripherin and GAP-43. During induction, both NF-66 and NF-L were expressed but in distinct patterns. NF-66 mRNA and protein were detected after 6 days of induction. In contrast, NF-L mRNA, but not protein, was expressed in both induced and control cells. Neither NF-M nor peripherin were expressed during induction. During differentiation of P19 cells, NF-66 mRNA levels rose markedly by the 1st day, reached a plateau between the 3rd-5th days and declined by the 7th day. NF-66 protein accumulation lagged slightly, reaching maximum abundance about the 5th day. The kinetics of NF-66 expression were similar to that of GAP-43. However, the pattern of NF-L expression was distinct from that of NF-66. NF-L mRNA, and some protein, was expressed in both RA-treated and control cells within 6 h after plating, but was down-regulated to baseline level thereafter in both populations. Neither NF-M or peripherin expression was detected during the differentiation. In summary, NF-66 was up-regulated most robustly among the four NF proteins during differentiation in P19 cells and was the major NF protein correlated with neurite extension.

Topics: Animals; Blotting, Northern; Carcinoma; Carrier Proteins; Cell Differentiation; Embryo, Mammalian; GAP-43 Protein; Gene Expression; Immunoblotting; Intermediate Filament Proteins; Membrane Glycoproteins; Models, Neurological; Nerve Tissue Proteins; Neurofilament Proteins; Neurons; Rats; Rats, Inbred Strains; Tretinoin; Tumor Cells, Cultured

We studied the effect of dietary retinoic acid (RA) in a two-stage protocol of skin carcinogenesis in female SENCAR mice. At 3 weeks of age mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA, 20 micrograms) and promoted with either 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 micrograms) once per week or mezerein (MEZ, 4 micrograms) twice per week for 20 weeks. At the week of DMBA initiation mice were also put on a purified diet containing either 3 (physiological dose) or 30 micrograms (pharmacological dose) of RA/g of diet. High dietary RA significantly inhibited papilloma yield but not incidence in the MEZ-promoted group. Papilloma incidence and yield were also lower in the MEZ- than in the TPA-treated groups. Cumulative carcinoma incidence and yield, and conversion efficiency (= (carcinomas/maximal papillomas) x 100%), were all decreased by high dietary RA in both MEZ- and TPA-treated groups. These results demonstrate that high dietary RA inhibited skin carcinogenesis in MEZ-promoted mice at the stages of tumor promotion and malignant conversion, while this inhibition occurred only at the malignant conversion stage in TPA-promoted mice.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma; Diet; Diterpenes; Female; Mice; Mice, Inbred SENCAR; Papilloma; Skin Neoplasms; Terpenes; Tretinoin

Using 7,12-dimethylbenz[a]anthracene as the initiator and 12-O-tetradecanoyl-13-acetate as the tumor promoter on the dorsal skin of Sencar mice, we previously showed that pharmacological dietary all-trans-retinoic acid and beta-carotene inhibit the conversion of papillomas to carcinomas in a two-stage system of chemical carcinogenesis. The purpose of this study was to determine the influence of dietary retinoic acid and beta-carotene on retinoid and beta-carotene concentrations in skin and other tissues. We were unable to measure tissue retinoic acid because of the relatively limited amount of tissue available for analysis and the fast rate of metabolism. Different dietary levels of retinoic acid or beta-carotene did not influence total retinol of skin, papilloma, and carcinoma tissues, which all showed a concentration of approximately 1 +/- 0.5 microgram/g wet wt. Equally refractory to dietary retinoic acid or beta-carotene was serum retinol concentration. In contrast, dietary retinoic acid protected loss of liver retinol and retinyl palmitate, and beta-carotene caused an increase in beta-carotene and retinyl palmitate in liver but did not affect serum and liver retinol. We further investigated metabolic and functional aspects of retinoic acid in cultured mouse epidermal keratinocytes (LC-8 cells) and found that these cells actively metabolized [10,11-14C]retinoic acid to polar compounds. Isomers of retinoic acid were a minor product in the presence of cells and the major product when incubated in serum-containing medium in the absence of cells. From the functional point of view, exposure of LC-8 cells to 3 x 10(-6) M all-trans-retinoic acid (RA) caused a 75-fold induction in tissue transglutaminase and an approximately 9-fold induction in 10(-6) M RA at three days of culture. We conclude that retinoic acid spares endogenous retinol and that beta-carotene greatly enhances liver retinyl palmitate levels. Moreover we show that although mouse epidermal cells metabolize retinoic acid at a very high rate, they respond functionally by induction of tissue transglutaminase activity. Because this enzyme has been suggested to be involved in programmed cell death, we are presently investigating the possibility that it may be involved in the inhibition of carcinogenesis in mice fed pharmacological doses of RA.

Topics: Animals; beta Carotene; Carcinoma; Carotenoids; Cell Division; Chromatography, High Pressure Liquid; Diet; Female; Keratinocytes; Liver; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Papilloma; Skin; Skin Neoplasms; Transglutaminases; Tretinoin; Tumor Cells, Cultured; Vitamin A

Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each class of these nuclear retinoid receptors is further subdivided into three species, namely alpha, beta, and gamma. Recent studies demonstrate that estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines and tumor samples exhibit significantly higher levels of RAR alpha than their ER-negative counterparts. ER-positive HBC cell lines are sensitive to, and ER-negative cell lines are resistant to, growth inhibitory effects of retinoic acid (RA). We previously demonstrated that the expression of functional ERs in an established ER-negative cell line resulted in higher levels of RAR alpha and sensitivity to growth inhibition by RA. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA in two RA-resistant ER-negative HBC cell lines. Analyses of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Utilizing RAR- and RXR-class selective retinoids, we further demonstrated that only the RAR alpha-selective retinoids mediated the growth inhibition in these cells, while the RXR-selective retinoids were biologically inert. We thus provide evidence that the molecular mechanisms of retinoid inhibition of HBC proliferation predominantly involve RAR alpha.

Topics: Base Sequence; Breast Neoplasms; Carcinoma; Cell Division; DNA, Complementary; Female; Genetic Vectors; Humans; Molecular Sequence Data; Receptors, Estrogen; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoids; Signal Transduction; Transfection; Tretinoin

Previously, we have shown that dietary retinoic acid (RA) at pharmacological doses (30 micrograms/g of diet) inhibited the malignant conversion of skin papillomas to carcinomas induced by a two-stage carcinogenesis protocol with 7,12-dimethylbenz[a]anthracene as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter (De Luca et al., Carcinogenesis, 14 (1993) 539-542). The purpose of this study was to determine the effect of dietary RA on skin papilloma and carcinoma formation induced by a complete carcinogenesis protocol with repeated DMBA treatment in female Sencar mice. Mice at 3 weeks of age were weaned onto a diet containing either 3 (control) or 30 (excess) micrograms of RA/g of diet and treated topically with DMBA (25.5 micrograms) once per week for 20 weeks. Mice fed excess dietary RA did not significantly differ from control mice in the following parameters: body weight, survival rate, papilloma incidence, cumulative carcinoma incidence (19.4% versus 23.7%), carcinoma yield (0.19 versus 0.26 per mouse), carcinoma conversion efficiency (5.2% versus 3.9%), and average age of carcinoma development (22.7 +/- 4.7 versus 23.3 +/- 2.8 weeks). However, papilloma yield was decreased by about 50% (i.e. 3.7 versus 7.0 at week 20, P < 0.01) between weeks 17 and 22 of age by excess dietary RA treatment. Contrary to other routes of administration (i.e. topical and systemic) of RA (Verma et al., Cancer Res., 42 (1982) 3519-3525), excess dietary RA did not enhance skin tumor formation. In addition, excess dietary RA failed to inhibit malignant conversion of papillomas to carcinomas in the complete carcinogenesis protocol. Thus, the modulation of RA on skin papilloma and carcinoma formation is dependent on carcinogenesis protocol and route of RA administration.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; Diet; Female; Mice; Papilloma; Skin Neoplasms; Tretinoin

The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines.

Topics: Antigens, CD; Breast Neoplasms; Calcitriol; Carcinoma; CD59 Antigens; Cell Membrane; Cytokines; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Flow Cytometry; Fluorescent Antibody Technique; Glioma; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Membrane Glycoproteins; Ovarian Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The T47D human breast carcinoma cell line has been shown to synthesize insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) and IGF-I receptors, and to exhibit a mitogenic response to exogenous IGF-I. We have used T47D cells to investigate the regulation of IGFBPs by IGF-I and retinoic acid (RA), agents that affect cell proliferation and have been shown to regulate IGFBP levels in other cell types. Exposure of T47D cells to IGF-I resulted in the appearance of IGFBP-2, -4, and -5 in conditioned medium but had no effect on the levels of IGFBPs in Triton X-100-extracted cells. This effect was most pronounced for IGFBP-5 and was also elicited by an IGF-I analog that retains affinity for IGFBPs but not by insulin or IGF analogs that have decreased affinity for IGFBPs. Additionally, this effect was not associated with a change in IGFBP-5 messenger RNA (mRNA) levels; however, the appearance of IGFBP-5 in the conditioned medium was inhibited by an anti-IGF-I receptor antibody (alpha IR-3). RA decreased IGFBP-5 mRNA levels and cell-associated IGFBP-5 in both the presence and absence of IGF-I and inhibited the IGF-I-stimulated secretion of IGFBP-5 into T47D cell conditioned medium. These results suggest that IGF-I increases IGFBP-5 levels in the T47D cell line both through direct interaction with IGFBP-5 as well as through a receptor-mediated process that does not require direct interaction with IGFBPs. The latter results are consistent with an effect of IGF-I on a factor that may modulate an IGFBP protease activity. The inhibitory effect of RA, on the other hand, appears to be due primarily to regulation of IGFBP-5 mRNA levels. Thus, IGFBP-5 accumulation appears to be positively regulated by IGF-I, potentially at the level of susceptibility to proteolysis, and negatively regulated at the level of gene expression by RA.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Culture Media; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Receptors, Somatomedin; Somatomedins; Tretinoin; Tumor Cells, Cultured

Expression of matrix Gla protein (MGP) gene and its regulation by retinoic acid (RA) and estrogen was investigated in eight human breast cancer cell lines. The promoter region of the MGP gene contains a consensus retinoic acid response element (RARE) and the MGP gene expression has been shown to be strongly induced by RA in other systems. Our results suggest that RA negatively regulates MGP mRNA expression in human breast cancer cells that have high levels of estrogen receptors (ER), i.e. MCF-7, ZR-75 and BT474 and positively regulates its expression in cells with either no ERs, i.e. MDA-MB-468 or very low levels of ERs, i.e. T47D. This indicates that ER levels may affect RA modulation of MGP gene expression in human breast cancer cells. The inhibitory effect of RA on MGP gene expression was abolished in RRO-I, the RA-resistant MCF-7 subline. We also demonstrate for the first time that estrogen strongly induces MGP gene expression in ER-positive cells and that estrogen-mediated induction of MGP is blocked by RA even in otherwise RA-resistant cells.

Topics: Amino Acid Sequence; Breast Neoplasms; Calcium-Binding Proteins; Carcinoma; Cycloheximide; Estradiol; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Matrix Gla Protein; Molecular Sequence Data; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Tretinoin

Previously we have shown that dietary retinoids are essential for papilloma formation induced by either an initiation-promotion or a complete skin carcinogenesis protocol. The present study was conducted to further determine the effect of dietary retinoic acid (RA) on papilloma formation and the conversion of papillomas to carcinomas. Skin tumors were induced in 3 week old female SENCAR mice by an initiation-promotion protocol with one application of 20 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA), followed by 20 weekly applications of 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice were fed RA at one of the three doses: 0.3 (nutritionally marginal dose), 3 (near physiological) and 30 (pharmacological) micrograms/g of diet. Mice fed 30 micrograms of RA/g of diet had the same survival rate as the other two groups despite a lower body weight and all three groups had similar papilloma incidence, which reached 100% at age 18 weeks. Mice fed 3 micrograms of RA/g of diet had the highest papilloma yield (approximately 14 papillomas/mouse) of all groups and it peaked between weeks 18 and 38 of age. These papillomas later regressed such that mice from all three groups had about the same papilloma yield at week 44 of age. Mice fed 30 micrograms of RA/g of diet failed to develop any visible carcinoma, while mice fed 0.3 or 3 micrograms/g showed 1.9% conversion of papillomas to carcinomas. Therefore, dietary RA at 30 micrograms/g of diet inhibited the conversion of papillomas to carcinomas without affecting papilloma incidence. In addition, dietary RA at 30 and 0.3 micrograms/g of diet lowered papilloma yield.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; Diet; Female; Mice; Papilloma; Skin Neoplasms; Tretinoin

Insulin-like growth factor binding protein (IGFBP) gene expression and IGFBP secretion were investigated in a retinoic acid (RA)-resistant subline (RROI) of MCF-7 human breast carcinoma cells. Our results demonstrate that RRO-I cells constitutively secrete higher levels of IGFBP-3 and IGFBP-5. In addition, we found that a 5-fold increase in IGFBP-5 mRNA levels observed in RRO-I cells, which eventually leads to a similar increase in the secreted levels of IGFBP-5, was partly due to an increase in the stability of IGFBP-5 mRNA. Actinomycin D and cycloheximide differentially stabilized IGFBP-5 mRNA in MCF-7 cells but not in RRO-I cells, indicating a difference in the control of IGFBP-5 gene regulation at the level of mRNA stability in these cell lines.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Dactinomycin; Drug Resistance; Gene Expression Regulation, Neoplastic; Insulin-Like Growth Factor Binding Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; Somatomedins; Time Factors; Tretinoin; Tumor Cells, Cultured

The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ectodermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endodermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) alpha, RAR beta, RAR gamma, jun A, jun D, c-fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.

Topics: Carcinoma; Carrier Proteins; Cell Differentiation; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Retinoids induce cellular differentiation and inhibit cellular proliferation. Proliferation of human breast carcinoma cells in vitro is markedly inhibited by these compounds. On the other hand, insulin-like growth factors (IGFs) and their receptors seem to be involved in the growth of certain breast carcinoma cells by autocrine or paracrine effects. Since the effects of both IGF-I and IGF-II may be modulated by specific binding proteins (IGF-BPs) we examined the possibility that one mechanism by which retinoic acid may inhibit cancer growth is by an alteration in these BPs, thereby blocking IGF's growth effect. Retinoic acid (RA; 1 microM) completely blocked the effect of IGF-I (50 ng/ml) on enhancing proliferation of MCF-7 cells in culture. This effect of RA was not associated with any significant change in specific IGF-I-binding sites on these cells. RA induced a 3-fold increase in IGF-binding activity in conditioned medium, measured using a polyethylene glycol-immunoglobulin precipitation assay and a charcoal absorption assay. This increase was associated with the appearance of 42- and 46-kDa IGF-BPs on ligand blotting. The effect of RA on these IGF-BPs was time and concentration dependent. In contrast, during some experiments the 27- and 36-kDa BPs actually decreased. These findings support the hypothesis that RA may inhibit the growth of certain breast carcinoma cells by increasing the secretion of certain IGF-BPs, which could directly modulate the growth effect of IGFs.

Topics: Breast Neoplasms; Carcinoma; Carrier Proteins; Cell Division; Culture Media; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Ligands; Retinoids; Somatomedins; Tretinoin; Tumor Cells, Cultured

It is becoming increasingly clear that cutaneous carcinogenesis in murine skin is a stepwise process comprising of initiation, promotion and progression. Most of the papillomas induced by an initiation-promotion protocol regress, while a few of them progress to malignant carcinomas. Progression of benign tumors into malignant cancer is critical since the latter lesions are capable of metastatic spread and eventual death. Inhibitors of the conversion process are therefore likely to be useful as cancer chemopreventive agents. All-trans retinoic acid (RA) is a known regulator of cellular proliferation and differentiation, and a known inhibitor of tumor promotion in murine skin. In this study we assessed the effect of topical application of RA on conversion of benign skin papillomas to malignant carcinomas. Papillomas were induced in SENCAR mice by topical application of 7,12-dimethylbenz[a]anthracene (DMBA) as tumor initiator followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter. In SKH-1 hairless mice papillomas were induced by thrice weekly exposure to ultraviolet B (UVB) radiation. At 18 (DMBA/TPA group) and 25 (UVB group) weeks papilloma yield stabilized and no new tumors developed. Beginning at the 20th week (DMBA/TPA group) and at the 27th week (UVB group), malignant conversion was achieved by twice weekly topical application of TPA or free radical-generating compounds benzoyl peroxide (BPO), 2,2-azobis(2-amidinopropane) (ABP) and tert-butyl peroxybenzoate (BPB). Application of RA (10 micrograms/animal) 1 h prior to skin application of TPA, BPO, ABP or BPB afforded significant protection (up to 70%) only against malignant conversion mediated by free radical-generating compounds in both chemically induced and UVB-induced benign skin papillomas. On the other hand, preapplication of RA was less effective in the suppression of spontaneous malignant conversion in vehicle-treated animals. These results suggest that, in addition to their anti-tumor promoting effects, retinoids may also act as anti-carcinogens by inhibiting the process of malignant conversion induced by free radical-generating compounds.

Topics: Animals; Carcinoma; Female; Mice; Neoplasms, Radiation-Induced; Papilloma; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Ultraviolet Rays

Retinoic acid (RA) treatment of F-9 embryonal carcinoma cells resulted in cell flattening and increased production of laminin B1 chain, both indicating differentiation to endoderm-like cells. In addition, RA caused a time- and dose-dependent decrease in growth rate in monolayer culture and a dose-dependent decrease in the ability of the cells to form colonies in soft agarose. Differentiation was accompanied by an increase in the fucosylation of specific high-molecular-weight cellular and cell-surface glycoproteins. The fucosylation of glycoproteins of Mr 175,000 (gp175), 250,000 (gp250), and 400,000 (gp400) increased as early as 24 hr after the addition of 5 x 10(-6) M RA to the culture medium. These changes preceded both growth inhibition and the induction of laminin B1 expression, which were detected 48 to 72 hr after addition of RA. The increased fucosylation of these glycoproteins showed a distinct dose-response relationship. Both gp175 and gp250 showed the greatest increase in fucosylation at 10(-5) M, which was also the dose at which RA induced laminin maximally, while the fucosylation of gp400 was greatest at 10(-8) M RA and declined at higher concentrations. The overall synthesis of large fucosylated glycopeptides decreased in RA-treated cells, in spite of the increases in the fucosylation of specific cellular glycoproteins. RA-induced differentiation of F-9 cells was also accompanied by a time- and dose-dependent increase in fucosyltransferase activity. Although the functions of these glycoproteins are not currently known, the early increase in their fucosylation can be considered as a marker of differentiation in this system.

Topics: Animals; Carcinoma; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fucose; Fucosyltransferases; Glycopeptides; Glycoproteins; Glycosylation; Immunoblotting; Mice; Molecular Weight; Neoplasm Proteins; Neoplasms, Germ Cell and Embryonal; Precipitin Tests; Time Factors; Tretinoin; Tumor Cells, Cultured

Medullary carcinoma of the thyroid (MCT), a tumor of calcitonin (CT)-secreting C cells, can display a variable malignant potential with poor prognosis linked to decreased cell differentiation. In vitro study of MCT has been hampered by the fact that few cell lines derived from this neoplasm have been available for study. Herein are reported the characteristics of two new lines derived from human MCT that are tumorigenic in athymic mice and do not secrete CT. After treatment with various concentrations of retinoic acid (a vitamin A derivative) and cyclic adenosine monophosphate, both lines exhibit the traits of more differentiated cells with a decrease in cellular proliferation and an increase in cytoplasmic CT content as shown by in situ immunoperoxidase staining. These cell lines should prove of great value in the study of the biology of MCT and the mechanisms underlying induced differentiation in this type of tumor.

Topics: Animals; Calcitonin; Carcinoma; Cell Line; Cyclic AMP; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Thyroid Neoplasms; Tretinoin

The human embryonal carcinoma cell lines Tera-2 clone 13 and NTera-2 clone D1 can be induced by retinoic acid to differentiate in vitro into neuroectodermal derivatives. The undifferentiated cells are rapidly proliferating and tumorigenic, whereas retinoic-acid-treated cells possess a decreased growth rate, lose their transformed phenotype and show a finite lifespan. Differentiation is accompanied by a marked increase in the levels of mRNA for TGF-beta 1 and TGF-beta 2 and the production of TGF-beta activity. Just like murine embryonal carcinoma cells the growth of Tera-2 clone 13 cells is not affected by the addition of either TGF-beta 1 or TGF-beta 2 to the culture medium. In contrast to published data on murine embryonal carcinoma cells, Tera-2 clone 13 and NTera-2 clone D1 cells bind TGF-beta 1 with high affinity, which is due to the presence of type-III TGF-beta receptors. Furthermore, and again in contrast to murine embryonal carcinoma cells, treatment of the human embryonal carcinoma cells with retinoic acid causes a nearly complete loss of TGF-beta 1 binding sites. These results are discussed in the light of similarities and differences in the regulation of growth and differentiation of human and murine embryonal carcinoma cell lines.

Topics: Carcinoma; Cell Line; Gene Expression Regulation; Humans; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured

Previous studies have shown dose-dependent growth inhibition of the human mammary carcinoma cell line MDA-MB-231 xenotransplanted in athymic mice using retinol. In this study, the growth inhibitory effect of retinoic acid (RA) and 13-cis-retinoic acid (13-cis-RA) was examined in vitro and in vivo. With both agents there was dose-related growth inhibition in monolayer culture. The MDA-MB-231 cell line was more sensitive in monolayer culture to 13-cis-RA than to RA. Anchorage-independent growth of the MDA-MB-231 cell line was also inhibited by both of these agents but only in a dose-dependent manner with 13-cis-RA. Athymic mice inoculated with MDA-MB-231 human mammary carcinoma cells were treated with various doses of RA and 13-cis-RA for 30 days. RA doses greater than 90 micrograms were clinically toxic to the animals. There was a decrease in tumor size with all doses of RA tested but not in a dose-related fashion. Response at the higher doses of RA may be related to subclinical toxicity. Doses of 13-cis-RA above 300 micrograms were clinically toxic. Unlike RA, there was no statistically significant decrease in tumor size with treatment with 13-cis-RA. These findings show that there is significant reduction in growth in vivo of the human mammary carcinoma cell line MDA-MB-231 after treatment with RA. However, in vivo response to the retinoids is not always predicted by in vitro methods.

Topics: Animals; Carcinoma; Cell Division; Dose-Response Relationship, Drug; Humans; Isotretinoin; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Transplantation; Retinoids; Transplantation, Heterologous; Tretinoin

The in vitro proliferation and colony-forming activity (plating efficiency) of Epstein-Barr virus (EBV) nuclear antigen-positive nasopharyngeal hybrid cells were inhibited by one of the retinoid, polyprenoic acid; E5166. It was dependent on retinoid concentration. Furthermore, an induction of EBV early antigen after treatment with 4 mM n-butyrate was markedly suppressed by E5166 and this was similar to the previously reported effects of retinoid acid on iododeoxyuridine (IUDR) and 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) induction. These results support to lead a clinical application of E5166 for combination chemotherapy in patients with nasopharyngeal carcinoma.

Topics: Antigens, Viral, Tumor; Butyrates; Butyric Acid; Carcinoma; Cells, Cultured; Fluorescent Antibody Technique; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Tretinoin; Tumor Virus Infections

A 2.4 kb RNA encoded by the murine Hox 1.1 (m6) homeobox gene is induced when F9 stem cells are differentiated with retinoic acid and dibutyryl cyclic AMP. The regulation of Hox 1.1 expression was probed by using cycloheximide, an inhibitor of protein synthesis. Production of the Hox 1.1 RNA in differentiating F9 cells was not blocked by treatment with cycloheximide, indicating that new protein synthesis is not required for its induction. On the contrary, this transcript was detected in F9 stem cells treated with cycloheximide, anisomycin, or emetine alone. Nuclear transcription assays indicated that the Hox 1.1 gene was transcribed in F9 stem cells and that the rate of transcription did not change early in the differentiation of F9 cells. These observations indicate that the induction of Hox 1.1 transcripts in F9 stem cells during differentiation is not regulated at the level of transcription initiation but results from stabilization of the transcript.

Topics: Animals; Bucladesine; Carcinoma; Embryonal Carcinoma Stem Cells; Gene Expression Regulation; Genes, Homeobox; Mice; Neoplastic Stem Cells; RNA Processing, Post-Transcriptional; Teratoma; Tretinoin

Dimethylsulfoxide, butyrate and retinoic acid, agents which induce differentiation of certain malignant cells, were examined for their effect on the activity of plasminogen activator (PA) of serumless conditioned medium (CM) of two human renal carcinoma cell lines. All three agents produced a decrease in PA activity. More than 90% of the PA was secreted in latent form, and this was not altered by the agents. Active PA components of Mr 52,000 and 93,000 were identified in cell secretions by zymography. In the presence of DMSO or butyrate the Mr 52,000 component was markedly reduced. Reversibility of the effect on PA was demonstrated for both DMSO and retinoic acid with activity of CM returning to control level after removal of the agent. That the effect was temporary agrees with most observations of the effects of these and similar agents on cells from solid tumors.

Topics: Butyrates; Butyric Acid; Carcinoma; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Kidney Neoplasms; Molecular Weight; Peptide Hydrolases; Plasminogen Activators; Tretinoin

In this paper we examined the effects of transforming growth factor beta (TGF beta) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGF beta inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGF beta causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGF beta induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGF beta enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGF beta induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGF beta, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGF beta in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGF beta, whereas growth of one carcinoma cell line was stimulated by TGF beta. These results indicate that a modified response to TGF beta could be one mechanism involved in the aberrant growth control of malignant cells.

Topics: Animals; Carcinoma; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial Cells; Growth Substances; Male; Peptides; Rabbits; Trachea; Tracheal Neoplasms; Transforming Growth Factors; Tretinoin

A panel of human cell lines was screened for production of colony-stimulating factor-1 (CSF-1) using a specific radioreceptor assay and criterion of macrophage colony growth in mouse bone marrow culture. The pancreatic carcinoma lines MIA PaCa and PANC were found to secrete high levels of CSF-1. In a bone marrow proliferation assay, the activities from these two lines were blocked by a CSF-1 specific neutralizing antiserum, confirming the predominant content of this macrophage growth factor. MIA PaCA cells stopped secreting CSF-1 when transferred to various serum-free media. Serum-free production could be reinitiated by phorbol myristic acetate (PMA). Purified CSF-1 from serum-free MIA PaCa cells stimulated the formation of 14-day colonies from total and nonadherent mononuclear human bone marrow cells. Most of the colonies consisted exclusively of large, dispersed macrophages that were intensely stained for nonspecific esterase. Although similar numbers of human 14-day colonies were stimulated by CSF-1 and other CSFs, more CSF-1 was required for the proliferation of human as compared with murine bone marrow progenitors. Northern analysis of mRNA from induced-MIA PaCa cells, using a human CSF-1 oligonucleotide probe, revealed multiple species of CSF-1 mRNA ranging from 1.5 to 4.5 kilobases (kb). Uninduced, serum-free cultures showed only the largest mRNA species, suggesting that serum removal interfered with CSF-1 mRNA processing related to synthesis and/or secretion of the protein. Regulation of the production of CSF-1 may be an important physiological process in hematopoiesis and macrophage functioning.

Topics: Animals; Bone Marrow Cells; Carcinoma; Cell Line; Colony-Stimulating Factors; Gene Expression Regulation; Humans; Macrophages; Mice; Pancreatic Neoplasms; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin

The cellular content of receptors for retinol (CRBP) and retinoic acid (CRABP) was measured in 148 human mammary carcinomas. High levels of CRABP were found in lobular carcinomas while those of the papillary subgroup had low levels of the receptor. Intermediate values for CRABP were observed for ductal, colloid and medullar carcinomas. The cellular levels of CRBP were high in ductal carcinomas and low in papillar carcinomas. A positive correlation was observed between the receptor for estradiol and CRABP. However, no significant correlation was found between the tumor cell DNA pattern and the content of CRABP and CRBP respectively. Recurrence of disease could be predicted by the nodal status and the level of estradiol receptor while the DNA pattern and the content of receptors for retinoic acid and retinol failed.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Papillary; Carrier Proteins; DNA, Neoplasm; Female; Humans; Middle Aged; Neoplasm Proteins; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin

Topics: Carcinoma; Cell Differentiation; Cell Division; Cell Line; Humans; Retinoids; Tretinoin

Topics: Butyrates; Butyric Acid; Carcinoma; China; Croton Oil; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Plant Extracts; Plants, Medicinal; Stimulation, Chemical; Tretinoin; Virus Activation; Vitamin A Deficiency

Syrian hamsters were given in a single dose of N-nitrosobis(2-oxopropyl)-amine (BOP) (40 mg/kg, s.c.) and 1 week later were fed 1 of 4 retinoid types (13-cis-retinoic acid (13-cis-RA), N-ethylretinamide (ERA), 2-hydroxyethylretinamide (OH-ERA), or 4-hydroxyphenylretinamide (PRA)) each at 3 levels (0.05, 0.1, 0.2 mM/kg diet). The pancreatic carcinoma incidence was not influenced significantly by feeding retinoids. The pancreatic adenoma incidence, however, was reduced by feeding each of the retinoids to female hamsters, with the reduction varying with the retinoid fed (13-cis-RA greater than ERA and OH-ERA greater than PRA). In male hamsters increased numbers of pancreatic adenomas were observed after feeding OH-ERA and PRA. Tumors induced in other tissues were reduced by retinoids in females, but not in males. Females fed 13-cis-RA and ERA had a lower incidence of gall bladder polyps, and feeding OH-ERA reduced the liver tumor incidence. Food consumption and serum alkaline phosphatase ans aspartate amino transferase activities were not influenced by BOP or retinoid type or level. Body and pancreas weight were influenced by retinoid level, but the effects were not consistently dose-related.

Topics: Animals; Carcinoma; Cricetinae; Female; Fenretinide; Male; Mesocricetus; Neoplasms, Experimental; Nitrosamines; Pancreatic Neoplasms; Sex Factors; Tretinoin

Male Syrian golden hamsters received 12 weekly intratracheal exposures to 0.5% N-methyl-N-nitrosourea with a special catheter. Following exposures, animals were randomized into 4 groups of 63 hamsters and placed on diets of lab meal or meal with 120 mg 13-cis-retinoid acid (CRA)/kg, 327 mg ethyl retinamide (ER)/kg, or 343 mg N-(2-hydroxyethyl)retinamide (HR)/kg for 6 months at which time all hamsters were killed. The observed incidences of tracheal epithelial neoplasms (No. of animals with tumors/total No. of animals) were 10/63 (lab meal), 22/61 (CRA), 24/63 (ER), and 17/62 (HR). The incidence of carcinomas (No. of animals with tumors/total No. of animals) were 4/63 (lab meal), 12/61 (CRA), 12/63 (ER), and 11/62 (HR). The weight loss and mortality relative to those in the group fed the lab meal were significantly in the group fed HR but not in the other retinoid-treated groups.

Topics: Amyloidosis; Animals; Carcinoma; Cricetinae; Isotretinoin; Male; Mesocricetus; Methylnitrosourea; Neoplasms, Experimental; Precancerous Conditions; Probability; Tracheal Neoplasms; Tretinoin

Blinded analyses of the concentrations of binding proteins for retinol and retinoic acid (CRABP) in homogenates of cancer and normal tissue aliquots obtained from human cervix, endometrium, ovary, breast, and lung were carried out by the sucrose gradient ultracentrifugation technique. In carcinomas of the cervix and endometrium, CRABP mean values of 50.4 and 123.2 pmol/g tissue, respectively were detected. Such concentrations represent a 3- and 4-fold increase over the mean values of CRABP in the normal cervix (16.9 pmol/g) and normal endometrium (30.8 pmol/g), respectively. In carcinomas of the ovary, the mean CRABP level was 128.6 pmol/g compared to the maximal mean value of less than or equal to 0.46 pmol/g in the normal ovary. Elevated levels of CRABP were also found in breast and lung carcinomas compared to the amounts detected in the same patient in normal tissue aliquots of the same organ. The differences between CRABP concentrations in cervical, endometrial, ovarian, and breast carcinomas and those in normal tissue are statistically significant. In contrast, cellular retinol-binding protein concentrations were reduced in the endometrial, ovarian, breast, and lung carcinomas compared to normal tissues. There were no significant differences between the log-mean concentrations of cellular retinol-binding proteins in the cytosols from tissue aliquots of carcinoma of the cervix and those in the cytosols from tissue aliquots of normal cervix.

Topics: Carcinoma; Carcinoma, Squamous Cell; Carrier Proteins; Endometrium; Female; Genital Neoplasms, Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Uterine Cervical Neoplasms; Uterine Neoplasms

Retinoic acid-binding protein (RABP), which is distinctly present in embryonic colon and lung, is below the limits of detection in adult mouse colon and lung. The binding protein is present in malignant murine colon tumors as well as in lungs of animals bearing subcutaneously implanted tumors. Primary cell cultures from 1 g of colon tumor 26 gave rise to about 10(7) tumor cells and yielded 30 mg of extractable protein. The lower limit for detection of RABP, based on the appearance of its specific 2S peak after sucrose density gradient sedimentation, was 0.1 mg of protein, which corresponds to 3.3 x 10(4) tumor cells. After subcutaneous implantation of colon tumor 26 in mice, no RABP peak was evident in the lung extracts up to the fourth day. From the fifth day onwards, RABP appeared in lung extracts, possibly as a consequence of pulmonary metastasis. Fragments of mouse lungs containing the metastatic tumor foci were reimplanted subcutaneously and produced tumors that contained RABP at levels comparable to those in colon tumor 26. The primary subcutaneous tumors and pulmonary metastatic tumors showed the same histologic appearance--an undifferentiated carcinoma. On the 15th day of subcutaneous implantation of colon tumor 26 in mice, RABP was detected in lung and brain but in none of the other tissues where the protein is normally undetectable. After intraperitoneal implantation of colon tumor 26 in mice, no well-defined RABP peaks were detected from their liver extracts. None of the three normal human colon extracts analyzed for RABP or a dihydrotestosterone-binding protein (DHTBP) contained any detectable amounts of either of the binding proteins. However, 70% of the human colon tumors contained RABP and 90% contained DHTBP. Both of these binding proteins were evident in the two human colon tissues adjoining colon tumors.

Topics: Adenocarcinoma; Animals; Brain Chemistry; Carcinoma; Carrier Proteins; Centrifugation, Density Gradient; Colonic Neoplasms; Dihydrotestosterone; Humans; Lung; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasms, Experimental; Tretinoin

The properties of a new aromatic retinoic acid analog are described. The compound: all-trans-N-ethyl-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraenamide = Ro 11-1430, exerts a therapeutic influence on chemically induced papillomas and carcinomas of the skin of mice. It leads to a marked regression of chemically induced epithelial tumors but does not inhibit the growth of transplantable tumors. The therapeutic use of retinoic acid and its analogs is limited by the appearance of the toxic side effects of the so-called hypervitaminosis A syndrome. The relationship between the anti-tumor activity and these toxic effects is considered a good inidicator for establishing the quality of a compound. The new retinoic acid analog posses a ten times more favorable therapeutic ratio than retinoic acid. The mechanism of action is discussed.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; Mice; Neoplasms, Experimental; Papilloma; Skin Neoplasms; Tretinoin; Vitamin A

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; Croton Oil; Female; Mice; Neoplasms, Experimental; Papilloma; Skin Neoplasms; Tretinoin; Vitamin A

Topics: Age Factors; Animals; Breast; Breast Neoplasms; Carcinoma; Epithelial Cells; Epithelium; Fetus; Humans; Lung; Lung Neoplasms; Molecular Weight; Neoplasm Proteins; Protein Binding; Rats; Tretinoin; Vitamin A