tretinoin and Cell-Transformation--Neoplastic

Vitamin A is an important trace essential nutrient. Vitamin A is present as a retinyl ester in animal foods and as β-carotene (provitamin A), which is a precursor of vitamin A, in plant foods such as green and yellow vegetables. After ingestion and absorption in the body, these are converted into retinol and stored as retinyl esters in stellate cells in the liver. The stored retinyl esters are decomposed into retinol as needed, and converted into the aldehyde retinal, which plays an important role in vision. Retinoic acid (RA) has a variety of effects. In particular, RA is used as a therapeutic agent for acute promyelocytic leukemia. This review will cover (1) elucidation of anti-refractory cancer effects of retinol (vitamin A) not mediated by RA receptors, (2) elucidation of anti-cancer effects of RA not mediated by RA receptors and (3) the development of candidate new anti-cancer agents that combine the actions of RA and retinol. Lessons learned from these findings are that vitamin A has anti-cancer activity not mediated by RA receptors; that nutritional management of vitamin A leads to prevention and treatment of cancer, and that new compounds developed from RA derivatives represent good anti-cancer drug candidates that are in various stages of clinical trials.

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Liver; Neoplasms; Receptors, Retinoic Acid; Retinyl Esters; Tretinoin; Vitamin A

Retinoic acid receptor-related orphan receptors (RORs) include RORα (NR1F1), RORβ (NR1F2), and RORγ (NR1F3). These receptors are reported to activate transcription through ligand-dependent interactions with co-regulators and are involved in the development of secondary lymphoid tissues, autoimmune diseases, inflammatory diseases, the circadian rhythm, and metabolism homeostasis. Researches on RORs contributing to cancer-related processes have been growing, and they provide evidence that RORs are likely to be considered as potential therapeutic targets in many cancers. RORα has been identified as a potential therapeutic target for breast cancer and has been investigated in melanoma, colorectal colon cancer, and gastric cancer. RORβ is mainly expressed in the central nervous system, but it has also been studied in pharyngeal cancer, uterine leiomyosarcoma, and colorectal cancer, in addition to neuroblastoma, and recent studies suggest that RORγ is involved in various cancers, including lymphoma, melanoma, and lung cancer. Some studies found RORγ to be upregulated in cancer tissues compared with normal tissues, while others indicated the opposite results. With respect to the mechanisms of RORs in cancer, previous studies on the regulatory mechanisms of RORs in cancer were mostly focused on immune cells and cytokines, but lately there have been investigations concentrating on RORs themselves. Thus, this review summarizes reports on the regulation of RORs in cancer and highlights potential therapeutic targets in cancer.

Topics: Animals; Carcinogenesis; Cell Transformation, Neoplastic; Circadian Rhythm; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Neoplasms; Orphan Nuclear Receptors; Receptors, Retinoic Acid; Tretinoin; Vitamin A

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

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Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; 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YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Acute promyelocytic leukemia (APL) is a distinct subset of acute myeloid leukemia (AML) associated with peculiar biologic and clinical features and requiring specific management. At the genetic level, APL is featured by a unique chromosome translocation t(15;17) which results in the PML-RARα gene fusion and chimeric protein. APL is the first example of differentiation therapy targeted to a defined genetic target i.e. PML-RARα. PML-RARα behaves as an altered retinoic acid receptor with an ability of transmitting oncogenic signaling leading to accumulation of undifferentiated promyelocytes. All-trans-retinoic acid (ATRA) induces disease remission in APL patients by triggering terminal differentiation of leukemic promyelocytes. More recently, arsenic trioxide (ATO) has been shown to contribute degradation of the PML-RARα oncoprotein through bonding the PML moiety and has shown excellent synergism with ATRA in clinical trials. Elucidating the oncogenic signaling of PML-RARα through various transcription factors and the study of APL mouse models have greatly helped to understand the molecular pathogenesis of APL. However, the precise molecular mechanism by which t(15;17) is formed and initiates leukemia remains unknown. While transforming oncogenic potential of PML-RARα has been described extensively, the mechanistic events important for the formation of t(15;17) have been taken from the model of Therapy-related APL (t-APL).

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Clinical Trials as Topic; Disease Models, Animal; DNA End-Joining Repair; Drug Synergism; Granulocyte Precursor Cells; Humans; Leukemia, Promyelocytic, Acute; Mice; Molecular Targeted Therapy; Neoplasm Proteins; Neoplasms, Second Primary; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Oxides; Signal Transduction; Topoisomerase II Inhibitors; Translocation, Genetic; Tretinoin

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by a chimeric oncoprotein containing the C terminus of the retinoic acid receptor-a (RARa) fused to an N-terminal partner, most commonly promyelocytic leukemia protein (PML). Mechanistically, PML-RARa acts as a transcriptional repressor of RARa and non-RARa target genes and antagonizes the formation and function of PML nuclear bodies that regulate numerous signaling pathways. The empirical discoveries that PML-RARa-associated APL is sensitive to both all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO), and the subsequent understanding of the mechanisms of action of these drugs, have led to efforts to understand the contribution of molecular events to APL cell differentiation, leukemia-initiating cell (LIC) clearance, and disease eradication in vitro and in vivo. Critically, the mechanistic insights gleaned from these studies have resulted not only in a better understanding of APL itself, but also carry valuable lessons for other malignancies.

Topics: Animals; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Mice; Oncogene Proteins, Fusion; Oxides; Remission Induction; Signal Transduction; Transcriptional Activation; Tretinoin

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The prognosis of APL is changing, from the worst among AML as it used to be, to currently the best. The application of all-trans-retinoic acid (ATRA) to the induction therapy of APL decreases the mortality of newly diagnosed patients, thereby significantly improving the response rate. Therefore, ATRA combined with anthracycline-based chemotherapy has been widely accepted and used as a classic treatment. It has been demonstrated that high doses of cytarabine have a good effect on the prevention of relapse for high-risk patients. However, as the indications of arsenic trioxide (ATO) for APL are being extended from the original relapse treatment to the first-line treatment of de novo APL, we find that the regimen of ATRA, combined with ATO, seems to be a new treatment option because of their targeting mechanisms, milder toxicities and improvements of long-term outcomes; this combination may become a potentially curable treatment modality for APL. We discuss the therapeutic strategies for APL, particularly the novel approaches to newly diagnosed patients and the handling of side effects of treatment and relapse treatment, so as to ensure each newly diagnosed patient of APL the most timely and best treatment.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Cell Transformation, Neoplastic; Central Nervous System Neoplasms; Consolidation Chemotherapy; Drug Synergism; Humans; Induction Chemotherapy; Leukemia, Promyelocytic, Acute; Maintenance Chemotherapy; Oxides; Recurrence; Tretinoin

Acute promyelocytic leukemia (APL) is a distinct subset of acute myeloid leukemia. An abnormal fusion gene, PML/RARA is detected in approximately 98% of patients with APL. PML/RARA confers long-term self-renewal properties to promyelocytes. All-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which are the major molecularly targeted therapies in APL, affect the PML/RARA fusion protein and cause differentiation and apoptosis of APL cells. Although the leukemia-initiating cells of APL may be present in a myeloid progenitor committed compartment, the precise population of those remains to be elucidated. However, recent studies have demonstrated the effect of ATRA and ATO on APL leukemia-initiating cells. Through these studies, we can understand more deeply how current clinical therapies lead to long-lasting remission of APL. ATRA and ATO have improved the prognosis of APL patients and have changed the role of hematopoietic stem cell transplantation (HSCT). At present, HSCT is not indicated for patients with APL in first complete remission, and considered for patients with relapsed APL. In this review, we discuss the three main topics as follows: the leukemia-initiating cells in APL, the current state-of-the-art treatment for newly diagnosed and relapsed APL, and the role of HSCT in APL patients.

Topics: Animals; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Glutamates; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Promyelocytic, Acute; Myeloid Progenitor Cells; Neoplastic Stem Cells; Nuclear Proteins; Oxides; Prodrugs; Promyelocytic Leukemia Protein; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

For decades, retinoic acid has been known to alter the proliferation and differentiation of myeloid cells. Currently, retinoic acid is a front-line agent in the treatment of certain forms of acute myelogenous leukemia. In this review, we focus on recent advances in our understanding of the mechanisms by which retinoids affect growth and proliferation of myeloid cells and contribute to the pathogenesis of leukemia. We have not attempted to summarize the related clinical literature.. The past 2 years have yielded important understanding of the mechanisms by which retinoids and their nuclear receptors interact with other signal transduction pathways and transcription factors to modify chromatin, alter gene expression, and participate in normal myeloid differentiation and leukemogenesis. Important advances regarding cell biology, molecular biology, biochemistry, and animal studies of retinoids and myeloid differentiation are reviewed.. Greater understanding of the role of retinoids and their receptors in myeloid cell growth and differentiation provides important insight into normal myelopoiesis. These findings have resulted in successful rational approaches to the treatment of acute leukemia and provide the promise of improved treatments in the near future.

Topics: Animals; Cell Differentiation; Cell Transformation, Neoplastic; Chromatin; Gene Expression Regulation; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Mice; Myeloid Cells; Myelopoiesis; Myeloproliferative Disorders; Oncogene Proteins, Fusion; Protein Processing, Post-Translational; Receptors, Retinoic Acid; Retinoids; Signal Transduction; Transcription Factors; Tretinoin

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

Topics: Animals; Anticarcinogenic Agents; Bronchi; Bronchial Diseases; Carcinoma, Embryonal; Cell Differentiation; Cell Transformation, Neoplastic; Cyclins; Cysteine Endopeptidases; Endopeptidases; Epithelial Cells; G1 Phase; Gene Expression Profiling; Gene Expression Regulation; Humans; Metaplasia; Mice; Models, Biological; Multienzyme Complexes; Neoplasms; Precancerous Conditions; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured; Ubiquitin; Vitamin A Deficiency

Proteasomes are the main non lysosomal proteolytic structures of the cells. They correspond to the major system eliminating abnormal proteins, short half-life proteins and proteins controlling the cell cycle. They are essential for the production of peptides subsequently presented by the MHC-I. They are formed by a proteolytic core (the 20S proteasome) made of 4 rings of 7 proteic subunits associated with regulatory complexes (namely the 19S complex forming the 26S proteasome). Using classical cell biology techniques (cytometry, immunofluorescence microscopy, Western blot) our group has particularly studied the proteasome expression of leukaemic cell lines (U937 and CCRF-CEM) during in vitro differentiation induced by PMA and Vitamin D plus retinoïc acid. During differentiation, the level of proteasome expression and its localization vary. The various monoclonal antibodies used provided different patterns according to the different subunits. There was a general trend to a disappearance of nuclear proteasome and to a decrease in their cytoplasmic expression. In contrast, proteosomal antigens were increased on the cell membrane and in culture supernatants. We derived an ELISA test to measure plasma proteasome concentrations. Preliminary results showed differences between patients with haemopoietic malignancies or solid tumors and normal donors. Proteasome levels varied under treatment. They were correlated with LDH levels. Taken together, these results argue in favor of a role for cellular proteasomes in malignant differentiation process, and emphasize the qualitative changes in proteasome expression. Plasma proteasomes do not only reflect tumor cell mass and could play a role in addition to their proteolytic activity. They seem to be a relevant tool for diagnosis, prognosis and therapeutic monitoring.

Topics: Biomarkers, Tumor; Cell Differentiation; Cell Membrane; Cell Nucleus; Cell Transformation, Neoplastic; Cysteine Endopeptidases; Cytoplasm; Enzyme-Linked Immunosorbent Assay; Hematologic Neoplasms; Humans; L-Lactate Dehydrogenase; Leukemia-Lymphoma, Adult T-Cell; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Proteasome Endopeptidase Complex; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; U937 Cells; Vitamin D

Topics: Animals; Cell Transformation, Neoplastic; Cytokines; Female; Gene Expression Regulation; Humans; Iodides; Mammary Glands, Animal; Pregnancy; Promoter Regions, Genetic; Symporters; Thyroglobulin; Thyroid Neoplasms; Thyrotropin; Tissue Distribution; Tretinoin

Acute progranulocytic leukemia (APL) is one of the most curable of all human cancers. Combination treatment with retinoic acid (RA) and anthracycline-based chemotherapy is safe and effective for the vast majority of patients, and several novel treatment approaches are under investigation for high-risk or relapsed patients. The APL-specific oncogenes PML-RAR alpha and PLZF-RAR alpha both bind nuclear corepressors and recruit histone deacetylase activity to promoters of RA target genes. The differential sensitivity of binding of these oncogenes to nuclear corepressors in the presence of RA appears to explain the resistance of PLZF-RAR alpha-related APL to RA and at the same time explains the effectiveness of RA in PML-RAR alpha-positive APL. Transcriptional repression of RA target genes, mediated by histone deacetylase activity, may thus be a key pathogenetic event in APL. Cure of the minority of resistant patients requires further refinement of current treatment approaches and appropriately timed incorporation of novel therapies, such as arsenic trioxide or histone deacetylase inhibitors.

Topics: Cell Transformation, Neoplastic; DNA-Binding Proteins; Histone Deacetylases; Humans; Interleukin-6; Kruppel-Like Transcription Factors; Leukemia, Promyelocytic, Acute; Models, Biological; Neoplasm Proteins; Oncogene Proteins, Fusion; Oncogenes; Promyelocytic Leukemia Zinc Finger Protein; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription Factors; Tretinoin

Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control

Topics: Acetylation; Acetyltransferases; Animals; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Chromatin; DNA-Binding Proteins; E2F Transcription Factors; Gene Expression Regulation; Growth Substances; Histone Acetyltransferases; Histone Deacetylases; Histones; Humans; Mice; Mice, Knockout; Models, Biological; Protein Processing, Post-Translational; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; Saccharomyces cerevisiae Proteins; Signal Transduction; Transcription Factor DP1; Transcription Factors; Transcription, Genetic; Tretinoin

The vast majority of cases of APL are associated with t(15; 17) leading to the formation of PML-RAR alpha, RAR alpha-PML and aberrant PML fusion products. PML-RAR alpha is invariably transcribed and is believed to mediate leukaemogenesis. PML was initially considered to be a transcription factor. However, characterisation of other RING finger containing proteins shows no direct evidence for DNA binding. The RING, B-box, and coiled-coil domains are more likely to represent sites of protein-protein interaction and may be critical for the stability of the multiprotein nuclear domains of which PML is an integral part. In APL the nuclear bodies become disrupted, presumably as a consequence of the presence of PML-RAR alpha and aberrant PML proteins that might render the structure unstable. PML-RAR alpha is capable of binding RXR and sequestering it into the disrupted nuclear domains. Sequestration of RXR would be expected to limit high affinity binding of VDR, TR and residual RARs to DNA response elements and might account for the block in myeloid differentiation at the promyelocyte stage that characterizes APL. Recently PML has been found to have growth suppressor/anti-oncogenic activity. It is unclear whether this is a property of PML itself or reflects a nonspecific function of the PML-associated nuclear domains. Hence the PML/RAR alpha rearrangement alone may be sufficient to cause APL. Abnormal PML function may prevent its growth-suppressor activity, leading to leukaemic transformation; concomitant disruption of retinoid pathways due to sequestration of RXR and/or an abnormal repertoire and character of response element activation mediated by the fusion protein, causing the block in myeloid differentiation (Fig. 3). Disruption of RAR alpha would be expected to account for the similar leukaemic phenotype associated with the t(5;17) and t(11;17) APL cytogenetic variants. Further characterisation of NPM and PLZF at the structural and functional level will determine whether PML and other proteins disrupted in APL associated translocations play an active or purely permissive role in leukaemogenesis and will help dissect the events leading to transformation from those causing blockade of myeloid differentiation and mediating the response to ATRA.

Topics: Adult; Antineoplastic Agents; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Middle Aged; Neoplasm Proteins; Nuclear Proteins; Oncogene Proteins, Fusion; Polymerase Chain Reaction; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Transcription Factors; Translocation, Genetic; Tretinoin; Tumor Suppressor Proteins

Topics: Animals; Arsenic; Artificial Gene Fusion; Cell Transformation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription Factors; Translocation, Genetic; Tretinoin; Tumor Suppressor Proteins

Tretinoin (all-trans-retinoic acid) is a retinol (vitamin A) derivative which has been evaluated as a topical treatment for the symptoms of photodamaged skin. In several well-controlled clinical trials, the proportion of patients showing improvement was significantly higher with 0.01 or 0.05% tretinoin cream than with placebo for criteria such as global assessment, fine and coarse wrinkling, pigmentation and roughness. Improvements in the overall severity of photodamage were also significantly greater with tretinoin than with placebo. The extent of clinical improvement with tretinoin has generally been moderate, but cytological and histological studies have shown that extensive changes in the epidermis and dermis occur during treatment. However, the permanency and clinical significance of these changes has yet to be fully evaluated. Topical tretinoin has also demonstrated potential for the treatment and eradication of premalignant skin growths such as actinic keratoses, and may be useful as combination therapy with fluorouracil in this indication. Dermatitis (the retinoid skin reaction) is the most common adverse event experienced by patients receiving topical tretinoin; this condition may persist for up to 3 months, but is usually mild or moderate in nature. Thus, topical tretinoin has been shown to be an effective form of treatment for the characteristic signs of photodamaged skin. Its ability to produce significant, albeit moderate, clinical improvements in symptoms such as fine wrinkling, roughness and pigmentation, together with its relatively mild or moderate adverse event profile, suggests that it is likely to be of considerable value in this indication. The treatment and eradication of potentially malignant growths such as actinic keratoses may also prove to be an important application for topical tretinoin.

Topics: Administration, Topical; Animals; Cell Transformation, Neoplastic; Clinical Trials as Topic; Drug Therapy, Combination; Fluorouracil; Humans; Skin; Skin Diseases; Sunlight; Tretinoin

Acute promyelocytic leukemia (APL), is a homogeneous subgroup of acute myelogenous leukemias characterized by phenotypic and genetic markers. APL is associated with a reciprocal chromosomal translocation t(15,17) which has been shown to disrupt the retinoic acid receptor alpha (RAR alpha) gene. As a result, a portion of the RAR alpha gene becomes fused with a chromosome 15 locus termed PML (promyelocytic myeloid leukemia) from which chimeric PML/RAR alpha fusion mRNAs are expressed. The presence of these fusion transcripts in APL patients strongly support the hypothesis that both the t(15;17), and thus PML/RAR alpha, play a crucial role in the leukemogenesis of this disease. APL cells are specifically responsive to all-trans retinoic acid (ATRA) and this characteristic has allowed the first differentiation therapy with retinoic acid. However, failure or partial responses are observed and, though this has most frequently been reported in patients at second or third relapse. The molecular basis of the absence of ATRA response in these patients has not been determined.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Multigene Family; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Translocation, Genetic; Tretinoin

In this article we have attempted to review the literature on the regulation of nuclear protooncogene expression by steroid hormones and other small molecules that interact with receptors of the steroid/thyroid superfamily. Until about 5 years ago, there were relatively few reports of steroidal regulation of cellular oncogenes, but hundreds of papers on this topic have appeared since then. This demonstrates the intense interest in this area that has developed recently. It now been demonstrated that all the major classes of steroid hormones control expression of nuclear protooncogenes in one or more systems. Given the actions of these proteins as transcription factors and their central role in cellular communications systems, it seems likely that they play a key role in mediating the biological effects of steroids on processes such as proliferation and differentiation. To date, most of the work in this general area has focused primarily on the regulation of three genes: c-fos, c-jun, and c-myc. However, a quick glance at the table of nuclear protooncogenes in the introduction of this article indicates that over 40 nuclear protooncogenes are now recognized. For the large majority of these, regulatory effects of steroids and related molecules have not yet been reported. Hence, we predict that reports in this general area of research will continue to appear at a very rapid rate over the next few years. In addition, we have tried to provide enough background information for readers to get an overview of the regulation of nuclear protooncogene expression by nonsteroidal factors. We felt this information was important to emphasize that steroid hormones represent only one of the many classes of regulatory molecules that control expression of nuclear protooncogenes. Thus, an important area for future research will be to understand how these multiple regulatory systems interact to control expression of this important class of cellular oncogenes and the biological processes that they mediate.

Topics: Animals; Cell Transformation, Neoplastic; DNA; DNA-Binding Proteins; Gene Expression Regulation; Genes, fos; Genes, jun; Genes, myc; Glucocorticoids; Gonadal Steroid Hormones; Hormones; Mammals; Microbodies; Nuclear Proteins; Oncogenes; Rats; Receptors, Steroid; Regulatory Sequences, Nucleic Acid; Signal Transduction; Steroids; Thyroid Hormones; Tretinoin

Topics: Animals; Cell Transformation, Neoplastic; Humans; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Topics: Carrier Proteins; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 12; Cisplatin; Combined Modality Therapy; Drug Resistance; Gene Expression Regulation, Neoplastic; Genes, ras; Genetic Markers; Humans; Male; Neoplasms, Germ Cell and Embryonal; Receptors, Retinoic Acid; Testicular Neoplasms; Tretinoin

Acute promyelocytic leukaemia has two highly specific particularities: a t(15;17) chromosomal translocation and the ability of a differentiation inducer all-trans-RA, to revert the malignant phenotype both in vitro and in vivo. Molecular characterization of the t(15;17) translocation has shown that it fuses a previously unknown zinc finger encoding gene, PML, to the RAR alpha, suggesting a link between the molecular mechanism of transformation and of RA dependent differentiation. The PML/RAR alpha fusion receptor--which is functionally altered--may block RA target genes, impair RA mediated differentiation and lead to transformation. Alternatively, or in addition, the PML transduction pathway may also be affected. Although it is clear that RA treatment must relieve APL cells from differentiation arrest, so far no model can satisfactorily account for this effect.

Topics: Carrier Proteins; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Genes; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Transcription Factors; Translocation, Genetic; Tretinoin; Tumor Suppressor Proteins

Topics: Cell Division; Cell Transformation, Neoplastic; Hemorrhagic Disorders; Humans; Leukemia, Promyelocytic, Acute; Prognosis; Risk Factors; Thrombosis; Tretinoin

Chronic hepatitis B virus (HBV) infection is etiologically related to human hepatocellular carcinoma (HCC). Most HCCs contain integrated HBV DNA in the liver cellular DNA, suggesting that the integration may be involved in carcinogenesis. From a comparison of a single HBV integration site present in a hepatoma with the corresponding unoccupied site in the non-tumourous tissue of the same liver, we have shown that HBV DNA inserted in a putative cellular exon with striking similarity to the DNA-binding domain of the thyroid/steroid hormone receptors. The corresponding cDNA has been isolated (hap gene) and shown to encode the retinoic acid receptor. In the original patient, integration took place so that the first codons of the viral surface protein gene became fused in frame with most of the hap gene. Because retinoic acid is known to regulate the transcription of target genes crucial for cellular growth and differentiation, it is most probable that consequent to the HBV insertion, hap, usually transcribed at a very low level in normal hepatocytes, became inappropriately expressed as an altered chimaeric retinoic acid receptor, thus contributing to the cell transformation. These results strongly support the possibility that HBV may play a direct role in liver carcinogenesis by insertional mutagenesis.

Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Carrier Proteins; Cell Transformation, Neoplastic; DNA; DNA, Neoplasm; DNA, Viral; Exons; Gene Expression Regulation, Neoplastic; Genes, Viral; Hepatitis B; Hepatitis B virus; Humans; Liver Neoplasms; Molecular Sequence Data; Mutation; Organ Specificity; Receptors, Retinoic Acid; Recombination, Genetic; Tretinoin

Transforming growth factor-beta (TGF-beta) plays an important role in controlling proliferation or differentiation in almost all epithelial tissues. The pathophysiology of TGF-beta during carcinogenesis is now an important area of investigation, since it appears that as the process of carcinogenesis progresses, epithelial cells often become refractory to the growth-regulatory actions of TGF-beta. In this article we consider the possible cellular and molecular bases for this phenomenon, and then discuss some pharmacological approaches to enhancing the synthesis or activity of TGF-beta. These approaches may provide new modalities for prevention of carcinogenesis, if they can be applied during the early stages of the disease process, before cells become refractory. We give particular attention to tamoxifen and retinoic acid, since it has been shown that these agents, which are of known efficacy for prevention of cancer, can markedly enhance the secretion of specific isotypes of TGF-beta by several types of cells.

Topics: Amino Acid Sequence; Animals; Blood Platelets; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Growth Inhibitors; Humans; Molecular Sequence Data; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Sequence Homology, Nucleic Acid; Species Specificity; Tamoxifen; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

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

Several key events in the multistep process of neoplastic transformation of rat tracheal epithelium (RTE) are described. Whether tracheal epithelium is exposed in vivo to carcinogenic agents or whether primary tracheal epithelial cells are exposed in vitro to carcinogens, initiated stem cells can be detected soon after the exposure by their ability to grow under selective conditions in culture. These initiated stem cells differ fundamentally from normal stem cells in their response to factors normally constraining proliferation and self-renewal. Thus, disruption of inhibitory control mechanisms of stem cell replication appears to be the first event in RTE cell transformation. While the probability of self-renewal (PSR) is clearly increased in initiated stem cells, most of the descendants derived from such stem cells differentiate and become terminal and do not express transformed characteristics. Progression from the first to the second stage of RTE cell transformation, the stage of the immortal growth variant (IGV), is characterized by loss of responsiveness to the growth-restraining effects of retinoic acid. In the third stage of neoplastic transformation, the stage during which neoplastic growth variants (NGV) appear, a growth factor receptor gene is inappropriately expressed in some of the transformants. Thus, it appears that loss of growth-restraining mechanisms may be an early event, and activation of a growth stimulatory mechanism a late event, in neoplastic transformation of RTE cells.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Neoplastic Stem Cells; Trachea; Tretinoin

Cancer appears to be a disease of altered maturation, with changes in genetic expression leading to a situation in which the physiological regulation of cellular proliferation and maturation are altered. Environmental factors as well as defined chemical agents have been demonstrated to have the capacity to convert neoplastic cells to end-stage forms with a finite life span through a process characteristic of cellular maturation. The correction of genetic defects by these inducers of differentiation does not appear to be required; the critical feature is that the differentiated cells assume a state in which they no longer possess the capability for continued cellular replication. The extrapolation of these advances, accomplished in experimental systems, to clinical practice should yield significant decreases in the neoplastic cell burden without the degree of morbidity produced by aggressive therapy with cytodestructive agents, especially when employed in multidrug combinations. The ultimate introduction of differentiation as a therapeutic approach to cancer treatment if attained, however, will require a variety of principles to be established, so that optimum efficacy may be obtained from each agent, the fabrication of new agents with major changes in the ratio of the concentrations required to produce cytotoxicity relative to those necessary to initiate maturation is attained, and the elucidation of non-antagonistic combinations of differentiation inducing agents with or without cytotoxic drugs is achieved to combat the problem of tumor cell heterogeneity.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Humans; Hydrocortisone; Leukemia, Experimental; Models, Biological; Naphthacenes; Phenotype; Thioguanine; Tretinoin

The myelodysplastic syndromes represent a preleukaemic state in which a clonal abnormality of haemopoietic stem cell is characterised by a variety of phenotypic manifestations with varying degrees of ineffective haemopoiesis. This state probably develops as a sequence of events in which the earliest stages may be difficult to detect by conventional pathological techniques. The process is characterised by genetic changes leading to abnormal control of cell proliferation and differentiation. Expansion of an abnormal clone may be related to independence from normal growth factors, insensitivity to normal inhibitory factors, suppression of normal clonal growth, or changes in the immunological or nutritional condition of the host. The haematological picture is of peripheral blood cytopenias: a cellular bone marrow, and functional abnormalities of erythroid, myeloid, and megakaryocytic cells. In most cases marrow cells have an abnormal DNA content, often with disturbances of the cell cycle: an abnormal karyotype is common in premalignant clones. Growth abnormalities of erythroid or granulocyte-macrophage progenitors are common in marrow cultures, and lineage specific surface membrane markers indicate aberrations of differentiation. Progression of the disorder may occur through clonal expansion or through clonal evolution with a greater degree of malignancy. Current attempts to influence abnormal growth and differentiation have had only limited success. Clinical recognition of the syndrome depends on an acute awareness of the signs combined with the identification of clonal and functional abnormalities.

Topics: Anemia, Refractory, with Excess of Blasts; Animals; Antineoplastic Agents; Blood Cell Count; Bone Marrow; Cell Transformation, Neoplastic; Cholecalciferol; Chromosome Aberrations; Chromosome Disorders; Colony-Forming Units Assay; Colony-Stimulating Factors; DNA; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Radiation-Induced; Mice; Myelodysplastic Syndromes; Oncogenes; Preleukemia; Rats; Tretinoin

Topics: Animals; Butyrates; Butyric Acid; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cholecalciferol; Cyclic AMP; Humans; Melanoma; Protein Kinases; Retinoids; Teratoma; Tretinoin; Vitamin A

Retinoids have been shown to inhibit tumour growth in several model systems. In this paper evidence that immune effectors are important for this effect is discussed. Injection of retinoic acid (RA) into mice before challenge with allogeneic or syngeneic tumour cells results in a strong increase in cell-mediated cytotoxicity specific for the respective tumour. This stimulation appears to be due to effects taking place before or during the induction phase rather than the effector phase of cell-mediated cytolysis. The effector cells responsible for cytotoxicity express the Thy 1 antigen, are H-2 specific and are therefore T killer cells. The induction of T cell-mediated cytotoxicity requires the participation of the lymphokine interleukin 2 (IL-2). The possibility was tested that RA directly or indirectly influences the production of IL-2 and thereby stimulates the induction of T killer cells. Results indeed show that RA-injected mice display an increased capacity to produce IL-2 upon stimulation of their splenocytes in a mixed lymphocyte reaction. It appears therefore that RA has an effect on T cells that are destined to produce IL-2 upon antigenic challenge. Since IL-2 plays a role not only in the induction of specific cytotoxic T cells but also in the induction of natural killer (NK) cells, RA was also tested in a model system in which NK cells appear to play an important protective role. Results showed that split-dose irradiated mice that lose their NK activity and subsequently develop leukaemia can be protected from leukaemogenesis either by reconstitution with NK cells or by injection with RA. The question of whether this effect is due to stimulation of immune effectors or is a direct effect on the preleukaemic cells is discussed.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cytotoxicity, Immunologic; Immunity, Cellular; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Transplantation, Isogeneic; Tretinoin

Most drugs available for cancer chemotherapy exert their effects through cytodestruction. Although significant advances have been attained with these cytotoxic agents in several malignant diseases, response is often accompanied by significant morbidity and many common malignant tumours respond poorly to existing cytotoxic therapy. Development of chemotherapeutic agents with non-cytodestructive actions appears desirable. Considerable evidence exists which indicates that (a) the malignant state is not irreversible and represents a disease of altered maturation, and (b) some experimental tumour systems can be induced by chemical agents to differentiate to mature end-stage cells with no proliferative potential. Thus, it is conceivable that therapeutic agents can be developed which convert cancer cells to benign forms. To study the phenomenon of blocked maturation, squamous carcinoma SqCC/Y1 cells were employed in culture. Using this system it was possible to demonstrate that physiological levels of retinoic acid and epidermal growth factor were capable of preventing the differentiation of these malignant keratinocytes into a mature tissue-like structure. The terminal differentiation caused by certain antineoplastic agents was investigated in HL-60 promyelocytic leukaemia cells to provide information on the mechanism by which chemotherapeutic agents induce cells to by-pass a maturation block. The anthracyclines aclacinomycin A and marcellomycin were potent inhibitors of N-glycosidically linked glycoprotein biosynthesis and transferrin receptor activity, and active inducers of maturation; temporal studies suggested that the biochemical effects were associated with the differentiation process. 6-Thioguanine produced cytotoxicity in parental cells by forming analog nucleotide. In hypoxanthine-guanine phosphoribosyltransferase negative HL-60 cells the 6-thiopurine initiated maturation; this action was due to the free base (and possibly the deoxyribonucleoside), a finding which separated termination of proliferation due to cytotoxicity from that caused by maturation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Leukemia, Myeloid; Models, Biological; Naphthacenes; Thioguanine; Tretinoin

Topics: Calcitriol; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Monokines; Phorbol Esters; Proteins; Tretinoin

Topics: Animals; Carcinogens; Cell Adhesion; Cell Communication; Cell Differentiation; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Cocarcinogenesis; Fibronectins; Humans; Neoplasms, Experimental; Ornithine Decarboxylase; Plasminogen Activators; Tetradecanoylphorbol Acetate; Tretinoin

EGF-Rs are cell membrane glycoproteins of wide distribution. They have not yet been fully characterized or purified but are probably molecules of 170-190,000 mol. wt. in most cells. The growth factor EGF binds and will saturate cell surface receptors with a KA of about 5 X 10(9) M-1 although a receptor class with an affinity in excess of 10(10) M-1 has been detected in some cells. The number of receptors on a cell does not determine the level of its response. Some cell types have receptors which bind EGF, but with no mitogenic response. The ways in which receptor affinity and/or number is modulated are described. This and other evidence is reviewed in a search for a suitable model of a mechanism of action on the cell, which best fits the current data. There is ample evidence that EGF binds to the receptor; that ligand-receptor complexes cluster or aggregate; and then are internalized and degraded, but evidence for a direct connection between internalization and the subsequent mitogenic response is lacking. Good correlations between internalization and mitogenic responses have been observed and developed into a theory of endocytic activation, but there is a body of evidence which cannot be accommodated by this theory. Instead, an alternative model is suggested.

Topics: Animals; Binding Sites; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Models, Biological; Molecular Weight; Organ Specificity; Peptides; Pregnancy; Receptors, Cell Surface; Species Specificity; Teratoma; Tissue Distribution; Tretinoin

Experimental investigations of the antineoplastic effects of retinoids are reviewed in this paper. In vitro studies have shown that the hyperplastic and metaplastic response to chemical carcinogens of mouse prostate cultures is suppressed by the addition of retinoids to the culture medium, that retinoids can partially inhibit the morphologic transformation of 10T 1/2 cells by physical or chemical carcinogens, and that the growth of some non-neoplastic and some neoplastic cell lines can be inhibited by retinoids. In vivo studies have shown that retinoids can suppress papilloma and carcinoma development (the promotion phase) in the two-stage skin carcinogenesis assay, inhibit mammary and bladder carcinogenesis in mice and rats, and inhibit the growth of some transplantabletumor lines. So far it has not been possible to inhibit predictably tumour formation in the intestinal tract or the respiratory tract of rodents. Almost all the synthetic retinoids have a higher therapeutic index than the natural retinoids in the prevention or treatment of cancer.

Topics: Animals; Cell Transformation, Neoplastic; Lung Neoplasms; Neoplasms, Experimental; Skin Neoplasms; Tretinoin; Vitamin A

The early and recent investigations in the field of retinoids and cancer are reviewed. The retinoids, including natural vitamin A compounds and their synthetic analogs, present a new class of substances exerting a prophylactic and a therapeutic effect both in certain experimental tumor models and in certain clinical conditions of preneoplastic and neoplastic lesions. Because of a particular physiological mechanism of action, the retinoids offer a new approach to the cancer problem, which is different from those of surgery, X-ray therapy, conventional chemotherapy, and immunotherapy.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Clinical Trials as Topic; Humans; Neoplasms; Neoplasms, Experimental; Tretinoin; Vitamin A

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

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

To evaluate the changes in differentiation markers and therapeutic effects in all-trans-retinoic acid (ATRA)-treated patients with dedifferentiated thyroid cancer.. Between September 2001 and July 2004 eleven patients were analysed retrospectively. They had dedifferentiated thyroid cancers (DTC) (four follicular, five papillary, two oxyphilic) and were selected for treatment with ATRA (1.00+/-0.09 mg x kg x d) for 30 or 60 days. All patients had advanced stage tumours with prior operative and radioiodine treatment. Extensive tumour invasion, distant metastatic spread, and insufficient or non-existent uptake of radioiodine precluded conventional therapeutic options. Changes in I uptake, response of target lesions, and serum thyroglobulin (Tg) levels were measured and compared in these patients before and after ATRA therapy.. In 11 patients with DTC, iodine uptake was increased in four and there was a partial response (PR) of target lesions in five patients as well as two patients with stable disease. Tg was assessed in eight patients, in whom two responders showed increased radioiodine uptake or no change and decreased Tg level, as well as PR after ATRA-induced differentiation therapy.. ATRA has an effect on the differentiation status of DTC and deserves further investigation.

Topics: Adult; Antineoplastic Agents; Cell Transformation, Neoplastic; Female; Humans; Iodine Radioisotopes; Male; Middle Aged; Radionuclide Imaging; Radiopharmaceuticals; Thyroglobulin; Thyroid Gland; Thyroid Neoplasms; Treatment Outcome; Tretinoin

Twenty-two patients with acute promyelocytic leukemia were treated with all-trans retinoic acid (RA, 45 mg/m2 per day) for 90 days. Of the 22, four patients were previously untreated, two were resistant after conventional chemotherapy, and 16 were in first (n = 11), second (n = 4), or third (n = 1) relapse. We observed 14 complete response, four transient responses, one failure, and three early deaths. Length of hospitalization and number of transfusions were notably reduced in complete responders. Correction of coagulation disorders and an increase of WBCs were the first signs of all-trans RA efficacy. Morphologic analysis performed at days 0, 15, 30, 45, 60, and 90 showed that complete remissions were obtained without bone marrow (BM) hypoplasia. Presence of Auer rods in the maturing cells confirmed the differentiation effect of the treatment. At remission, the t(15;17) initially present in 20 patients was not found. The in vitro studies showed a differentiation in the presence of all-trans RA in 16 of the 18 tested cases. The single nonresponder to all trans RA in vitro did not respond in vivo. Adverse effects of RA therapy--skin and mucosa dryness, hypertriglyceridemia, and increase of hepatic transaminases--were frequently noted. We also observed bone pain in 11 patients and hyperleukocytosis in four patients. Whether maintenance treatment consisted of low-dose chemotherapy or all-trans RA, early relapses were observed. Five patients are still in complete remission (CR) at 4 to 13 months. Our study confirms the major efficacy of all-trans RA in M3, even in relapsing patients. Remissions are obtained by a differentiation process.

Topics: Adult; Aged; Aged, 80 and over; Cell Transformation, Neoplastic; Female; Follow-Up Studies; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Tretinoin

Internal dosimeters that can provide information about responses to chemopreventive agents in a short time would be invaluable for planning treatment protocols for large-scale intervention trials. Micronuclei meet many of the prerequisites of a good intermediate endpoint. They can be quantified in cultured cells, animal tissues and human exfoliated cells and biopsies. With image scanning, up to 10(5) cells can be screened for micronuclei within a few minutes. The predictive value of micronuclei has been demonstrated using cultured cells exposed to carcinogens and chemopreventive agents and using oral mucosa of betel-quid chewers. DNA adducts, as detected by 32P-postlabelling techniques, could conceivably be another potentially useful marker. However, prior to their use in intervention trials, interindividual variations in their levels in primary, secondary and nontarget tissues and the relationship with doses of carcinogens must be established. The wide scatter of DNA adduct levels in the bronchial mucosa of smokers and of nonsmokers reveals one difficulty that can be encountered using this marker in intervention trials.

Topics: Animals; Antineoplastic Agents; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Clinical Trials as Topic; DNA; Humans; Leukoplakia; Micronucleus Tests; Smoking; Tretinoin

The early and recent investigations in the field of retinoids and cancer are reviewed. The retinoids, including natural vitamin A compounds and their synthetic analogs, present a new class of substances exerting a prophylactic and a therapeutic effect both in certain experimental tumor models and in certain clinical conditions of preneoplastic and neoplastic lesions. Because of a particular physiological mechanism of action, the retinoids offer a new approach to the cancer problem, which is different from those of surgery, X-ray therapy, conventional chemotherapy, and immunotherapy.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Clinical Trials as Topic; Humans; Neoplasms; Neoplasms, Experimental; Tretinoin; Vitamin A

Topics: Animals; beta Catenin; Carcinogenesis; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Humans; Mice; Retinoic Acid 4-Hydroxylase; Tretinoin; Wnt Signaling Pathway

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Humans; Kruppel-Like Factor 4; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Transcription Factors; Tretinoin

HOX proteins are transcription factors that regulate stem cell (SC) function, but their role in the SC origin of cancer is under-studied. Aberrant expression of HOX genes occurs in many cancer types. Our goal is to ascertain how retinoic acid (RA) signaling and the regulation of

Topics: Aldehyde Dehydrogenase; Cell Proliferation; Cell Transformation, Neoplastic; Colonic Neoplasms; Colorectal Neoplasms; Homeodomain Proteins; Humans; Neoplastic Stem Cells; Population Density; Stem Cells; Tretinoin

Melanoma originates from the malignant transformation of melanocytes. Compared with other skin cancers, melanoma has a higher fatality rate. The 5-year survival rate of patients with early-stage primary melanoma through surgical resection can reach more than 90%. However, the 5-year survival rate of patients with metastatic melanoma is only 25%. Therefore, accurate assessment of melanoma progression is critical. Previous studies have found that Retinoic Acid Induced 14(RAI14) is critical in tumorigenesis. However, the biological function of RAI14 for the development of melanoma is unclear. In this study, RAI14 is highly expressed in melanoma and correlated with prognosis. The expression of RAI14 can affect the proliferation, migration and invasion of melanoma cells. F-Box Protein 32(FBXO32) is an E3 ubiquitin ligase of c-MYC. We found that RAI14 affects the transcriptional expression of FBXO32 and regulates the stability of c-MYC. These results suggest that RAI14 play an important role in the growth of melanoma and is expected to be a therapeutic target for melanoma.

Topics: Cell Proliferation; Cell Transformation, Neoplastic; Cytoskeletal Proteins; F-Box Proteins; Humans; Melanoma; Muscle Proteins; Proto-Oncogene Proteins c-myc; Skin Neoplasms; SKP Cullin F-Box Protein Ligases; Transcription Factors; Tretinoin; Ubiquitin-Protein Ligases

SH-SY5Y human neuroblastoma cells are commonly used as neuronal models. Here, we examined different aspects of SH-SY5Y cell differentiation. Various differentiation protocols have been proposed previously, including treatments with retinoic acid, brain-derived neurotrophic factor (BDNF), cholesterol and oestradiol. We examined undifferentiated SH-SY5Y cells (UNDIFF); cells differentiated by the treatment with retinoic acid (RA); retinoic acid + BDNF (RB); and retinoic acid + BDNF + cholesterol + oestradiol (RBCE). We performed whole-cell patch-clamp recordings from these cells and nanomechanically characterised them by using atomic force microscopy (AFM). Our results indicated that Na

Topics: Biomechanical Phenomena; Brain-Derived Neurotrophic Factor; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Electrophysiological Phenomena; Humans; Microscopy, Atomic Force; Neurons; Tretinoin

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis

Topics: Adenomatous Polyposis Coli; Adult Stem Cells; Aldehyde Dehydrogenase 1 Family; Animals; Biomarkers; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromogranin A; Colon; Colorectal Neoplasms; Feedback, Physiological; Genes, APC; Glucagon-Like Peptide 2; Glucagon-Like Peptide-2 Receptor; HCT116 Cells; HT29 Cells; Humans; Mice; Models, Genetic; Mutation; Neuroendocrine Cells; Receptors, Somatostatin; Signal Transduction; Somatostatin; Stem Cell Niche; Tretinoin; Wnt Signaling Pathway

Primary myelofibrosis (PMF) is a type of cloned myeloproliferative neoplasm stemming from haematopoietic stem cells, and tends to transform to acute myeloid leukaemia (AML) in approximately 10-20% of cases over a 10-year period. The transformation into AML has a poor prognosis, with a median overall survival of only 2.6 months in patients receiving supportive treatment. To date, treatment of AML transformation remains poor. The case of a 58-year-old female patient with AML transformed from PMF, who was treated with decitabine combined with all-trans retinoic acid, is reported. The patient had complete remission and a 17-month overall survival from initial diagnosis of transformed AML, with tolerated haematologic toxicity during the treatment period.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Decitabine; Female; Humans; Leukemia, Myeloid, Acute; Middle Aged; Primary Myelofibrosis; Prognosis; Tretinoin

The introduction of all-trans retinoic acid (ATRA) has made acute promyelocytic leukemia (APL) a curable disease; however, early death prior to the completion of treatment remains a problem. In quantitative evaluation of response to ATRA treatment, lymphocytes must be excluded as they do not originally have t(15;17). We categorized peripheral blood leukocytes by nuclear morphology into polymorphonuclear cells (PMNs) comprising segmented granulocytes, and non-polymorphonuclear cells (NPMs) which includes lymphocytes, monocytes, band cells, and immature myeloid cells. We consecutively evaluated the ratio of t(15;17)-positive cells using fluorescence in situ hybridization in eight newly diagnosed patients with APL. We confirmed the differentiation of APL cells until cytogenetic complete remission; the association of a decrease of t(15;17)-positive NPMs and an increase of t(15;17)-positive PMNs was followed by a decrease of t(15;17)-positive PMNs. The kinetic pattern of t(15;17)-positive NPMs and PMNs was consistent in most patients, irrespective of leukocyte counts at diagnosis, additional chromosomal changes, and ATRA with or without chemotherapies. Kinetic analysis enables us to evaluate treatment response and the recovery of normal hematopoiesis in individuals.

Topics: Adult; Aged; Aged, 80 and over; Cell Transformation, Neoplastic; Female; Humans; In Situ Hybridization, Fluorescence; Karyotype; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Promyelocytic Leukemia Protein; Retinoic Acid Receptor alpha; Translocation, Genetic; Tretinoin

Insulin resistance (IR) is closely associated with the progression of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR) targets retinoid X receptor α and reportedly prevents HCC recurrence in clinical practice. Angiotensin-II receptor blocker (ARB) can also inhibit experimental hepatocarcinogenesis and HCC development. These are reported to suppress IR-based hepatocarcinogenesis; however, limited data are available regarding the combined effects of both these agents. This study aimed to investigate the combined chemopreventive effect of ACR and ARB on liver tumorigenesis on rats with congenital diabetes.. Male diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats underwent 70% partial hepatectomy following a single intraperitoneal injection of diethylnitrosamine to induce hepatocarcinogenesis and the administration of ACR (peretinoin, 40 mg/kg/day), ARB (losartan, 30 mg/kg/day), and a combination of ACR and ARB. Six weeks thereafter, we assessed the size and number of the pre-neoplastic lesions (PNL) as well as the altered angiogenesis, oxidative stress, and chronic inflammation in the liver. Moreover, we assessed the effects exerted by ACR and ARB on in vitro cell growth in human HCC cell lines and human umbilical vascular endothelial cells (HUVECs).. OLETF rats showed increase in the size and number of PNLs compared to LETO rats. ACR suppressed the augmentation in size and number of PNLs in the OLETF rats with suppression of cell growth, intrahepatic angiogenesis, lipid peroxidation, oxidative DNA damage, and proinflammatory cytokine production. Combining ACR with ARB enhanced the tumor-suppressive effect and ameliorated intrahepatic angiogenesis, lipid peroxidation, and proinflammatory status; however, cell growth and oxidative DNA damage remained unchanged. IR-mimetic condition accelerated in vitro proliferative activity in human HCC cells, while ACR inhibited this proliferation with G0/G1 arrest and apoptosis. Furthermore, ACR and ARB significantly attenuated the HUVECs proliferation and tubular formation under the IR-mimetic condition, and a combination of both agents demonstrated greater inhibitory effects on HUVEC growth than each single treatment.. ACR and ARB exert a combined inhibitory effect against IR-based hepatocarcinogenesis by the inhibition of cell growth, intrahepatic angiogenesis, and oxidative stress. Thus, this combination therapy appears to hold potential as a chemopreventive treatment therapy against HCC.

Topics: Angiotensin Receptor Antagonists; Animals; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Diethylnitrosamine; Disease Models, Animal; DNA Damage; Drug Synergism; Humans; Lipid Peroxidation; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Oxidative Stress; Protective Agents; Rats; Rats, Inbred OLETF; Tretinoin

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Genes, Homeobox; Humans; Leukemia, Myeloid, Acute; Octamer Transcription Factor-3; Tretinoin

Topics: Alitretinoin; Antineoplastic Agents; Cell Transformation, Neoplastic; Humans; Ki-1 Antigen; Male; Middle Aged; Mycosis Fungoides; Skin Neoplasms; Tretinoin

Topics: Adenocarcinoma; Adult; Antineoplastic Agents; Cell Transformation, Neoplastic; Humans; Maintenance Chemotherapy; Male; Radiography; Retroperitoneal Neoplasms; Teratoma; Treatment Outcome; Tretinoin

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Down-Regulation; Gene Silencing; Hematopoietic Stem Cells; Leukemia, Myeloid, Acute; Mice, Inbred C57BL; Mutant Proteins; Mutation; Polycomb Repressive Complex 1; Polycomb-Group Proteins; Protein Binding; Tretinoin

Chronic intestinal inflammation accompanies familial adenomatous polyposis (FAP) and is a major risk factor for colorectal cancer in patients with this disease, but the cause of such inflammation is unknown. Because retinoic acid (RA) plays a critical role in maintaining immune homeostasis in the intestine, we hypothesized that altered RA metabolism contributes to inflammation and tumorigenesis in FAP. To assess this hypothesis, we analyzed RA metabolism in the intestines of patients with FAP as well as APC

Topics: Adenoma; Adenomatous Polyposis Coli; Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dendritic Cells; Enterocolitis; Genes, APC; Humans; Mice; Phenotype; Th17 Cells; Tretinoin; Tumor Burden; Vitamin A; Vitamin A Deficiency

While the nucleoporin 98-retinoic acid receptor gamma (NUP98-RARG) is the first RARG fusion protein found in acute leukemia, its roles and the molecular basis in oncogenic transformation are currently unknown. Here, we showed that homodimeric NUP98-RARG not only acquired unique nuclear localization pattern and ability of recruiting both RXRA and wild-type NUP98, but also exhibited similar transcriptional properties as RARA fusions found in acute promyelocytic leukemia (APL). Using murine bone marrow retroviral transduction/transformation assay, we further demonstrated that NUP98-RARG fusion protein had gained transformation ability of primary hematopoietic stem/progenitor cells, which was critically dependent on the C-terminal GLFG domain of NUP98 and the DNA binding domain (DBD) of RARG. In contrast to other NUP98 fusions, cells transformed by the NUP98-RARG fusion were extremely sensitive to all-trans retinoic acid (ATRA) treatment. Interestingly, while pan-RXR agonists, SR11237 and LGD1069 could specifically inhibit NUP98-RARG transformed cells, mutation of the RXR interaction domain in NUP98-RARG had little effect on its transformation, revealing that therapeutic functions of rexinoid can be independent of the direct biochemical interaction between RXR and the fusion. Together, these results indicate that deregulation of the retinoid/rexinoid signaling pathway has a major role and may represent a potential therapeutic target for NUP98-RARG-mediated transformation.

Topics: Animals; Cell Nucleus; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; HEK293 Cells; HeLa Cells; Humans; Leukemia; Mice; Microscopy, Fluorescence; Nuclear Pore Complex Proteins; Protein Binding; Protein Interaction Mapping; Protein Multimerization; Protein Structure, Tertiary; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor gamma; Retroviridae; Signal Transduction; Tretinoin

Aldehyde dehydrogenase 1 (ALDH1) is a cancer stem-like cell (CSC) marker in human cancers; however, the specific ALDH1-regulated function and its underlying signalling pathways have not been fully demonstrated. Here, we investigated the ALDH1-regulated function and its underlying signalling and tested whether all-trans retinoic acid (ATRA) can suppress ALDH1-regulated tumour behaviour in ovarian cancer cells. By modulating ALDH1 expression using flow cytometry enrichment and exogenous overexpression or knockdown, we showed that the ALDH1 activity is positively correlated with stemness in ovarian cancer cells according to measures such as sphere formation and CSC marker expression as well as tumourigenesis in a mouse xenograft model. The findings indicate that the ALDH1 directly regulates the functions of ovarian cancer cells. We also showed that ALDH1 can regulate the expression of FoxM1 and Notch 1, which are involved in the downstream signalling of ALDH1-mediated biofunctions. Inhibition of FoxM1 by Thiostrepton and of Notch1 by DAPT downregulated the sphere formation ability of cells. ATRA reduced ALDH1 expression, suppressed tumour formation and inhibited sphere formation, cell migration and invasion in ALDH1-abundant ovarian cancer cells. We conclude that ATRA downregulates ALDH1/FoxM1/Notch1 signalling and suppresses tumour formation in ovarian cancer cells.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Cell Movement; Cell Transformation, Neoplastic; Dipeptides; Down-Regulation; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Humans; Isoenzymes; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Neoplastic Stem Cells; Ovarian Neoplasms; Receptor, Notch1; Retinal Dehydrogenase; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Thiostrepton; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The retinoic acid (RA)-metabolizing enzyme CYP26A1 has been shown to efficiently enhance the oncogenic potential of breast cancer, suggesting a potential oncogenic function. We previously demonstrated that CYP26A1 confers unique cell survival properties by modulating the expression of a variety of genes and identified a number of genes that drive the cells into the oncogenic state. Accumulating evidence suggested that fascin is overexpressed in various types of cancer, primarily leading to increased cell motility. Therefore, in the present study, we examined fascin, an actin-bundling protein, using immunohistochemical and SA-β-gal staining as well as TUNEL and colony forming assays. The results of the present study showed that the expression levels of fascin increased significantly in response to CYP26A1 overexpression and, conversely, treatment with all-trans RA downregulated the expression of fascin. In addition, primary breast carcinoma samples, particularly hormone receptor-negative carcinomas and CYP26A1-overexpressing cancers, expressed elevated levels of fascin. Notably, fascin contributed to the ability of breast carcinoma cells to escape premature senescence and exhibit enhanced cell apoptotic resistance, promoting anchorage-independent growth properties. Fascin also promoted cell motility and the invasiveness of CYP26A1-expressing breast carcinoma cells. These data suggest that fascin expression is modulated by the intracellular RA status regulated by the expression of CYP26A1 and plays a significant role in the malignant behavior of CYP26A1-expressing breast carcinoma cells. CYP26A1 exerts oncogenic functions during breast carcinogenesis. Therefore, CYP26A1-mediated oncogenic characteristics may be partially responsible for the elevated expression of fascin.

Topics: Breast Neoplasms; Carrier Proteins; Cell Survival; Cell Transformation, Neoplastic; Cytochrome P-450 Enzyme System; Female; Humans; MCF-7 Cells; Microfilament Proteins; Retinoic Acid 4-Hydroxylase; Tretinoin

The transcription factor Kruppel-like factor 2 (KLF2) displays anticarcinogenic activities but the mechanism that underlies this activity is unknown. We show here that KLF2 is markedly downregulated in human breast cancers and that its expression positively correlates with breast cancer patient survival. We show further that KLF2 suppresses tumor development by controlling the transcriptional activity of the vitamin A metabolite retinoic acid (RA). RA regulates gene transcription by activating two types of nuclear receptors: RA receptors (RARs), which inhibit tumor development, and peroxisome proliferator-activated receptor β/δ (PPARβ/δ), which promotes tumorigenesis. The partitioning of RA between these receptors is regulated by two carrier proteins: cellular retinoic acid-binding protein 2 (CRABP2), which delivers RA to RARs, and fatty acid-binding protein 5 (FABP5), which shuttles ligands to PPARβ/δ. We show that KLF2 induces the expression of CRABP2 and RARγ and inhibits the expression FABP5 and PPARβ/δ thereby shifting RA signaling from the pro-carcinogenic FABP5/PPARβ/δ to the growth-suppressing CRABP2/RAR path. The data thus reveal that KLF2 suppresses tumor growth by controlling the transcriptional activities of RA.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Fatty Acid-Binding Proteins; Female; Heterografts; Humans; Kruppel-Like Transcription Factors; MCF-7 Cells; Mice; Mice, Nude; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Signal Transduction; Transcription, Genetic; Tretinoin

The recent finding that almost all patients with acute promyelocytic leukaemia (APL) may be cured using a combination of retinoic acid (RA) and arsenic trioxide (As(2)O(3)) (N Engl J Med, 369, 2013 and 111) highlights the progress made in our understanding of APL pathogenesis and therapeutic approaches over the past 25 years. The study of APL has revealed many important lessons related to transcriptional control, nuclear organization, epigenetics and the role of proteolysis in biological control. Even more important has been the clinical demonstration that molecularly targeted therapy can eradicate disease.

Topics: Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Cell Transformation, Neoplastic; Epigenesis, Genetic; Humans; Leukemia, Promyelocytic, Acute; Molecular Targeted Therapy; Oxides; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptor alpha; Signal Transduction; Tretinoin

Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 µM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 µM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 µM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 µM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 µM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to inhibit the progression of premalignant lesions of the breast.

Topics: Breast; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Epithelial Cells; Estradiol; Female; Gene Expression Regulation; Humans; Tretinoin

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34(+) progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Gene Expression; Gene Expression Regulation, Leukemic; Granulocytes; Humans; Leukemia, Myeloid, Acute; Sequestosome-1 Protein; Tretinoin; Ubiquitination; Up-Regulation

PML/RARA is the oncoprotein driving acute promyelocytic leukemia (APL). It suppresses genes expression by recruitment of a number of transcriptional repressors, resulting in differentiation block and malignant transformation of hematopoietic cells. Here, we found that mice primary hematopoietic progenitor cells (HPCs), transduced by DNA-binding-defective PML/RARA mutants, were deficient in colony formation. Further experiments showed that DNA-binding-defective PML/RARA mutants could not repress the transcription of retinoic acid regulated genes. Intriguingly, there were no significant differences of the micro-speckled intracellular distribution between the mutants and wild-type PML/RARA. Some retinoic acid target genes regulated by PML/RARA are involved in not only differentiation block but also hematopoietic cell self-renewal. Altogether, our data demonstrate that direct DNA-binding is essential for PML/RARA to immortalize hematopoietic cells, while disruption of PML-nuclear body does not seem to be a prerequisite for hematopoietic cell transformation.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred C57BL; Mutation; Nuclear Proteins; Oncogene Proteins, Fusion; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

PML/RARA, a potent transcriptional inhibitor of nuclear receptor signaling, represses myeloid differentiation genes and drives acute promyelocytic leukemia (APL). Association of the retinoid X receptor-α (RXRA) coreceptor to PML/RARA is required for transformation, with RXRA promoting its efficient DNA binding. APL is exquisitely sensitive to retinoic acid (RA) and arsenic trioxide (arsenic), which both trigger cell differentiation in vivo. Whereas RA elicits transcriptional activation of PML/RARA targets, how arsenic triggers differentiation remains unclear. Here we demonstrate that extinction of PML/RARA triggers terminal differentiation in vivo. Similarly, ablation of retinoid X receptors loosens PML/RARA DNA binding, inducing terminal differentiation of APL cells ex vivo or in vivo. RXRA sumoylation directly contributes to PML/RARA-dependent transformation ex vivo, presumably by enhancing transcriptional repression. Thus, APL differentiation is a default program triggered by clearance of PML/RARA-bound promoters, rather than obligatory active transcriptional activation, explaining how arsenic elicits APL maturation through PML/RARA degradation.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Chlorocebus aethiops; COS Cells; Gene Expression Profiling; Humans; Leukemia, Promyelocytic, Acute; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Nude; Mice, Transgenic; Oncogene Proteins, Fusion; Oxides; Promoter Regions, Genetic; Protein Binding; Retinoid X Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sumoylation; Transcriptional Activation; Tretinoin

In PML/RARA-driven acute promyelocytic leukemia (APL), retinoic acid (RA) induces leukemia cell differentiation and transiently clears the disease. Molecularly, RA activates PML/RARA-dependent transcription and also initiates its proteasome-mediated degradation. In contrast, arsenic, the other potent anti-APL therapy, only induces PML/RARA degradation by specifically targeting its PML moiety. The respective contributions of RA-triggered transcriptional activation and proteolysis to clinical response remain disputed. Here, we identify synthetic retinoids that potently activate RARA- or PML/RARA-dependent transcription, but fail to down-regulate RARA or PML/RARA protein levels. Similar to RA, these uncoupled retinoids elicit terminal differentiation, but unexpectedly fail to impair leukemia-initiating activity of PML/RARA-transformed cells ex vivo or in vivo. Accordingly, the survival benefit conferred by uncoupled retinoids in APL mice is dramatically lower than the one provided by RA. Differentiated APL blasts sorted from uncoupled retinoid-treated mice retain PML/RARA expression and reinitiate APL in secondary transplants. Thus, differentiation is insufficient for APL eradication, whereas PML/RARA loss is essential. These observations unify the modes of action of RA and arsenic and shed light on the potency of their combination in mice or patients.

Topics: Animals; Antineoplastic Agents; Arsenic; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Mice; Nuclear Proteins; Promyelocytic Leukemia Protein; Proteolysis; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Suppressor Proteins

To explore the effect and mechanism of recombined adenovirus carrying NLS-RARalpha gene on proliferation of HL-60 cells and the differentiation of HL-60 cells induced by ATRA.. HL-60 cells was infected with Ad-NLS-RARalpha and control virus Ad-KZ. The efficiency of infection was detected by FCM. The mRNA and protein levels of NLS-RARalpha were assessed by Real-time PCR (RT-PCR) and Western blot, respectively. MTT assay were applied to determine proliferation of HL-60 cells. Cell surface differentiation antigen CD11b of infected HL-60 cell induced by ATRA was examined by FCM. The mRNA and protein levels of C-MYC of infected HL-60 cell induced by ATRA were determined by Real-time PCR (RT-PCR) and Western blot assay.. The efficiency of infection of Ad-NLS-RARalpha and Ad-KZ on HL-60 cell was 70%-80%. The mRNA and protein levels of NLS-RARalpha gene of HL-60 cells which infected with Ad-NLS-RARalpha were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05). The proliferation ability of HL-60 cell infected with Ad-NLS-RARalpha was significantly increased (P < 0.05). The level of CD11b of HL-60 cell infected with Ad-NLS-RARalpha and induced by ATRA was clearly decreased than control groups (P < 0.05). The mRNA and protein levels of C-MYC gene of HL-60 cells infected with Ad-NLS-RARalpha and induced by ATRA were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P < 0.05).. The recombined adenovirus Ad-NLS-RARalpha can increase the proliferation ability of HL-60 cell, and inhibit the differentiation of HL-60 cell through reduce the expression level of C-MYC gene.

Topics: Adenoviridae; alpha Karyopherins; Cell Proliferation; Cell Transformation, Neoplastic; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

The reactivation of the INK4-ARF locus, which is epigenetically repressed by Polycomb proteins in healthy cells, is a hallmark of senescence. One mechanism of reactivating Polycomb-silenced genes is mediated by the epigenetic factor ZRF1, which associates with ubiquitinated histone H2A. We show that cells undergoing senescence following oncogenic Ras expression have increased ZRF1 levels, and that this binds to the p15INK4b, ARF and p16INK4a promoters. Furthermore, ZRF1 depletion in oncogenic Ras-expressing cells restores proliferation by preventing Arf and p16Ink4a expression, consequently bypassing senescence. Thus, ZRF1 regulates the INK4-ARF locus during cellular proliferation and senescence, and alterations in ZRF1 may contribute to tumorigenesis.

Topics: Animals; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, ras; Humans; Mice; Mice, Inbred C57BL; Molecular Chaperones; Oncogene Proteins; RNA-Binding Proteins; Tretinoin

TRPC channels are Ca²⁺-permeable cationic channels controlling Ca²⁺ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca²⁺ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1(E9del)) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1(E9del), TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy.

Topics: Adenocarcinoma; Alternative Splicing; Amino Acid Sequence; Antibodies, Blocking; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Membrane Transport Modulators; Molecular Sequence Data; Neoplasm Proteins; Ovarian Neoplasms; Ovary; Protein Isoforms; RNA, Messenger; RNA, Small Interfering; Tretinoin; TRPC Cation Channels

The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα knock-in mice did not show altered DNA methylation and the expression of PML-RARα in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. Treatment of APL blasts with all-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα-mediated initiation of leukemogenesis.

Topics: Animals; Cell Transformation, Neoplastic; Chromosomes, Human; CpG Islands; Disease Progression; DNA Methylation; DNA, Neoplasm; Gene Expression Regulation, Leukemic; Gene Knock-In Techniques; Hematopoietic Stem Cells; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Phenotype; Polycomb Repressive Complex 2; Preleukemia; Recombinant Fusion Proteins; Repressor Proteins; Transcription Factors; Tretinoin

Topics: Animals; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Leukemia; Leukemia, Myeloid, Acute; Mice; Neoplasm Proteins; Trans-Activators; Tretinoin; Tumor Suppressor Proteins

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

Topics: Acyltransferases; Alanine Transaminase; Animals; Apoptosis; Carcinogens; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytochrome P-450 Enzyme System; Diethylnitrosamine; DNA Modification Methylases; DNA Repair Enzymes; Hepatic Stellate Cells; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; O(6)-Methylguanine-DNA Methyltransferase; Retinoids; Signal Transduction; Tretinoin; Tumor Suppressor Proteins

To evaluate disease dynamics, treatment results, and frequency of malignant transformation. Ten-year single center retrospective study. The study included 171 patients, 28-99 years old. Follow-up was 1-16 years. 49.5% exhibited changes in clinical presentation, with 19% yearly increase of probability for type shift. Index of extent (number of oral locations) showed a mean 40% decrease and 94.1% reported improvement. There were significant differences between treated and untreated patients (P=0.012). Patients with or without systemic diseases had identical treatment requirements for oral lesions. The prevalence of SCC was 5.8%. Oral lichen planus constantly changes presentation and extent of involvement. The effect of systemic diseases was insignificant in the present study. There is a clear value for treatment to reduce the extent of lesions. The results indicate that all clinical forms of the disease need to be equally followed since the clinical presentation typically changes over time, while malignant transformation can occur in all forms.

Topics: Adult; Aged; Aged, 80 and over; Anti-Inflammatory Agents; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Clobetasol; Dexamethasone; Female; Humans; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Precancerous Conditions; Prednisone; Prevalence; Retrospective Studies; Tacrolimus; Tretinoin; Triamcinolone

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

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

Aberrant histone acetylation was physiopathologically associated with the development of acute myeloid leukemias (AMLs). Reversal of histone deacetylation by histone deacetylase inhibitor (HDACis) activates a cell death program that allows tumor regression in mouse models of AMLs. We have used several models of PML-RARA-driven acute promyelocytic leukemias (APLs) to analyze the in vivo effects of valproic acid, a well-characterized HDACis. Valproic acid (VPA)-induced rapid tumor regression and sharply prolonged survival. However, discontinuation of treatment was associated to an immediate relapse. In vivo, as well as ex vivo, VPA-induced terminal granulocytic differentiation. Yet, despite full differentiation, leukemia-initiating cell (LIC) activity was actually enhanced by VPA treatment. In contrast to all-trans retinoic acid (ATRA) or arsenic, VPA did not degrade PML-RARA. However, in combination with ATRA, VPA synergized for PML-RARA degradation and LIC eradication in vivo. Our studies indicate that VPA triggers differentiation, but spares LIC activity, further uncouple differentiation from APL clearance and stress the importance of PML-RARA degradation in APL cure.

Topics: Acetylation; Animals; Anticonvulsants; Antineoplastic Agents; Cell Differentiation; Cell Transformation, Neoplastic; Flow Cytometry; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Recurrence, Local; Oncogene Proteins, Fusion; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Valproic Acid; Xenograft Model Antitumor Assays

Although microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34(+) hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate-induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-β-activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.

Topics: Antineoplastic Agents; Carcinogens; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; HEK293 Cells; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Myeloid Cells; Tetradecanoylphorbol Acetate; Transfection; Tretinoin

Although the concept of cancer stem cells (CSCs) is well-accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Aldehyde Oxidoreductases; Animals; Apoptosis; Cell Transformation, Neoplastic; Dacarbazine; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Isoenzymes; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neoplastic Stem Cells; Response Elements; Retinal Dehydrogenase; RNA, Small Interfering; Skin Neoplasms; Temozolomide; Tretinoin

We assessed whether imaging α(v)β(3) integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses.. Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8 weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles). (18)F-Fluorodeoxyglucose ((18)F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8 weeks with cisplatin+ATRA). Imaging of α(v)β(3) expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [(99m)Tc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse α(v)β(3) expression were performed.. Cisplatin+ATRA induced differentiation of the xenografts. After 8 weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for (18)F-FDG [mean standardised uptake value (SUV(mean)) = 0.48 ± 0.05] compared to untreated embryonal carcinoma (SUV(mean) = 0.92 ± 0.13) (p = 0.005). α(v)β(3) imaging accurately distinguished mature teratoma (tumour to muscle ratio = 4.29 ± 1.57) from necrosis (tumour to muscle ratio = 1.3 ± 0.26) (p = 0.0002). Immunohistochemistry studies showed that α(v)β(3) integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma.. Imaging α(v)β(3) integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cisplatin; Diagnosis, Differential; Fluorodeoxyglucose F18; Humans; Integrin alphaVbeta3; Male; Molecular Imaging; Necrosis; Neoplasm, Residual; Rats; Teratoma; Testicular Neoplasms; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Tretinoin

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

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

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis in animal models and elevated risk for a number of human cancers. Here, we found that CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, is highly expressed in 42% (27/65) of primary breast cancers. We also showed that enhanced expression of CYP26A1 suppresses cellular responses to anoikis and consequently promotes anchorage-independent growth. This transformed phenotype was sufficient to markedly increase tumorigenic and metastatic potential. Suppression of CYP26A1 significantly reversed the CYP26A1-mediated oncogenic characteristics, suggesting a direct link between intracellular RA status and tumorigenicity. Our observations provide strong evidence for oncogenic and cell survival properties of CYP26A1 in carcinogenesis, and suggest mechanisms whereby VAD might promote cancer development.

Topics: Animals; Anoikis; Breast Neoplasms; Carcinogenicity Tests; Carcinogens; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cytochrome P-450 Enzyme System; Disease Models, Animal; Drug Interactions; Gene Expression Profiling; Humans; Mice; Mice, Knockout; Neoplasms; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured; Vitamin A; Vitamin A Deficiency

To investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during ATRA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules.. K562 cells cultured with or without ATRA (1 micromol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward granulocyte lineage was confirmed by microscopic study (Wright's staining) and flowcytometry. Expression levels of MtF, TfR1 and Fn were evaluated with semiquantitative RT-PCR, while K562 cells cultured without ATRA as control.. Over 21.2% of K562 cells demonstrated features of granulocyte, and the expression of CD13 on cell surface increased significantly at day 5 with ATRA treatment (P < 0.05, compared with control). K562 cells could express a certain level of MtF before ATRA-induced differentiation. With increase of ATRA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 86.5% and 79.2% of that before ATRA treatment. While Fn mRNA expression increased to 1.21 folds of that before ATRA treatment.. MtF expression is downregulated during ATRA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.

Topics: Antigens, CD; Antineoplastic Agents; Cell Transformation, Neoplastic; Ferritins; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Mitochondrial Proteins; Receptors, Transferrin; RNA, Messenger; Tretinoin

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) translocation that yields a PML/RARA fusion protein. Expression of PML/RARA, a potent transcriptional repressor, induces APL in mice. Both retinoic acid (RA) and arsenic trioxide directly target PML/RARA-mediated transcriptional repression and protein stability, inducing rapid differentiation of the promyelocytes and clinical remission in most APL patients. RA also triggers growth arrest and progressive clearance of leukemia initiating cells (LIC), both ex vivo and in vivo. Suboptimal RA concentrations or expression of the PLZF/RARA variant allows complete RA-induced differentiation, but neither LIC clearance nor disease remission. Thus, RA-induced differentiation and LIC clearance may be uncoupled. The RA/arsenic trioxide association, which dramatically synergizes for PML/RARA degradation but not for differentiation, rapidly clears LIC in a proteasome-dependent manner, resulting in APL eradication in murine models and patients. Collectively, these results demonstrate that LIC clearance, which mirrors PML/RARA degradation, is the primary basis for APL cure by the RA/arsenic trioxide association, rather than differentiation. Oncogene degradation could be a generally applicable therapeutic strategy to clear LICs in several types of tumors.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Granulocyte Precursor Cells; Humans; Leukemia, Promyelocytic, Acute; Mice; Oncogene Proteins, Fusion; Oncogenes; Oxides; Tretinoin

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Transformed; Cell Line, Tumor; Cell Shape; Cell Transformation, Neoplastic; Dihydroorotate Dehydrogenase; DNA Fragmentation; Electron Transport Complex IV; Epithelial Cells; Fenretinide; Humans; Hydroxides; Mitochondria; Oxidoreductases Acting on CH-CH Group Donors; Permeability; Reactive Oxygen Species; Tretinoin

Topics: Animals; Antineoplastic Agents; Arsenic; Cell Differentiation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Mice; Oncogene Proteins, Fusion; Tretinoin

Retinoids have been studied extensively for their potential as therapeutic and chemopreventive agents for a variety of cancers, including nonmelanoma skin cancer (NMSC). Despite their use for many years, the mechanism of action of retinoids in the prevention of NMSC is still unclear. In this study we have attempted to understand the chemopreventive mechanism of all-trans retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic.. We have used the 2-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model to investigate the chemopreventive effects of ATRA. We have compared the gene expression profiles of control skin to skin subjected to the 2-stage protocol, with or without ATRA, using Affymetrix 430 2.0 DNA microarrays. Approximately 49% of the genes showing altered expression with TPA treatment are conversely affected when ATRA is co-administered. The activity of these genes, which we refer to as 'counter-regulated', may contribute to chemoprevention by ATRA. The counter-regulated genes have been clustered into functional categories and bioinformatic analysis has identified the B-Raf/Mek/Erk branch of the MAP kinase pathway as one containing several genes whose upregulation by TPA is blocked by ATRA. We also show that ATRA blocks signaling through this pathway, as revealed by immunohistochemistry and Western blotting. Finally, we found that blocking the B-Raf/Mek/Erk pathway with a pharmacological inhibitor, Sorafenib (BAY43-9006), induces squamous differentiation of existing skin SCCs formed in the 2-stage model.. These results indicate that ATRA targets the B-Raf/Mek/Erk signaling pathway in the 2-stage mouse skin carcinogenesis model and this activity coincides with its chemopreventive action. This demonstrates the potential for targeting the B-Raf/Mek/Erk pathway for chemoprevention and therapy of skin SCC in humans. In addition our DNA microarray results provide the first expression signature for the chemopreventive effect of ATRA in a mouse skin cancer model. This is a potential source for novel targets for ATRA and other chemopreventive and therapeutic agents that can eventually be tested in the clinic.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Immunohistochemistry; MAP Kinase Kinase Kinases; Mice; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins B-raf; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA and PLZF/RARA fusion proteins through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is still poorly understood. Here we show that, like PML/RARA, PLZF/RARA expression leads to recruitment of the Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements. Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. We further show that Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1 in PLZF/RARA-mediated myeloid leukemic development.

Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Chromatin; Humans; Leukemia; Nuclear Proteins; Oncogene Proteins, Fusion; Polycomb Repressive Complex 1; Polycomb-Group Proteins; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins; Repressor Proteins; Tretinoin; U937 Cells

Rex1 (zfp42) is a zinc finger protein expressed primarily in undifferentiated stem cells, both in the embryo and the adult. Upon all-trans retinoic acid induced differentiation of murine embryonic stem (ES) cells, Rex1 mRNA levels decrease several fold. To characterize the function(s) of Rex1 more extensively, we generated Rex1 double knockout ES cell lines. The disruption of the Rex1 gene enhanced the expression of ectoderm, mesoderm, and endoderm markers as compared to wild-type (Wt) cells. We propose that Rex1 acts to reduce retinoic acid induced differentiation in ES cells. We performed microarray analyses on Wt and Rex1-/- cells cultured in the presence or absence of LIF to identify potential Rex1 targets. We also evaluated gene expression in a Wt line that overexpresses Rex1 and in a Rex1-/- line in which Rex1 expression was restored. These data, taken together, suggest that Rex1 influences differentiation, cell cycle regulation, and cancer progression.

Topics: Animals; Base Sequence; Cell Cycle; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; DNA Primers; Embryonic Stem Cells; Gene Expression; Mice; Mice, Knockout; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Transcription Factors; Transfection; Tretinoin

Mechanisms by which the inhibitory effect of retinoic acid on tumor growth is attenuated as tumors progress to more advanced stages are unclear.. This study utilizes a novel cell culture system of human breast epithelial cells (HBEC). Immortal (M13SV1), weakly tumorigenic (M13SV1-R2), and highly tumorigenic (M13SV1-R2N1) transformed Type I HBEC were derived sequentially from the same parental Type I HBEC (stem cells) developed from reduction mammoplasty of healthy women. Effects of all-trans retinoic acid (AT-RA) on the growth, protein expression of RAR-alpha, beta and gamma, and RARE transcriptional activation were determined.. AT-RA reduces proliferation rates of immortal and weakly tumorigenic cells, but not highly tumorigenic cells. This loss of response of highly tumorigenic cells to AT-RA is associated with overexpression of p185(c-erbB2/neu). It is not associated with decreased RAR-alpha, beta or gamma expression, or activation by AT-RA; RAR-alpha, beta and gamma are expressed and AT-RA increases RARE transcriptional activity in all cell lines tested in this study.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Luciferases; Receptor, ErbB-2; Receptors, Retinoic Acid; Regulatory Elements, Transcriptional; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Transcriptional Activation; Tretinoin

Squamous cell carcinoma (SCC) of the skin is the most clinically aggressive form of nonmelanoma skin cancer. We have determined the effects of all-trans retinoic acid (ATRA), a naturally occurring chemopreventive retinoid, on signal transducer and activator of transcription 3 (Stat3) signaling during the development of skin SCC. Stat3 is a transcription factor that plays a critical role in cell proliferation and survival, and it is constitutively active in several malignant cell types. We have previously shown that Stat3 is required for the initiation, promotion, and progression of skin SCC. ATRA is a highly efficient suppressor of tumor formation in the two-stage mouse skin carcinogenesis model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/Erk signaling pathway. In this study, we have determined the pattern of Stat3 phosphorylation throughout the course of the two-stage protocol, both in the presence and absence of ATRA. We have used both SENCAR mice and K5.Stat3C transgenic mice, which express the Stat3C protein, a constitutively active form of Stat3, in the skin. Using Western blotting and immunohistochemical staining with phosphospecific antibodies, we show that coadministration of ATRA suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced phosphorylation of Stat3 in both models, but was only able to suppress tumor formation in the SENCAR mice. Surprisingly, ATRA actually enhanced tumor formation in 12-O-tetradecanoylphorbol-13-acetate-treated K5.Stat3C mice. We hypothesize that ATRA blocks tumor formation, at least in part, by targeting events upstream of Stat3, such as the B-Raf/Mek/Erk pathway, and that in the K5.Stat3C mice, in which Stat3 activity is constitutive, it cannot suppress tumor formation.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Female; Immunohistochemistry; MAP Kinase Kinase Kinases; Mice; Mice, Inbred SENCAR; Mice, Transgenic; raf Kinases; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate; Tretinoin

By using an shRNA approach to knockdown the expression of the prostaglandin (PG)-F(2alpha) receptor (FP-R), the role of PGF(2alpha) in the process of phenotypic transformation of normal rat kidney (NRK) fibroblasts has been studied. Our data show that PGF(2alpha) up-regulates Cox-2 expression both at the mRNA and protein level, indicating that activation of FP-R in NRK fibroblasts induces a positive feedback loop in the production PGF(2alpha). Knockdown of FP-R expression fully impaired the ability of PGF(2alpha) to induce a calcium response and subsequent depolarization in NRK cells. However, these cells could still undergo phenotypic transformation when treated with a combination of EGF and retinoic acid, but in contrast to the wild-type cells, this process was not accompanied by a membrane depolarization to -20 mV. Knockdown of FP-R expression also impaired the spontaneous firing of calcium action potentials by density-arrested NRK cells. These data show that a membrane depolarization is not a prerequisite for the acquisition of a transformed phenotype. Furthermore, our data provide the first direct evidence that activity of PGF(2alpha) by putative pacemaker cells underlies the generation of calcium action potentials in NRK monolayers.

Topics: Action Potentials; Animals; Cell Count; Cell Line, Transformed; Cell Proliferation; Cell Transformation, Neoplastic; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprost; Down-Regulation; Enzyme Induction; Fibroblasts; Humans; Membrane Proteins; Phenotype; Rats; Receptors, Prostaglandin; RNA, Small Interfering; Transforming Growth Factor beta; Tretinoin

To explore the effect of all-trans-retinoic acid (ATRA) on the growth inhibition and cellular differentiation of C6 glioma cells.. Human glioma C6 cells were treated with 5 mg/L ATRA,and the inhibition of cell growth was assessed by methyl thiazolyl tetrazolium assay. The differentiation of C6 cells was determined by flow cytometry, microscopy,transmission electron microscope, and immunohistochemical technique.. Treatment of ATRA could result in the growth inhibition of C6 cells, and the cell density significantly decreased(P<0.01). The cell cycle distribution was changed, G0/G1 phase was prolonged, and cells at S phase decreased(P<0.01). The C6 glioma cells displayed normal fibroblast-like morphology under the microscope before the induction, and the ATRA-treated C6 cells became slightly long, turned into round in the middle, and had protrusions at both ends. The ATRA-treated C6 cells did not display obvious apoptosis by flow cytometry(P>0.05).Whereas, early apoptosis was observed under the transmission electron microscope, the vacuoles increased, the mitochondria and endoplasmic reticulum were abundant in the cytoplasm, and the cellular structures tended to be normal.The expression of glial fibrillaryacidic protein in C6 cells increased in the treatment group.. ATRA can inhibit the proliferation, and induce the differentiation of C6 glioma cells.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Proliferation; Cell Transformation, Neoplastic; Glioma; Humans; Mice; Tretinoin; Tumor Cells, Cultured

Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Nude; Oncogene Proteins, Fusion; Phosphorylation; Serine; Signal Transduction; Tretinoin; Xenograft Model Antitumor Assays

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

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

9-cis-Retinoic acid (9cRA) and 1alpha,25-dihydroxyvitamin D3 (1,25D) show promise as potential chemopreventive agents. We examined 9cRA and 1,25D, alone and in combination, for their potential to inhibit carcinogen (NNK)-induced lung carcinogenesis in A/J mice. A/J mice (n=14/group) were treated with 9cRA (7.5, 15, or 30 mg/kg diet), 1,25D (2.5 or 5.0 microg/kg diet), or a combination of 9cRA (15 mg/kg diet) plus 1,25D (2.5 microg/kg diet) for 3 weeks before and 17 weeks after carcinogen injection. Lung tumor incidence, tumor multiplicity, plasma 1,25D levels and kidney expression of vitamin D 24-hydroxylase (CYP24) were determined. Compared to carcinogen-injected controls, mice receiving 9cRA supplementation had significantly lower tumor multiplicity at all doses (decreased 68-85%), with body weight loss at the higher doses of 9cRA. Mice receiving 1,25D supplementation had significantly lower tumor incidence (decreased 36 and 82%) and tumor multiplicity (decreased 85 and 98%), but experienced significant body weight loss, kidney calcium deposition, elevated kidney CYP24 expression and decreased fasting plasma 1,25D levels. Although, there was no apparent influence on chemopreventive efficacy, addition of 9cRA to 1,25D treatment effectively prevented the weight loss and kidney calcification associated with 1,25D treatment alone. These data demonstrate that 9cRA and 1,25D, alone or combined, can inhibit lung tumor promotion in the A/J mouse model. Combining 1,25D with 9cRA has the potential to mitigate the toxicity of 1,25D, while preserving the significant effect of 1,25D treatment against lung carcinogenesis. The underlying mechanism behind this effect does not appear to be related to retinoid modulation of vitamin D catabolism.

Topics: Adenoma; Alitretinoin; Animals; Antineoplastic Agents; Calcitriol; Carcinogens; Cell Transformation, Neoplastic; Dietary Supplements; Disease Models, Animal; Drug Therapy, Combination; Lung Neoplasms; Male; Mice; Mice, Inbred A; Nitrosamines; Retinoid X Receptors; Steroid Hydroxylases; Tretinoin; Vitamin D3 24-Hydroxylase; Vitamins

Transcriptional activation of the nuclear receptor RAR by retinoic acid (RA) often leads to inhibition of cell growth. However, in some tissues, RA promotes cell survival and hyperplasia, activities that are unlikely to be mediated by RAR. Here, we show that, in addition to functioning through RAR, RA activates the "orphan" nuclear receptor PPARbeta/delta, which, in turn, induces the expression of prosurvival genes. Partitioning of RA between the two receptors is regulated by the intracellular lipid binding proteins CRABP-II and FABP5. These proteins specifically deliver RA from the cytosol to nuclear RAR and PPARbeta/delta, respectively, thereby selectively enhancing the transcriptional activity of their cognate receptors. Consequently, RA functions through RAR and is a proapoptotic agent in cells with high CRABP-II/FABP5 ratio, but it signals through PPARbeta/delta and promotes survival in cells that highly express FABP5. Opposing effects of RA on cell growth thus emanate from alternate activation of two different nuclear receptors.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Fatty Acid-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; PPAR-beta; Receptors, Retinoic Acid; Transcriptional Activation; Tretinoin

Signal transducers and activators of transcription 3 (STAT3) was originally identified as a transcription factor that mediates cytokine-induced responses. In these pathways, Janus-activated kinase (JAK)-induced transient tyrosine phosphorylation of STAT3 promotes gene expression in response to a number of cytokines, which is inhibited by feedback mechanisms. A number of studies have shown that STAT3 is constitutively activated in human cancer cells, leading to cell proliferation. It is unclear, apart from a chronic tyrosyl phosphorylation of STAT3, what mechanisms contribute to the STAT3 deregulation in tumors. Earlier, we have isolated a novel growth inhibitory gene product, gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), using a genetic approach. GRIM-19 is an IFN/retinoic acid-regulated growth suppressor. Subsequent analyses have shown that GRIM-19 binds to STAT3 and prevents interleukin-6-induced transcription of cellular genes. However, its effects on a constitutively active STAT3 and cellular transformation are unknown. In this study, we show that GRIM-19 suppresses constitutive STAT3-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of cellular genes involved in cell proliferation and apoptosis.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Death; Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Mice; NADH, NADPH Oxidoreductases; Phosphorylation; Rats; STAT3 Transcription Factor; Transcription, Genetic; Tretinoin

In an attempt to develop effective and non-cytotoxic cancer chemopreventive derivatives of retinoids, a novel acyclic retinoid has previously been synthesized. This acyclic retinoid, NIK-333, had been reported to suppress recurrence of primary hepatocellular carcinomas. We explored the molecular mechanisms by which NIK-333 exerts anti-proliferative effects.. We employed Balb/c 3T3 cells, since they can be used as a quantitative transformation assay and since we can study a possible involvement of gap-junctional intercellular communication (GJIC) in their growth control. We included all-trans-retinoic acid (ATRA) for comparison.. We first confirmed that these cells express the retinoid receptors, RARalpha, gamma and RXRalpha, suggesting that they respond to NIK-333 and ATRA. When NIK-333 or ATRA was added to Balb/c 3T3 cells during the induction of cell transformation by a standard (3-methylcholanthrene (MCA) alone) or two-stage (low dose of MCA plus 12-O-tetradecanoylphorbol-13-acetate (TPA)) protocol, there was a significant decrease in the number of transformed foci. The extent of inhibition of transformation by NIK-333 was similar to that exerted by ATRA. Employing the microinjection dye-transfer assay, we found that both retinoids increase GJIC level when measured 24h after treatment. The extent of GJIC increase by NIK-333 was similar to that of ATRA. These retinoids also increased the amount of connexin 43 (Cx43) on the plasma membrane as revealed by immunostaining.. These data indicate that NIK-333 suppresses chemical carcinogenesis in vitro and support the hypothesis that enhancement of GJIC is involved in this process.

Topics: Animals; BALB 3T3 Cells; Cell Communication; Cell Transformation, Neoplastic; Connexin 43; Gap Junctions; Mice; Retinoids; Tretinoin

Leukemias are differentially sensitive to histone deacytelase inhibitor (HDI)-induced apoptosis, but molecular reasons for this remain unclear. We here show that BCR/ABL-, but not FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-transformed 32D cells or primary acute myeloid leukemia (AML) blasts undergo apoptosis after treatment with the HDI valproic acid (VPA) plus all-trans retinoic acid (VPA/ATRA). A particular VPA/ATRA responsiveness of Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (ALL) was confirmed in a therapy-refractory patient in vivo. HDI-stimulated apoptosis in Ph+ cells was caspase dependent, but independent from Akt pathway inhibition. Conversely, separate blockage of the Akt/mTor-signaling pathway was a prerequisite for overcoming apoptosis resistance to VPA/ATRA in FLT3-ITD cells, and primary AML blasts (n = 9). In conclusion, constitutive Akt activation causes apoptosis resistance to VPA/ATRA in AML, but not in Ph+ leukemia. This warrants the application of HDI-based therapies in poor-risk Ph+ ALL, and the use of Akt/mTor inhibitors to overcome HDI resistance in AML.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Fusion Proteins, bcr-abl; Histone Deacetylase Inhibitors; Humans; Leukemia, Myelomonocytic, Acute; Mutation; Oncogene Protein v-akt; Philadelphia Chromosome; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Tretinoin; Tumor Cells, Cultured; Valproic Acid

Recurrent chromosomal translocations involving the RAR alpha locus on chromosome 17 are the hallmark of acute promyelocytic leukemia (APL). The RAR alpha gene fuses to variable partners (PML, PLZF, NPM, NuMA and STAT5B: X genes) leading to the expression of APL-specific fusion proteins with identical RAR alpha moieties. To analyse whether the variable X moiety could affect the activity of the fusion protein in vivo, we generated and characterized, on a comparative basis, NPM/RAR alpha transgenic mice (TM) in which the fusion gene is expressed under the control of a human Cathepsin G (hCG) minigene. We compared the features of the leukemia observed in these TM with those in hCG-PML/RAR alpha and hCG-PLZF/RAR alpha TM. In all three transgenic models, leukemia developed after a variably long latency, with variable penetrance. However, the three leukemias displayed distinct cytomorphological features. hCG-NPM/RAR alpha leukemic cells resembled monoblasts. This phenotype contrasts with what was observed in the hCG-PML/RAR alpha TM model in which the leukemic phase was characterized by the proliferation of promyelocytic blasts. Similarly, hCG-PLZF/RAR alpha TM displayed a different phenotype where terminally differentiated myeloid cells predominated. Importantly, the NPM/RAR alpha oncoprotein was found to localize in the nucleolus, unlike PML/RAR alpha and PLZF/RAR alpha, thus possibly interfering with the normal function of NPM. Similarly to what was observed in human APL patients, we found that NPM/RAR alpha and PML/RAR alpha, but not PLZF/RAR alpha leukemia, was responsive to all-trans retinoic acid (ATRA) or As2O3 treatments. Taken together, our results underscore the critical relevance of the X moiety in dictating the biology of the disease and the activity of the APL fusion oncoprotein.

Topics: Animals; Antineoplastic Agents; Cathepsin G; Cathepsins; Cell Proliferation; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Fusion; Humans; Kruppel-Like Transcription Factors; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Neoplasm Proteins; Nuclear Proteins; Phenotype; Promyelocytic Leukemia Protein; Promyelocytic Leukemia Zinc Finger Protein; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Serine Endopeptidases; Transcription Factors; Translocation, Genetic; Tretinoin; Tumor Suppressor Proteins

9-cis-Retinoic acid (9cRA) binds both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) and has been shown to be a potential chemopreventive agent both in lung cancer cell culture studies and in clinical trials studying former smokers. However, direct evidence of the efficacy of 9cRA against lung tumor development in vivo is lacking. In the present study, we determined whether treatment with 9cRA has the potential to inhibit lung carcinogenesis by upregulating RAR-beta and down-regulating COX-2 expression in the A/J mouse lung cancer model. A/J mice (n=14-15/group) were treated as follows: (1) Control (Sham treated); (2) NNK (100mg NNK/kg body weight); (3) NNK+9cRA (15mg/kg diet); and (4) NNK+celecoxib (a COX-2-specific inhibitor, 500mg/kg diet). Tumor incidence, tumor multiplicity, RAR-beta mRNA, COX-2 mRNA, and COX-2 protein levels in lung samples of mice were determined 4 months after carcinogen injection. The results showed that mice receiving 9cRA supplementation had significantly lower tumor multiplicity (48% reduction, P<0.05) and showed a trend toward lower tumor incidence (40% reduction, P=0.078), as compared with the mice given NNK alone. Although, celecoxib treatment resulted in greater declines in tumor incidence and tumor multiplicity (75 and 88%, respectively, P<0.05), the chemoprotective effects of celecoxib were accompanied by increased mortality while 9cRA treatment resulted in no weight-loss associated toxicity or mortality. Supplementation with 9cRA was effective in increasing RAR-beta mRNA, but this increase was not accompanied by decreased levels of COX-2 mRNA or protein. These results suggest that 9cRA supplementation may provide protection against lung carcinogenesis and this effect may be mediated in part by 9cRA induction of RAR-beta, but not inhibition of COX-2 transcription.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Carcinogens; Celecoxib; Cell Transformation, Neoplastic; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dietary Supplements; Disease Models, Animal; Lung; Lung Neoplasms; Male; Membrane Proteins; Mice; Mice, Inbred A; Nitrosamines; Pyrazoles; Receptors, Retinoic Acid; Sulfonamides; Tretinoin

Retinoids are known to suppress carcinogenesis in various epithelial tissues. Among them, all-trans-retinoic acid (atRA) is recognized as one such active retinoid. However, despite the known anticarcinogenic activity of atRA, it exhibits its short plasma half-life during repeated oral administration due to the "acute retinoid resistance" in the liver. This has been the major limitation in clinical applications of atRA. Therefore, in order to render atRA more suitable for clinical uses, sustained delivery of atRA using biodegradable microspheres is suggested in this study. When 50 mg atRA/kg of atRA-loaded microspheres were subcutaneously administered to rats once, the atRA concentration in plasma was maintained around 6.5 ng/ml for 7 weeks, with only minor signs of toxicity. When the chemopreventive efficacy of atRA-loaded microspheres was evaluated using a model of 4-nitroquinoline 1-oxide-induced oral carcinogenesis in F344 rats, a single injection of atRA-loaded microspheres significantly suppressed oral carcinogenesis. Additional injections of atRA-loaded microspheres, however, did not indicate further suppression of carcinogenesis.

Topics: 4-Nitroquinoline-1-oxide; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Delayed-Action Preparations; Drug Carriers; Drug Compounding; Male; Microspheres; Mouth Neoplasms; Neoplasms, Experimental; Palatal Neoplasms; Polyesters; Polyethylene Glycols; Precancerous Conditions; Rats; Rats, Inbred F344; Solubility; Tongue Neoplasms; Tretinoin

This study was purposed to investigate whether aminopeptidase inhibitor, bestatin, can potentiate all-trans retinoic acid (ATRA)-inducing differentiation in NB4 cells, and to explore its mechanism. The NB4 cells were exposed to either bestatin and ATRA alone or in combination, the morphological changes of NB4 cells were observed by optical microscopy, the CD11b expression was measured by flow cytometry, the function of defferentiation cells was analyzed by nitroblue-tetrazolium (NBT) reduction assay, the mRNA expressions of c-myc and c-EBPepsilon in NB4 cells were detected by RT-PCR, the c-Myc protein expression was determined by Western blot. The results showed that treatment with bestatin alone induced no significant changes in morphology, NBT reduction activity and CD11b expression in NB4 cells. NB4 cells incubated with 10 nmol/L ATRA plus 100 microg/ml bestatin showed more morphologic feature of metamyelocyte and band neutrophil than ATRA alone treated cells. 100 microg/ml bestatin enhanced the NBT reduction activity in NB4 cells induced by various concentrations of ATRA (10, 20, 40 nmol/L). The effects of various concentrations of ATRA in combination with 100 microg/ml bestatin were statistically different from the effect of ATRA alone (P < 0.01). From 48 to 96 hours, 100 microg/ml bestatin time-dependently increased NBT reduction in NB4 cells induced by 10 nmol/L ATRA (P < 0.01). 10 nmol/L ATRA plus 100 microg/ml bestatin for 72 hours prominently elevated CD11b expression in NB4 cells as compared with ATRA alone treated NB4 cells (P < 0.01). There was a substantial decrease in c-myc mRNA levels when 100 microg/ml bestatin was added to 10 nmol/L ATRA (P < 0.05). Various concentrations (50, 75, 100 microg/ml) of bestatin combined with 10 nmol/L ATRA down-regulated the expression of c-Myc protein, which was negatively correlated with the NBT reduction activity of NB4 cells induced by 10 nmol/L ATRA alone or plus bestatin at various concentrations (r = -0.940, P = 0.017). However, 100 microg/ml bestatin plus 10 nmol/L ATRA could not induce any significant changes in the levels of c-EBPepsilon mRNA as compared with ATRA alone treated NB4 cells. It is concluded that an aminopeptidase inhibitor bestatin can potentiate ATRA-inducing differentiation of NB4 cells, possibly by down-regulating c-myc expression in synergy with ATRA.

Topics: Aminopeptidases; Antibiotics, Antineoplastic; Cell Transformation, Neoplastic; Down-Regulation; Drug Synergism; Humans; Leucine; Leukemia, Promyelocytic, Acute; Proto-Oncogene Proteins c-myc; Tretinoin; Tumor Cells, Cultured

Occludin is the first identified integral protein for the tight junction (TJ), and its long COOH-terminal domain is considered to have functions in receiving and transmitting cell survival signals. Loss of TJ-associated molecules, such as occludin, has been correlated with tumor progression in carcinogenesis; however, the precise molecular mechanisms explaining its loss of expression and whether occludin expression has any effects on cancer phenotypes remain to be clarified. Here, we show that forced expression of occludin in cancer cells exhibits enhanced sensitivity to differently acting apoptogenic factors, and thus inhibits the tumorigenicity of transformed cells, via modulation of unique sets of apoptosis-associated genes. In addition, studies using deletion mutants of occludin constructs show that 44 amino acids at the COOH-terminal end play a critical role in modifying the cellular phenotypes. Interestingly, occludin decreases cellular invasiveness and motility, thereby abrogating metastatic potencies of cancer cells. We also found that occludin expression is silenced by CpG island hypermethylation on its promoter region. Synergy with a demethylator and histone deacetylase inhibitor or retinoids that stimulate retinoic acid receptor alpha induces endogenous occludin, which is sufficient for apoptotic sensitization. Our results show the functional diversity of occludin and suggest that methylator phenotype of occludin provides enhanced tumorigenic, invasive, and metastatic properties of cancer cells, identifying occludin as a likely candidate for a tumor-suppressor gene in certain types of cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Female; Gene Silencing; Genes, Tumor Suppressor; HeLa Cells; Humans; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Neoplasms; Occludin; Rats; RNA, Messenger; Signal Transduction; Transfection; Tretinoin

An improvement of the Waliszewski and Konarski approach ([2002] Synapse 43:252-258) to determine the temporal fractal dimension b(t) and scaling factor a(t) for the process of neuronal differentiation and synapse formation in the fractal space-time is presented. In particular the analytical formulae describing the time-dependence of b(t)(t) and a(t)(t), which satisfy the appropriate boundary conditions for t-->0 and t-->infinity, are derived. They have been used to determine the temporal fractal dimension and scaling factor from the two-parametric Gompertz function fitted to experimental data obtained by Jones-Villeneuve et al. ([1982] J Cell Biol 94:253-262) for embryonal carcinoma P19 cells treated by retinoic acid. The results of the calculations differ from those obtained previously by making use of the three- and four-parametric Gompertz function as well as other S-shape functions (Chapman, Hill, Logistic, Sigmoid) evaluated by the fitting of the experimental curve. The temporal fractal dimension can be used as a numerical measure of the neuronal complexity emerging in the process of differentiation, which can be related to the morphofunctional cell organization. A hypothesis is formulated that neuronal differentiation and synapse formation have a lot in common with the process of tumorigenesis. They are qualitatively described by the same Gompertz function of growth and take place in the fractal space-time whose mean temporal fractal dimension is lost during progression.

Topics: Algorithms; Animals; Brain; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Embryonal Carcinoma Stem Cells; Fractals; Mice; Models, Biological; Neoplasm Invasiveness; Neoplastic Stem Cells; Nerve Growth Factors; Neural Pathways; Neurons; Stem Cells; Synapses; Time Factors; Tretinoin

Retinoic acid (RA) is a master epigenetic regulator that plays a pivotal role in both breast morphogenesis and development. Here, we show for the first time that RA, via the RA receptor alpha (RARalpha), epigenetically regulates in a concerted fashion the transcription of two RA-responsive genes, the RA receptor beta2 (RARbeta2) and the cellular retinol-binding protein 1 (CRBP1). Specifically, an impaired RA signal through RARalpha in human breast epithelial cells triggers a repressive epigenetic domino effect, involving first RARbeta2 and second CRBP1. The phenotype acquired by breast epithelial cells clearly implies that the resistance to RA-mediated growth inhibition precedes the acquisition of morphological epithelial transformation, thus supporting the occurrence of sequential transcriptional silencing of first RARbeta2 and second CRBP1. The identification of this epigenetic network mechanistically linking RARbeta2 and CRBP1 transcription provides the basis for devising more accurate epigenetic tests for the prediction of breast cancer risk.

Topics: Breast; Breast Neoplasms; Cell Transformation, Neoplastic; Epigenesis, Genetic; Epithelial Cells; Female; Gene Silencing; Genetic Predisposition to Disease; Humans; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Risk; Tretinoin

It is well known that the differentiation of acute promyelocytic leukaemia (APL) cells by all-trans-retinoic acid (ATRA) may be enhanced by myeloid growth factors, but the requirement for growth factors in this process is unclear. Our previous studies in multiple myeloma and non-APL acute myeloid leukaemia demonstrated that lineage-specific growth factors are required for the maximal activity of many pharmacologic differentiating agents in vitro. Thus, we studied whether the differentiation of APL is similarly dependent on growth factors. We found that the myeloid growth factors granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor markedly increased the differentiation of NB4 cells or APL blasts from clinical samples treated with ATRA, arsenic trioxide (ATO), or bryostatin-1 as evidenced by the enhanced expression of myeloid surface antigens and the inhibition of clonogenic growth. Furthermore, myeloid growth factors were necessary for the differentiation of APL cells since the activity of each pharmacologic agent could be blocked by specific growth factor-neutralizing antibodies. Each differentiating agent was active only at concentrations that inhibited cell cycling, suggesting that this property is also required for differentiation. These data demonstrate that both pharmacologic differentiating agents and myeloid growth factors are required, but neither sufficient, for the differentiation of APL cells. The combined use of pharmacologic differentiating agents and growth factors may improve the clinical efficacy of differentiation therapy in APL.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Bryostatins; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Macrolides; Myelopoiesis; Oxides; Tretinoin

Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs.. The IL-4/GM-CSF (granulocyte-macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells.. Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR-Lin- cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-gamma production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR-Lin- cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid.. These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest.

Topics: Aged; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Carcinoma, Renal Cell; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dendritic Cells; Female; Humans; Kidney Neoplasms; Middle Aged; Phenotype; Putrescine; T-Lymphocytes; Tretinoin

D-type cyclins (cyclins D1, D2, and D3) promote G1-S progression and are aberrantly expressed in cancer. We reported previously that all-trans-retinoic acid chemo-prevented carcinogenic transformation of human bronchial epithelial (HBE) cells through proteasomal degradation of cyclin D1. Retinoic acid is shown here to activate distinct mechanisms to regulate different D-type cyclins in HBE cells. Retinoic acid increased cyclin D2, decreased cyclin D3 and had no effect on cyclin D1 mRNA expression. Retinoic acid decreased cyclin D1 and cyclin D3 protein expression. Repression of cyclin D3 protein preceded that of cyclin D3 mRNA. Proteasomal inhibition prevented the early cyclin D3 degradation by retinoic acid. Threonine 286 (T286) mutation of cyclin D1 stabilized cyclin D1, but a homologous mutation of cyclin D3 affecting threonine 283 did not affect cyclin D3 stability, despite retinoic acid treatment. Lithium chloride and SB216763, both glycogen synthase kinase 3 (GSK3) inhibitors, inhibited retinoic acid repression of cyclin D1, but not cyclin D3 proteins. Notably, phospho-T286 cyclin D1 expression was inhibited by lithium chloride, implicating GSK3 in these effects. Expression of cyclin D1 and cyclin D3 was deregulated in retinoic acid-resistant HBE cells, directly implicating these species in retinoic acid response. D-type cyclins were independently targeted using small interfering RNAs. Repression of each D-type cyclin suppressed HBE growth. Repression of all D-type cyclins cooperatively suppressed HBE growth. Thus, retinoic acid repressed cyclin D1 and cyclin D3 through distinct mechanisms. GSK3 plays a key role in retinoid regulation of cyclin D1. Taken together, these findings highlight these cyclins as molecular pharmacologic targets for cancer chemoprevention.

Topics: Bronchi; Cell Line; Cell Transformation, Neoplastic; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Epithelial Cells; Glycogen Synthase Kinase 3; Humans; Lung Neoplasms; RNA, Messenger; RNA, Small Interfering; Transfection; Tretinoin

Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Animals; Bacteria; Breast Neoplasms; Carcinogenicity Tests; Cell Transformation, Neoplastic; Ethanol; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Inactivation, Metabolic; Neoplasms; Polymorphism, Genetic; Rats; Tretinoin

The LYL1 gene encodes a basic helix-loop-helix transcription factor involved in T-cell acute lymphoblastic leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of LYL1 was at higher levels in the majority cases of acute myeloblastic leukemia (AML) or myelodysplastic syndrome when compared to normal bone marrow. Our study also showed that LYL1 was highly expressed in most AML cell lines and in CD34+ AML cells. To determine whether LYL1 had an affect on the phenotype and behavior of myeloid cells, we introduced full-length LYL1 cDNA into K562 cells using electroporation and U937 cells with retroviral infection. Both of the derivative cell lines with overexpression of LYL1 had an increased growth rate and clonogenecity. Forced expression of LYL1 in K562 cells enhanced spontaneous and hemin-induced erythroid differentiation but blocked spontaneous as well as PMA-induced megakaryocytic differentiation. Overexpression of LYL1 in U937 cells blocked all-trans retinoic acid-induced monocytic differentiation. The LYL1-transfected U937 cells were also more resistant to the cytotoxic drug cytarabine. These results demonstrate that LYL1 may play a role in early hematopoiesis and may be a potential oncogenic factor in AML.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow; Cell Transformation, Neoplastic; Cytarabine; DNA-Binding Proteins; DNA, Complementary; Drug Resistance, Neoplasm; Electroporation; Gene Expression Profiling; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Neoplasm Proteins; Phenotype; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

To study the effect of Zhengganfang (ZGF) drug serum in inducing differentiation of Bel-7402 human hepatocarcinoma cell (HHC) and its influence of the telomerase activity.. Rats were fed with ZGF decoction to prepare the drug-serum of ZGF, and the drug-serum was used for Bel-7402 HHC culture. Serum of normal rat, was taken as negative control and retinoic acid (RA) as the positive control. Cell proliferation was detected by MTT colorimetric assay, nucleocytoplasm ratio by image assay, alpha-fetoprotein (AFP) by RIA, alkaline phosphatase (ALP) activity and gamma-glutamyl transpeptidase (gamma-GT) by dynamic colorimetric assay and telomerase activity of Bel-7402 cell by PCR-ELISA.. Drug-serum of ZGF could inhibit the proliferation of Bel-7402 cell, markedly reduce the nucleocytoplasm ratio and the secretion of AFP, decrease gamma-GT activity, increase ALP activity and suppress the telomerase activity of Bel-7402 cell.. ZGF can induce the differentiation of Bel-7402 HHC, the main mechanism maybe through the suppression of telomerase activity.

Topics: Alkaline Phosphatase; alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Drugs, Chinese Herbal; gamma-Glutamyltransferase; Hepatocytes; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Serum; Telomerase; Tretinoin; Tumor Cells, Cultured

Squamous cell carcinoma (SCC) is the most prevalent form of epithelial cancer. SCC results when normal epithelial cells undergo multiple neoplastic changes that culminate in the evolution of an invasive cancer. Retinoids are commonly used as chemopreventive and treatment agents in skin cancer; however, SCC progression is accompanied by a gradual loss of retinoid responsiveness. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has shown promising anti-neoplastic activity in a variety of tumor cells, including those that are resistant to all-trans retinoic acid (t-RA). We investigated the effect of HPR on growth and apoptosis of squamous cells at different stages of carcinogenesis. We then determined if retinoic acid receptor (RAR) overexpression affected the outcome of HPR treatment. To model SCC malignant progression, we used a panel of murine keratinocytes representing different stages of squamous cell carcinogenesis. This panel consisted of primary keratinocytes, SP1 and 308 papilloma cell lines, the PAM-212 squamous carcinoma cell line, and the spindle I7 cell line. With the exception of the primary keratinocytes, all cells were unresponsive to t-RA treatment. Pharmacological concentrations of HPR were non-cytotoxic to all keratinocytes tested and HPR sensitivity was stage-dependent, with the papilloma cell lines being the most sensitive, and the spindle cells being the most resistant. Overexpression of RARgamma in SP1 papilloma cells enhanced growth suppression and apoptosis induction by HPR. HPR-induced growth suppression was accompanied by a simultaneous block in the G(1) phase of the cell cycle in RAR-transduced and control SP1 cells and differential regulation of cell cycle and apoptotic mediators.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Fenretinide; G1 Phase; Keratinocytes; Mice; Mice, Inbred BALB C; Neoplasm Staging; Nevus, Spindle Cell; Papilloma; Propidium; Receptors, Retinoic Acid; Ribonucleases; Tretinoin

The specific signals required for actin polymerization in response to extracellular factors remain unknown. However, in many cell types, there is a correlation between actin polymerization, activation of phosphatidylinositol 3-kinase (PI 3-kinase), and the production of the second messenger phosphatidylinositol-3,4,5-triphosphate. Increased levels of PI 3-kinase have been detected during cell growth and transformation. However, PI 3-kinase is also activated during differentiation, suggesting that PI 3-kinase and its lipid products also play a role in the regulation of cellular differentiation. The newly characterized CAC-1 cell line established from a poorly differentiated human endometrial adenocarcinoma (Exp. Mol. Pathol. 69 (2000), 175) was used as a model to investigate the role of PI 3-kinase in differentiation induction. CAC-1 cells differentiated upon treatment with pharmacological doses of retinoids (1 micro M of 13-cis or all-trans), evidenced by actin filament reorganization, and cell enlargement. PI 3-kinase staining is primarily localized to perinuclear regions in untreated cells. However, retinoic acid treatment induced PI 3-kinase to relocalize throughout the cytoplasm. Subcellular fractionation and Western blotting confirmed that PI 3-kinase decreased in the particulate fraction, concurrent with retinoid-induced differentiation. Interestingly, pretreatment with the PI 3-kinase inhibitor wortmannin (100 nM) prior to retinoic acid treatment prevented retinoic acid-induced actin reorganization and cell enlargement. To distinuish whether retinoid regulation of PI 3-kinase is mediated through traditional nuclear retinoic acid receptors, the levels of retinoic acid receptor-beta (RAR-beta) protein were evaluated. Retinoid treatment did not alter RAR-beta protein levels compared to controls. These data suggest that PI 3-kinase activity and cytoplasmic relocalization are required for retinoid-induced differentiation of poorly differentiated human endometrial adenocarcinoma cells.

Topics: Actins; Adenocarcinoma; Androstadienes; Antineoplastic Agents; Blotting, Western; Cell Transformation, Neoplastic; Endometrial Neoplasms; Enzyme Inhibitors; Female; Humans; Immunohistochemistry; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Transport; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured; Wortmannin

It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage. In this study, we compared the expression profiles of cancer-related genes by cDNA microarray analysis during the differentiation of human promyelocytic leukemia HL-60 cells into either monocytes or granulocytes. RNA was isolated at times 0, 6, 12, 24, 36, 48, and 72 h following stimulation of differentiation with all-trans retinoic acid (all-trans RA) or 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and hybridized to the microarray gene chips containing 872 genes related to cell-cycles, oncogenes and leukemias. Several genes were commonly or differentially regulated during cell differentiation into either lineage, as demonstrated by both hierarchical and self-organizing map clustering analysis. At 72 h the expression levels of 45 genes were commonly up- or down-regulated at least a twofold in both lineages. Most importantly, 32 genes including alpha-L-fucosidase gene and adducin gamma subunit gene were up- or down-regulated only in all-trans RA-treated HL-60 cells, while 12 genes including interleukin 1beta and hypoxia-inducible factor 1alpha were up- or down-regulated only in 1,25-(OH)(2)D(3)-treated HL-60 cells. The expression of selected genes was confirmed by Northern blot analysis. As expected, some genes identified have not been examined during HL-60 cell differentiation into either lineage. The identification of genes associated with a specific differentiation lineage may give important insights into functional and phenotypic differences between two lineages of HL-60 cell differentiation.

Topics: Calcitriol; Cell Division; Cell Lineage; Cell Survival; Cell Transformation, Neoplastic; DNA, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Granulocytes; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Monocytes; Oligonucleotide Array Sequence Analysis; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Retinoids can regulate the proliferation and differentiation of various tumor cells. It is thought that nuclear retinoid receptors mediate these effects by regulating gene transcription. The identity of specific retinoid target genes is only beginning to be unraveled. One candidate for mediating retinoid-induced growth suppression is the novel class II tumor suppressor gene tazarotene-induced gene 3 (TIG3). We examined the constitutive and all-trans retinoic acid (ATRA)-inducible expression of TIG3 mRNA in five head and neck squamous cell carcinoma (HNSCC) and five nonsmall cell lung carcinoma (NSCLC) cell lines to determine whether it is associated with their responsiveness to ATRA. The expression patterns of retinoic acid receptor beta (RARbeta), another putative retinoid-inducible tumor suppressor gene, were also examined. The constitutive TIG3 expression was high in one HNSCC cell line and two NSCLC cell lines, and moderate to very low in the other cells. Some RARbeta-expressing cells had either low or undetectable TIG3 levels and vice versa. ATRA (1 microM; 48 h) increased TIG3 mRNA in 4/5 HNSCCs and 3/5 NSCLCs and RARbeta mRNA in some of the same cell lines, but also in cells that did not show TIG3 induction. TIG3 mRNA was induced by ATRA between 6 and 12 h in most of the responsive cells. ATRA concentrations required for TIG3 induction ranged from 1 to 500 nM depending on the cell line. The pan-RAR antagonists AGN193109 and the RARalpha antagonist Ro 41-5253 blocked TIG3 induction by ATRA. ATRA suppressed anchorage-independent colony formation in most cells that had a high or moderate constitutive or induced TIG3 expression level. In contrast, RARbeta mRNA expression pattern was not correlated with sensitivity to ATRA. These results suggest that TIG3 is regulated by ATRA via retinoid receptors in certain aerodigestive tract cancer cells, and its induction by ATRA is associated with the suppression of anchorage-independent growth.

Topics: Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; DNA, Complementary; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Lung Neoplasms; Phenotype; Receptors, Retinoic Acid; RNA, Messenger; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have developed a nontumorigenic epithelial cell line, DP-153, from the dorsal prostate of a Lobund/Wistar rat treated with N-methyl-N-nitrosourea and testosterone propionate. DP-153 cells express cytokeratins 5 and 14, but not cytokeratin 18, consistent with a basal epithelial cell phenotype. Similar to the nontumorigenic NRP-152 prostatic cell line, DP-153 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic cells. They express prostatic acid phosphatase and androgen receptors and require several mitogens (epidermal growth factor, insulin, dexamethasone, and cholera toxin) for sustained growth in culture under serum-containing conditions. DP-153 cells are also growth-stimulated by keratinocyte growth factor and basic fibroblast growth factor and growth-inhibited by all-trans-retinoic acid, 1,25-dihydroxyvitamin D(3), and transforming growth factor (TGF)-beta1. We demonstrate that expression of dominant-negative TGF-beta receptor type II by retroviral transduction of DP-153 cells leads to complete loss of TGF-beta1-induced growth inhibition. When transplanted s.c. in athymic mice, DP-153 cells expressing dominant-negative TGF-beta receptor type II form tumors as early as 4 weeks, in contrast to the vector control and parental cell line, which do not form tumors even 8 months after transplantation, supporting the observation that TGF-beta functions as a tumor suppressor in these cells. Our data further support that DP-153 is a suitable cell line for analysis of normal prostatic growth and carcinogenesis.

Topics: Animals; Calcitriol; Cell Division; Cell Transformation, Neoplastic; Growth Substances; Isoenzymes; Karyotyping; Keratins; Male; Mice; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type II; Receptors, Androgen; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 M 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of -casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR , as well as RXR a, and g expression was low in both HC11 and HC11ras cells. In addition, RAR mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Topics: Animals; Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; Female; Gene Expression Regulation; Genes, ras; Mammary Glands, Animal; Mice; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Vitamin D

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Androstadienes; Antigens, CD; Antigens, Differentiation; Antineoplastic Agents; Cell Transformation, Neoplastic; Chromones; Enzyme Inhibitors; Hematopoietic Stem Cells; HL-60 Cells; Humans; Membrane Glycoproteins; Morpholines; NAD+ Nucleosidase; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Tretinoin; Up-Regulation; Wortmannin

We previously showed that the expression of p16(ink4a) (p16), in conjunction with retinoic acid (RA) treatment in the p16-deficient astrocytoma cell line, U343 MG-A, induced a potent cell cycle arrest in G(1) associated with changes in morphology. In this study, we investigated the effects of p16 expression and RA treatment on the expression and distribution of actin, glial fibrillary acidic protein (GFAP), and vimentin within the U343 MG-A astrocytoma cytoskeleton. Changes in expression and location of the small GTPase, rhoA, were also examined after p16 expression and RA treatment. We showed that p16 expression and RA treatment led to an increase in the expression of GFAP, as well as its reorganization but that it did not significantly affect actin or vimentin expression. p16 induction in combination with RA treatment resulted in a decreased expression and activation of rhoA as determined by immunocytochemistry and Western blot analysis of soluble and insoluble fractions of cell lysates. Endogenous levels of rhoA expression varied among samples in a panel of astrocytoma cell lines as determined by Western blot analysis. Introduction of a dominant active rhoA mutant into p16-induced, RA-treated U343 MG-A astrocytoma cells was associated with the loss of long astrocytic processes and stellate morphology. These data are among the first to report the pattern of rhoA expression in human astrocytoma cell lines. They furthermore suggest that the stellate cell phenotype observed in U343 MG-A astrocytoma cells after cyclin-dependent kinase inhibitor (CKI) induction and RA treatment is accompanied by an inhibition and inactivation of rhoA in this cell system.

Topics: Actin Cytoskeleton; Astrocytoma; Brain Neoplasms; Cell Compartmentation; Cell Differentiation; Cell Membrane; Cell Movement; Cell Size; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Phenotype; Protein Transport; rhoA GTP-Binding Protein; Tretinoin; Tumor Cells, Cultured

Brain injuries trigger physiological reactions which are mediated by a number of cytokines including interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF). Astrocytes and microglia, the protagonists in these traumatic responses, are known to secrete a variety of paracrine signals. Oligodendrocytes are involved as well and constitute another possible source of cytokines. Here we show the expression of IL-6, CNTF, and LIF in OLN-93 cells, derived from rat oligodendrocyte primary cultures. While differential gene transcription after injury has been described for many cytokines, the regulation of these physiological responses is unknown in many instances. Recent experiments indicate that the transcriptional activator retinoic acid (RA) plays a role in peripheral nerve regeneration. Transcripts of the retinoic acid receptors and the retinoid X receptors were also detected in OLN-93 oligodendrocytes. Using quantitative RT-PCR, we have therefore investigated the effect of RA on the expression of neuropoietic cytokines in these cells. Treatment with 1 microM all- trans RA for 24 h increased the mRNA concentration of LIF by a factor of 3.1 ( P<0.01). In contrast, RA had no significant effect on the expression of CNTF. The results suggest RA as a possible regulator of cytokine signaling in the CNS.

Topics: Animals; Cell Line; Cell Size; Cell Transformation, Neoplastic; Ciliary Neurotrophic Factor; Gene Expression Regulation; Growth Inhibitors; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Neuroglia; Oligodendroglia; Rats; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin

Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation.

Topics: Antineoplastic Agents; Biomarkers; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cyclin A; Cyclin D1; DNA-Binding Proteins; Humans; Neuroblastoma; Pregnancy-Specific beta 1-Glycoproteins; rho GTP-Binding Proteins; Trans-Activators; Tretinoin

Von Hippel-Lindau (VHL) tumor suppressor protein is expressed in neurons of the central nervous system and plays an important role during the neuronal differentiation of central nervous system progenitor cells. To elucidate the neuronal differentiating potential of VHL protein in neuroblastoma cells, we overexpressed or inhibited VHL protein in human neuroblastoma cells (SY-SH5Y), and examined the morphological change, expressions of neuronal markers, and electrophysiological functions. Here we show that with VHL gene transduction SY-SH5Y cells stably expressing the VHL protein had neurite-like processes with varicosities, showed the distinct expression of the neuronal markers neuropeptide Y and neurofilament 200, acquired regulated neurosecretion competence in response to depolarizing and cholinergic stimuli, and had large voltage-gated fast sodium currents and delayed rectifier potassium (Kv) currents compatible with those of functional neurons. In addition, they displayed inactivated ether-á-go-go potassium channels related to the promotion of the cell cycle and to the termination of differentiation. Also, by treatment with retinoic acid, they rapidly underwent cell death related to apoptosis. These findings suggest that the induction of neuronal function by VHL protein is associated with down-regulation of the cell cycle. In contrast, the inhibition of endogenous expression of VHL protein with antisense-orientated VHL gene transduction reduced such neuronal properties inherent to these cells, including the capacity for activation of ether-á-go-go channels. In conclusion, VHL protein has a neuronal differentiating potential to transform neuroblastoma cells into functional neuron-like cells. Our finding of the neuronal differentiation of neuroblastoma cells under the control of the VHL gene may contribute to the development of clinical techniques for neuronal regeneration in the case of intractable neuronal diseases and for differentiation therapy against neuroblastomas.

Topics: Apoptosis; Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Ether-A-Go-Go Potassium Channels; Genetic Therapy; Humans; Ion Channel Gating; Ligases; Neuroblastoma; Neurofilament Proteins; Neurons; Neuropeptide Y; Potassium Channels; RNA, Messenger; Sodium Channels; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Telomerase, a ribonucleoprotein complex of hTERT, hTR, and TP1, has been reported to be associated with carcinogenesis and multidrug resistance (MDR). This study used our in vitro human cervical multistep carcinogenesis/MDR model system in which normal human ectocervical and endocervical (HEN) cells were immortalized by HPV18 or 16, respectively, and subsequently transformed. The first evidence was found that immortalization and telomerase activation were correlated with increased expression specifically of two of the hTERT alternatively spliced mRNAs, one encoding wild-type protein containing the full-length functional reverse transcriptase (RT) region and one encoding a defective RT protein. Expression of neither hTERT mRNA containing full-length functional or defective RT motif was affected by transformation/MDR. All-trans-retinoic acid (ATRA) treatment of HPV-immortalized HEN-16-2 cells and transformed/MDR HEN-16-2/CDDP cells inhibited telomerase activity and downregulated expression of hTERT mRNAs containing full-length functional and a defective RT motif, but there were no changes in hTR and TP1 expression. Moreover, ATRA inhibited cell growth rate of HEN-16-2 and HEN-16-2/CDDP cells equally. These results provided the first evidence that ATRA equally in both immortalized and transformed/MDR cell lines inhibits telomerase activity and downregulates expression, but not splicing, of hTERT, and this is correlated with cell growth rate inhibition; the potential is implicated for applying ATRA to hTERT-targeted treatment of cervical cell carcinogenesis/MDR.

Topics: Alternative Splicing; Antineoplastic Agents; Carrier Proteins; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Cervix Uteri; DNA-Binding Proteins; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Activation; Female; Gene Expression Profiling; HeLa Cells; Humans; Models, Biological; RNA-Binding Proteins; Telomerase; Tretinoin; Uterine Cervical Neoplasms

There is a need to identify lung cancer prevention mechanisms. All-trans-retinoic acid (RA) was reported previously to inhibit N-nitrosamine-4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) carcinogenic transformation of BEAS-2B human bronchial epithelial cells (J. Langenfeld et al., Oncogene, 13: 1983-1990, 1996). This study was undertaken to identify pathways targeted during this chemoprevention.. Because epidermal growth factor receptor (EGFR) overexpression is frequent in non-small cell lung cancers (NSCLC) and bronchial preneoplasia, BEAS-2B cells, carcinogen-transformed BEAS-2B(NNK) cells, and retinoid chemoprevented BEAS-2B(NNK RA) cells were each examined for EGFR expression. Whether RA treatment regulated directly EGFR expression or reporter plasmid activity was studied. RA effects on epidermal growth factor (EGF) induction of EGFR-phosphotyrosine levels, cyclin D1 expression and mitogenesis were examined in BEAS-2B cells.. Findings reveal that NNK-mediated transformation of BEAS-2B cells increased EGFR expression. RA treatment repressed EGFR expression and reporter plasmid activity in these cells. This treatment reduced EGF-dependent mitogenesis as well as EGFR-associated phosphotyrosine levels and cyclin D1 expression. These findings extend prior work by highlighting EGFR as a chemoprevention target in the lung. Notably, RA treatment prevented transformation as well as outgrowth of EGFR overexpressing bronchial epithelial cells, despite NNK exposure. After acute NNK exposure, p53-induced species that appear after DNA damage or oxidative stress were evident before an observed increase in EGFR expression.. These findings indicate how effective chemoprevention prevents carcinogenic transformation of bronchial epithelial cells when repair of genomic damage does not select against EGFR overexpressing cells. This implicates EGFR as a chemoprevention target in the carcinogen-exposed bronchial epithelium.

Topics: Antineoplastic Agents; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Cyclin D1; DNA Primers; ErbB Receptors; Gene Expression Regulation; Humans; Lung Neoplasms; Mitosis; Nitrosamines; Phosphotyrosine; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin

Topics: Azacitidine; Cell Division; Cell Transformation, Neoplastic; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Gene Silencing; Genes, Tumor Suppressor; Humans; Leukemia, Promyelocytic, Acute; Leukocytes; Mutation; Neoplasm Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tretinoin; Zinc

DNA methylation of tumor suppressor genes is a frequent mechanism of transcriptional silencing in cancer. The molecular mechanisms underlying the specificity of methylation are unknown. We report here that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters and that hypermethylation contributes to its leukemogenic potential. Retinoic acid treatment induces promoter demethylation, gene reexpression, and reversion of the transformed phenotype. These results establish a mechanistic link between genetic and epigenetic changes during transformation and suggest that hypermethylation contributes to the early steps of carcinogenesis.

Topics: Azacitidine; Binding Sites; Cell Differentiation; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cloning, Molecular; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Exons; Gene Expression; Gene Silencing; Histone Deacetylases; Humans; Leukemia, Promyelocytic, Acute; Mutation; Neoplasm Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Zinc

Activation of telomerase is an early event in the development of breast and other cancers that may lead to cell immortalization, a critical and rate-limiting step in cancer progression. Breast epithelial cells from women with Li-Fraumeni syndrome (LFS) immortalize spontaneously and reproducibly in culture. We, therefore, tested whether immortalization of these cells could be prevented by treating them with chemopreventive agents and by inhibiting telomerase activity.. Noncancerous, preimmortal breast epithelial cells derived from a patient with LFS were treated for 3 months with nontoxic concentrations of the chemopreventive agents oltipraz, difluoromethylornithine, tamoxifen, and retinoic acid or with two different telomerase inhibitors. The frequency of spontaneous immortalization of LFS-derived cells was estimated by an approach based on fluctuation analyses. Statistical analyses were two-sided.. The frequency of spontaneous immortalization events of LFS-derived breast epithelial cells was reduced by long-term treatment with retinoic acid (P<0.001) or tamoxifen (P<0.05) compared with solvent-treated cells. The frequency of immortalization was also reduced by treating LFS-derived cells with an antitelomerase antisense oligonucleotide (P<0.001) or by inducing the cells to express a dominant negative mutant of telomerase (P<0.025) compared with cells treated with a control oligonucleotide or with empty vector, respectively.. Treatment of preimmortal LFS breast epithelial cells with chemopreventive and antitelomerase agents decreased the frequency of spontaneous immortalization in vitro. These studies validate the application of a new cell culture model system to screen the effects of novel chemopreventive agents by use of cell immortalization as an end point. The results also suggest that the telomerase ribonucleoprotein complex may be an important molecular target for breast cancer prevention.

Topics: Antineoplastic Agents; Breast; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Disease Progression; DNA, Complementary; Eflornithine; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Female; Humans; Li-Fraumeni Syndrome; Oligodeoxyribonucleotides, Antisense; Point Mutation; Pyrazines; Reverse Transcriptase Inhibitors; Tamoxifen; Telomerase; Thiones; Thiophenes; Transformation, Genetic; Tretinoin

All-trans-retinoic acid (ATRA) has significantly improved the treatment results in acute promyelocytic leukemia (M3). In non-M3 acute myeloid leukemia (AML), the effects are less clear, and there is a pronounced heterogeneity in the sensitivity to the growth-inhibitory effects of retinoids in leukemic cells from different non-M3 AML patients. Retinoids exert their effects through a number of nuclear receptors [retinoic acid receptors (RARs) and retinoid X receptors (RXRs)]. In this study, we determined the expression of RAR alpha, RAR beta, RAR gamma, and RXR alpha by real-time PCR in four cell lines and in blast cells from patients with non-M3 AML before and after ATRA incubation. All four receptors were expressed in cells from all 18 tested patient samples and in four myeloid cell lines. In the majority of the patient samples as well as in the cell lines, there was a pattern of high expression of RAR alpha and RXR alpha and low expression of RAR beta and RAR gamma. There was no correlation between the basal expression of any of the retinoid receptors and sensitivity to ATRA. A 24-h exposure to ATRA increased the expression of RAR alpha, RAR beta, RAR gamma, and RXR alpha in 46%, 77%, 30%, and 38% of the samples, respectively. The mean increase in receptor expression was most pronounced for RAR beta and RXR alpha. There was a significant correlation between an increase in RAR beta expression in response to ATRA and sensitivity to ATRA (P < 0.014). No such correlations were found for RAR alpha, RAR gamma, and RXR alpha. The expression of the monocytoid marker CD14 was significantly correlated with increased expression of RAR alpha (P = 0.03). We conclude that RAR alpha, RAR beta, RAR gamma, and RXR alpha are expressed in non-M3 AML blast cells and that ATRA-induced expression of RAR beta may be a marker for retinoid sensitivity.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Cell Division; Cell Transformation, Neoplastic; DNA Primers; Dose-Response Relationship, Drug; Female; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Lipopolysaccharide Receptors; Male; Middle Aged; Polymerase Chain Reaction; Receptors, Retinoic Acid; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Neuroblastoma, the most common extracranial solid tumor in children, arises from precursors of the sympathetic nervous system. Neuroblastoma cell lines are responsive to the differentiation agent retinoic acid, which induces its effects by altering transcription rates of specific target genes. We identified autotaxin (ATX), which encodes an autocrine tumor motility-stimulating factor, as a gene whose expression is significantly induced by retinoic acid in neuroblastoma cells. ATX induction was specific for neuroblastoma cell lines that contain N-myc amplification, a cytogenetic feature commonly associated with aggressive neuroblastomas. Although ATX expression was associated with amplification of the N-myc locus, N-myc itself was neither sufficient nor required for ATX expression, suggesting that a coamplified gene is responsible. ATX induction by retinoic acid was due to increased transcription and required new protein synthesis.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Amplification; Gene Expression; Genes, myc; Glucose-6-Phosphate Isomerase; Glycoproteins; Humans; Multienzyme Complexes; Neuroblastoma; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Repressor Proteins; Retroviridae; RNA, Messenger; Tretinoin

Retinoic acid (RA) is the form of vitamin A that controls differentiation and proliferation of epithelia. Our previous work established that normal breast epithelia synthesize RA from retinol, an ability retained by three immortalized but nontumorigenic cell lines but lost in five of six breast cell lines. In this work, we characterize the cause of this defect in one of the lines, the MCF-7 line. We have determined that the immortalized but nontumorigenic cell line, MTSV1.7, capable of synthesizing RA from both retinol and retinal, contains a retinaldehyde dehydrogenase activity for the second step in RA biosynthesis. We have identified it, after isolation, as a previously described enzyme, aldehyde dehydrogenase 6 (ALDH6). Immunohistochemical analysis of normal human breast with antibodies to ALDH6 showed expression of this enzyme in the glandular epithelia colocalized with cellular RA-binding protein type II, a possible marker for certain cells able to synthesize RA. ALDH6 was not present in MCF-7 cells, and these cells were unable to oxidize retinal to RA in culture. When MCF-7 cells were then transfected with ALDH6, they (re)gained the ability to oxidize retinal to RA as well as some ability to synthesize RA when provided with retinol. This suggests that loss of ALDH6 expression is the defect in RA biosynthesis in these cells. Identification of ALDH6 as the retinaldehyde dehydrogenase present in normal human breast epithelia provides the first tool necessary for studying the loss of RA synthetic ability in cancer cells and the relationship of this process to malignant transformation.

Topics: Aldehyde Dehydrogenase; Aldehyde Oxidoreductases; Blotting, Western; Breast; Cell Line; Cell Transformation, Neoplastic; Epithelium; Humans; Immunohistochemistry; Isoenzymes; Oxidation-Reduction; Retinal Dehydrogenase; Retinaldehyde; Transfection; Tretinoin

Human SEC14L1 shows partial sequence homology to the budding yeast SEC14 protein and the Japanese flying squid retinal-binding protein and was previously generally localized to 17q25. We more precisely mapped SEC14L1 within a discrete region of 17q25 that likely harbors at least one putative breast and ovarian tumor suppressor gene. We determined that this gene consists of 18 exons ranging in size from 70 bp (exon 11) to 3088 bp (exon 17) and spanning at least 58 kb of DNA. Exon 17 contained a highly polymorphic variable number of tandem repeats (VNTR) and was present only in the larger ubiquitously expressed 5.5-kb transcript. The 3.0-kb ubiquitously expressed transcript included sequences at the beginning of exon 17 (designated exon 17a) and the end of exon 17 (designated exon 18), but lacked the internal 2439 bp of exon 17, including the VNTR. This alternative splicing resulted in a predicted protein of 719 residues from the smaller transcript with four more terminal amino acids than the 715 residue protein predicted from the larger transcript. EST H49244 spanned exon 11 of SEC14L1 and was specifically expressed in human peripheral blood leukocytes. One intragenic single nucleotide polymorphism (SNP) was confirmed. SEC14L1 contained the CRAL/TRIO domain also found in alpha-tocopherol transfer protein (TTPA) and cellular retinaldehyde-binding protein (CRALBP). As retinoids have been shown to inhibit the growth of breast cancer cells, loss of the proposed SEC14L1 retinal-binding function may contribute to breast tumorigenesis. As TTPA and CRALBP have been implicated in retinitis pigmentosa (RP), altered SEC14L1 expression may contribute to RP in previously unlinked families. Coding exon-specific PCR primers were designed to aid in future expression and mutational analyses.

Topics: Adult; Breast; Breast Neoplasms; Carrier Proteins; Cell Line; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 17; Contig Mapping; DNA, Neoplasm; Exons; Expressed Sequence Tags; Female; Fetal Proteins; Genes, Tumor Suppressor; Glycosylation; Golgi Apparatus; Humans; Leukocytes; Minisatellite Repeats; Molecular Sequence Data; Multigene Family; Organ Specificity; Polymorphism, Genetic; Protein Processing, Post-Translational; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing; Tretinoin; Tumor Cells, Cultured

Research into molecular and genetic mechanisms underlying prostate carcinogenesis would be greatly advanced by in vitro models of prostate tumors representing primary tumors. We have successfully established an immortalized human prostate epithelial (HPE) cell culture derived from a primary tumor with telomerase. The actively proliferating early passaged RC-58T cells were transduced through infection with a retrovirus vector expressing the human telomerase catalytic subunit (hTERT). A high level of telomerase was detected in RC-58T/hTERT cells but not RC-58T cells. RC-58T/hTERT cells are currently growing well at passage 50, whereas RC-58T cells senesced at passage 7. RC-58T/hTERT cells exhibit transformed morphology. More importantly, these immortalized cells showed anchorage-independent growth as they formed colonies in soft agar and grew above the agar layer. Expression of androgen-regulated prostate specific gene NKX3.1 and epithelial specific cytokeratin 8 (CK8) but not prostate specific antigen (PSA) and androgen receptor was detected in RC-58T/hTERT cells. Prostate stem cell antigen (PSCA) and p16 were also expressed in this cell line. RC-58T/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor (TGF)-beta1 known potent inhibitors of prostate epithelial cell growth. A number of chromosome alterations were observed including the loss of chromosomes Y, 3p, 10p, 17p, 18q and the gain of chromosomes 16 and 20. These results demonstrate that this primary tumor-derived HPE cell line retained its transformed phenotypes and should allow studies to elucidate molecular and genetic alterations involved in prostate cancer. This is the first documented case of an established human prostate cancer cell line from a primary tumor of a prostate cancer patient with telomerase.

Topics: Agar; Cell Culture Techniques; Cell Division; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Chromosome Aberrations; DNA-Binding Proteins; Epithelial Cells; Humans; Karyotyping; Male; Models, Biological; Prostatic Neoplasms; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Telomerase; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured

The most common chromosomal translocation in acute promyelocytic leukemia (APL), t15;17(q22;q21), creates PMLRARalpha and RARalphaPML fusion genes. We previously developed a mouse model of APL by expressing PMLRARalpha in murine myeloid cells. In order to examine the mechanisms by which PMLRARalpha can initiate leukemia, we have now generated transgenic mice expressing PMLRARalpham4 and RARalpham4, proteins that are unable to activate transcription in response to retinoic acid. PMLRARalpham4 transgenic mice developed myeloid leukemia, demonstrating that transcriptional activation by PMLRARalpha is not required for leukemic transformation. The characteristics of the leukemias arising in the PMLRARalpham4 transgenic mice varied from those previously observed in our PMLRARalpha transgenic mice, indicating that ligand responsiveness may influence the phenotype of the leukemic cells. The leukemias that arose in PMLRARalpham4 transgenic mice did not differentiate in response to retinoic acid therapy. This result supports the hypothesis that a major therapeutic effect of retinoic acid is mediated directly through the PMLRARalpha protein. However, a variable effect on survival suggested that this agent may be of some benefit in APL even when leukemic cells are resistant to its differentiative effects. Transgenic mice expressing high levels of RARalpham4 have not developed leukemia, providing evidence that the PML domain of PMLRARalpha plays a specific and critical role in the pathogenesis of APL. (Blood. 2000;95:1541-1550)

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Transformation, Neoplastic; Chlorocebus aethiops; Disease Progression; Gene Expression Regulation, Leukemic; Genes, Dominant; Humans; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Mutagenesis; Neoplasm Proteins; Oncogene Proteins, Fusion; Phenotype; Protein Structure, Tertiary; Radiation Chimera; Recombinant Fusion Proteins; Repressor Proteins; Transcriptional Activation; Transfection; Tretinoin

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.

Topics: Animals; Calcium; Calcium Channels; Cell Differentiation; Cell Membrane Permeability; Cell Survival; Cell Transformation, Neoplastic; Fibroblasts; HL-60 Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Mice; NADPH Oxidases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; Tretinoin

Many patients with liver cirrhosis are in a state of protein and energy malnutrition and require careful nutritional support. Our research has revealed that approximately 30% of the patients have protein-energy malnutrition, 40% protein malnutrition, and 10% energy malnutrition; 20% are in a normal nutritional state. Supplementation with branched-chain amino acids alleviates chronic liver failure, improves the protein nutritional state, and subsequently prolongs survival. In contrast, therapeutic modalities for energy malnutrition have not yet been fully elucidated and await further studies. Improved survival of the cirrhotic patients essentially brings a higher incidence of hepatocellular carcinoma (HCC). A synthetic analogue of vitamin A (acyclic retinoid or 4,5-dehydrogeranyl geranoic acid) prevents at least the development of second primary tumors after curative treatment of preceding HCC. The mechanism of this cancer chemo-prevention is clonal deletion of premalignant and latent malignant cells by the retinoid. We describe our clinical experiences with these two nutritional pharmacotherapies of chronic liver diseases and review their basic mechanisms.

Topics: Amino Acids, Branched-Chain; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Combined Modality Therapy; Humans; Liver Cirrhosis; Liver Failure; Liver Neoplasms; Nutritional Support; Protein-Energy Malnutrition; Randomized Controlled Trials as Topic; Survival Rate; Treatment Outcome; Tretinoin

The t(15;17) chromosomal translocation in acute promyelocytic leukemia (APL) generates the PML-RARalpha fusion protein. The recruitment of nuclear receptor corepressor SMRT/N-CoR and subsequent repression of retinoid target genes is critical for the oncogenic function of PML-RARalpha. Here we show that the ability of PML-RARalpha to form homodimers is both necessary and sufficient for its increased binding efficiency to corepressor and inhibitory effects on hormonal responses in myeloid differentiation. We further provide evidence that altered stoichiometric interaction of SMRT with PML-RARalpha homodimers may underlie these processes. Finally, we demonstrate that a RXR AF2 mutant recapitulates many biochemical and functional properties of PML-RARalpha. Taken together, our results provide an example that altered dimerization of a transcription factor can be directly linked to cellular transformation and implicate dimerization interfaces of oncogenes as potential drug targets.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Cholecalciferol; Dimerization; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; Leukemia, Promyelocytic, Acute; Models, Genetic; Monocytes; Neoplasm Proteins; Nuclear Receptor Co-Repressor 2; Oncogene Proteins, Fusion; Protein Binding; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Repressor Proteins; Retinoid X Receptors; Signal Transduction; Transcription Factors; Transcription, Genetic; Translocation, Genetic; Tretinoin

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Topics: Cell Transformation, Neoplastic; Core Binding Factor Alpha 2 Subunit; Histone Deacetylases; Humans; Leukemia; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Oncogene Proteins, Fusion; Peptide Fragments; Protein Binding; Protein Structure, Quaternary; Repressor Proteins; Response Elements; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Transcription, Genetic; Tretinoin

Topics: Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Signal Transduction; Transcription, Genetic; Translocation, Genetic; Tretinoin

Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.

Topics: Alitretinoin; Animals; Anticarcinogenic Agents; Antigens, Polyomavirus Transforming; Cell Transformation, Neoplastic; Female; Gene Expression; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Tretinoin

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cell Survival; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Hematopoiesis; Humans; Kruppel-Like Transcription Factors; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Mutation; Neoplasm Proteins; Oncogene Proteins, Fusion; Promyelocytic Leukemia Zinc Finger Protein; Repressor Proteins; Stem Cells; Transcription Factors; Transcription, Genetic; Transgenes; Translocation, Genetic; Tretinoin

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; Cell Transformation, Neoplastic; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endoderm; Enzyme Activation; Mice; Parathyroid Hormone; ras Proteins; Rats; Tretinoin; Tumor Cells, Cultured

Retinoic acid inhibits transformation of cells by polyoma virus middle T oncoprotein. Inhibition of transformation results from a retinoic acid-dependent failure of cells to fully express the c-fos proto-oncogene. Retinoic acid prevents transactivation of the c-fos promoter by disrupting signaling between tyrosine kinases at the plasma membrane and trans-acting factors at the c-fos promoter. We used complementary genetic, biochemical and molecular approaches to demonstrate that: (1) phosphatidylinositol 3-kinase signaling is the principle mechanism of polyoma virus middle T oncoprotein activation of c-fos expression; (2) middle T/phosphatidylinositol 3-kinase transactivation of the c-fos promoter and transformation of cells requires activation of both the small GTP-binding protein Rac and Jun N-terminal kinase; (3) retinoic acid inhibits activation of Jun N-terminal kinase, thereby preventing c-fos transactivation and transformation; and (4) middle T activation of c-fos transcription requires both the serum response element and the promoter proximal cyclic AMP response element. These studies identify a novel target through which retinoids prevent oncogenic transformation.

Topics: Antigens, Polyomavirus Transforming; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; rac GTP-Binding Proteins; Transcriptional Activation; Tretinoin

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformati

Topics: Antigens, Viral, Tumor; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Enzyme Activation; HL-60 Cells; Humans; Isoenzymes; Mitogen-Activated Protein Kinase 1; Mutation; Phosphatidylinositol 3-Kinases; Phospholipase C gamma; Polyomavirus; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; src-Family Kinases; Transcription Factors; Transfection; Tretinoin; Type C Phospholipases

Retinoic acid syndrome is a serious condition that may complicate the treatment of acute promyelocytic leukaemia patients. This syndrome may be treated effectively with high-dose corticosteroid therapy and, as a result, many patients with acute promyelocytic leukaemia receive dexamethasone at some point during treatment. We investigated whether dexamethasone would also antagonize the beneficial effects of retinoic acid. In t(15;17)-positive NB4 cells, dexamethasone did not affect the retinoic acid induced differentiation, normalization of PML-nuclear bodies or the induction of thrombomodulin mRNA. Finally, dexamethasone did not inhibit the anti-proliferative effect of retinoic acid but rather showed anti-proliferative activity itself.

Topics: Antineoplastic Agents, Hormonal; Blotting, Northern; CD18 Antigens; Cell Division; Cell Transformation, Neoplastic; Dexamethasone; Humans; Leukemia, Promyelocytic, Acute; Thrombomodulin; Tretinoin; Tumor Cells, Cultured

PML and Tif1a are fused to RARA and Braf, respectively, resulting in the production of PML-RARalpha and Tif1alpha-B-Raf (T18) oncoproteins. Here we show that PML, Tif1alpha and RXRalpha/RARalpha function together in a transcription complex that is dependent on retinoic acid (RA). We found that PML acts as a ligand-dependent coactivator of RXRalpha/RARalpha. PML interacts with Tif1alpha and CBP. In Pml-/- cells, the RA-dependent induction of genes such as RARB2 and the ability of Tif1alpha and CBP to act as transcriptional coactivators on RA are impaired. We show that both PML and Tif1alpha are growth suppressors required for the growth-inhibitory activity of RA. T18, similar to PML-RARalpha, disrupts the RA-dependent activity of this complex in a dominant-negative manner resulting in a growth advantage. Our data define a new pathway for the control of cell growth and tumorigenesis, and provide a new model for the pathogenesis of acute promyelocytic leukaemia (APL).

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; CREB-Binding Protein; DNA; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Leukemia, Promyelocytic, Acute; Mutation; Neoplasm Proteins; Nuclear Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Receptors, Retinoic Acid; Retinoid X Receptors; Trans-Activators; Transcription Factors; Transfection; Tretinoin

To use NB4, an authentic human acute promyelocytic leukemia cell line, as well as the marrow cells from patients with acute promyelocytic leukemia (APL), containing the PML/RAR alpha fusion gene and fused protein to examine the growth inhibition and cytodifferentiation induced by all-trans retinoic acid (ATRA), butyric acid (BA) and its prodrug tributyrin (TB) either as a single agent or in combinations.. NB4 and APL cells were cultured in presence of ATRA, BA and TB respectively either as a single agent or in combinations at various concentration ratio. Cell growth was measured and myeloid differentiation was determined by morphology and the percentage of positive nitroblue tetrazolium reduction (NBT) on consecutive days over the whole process of culture.. NB4 cells can be induced by ATRA alone and synergistically induced by the combinations of BA or TB with ATRA to differentiate. The synergy was reflected by a remarkable decrease in the effective concentration of ATRA required in the combinations in comparison with it as a sole agent. The combinations also shortened the time for the cells to reach the same level of maturation as that needed for ATRA alone. The potentiation on ATRA-induced differentiation of NB4 cells seemed depending on an appropriate concentration ratio of each inducer in the combinations and the time of action. A preliminary result of in vitro induction of primarily cultured leukemic cells from APL patients by the combined inducers was promising.. The combinations of ATRA with BA or TB at an appropriate ratio may improve the clinical outcome of differentiation therapy for APL patients.

Topics: Butyric Acid; Cell Transformation, Neoplastic; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Prodrugs; Stereoisomerism; Translocation, Genetic; Tretinoin; Triglycerides; Tumor Cells, Cultured

To investigate the role of CDKs in the RA inducing HL-60 cell differentiation.. The effects of all-trans retinoic acid (ATRA) and arotinoid ethylester(AE) at the dosage of 5 x 10(-6) mol/L on the proliferation and differentiation of HL-60 cells were examined by NBT reduction test and cytometry analysis. Meanwhile, histone H1 kinase assay was used to observe the changes of CDK2 activities in HL-60 cells treatment with ATRA and AE. The amounts of both cyclin E/CDK2 and cyclin D1/CDK4 complexes were observed with immunoprecipitates.. Both ATRA and AE inhibited the growth of HL-60 cells, arrested cells in G1/G0 phase, and induced cells to differentiation. The activities of cyclin E/CDK2 and the amounts of cyclin D1/CDK4 complexes were decreased in ATRA- or AE-treated HL-60 cells.. The effects of RA on the proliferation and differentiation of HL-60 cells may be associated with significant decreases in the activities of CDKs.

Topics: Antineoplastic Agents; CDC2-CDC28 Kinases; Cell Division; Cell Transformation, Neoplastic; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; HL-60 Cells; Humans; Protein Serine-Threonine Kinases; Tretinoin

To assess the safety of adenovirus-mediated transfer of the RA538 (Ad-RA538) for the treatment of cancer and to furthermore in preparation for a clinical trial of Ad-RA538.. RT-PCR was used to detect the transcription of Ad-RA538 in HeLa cells infected with extracts from HeLa cells previously infected with Ad5-RA538. Cell counting was made to observe the effects of Ad-RA538 on the growth of the normal human fetal lung cell line 2BS. The virus was intraperitoneally injected into 2 groups of BalB/C mice at a dosage of 10(7) pfu and 10(9) pfu. Blood samples were taken from the mice to test the liver and renal function. PCR were used to screen the vital organs for the presence of adenovirus DNA. Microscopic examination of the vital organs was performed to observe the pathogenicity of Ad-RA538.. Ad-RA538 was a replication-defective virus. It could infect 2BS cells effectively, but could not inhibit 2BS cell growth. No mouse died and no signs of general toxicity were seen following intraperitoneal injection of Ad-RA538. The adenoviral vector was present in the liver, spleen, kidney and stomach of mice injected with 10(9) pfu Ad-RA538. Six and 12 days after injection, mild inflammation was observed in the liver of mice received 10(9) pfu Ad-RA538.. Ad-RA538 is safe both in vivo and in vitro, and clinical trials of Ad-RA538 can be performed.

Topics: Adenovirus E1 Proteins; Adenoviruses, Human; Animals; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Complementary; DNA, Recombinant; DNA, Viral; Esophageal Neoplasms; Genetic Vectors; HeLa Cells; Humans; Male; Mice; Mice, Inbred BALB C; Tretinoin; Tumor Cells, Cultured

The PML gene is fused to the retinoic acid receptor alpha (RARalpha) gene in chromosomal translocations associated with acute promyelocytic leukemia (APL). Ablation of murine PML protein by homologous recombination revealed that PML regulates hemopoietic differentiation and controls cell growth and tumorigenesis. PML function was essential for the tumor-growth-suppressive activity of retinoic acid (RA) and for its ability to induce terminal myeloid differentiation of precursor cells. PML was needed for the RA-dependent transactivation of the p21WAF1/CIP1 gene, which regulates cell cycle progression and cellular differentiation. These results indicate that PML is a critical component of the RA pathway and that disruption of its activity by the PML-RARalpha fusion protein may be important in APL pathogenesis.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Fibroblasts; Gene Targeting; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Leukemia, Promyelocytic, Acute; Male; Mice; Monocytes; Neoplasm Proteins; Neoplasms, Experimental; Nuclear Proteins; Oncogene Proteins, Fusion; Promyelocytic Leukemia Protein; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Suppressor Proteins

Retinoic acid (RA) was topically applied to the skin of Sencar mice during the promotion phase of specific tumor induction protocols that produce papillomas at low (12-O-tetradecanoylphorbol-13-acetate promoted, TPA) or high (mezerein-promoted) risk for premalignant progression and malignant conversion. RA consistently reduced the yield of papillomas and carcinomas in both protocols, but the frequency of malignant conversion in papillomas that emerged during RA treatment was not reduced. When TPA was reapplied after cessation of RA treatment, the number of papillomas increased 2-fold, suggesting that RA had not eliminated initiated cells. In vitro, RA prevented the emergence of transformed keratinocytes in an assay that mimics malignant conversion, suggesting that RA can suppress conversion if applied during the stage of premalignant progression. Examination of tumor markers at weeks 14 and 22 of the tumor-induction experiments in vivo indicated that papillomas evolving during RA treatment exhibited a phenotype of high progression risk, even in the TPA-promoted groups. In the majority of these tumors, the alpha6beta4 integrin and retinoid X receptor alpha transcripts were detected suprabasally, indicating an advanced state of premalignant progression. RA-treated tumors also expressed higher levels of transcripts for transforming growth factor (TGF)-beta1 and localized TGF-beta1 peptide in the basal portions of the tumor fronds. Because up-regulated expression of TGF-beta1 suppresses papilloma formation, these studies suggest a mechanism whereby RA can prevent papilloma eruption via a TGF-beta intermediate, but papillomas resistant to RA may have altered TGF-beta signaling and progress to carcinomas at an increased frequency.

Topics: Administration, Topical; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Biomarkers, Tumor; Carcinogens; Carcinoma, Basal Cell; Cell Transformation, Neoplastic; Disease Progression; Diterpenes; Female; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Papilloma; Phenotype; Precancerous Conditions; Receptors, Retinoic Acid; Retinoid X Receptors; Risk Factors; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor beta; Tretinoin

In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth--e.g. containing cells in all phases of the growth cycle--as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

Topics: Animals; Anticarcinogenic Agents; Carcinogens; Cell Cycle Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Dose-Response Relationship, Radiation; Linear Energy Transfer; Mice; Mice, Inbred C3H; Microtubule-Associated Proteins; RNA, Messenger; S Phase; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Suppressor Proteins

The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-myb acts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RAR alpha functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor alpha inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by the mim-1 gene. Conversely, v-Myb protein interferes with RAR alpha transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other's functions.

Topics: Acetyltransferases; Animals; Apoptosis; Cell Differentiation; Cell Transformation, Neoplastic; Chlorides; Coturnix; Fibroblasts; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Hematopoiesis; Hematopoietic Stem Cells; HL-60 Cells; Humans; Oncogene Proteins v-myb; Protein Biosynthesis; Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retroviridae Proteins, Oncogenic; Transcription, Genetic; Tretinoin; Zinc Compounds

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Promyelocytic, Acute; Oxides; Tretinoin; Tumor Cells, Cultured

Following injection into sublethally irradiated DBA/2 or BALB/c mice, factor-dependent FDC-P1 cells undergo leukemic transformation due to oncogene activation by insertion of intracisternal A-particle (IAP) genetic elements. Similar events are observed in vitro during coculture of FDC-P1 cells with irradiated bone marrow stroma cells. To elucidate the mechanism of IAP transposition, we studied the level of IAP expression under the growth conditions preceding cell transformation. In vitro experiments showed that the type of growth factor, FDC-P1 cell density, costimulation with steroid hormones or abrupt growth factor withdrawal had no effect on IAP mRNA levels (major transcripts of 7.4, 4.0 and 1.9 kb). By contrast, stimulation with suboptimal concentrations of GM-CSF or IL-3 induced a mean 2. 5-fold increase in the intensity of the 7.4 kb band, and induction of macrophage differentiation with retinoic acid resulted in an increased stability of the 4.0 kb band. Although suboptimal growth stimulation and incipient macrophage differentiation have previously been shown to occur in the process of FDC-P1 cell transformation, an increase in IAP expression could not convincingly be demonstrated in FDC-P1 cell populations isolated from irradiated BALB/c mice or stroma cell cocultures. Further experiments are required to define the role of suboptimal growth stimulation and/or macrophage differentiation in this transformation model.

Topics: Animals; Antineoplastic Agents; Bone Marrow Cells; Cell Transformation, Neoplastic; Cells, Cultured; Dexamethasone; Female; Genes, Intracisternal A-Particle; Gonadal Steroid Hormones; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-3; Mice; Mice, Inbred BALB C; RNA, Messenger; Tretinoin; Whole-Body Irradiation

The fact that treatment of leukemia (Acute Promyelocytic Leukemia) with ATRA (All-Trans Retinoic Acid) was so succeeded that it was considered as a good example for tumor therapy. In the treatment of solid tumors by means of induced differentiation, however, has not been yet so broken-through. DMSO (Dimethylsulfoxide) was a common and simple organic compound, which comprised a variety of biological activities. For example, DMSO induced differentiation of leukemia in many reports. However, the effect of DMSO on solid tumors was to be explored further. In the present study, DMSO was used to human esophageal cancer cell lines in vitro in comparison with the classical inducer ATRA. From the view of morphology, cell cycle, growth inhibition, cytokeratin 4 expression, dye transfer and tumorigenecity, the results demonstrated that DMSO as well as ATRA could induce differentiation of human esophageal cancer cells. Interestingly, DMSO was confirmed to be more effective in inducing differentiation of esophageal cancer cells than ATRA. It suggests that DMSO showed some good prospects for the treatment of solid tumors.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Esophageal Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

Phorbol 12-myristate 13-acetate (PMA), N,N'-hexamethylenebisacetamide (HMBA) and retinoic acid induce cell differentiation in U-937 promonocytic cells. This report examines the effects of these agents on DNA topoisomerase I activity. A decrease in enzyme activity could be detected as early as 30 min after treatment with all three differentiating compounds and lasted at least 48 h. No alteration in the levels of DNA topoisomerase I transcript or protein was observed during these treatments. The results might be explained by post-translational events that render DNA topoisomerase type I less active.

Topics: Acetamides; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; DNA Topoisomerases, Type I; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Neuropeptide Y (NPY) acts through specific receptors to inhibit adenyl cyclase and may have a role in neuroblastomas and neuroepitheliomas (NE) as a regulator of cell growth and differentiation. The authors have examined the status of NPY receptors in the NE cell line SK-N-MC and the effect of retinoic acid (RA), a known differentiating agent, on their expression and function.. Competitive NPY binding studies were performed on normal and RA-treated cells, followed by Scatchard analysis. NPY receptor function in the absence of or following RA treatment was determined by the ability of various concentrations of NPY to attenuate the forskolin-stimulated accumulation of intracellular cAMP. The mitogenic effect of NPY was evaluated by growing normal or RA-treated cell in the presence of various concentrations of NPY.. Scatchard analysis showed a Kd of 2.3 nmol/L and a Bmax of 91,000 receptors per cell for untreated cells. RA treatment decreased receptor expression to 11,700 per cell without a significant change in receptor affinity (3.6 nmol/L). The effect of forskolin was inhibited by NPY in a dose-dependent fashion in both untreated and treated cells indicating functional receptors in both NPY stimulates the growth of normal SK-N-MC cells. NPY stimulated growth was significantly attenuated after RA treatment, possibly as a result of decreased NPY receptor expression.. Treatment of SK-N-MC cells with RA, a known differentiating agent, leads to decreased expression of functional NPY receptors and a concomitant decrease in the mitogenic effect of NPY. This supports the role for NPY in the pathogenesis of NE.

Topics: Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Humans; Mitogens; Neuroectodermal Tumors, Primitive, Peripheral; Neuropeptide Y; Receptors, Neuropeptide Y; Tretinoin; Tumor Cells, Cultured

A 30-year-old man with a diagnosis of acute promyelocytic leukemia (APL) was admitted. Laboratory findings were as follows: WBC 32,900/microliter with 88% promyelocytes, Hb 10.4 g/dl, platelets 2.6 x 10(4)/microliter. Coagulation tests revealed DIC. Bone marrow was hypercellular with 91.8% promyelocytes which were strongly positive for peroxidase and positive for alpha-naphthyl butyrate esterase. Cytogenetic study revealed 46, XY, t(15;17) (q22:q11). He was treated with all-trans retinoic acid (ATRA) along with hydroxyurea (HU) and low-molecular weight heparin (LMH). Because his WBC increased to 93,700/microliter on day 6 of ATRA therapy, DCMP chemotherapy was given, while ATRA was withheld. He developed enterocolitis due to myelosuppression. ATRA was restarted along with granulocyte-colony stimulating factor (G-CSF). His WBC rose to 10,400/microliter with a marked, but temporary predominance of myelomonocytes both in peripheral blood and in bone marrow. These myelomonocytoid cells were positive for specific and nonspecific esterase double stainings. Then he entered complete remission. It was of interest that myelomonocytoid differentiation of APL cells was induced by ATRA. The etiology was discussed.

Topics: Adult; Cell Transformation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Male; Remission Induction; Tretinoin

Growth factor-independent proliferation and loss of response to differentiation factors are believed to be critical elements in carcinogenesis. We have developed an in vitro model of human prostatic carcinogenesis by the introduction of SV40 DNA into normal prostatic epithelial cells to create a transformed, immortal cell line, pRNS-1-1. This non-tumorigenic cell line responded similarly to normal prostatic epithelial cells to most growth- and differentiation-regulatory factors, with the notable exception of loss of response to the inhibitory factor 1,25-dihydroxyvitamin D3. In this study, we describe the introduction of the ras oncogene into pRNS-1-1 cells to create a tumorigenic cell line, pRNS-1-1/ras. In addition to an attenuated response to 1,25-dihydroxyvitamin D3, these cells also became unresponsive to retinoic acid and gained the ability to undergo clonal proliferation in the absence of epidermal growth factor (EGF). EGF-independent growth could not be linked to the production of autocrine transforming growth factor-alpha, but instead was likely due to sustained signaling by the ras oncogene, bypassing ligand-activation of the EGF receptor. Ligand-independent proliferation, coupled with the loss of response to the growth-inhibitory and differentiation agent retinoic acid, may be important elements in the conversion of human prostatic epithelial cells to tumorigenicity.

Topics: Animals; Calcitriol; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Epithelium; Fibroblast Growth Factor 1; Genes, ras; Humans; Keratins; Male; Mice; Mice, Nude; Neoplasm Transplantation; Prostate; Somatomedins; Transforming Growth Factor alpha; Tretinoin; Tumor Necrosis Factor-alpha

Myeloperoxidase (MPO) catalyzes a reaction between chloride and hydrogen peroxide to generate hypochlorous acid and other reactive compounds that have been linked to DNA damage. The MPO gene is expressed at high levels in normal myeloid precursors and in acute myeloid leukemias (AMLs) which are clonal derivatives of myeloid precursors that have lost the ability to differentiate into mature blood cells. Two MPO alleles differ at -463 G/A within a cluster of nuclear receptor binding sites in an Alu element. The -463 G creates a stronger SP1 binding site and retinoic acid (RA) response element (RARE) in the allele termed Sp. In this study, we investigate potential links between MPO genotype, MPO expression level, and myeloid leukemia. The SpSp MPO genotype is shown to correlate with increased MPO mRNA levels in primary myeloid leukemia cells. This higher-expressing SpSp genotype is further shown to be overrepresented in acute promyelocytic leukemia-M3 (APL-M3) and AML-M4, suggesting that higher levels of MPO are associated with an increased risk for this subset of leukemias.

Topics: Alleles; Binding Sites; Cell Transformation, Neoplastic; Disease Susceptibility; DNA Damage; Female; Gene Frequency; Genotype; Humans; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Male; Neoplasm Proteins; Peroxidase; Polymerase Chain Reaction; Risk; RNA, Messenger; RNA, Neoplasm; Tretinoin

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation.

Topics: Bronchi; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Cyclin D1; Cysteine Endopeptidases; Epithelial Cells; Gene Expression Regulation; Humans; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Signal Transduction; Tretinoin

Retinoylation (retinoic acid acylation) is a post-translation modification of proteins occurring in a variety of mammalian cell lines and in vivo. To gain further knowledge of the role of retinoylation we studied it in NIH 3T3 cells and NIH 3T3 cells transformed by an activated Ha-ras oncogene (NIH Ha-ras-3T3 cells). In serum-free medium retinoic acid (RA) inhibited growth of NIH 3T3 cells but did not inhibit growth of NIH Ha-ras-3T3 cells. After incubation with [3H]RA, the level of retinoylated protein in NIH 3T3 cells was about 1.5-fold greater than in NIH Ha-ras-3T3 cells. On one-dimensional polyacrylamide gel electrophoresis, both the rate and the extent of retinoylation were greater in NIH 3T3 cells. We detected about 40 retinoylated proteins in NIH 3T3 cells by two-dimensional polyacrylamide gel electrophoresis. Only about 15 proteins were retinoylated, but at reduced levels, in NIH Ha-ras-3T3 cells. These results suggest that the activated ras oncogene inhibits retinoylation. This inhibition may in turn be related to the loss of other RA responses of NIH 3T3 cells, including growth inhibition, retinoic acid catabolism, down-regulation of fibronectin biosynthesis, and induction of tissue-type transglutaminase, which are not seen to the same extent in NIH Ha-ras-3T3 cells.

Topics: 3T3 Cells; Animals; Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Fibronectins; Genes, ras; GTP Phosphohydrolases; GTP-Binding Proteins; Mice; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tretinoin

We compared the ability of all-transretinoic acid (RA), all-trans-retinoyl-beta-D-glucuronide (RAGL), and all-trans-beta-carotene (BC) to inhibit growth and to induce differentiation of the human promyelocytic leukemia cell line HL-60 into morphologically mature granulocytes. BC was made water-soluble by the solutol-solvent-system. RA (1 microM) could induce differentiation of 85% of the HL-60 cells after a total incubation time of 180 hours, RAGL (5 microM) induced 64% of the cells, whereas 33% of the HL-60 cells were differentiated after incubation with BC (10 microM), which was determined by assessing cell functional capacity to reduce nitroblue tetrazolium dye in response to phorbolesters. The absence of RA in RAG and BC treated cells gives strong evidence that RAG and BC exert intrinsic biological effects.

Topics: Antioxidants; beta Carotene; Cell Division; Cell Survival; Cell Transformation, Neoplastic; HL-60 Cells; Humans; Time Factors; Tretinoin

Human neuroblastoma cells contain a 260 kDa surface-associated antigen (NB-p260) that is recognised by natural cytotoxic IgM antibodies. In this study we demonstrate that NB-p260 is expressed in vivo in a neuroblastoma tumour specimen but not in normal human tissues of neuronal origin. Since MYCN amplification is a clinical marker of neuroblastoma disease progression, we analysed the expression of NB-p260 in human neuroblastoma cell lines with different MYCN amplification status. However, both amplified and non-amplified neuroblastoma cell lines exhibited comparable NB-p260 expression. Treatment of neuroblastoma cells with the differentiation-inducing agent retinoic acid (RA) also had no effect on the expression of NB-p260. Collectively, the data suggest that expression of NB-p260 on human neuroblastoma cells is independent of malignancy and differentiation status of neuroblastoma.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Transformation, Neoplastic; Gene Amplification; Gene Expression; Genes, myc; Humans; Immunoblotting; Immunoglobulin M; Immunohistochemistry; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

To study the relation between the PML-RAR alpha gene and the effects of ATRA on proliferation and differentiation in acute promyelocytic leukemia cell line NB4 cells.. ASODN-mediated inactivation of the PML-RAR alpha mRNA was measured by RT-PCR. The proliferation and differentiation of NB4 cells were determined by proliferation curve, morphology, CD antigen of membrane and NBT test. NB4 Cell cycle was analyzed by FACS.. The inactivation of PML-RAR alpha mRNA by ASODN could egnhance the maturation effects and growth suppression of ATRA on NB4 cells. The percentage of S phase cell was decreased from 52% to 29%, and NBT rate increased. All suggest that the sensitivity of NB4 cell to ATRA was improved.. The PML-RAR alpha gene, as a molecular marker of APL, is responsible for pathogenesis of APL, and it also decreases the sensitivity of ATRA to APL cells.

Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Tretinoin; Tumor Cells, Cultured

Changes of [Ca2+]i in CCL229 cells induced by retionoic acid (RA), 1,25(OH)2VD3 and PMA were measured by spectrofluorometry. The effects of endoplasmic reticulum (ER)-specific Ca(2+)-ATPase inhibitor thapsigargin (TG) and IP3 receptor inhibitor heparin on RA-induced changes of [Ca2+]i were observed and the relationship between RA-induced changes of [Ca2+]i and ER was also investigated. The results showed that [Ca2+]i increased markedly in several seconds after treated by RA and 1,25(OH)2VD3. When cells were pretreated with EGTA and verapamil (Ca2+ entry blocker drug), TG could not inhibit RA-stimulated Ca2+ release from intracellular calcium pools and TG could increase [Ca2+]i after pretreated by RA. In addition, heparin could not completely inhibit RA-induced [Ca2+]i increase. The results suggest that RA might stimulate IP3-sensitive pool or IP3-insensitive pool on ER to increase [Ca2+]i, or there might be RA-sensitive calcium pools except ER in cells.

Topics: Antineoplastic Agents; Calcium; Cell Transformation, Neoplastic; Colorectal Neoplasms; Endoplasmic Reticulum; Humans; Tretinoin; Tumor Cells, Cultured

We detected the antiproliferative effect with MTT test and investigated the changes in biological properties, cytomorphology and ultrastructure through cytopathology and electronic microscopy. Cell growth of JF-305 was inhibited by all-trans retinoic acid (ATRA). The maximal inhibitory rate was 34.7%. The number of proliferative cells reduced (P < 0.01). Cell metabolism slowed down, secretory functions recovered, and malignant degree decreased. ATRA can inhibit the proliferation and induce the differentiation of human pancreatic adenocarcinoma JF-305 cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Division; Cell Transformation, Neoplastic; Humans; Pancreatic Neoplasms; Tretinoin; Tumor Cells, Cultured

Retinoic acid and its analogues play important roles in modulating cell growth, differentiation, immunity and apoptosis. Clinically they are used for cancer chemoprevention and chemotherapy. Based upon the moiety of 3,5-di-t-butyl-4-hydroxy phenyl ring, a series of substituted aromatic amide, ester and chalcones were designed and synthesized, which mimic the molecular shape, size, and spacial disposition of functional groups of retinoic acid. The general structure is as follows: [formula: see text] where R stands for hydrogen atom or methyl group, Y is the linkage -CONH-, -NHCO-, -COO-, -COCH = CH-, or a member of a heterocycle, X represents various substituents at different positions. The SAR indicates that the presence of hydrophobic group(s) at one end of the molecule, and a carboxyl group at the other end, and a conjugative system of molecule are necessary and full prerequisite for exhibiting activity. Loss of any one factor of them will abolish the activity. Being obligatory for anti-oxidative effect, the phenolic hydroxy group does not convey biological activity, because after methylation of the hydroxy group the compound increases the differentiation-inducing activity and loses the anti-oxidative effect, indicating that there is no correlation between the two activities. With a stable conformation of two phenyl rings with cis-conformation N-methylated acyl amide (No. 30) features in bent shape of the molecule, instead of an extended conformer, which is taken by the non-N-methylated partner and all-trans retinoic acid. A bent conformer of No. 30 accounts for the inactivity. In this paper compounds No. 4f, 4g, 5a, 7, 13, 32, 37, and 38 exhibited significant activity among them 4-[3-(3, 5-di-t-4-methoxyphenyl)-3-oxo-1-propenyl] benzoic acid (No. 38) showed high activity comparable to that of retinoic acid. The pharmacological action of No. 38 is under investigation.

Topics: Benzoates; Cell Transformation, Neoplastic; Crystallography, X-Ray; HL-60 Cells; Humans; Molecular Conformation; Molecular Structure; Structure-Activity Relationship; Tretinoin

Both retinoic acid (RA) treatment and dominant-negative c-Jun mutant expression effectively inhibit phorbol ester-induced AP-1 activity and induced neoplastic transformation in mouse epidermal JB6 cells. However, both reagents also target non-AP-1 molecules in addition. Because liganded retinoic acid receptors interact with and transactivate RA response elements (RAREs) on DNA, as well as interact with Jun protein to block AP-1 activity, the question arises as to which of these two activities of retinoids is responsible for antitumor-promoting activity. To address this question we generated JB6 promotion-sensitive (P+) cell lines that are stably transfected with a construct containing the collagenase promoter bearing one AP-1-binding site that drives a luciferase reporter gene. The stable collagenase-luciferase-transfected cell lines showed 1.5-3.5-fold enhanced AP-1 activity when treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Up to 90% of TPA-induced AP-1 activity was blocked by retinoids SR11238, SR11302, or trans-RA, but not by retinoid SR11235. Of these retinoids, only RA and SR11235 were able to transactivate RARE-dependent gene expression. Transrepression of TPA-induced AP-1 and transactivation of RARE by RA, SR11238, and SR11302 were concentration dependent at 10(-10) to 10(-6) M retinoid. When tested for activity in inhibiting tumor promoter-induced transformation in JB6 P+ cells, the retinoids specific for AP-1 transrepression were inhibitory, whereas SR11235, which only activated RARE, showed little effect. We thus conclude that the AP-1-blocking activity of retinoids is likely to be responsible for the antitumor-promoting activity. This result, together with the observation that dominant-negative Jun blocks transformation, argues for a requirement of induced AP-1 in the tumor promoter-induced transformation process.

Topics: Animals; Anticarcinogenic Agents; Base Sequence; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Gene Expression; Genes, Reporter; Luciferases; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Retinoids; Sensitivity and Specificity; Skin; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcriptional Activation; Transfection; Tretinoin

Human papillomaviruses (HPVs) and cigarette smoking are epidemiologically associated with cervical cancer. We recently found that HEN-16 and HEN-16-2 HPV type 16-immortalized endocervical cells form tumors after treatment with cigarette smoke condensate and derived 2 tumor cell line cultures, HEN-16T and HEN-16-2T, respectively. Here, we examine the molecular pathologic effect of tumorigenesis. HEN-16T and HEN-16-2T exhibit unchanged status and expression of integrated HPV 16 DNA. However, the expression of the cytokeratin CK7 and CK13 endocervical cell markers is more homogeneous in monolayer and organotypic raft cultures after tumorigenesis. For the effect of retinoic acid on monolayers for growth inhibition, HEN-16T were significantly less sensitive than the normal and immortalized non-tumorigenic cells. HEN-16-2T were completely resistant. Moreover, the rafts from both tumorigenic cell line cultures were resistant to retinoic acid and continued to display thick rafts and homogeneous severe dysplasia/carcinoma in situ. In contrast, the non-malignant HEN-16 and HEN-16-2 rafts were thinner, and treatment with retinoic acid blocked the formation of severe dysplasia, reconstructing an epithelium resembling that of the normal endocervix. Our results support the significance of non-viral factors in the mechanism by which cigarette smoking induces tumorigenesis in the late stages of HPV-initiated progression to cervical cancer. Importantly, our data indicate that the sensitivity to retinoic acid of the HPV-containing endocervical cells is lost following tumorigenesis in vitro and possibly in women.

Topics: Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; Drug Resistance; Female; Humans; Keratins; Keratolytic Agents; Papillomaviridae; Tretinoin; Uterine Cervical Neoplasms

All-trans-retinoic acid (RA) markedly reduced the level of intracellular fibronectin (FN) in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Pulse/chase experiments indicated that RA affects FN biosynthesis rather than its turnover rate. Steady state levels of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, suggesting that RA acts at the translational level. Similar effects were observed in other fibroblasts. In NIH-3T3 cells, RA had distinct effects on different receptors; it down-modulated retinoic acid receptor (RAR) a protein and transcript levels, it up-regulated RAR beta transcripts, and it had no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene down-modulated RAR expression and abolished responsiveness to RA. We identified the retinoid signal transduction pathways responsible for the effects of RA on FN and RAR alpha proteins by the use of the retinoid X receptor-selective compound, SR11237, by stable over-expression of a truncated form of the RAR alpha gene, RAR alpha 403 with strong RAR dominant negative activity, and by overexpression of RAR alpha. We conclude that: 1) RA-dependent FN down-modulation is mediated by RARs, 2) retinoid X receptors mediate the observed reduction of RAR alpha by RA, and 3) the block of RA responsiveness in Ha-ras cells cannot be overcome by overexpression of RAR alpha. These studies have defined fibronectin and RAR alpha as targets of RA in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Down-Regulation; Fibronectins; Gene Expression; Mice; Protein Biosynthesis; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Transformation, Genetic; Tretinoin

Topics: Carcinoma, Hepatocellular; Carrier State; Cell Transformation, Neoplastic; DNA, Viral; Hepatitis B virus; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Retinoids; Tretinoin

To localize the differentiation-relevant cDNA RA538 (3.8kb) onto the human chromosome 8q2.. A modified non-isotopic labeling technique was used.. The cDNA RA538 was obtained by subtraction hybridization from human esophageal cancer cell line EC8712 induced by retinoic acid. Fluorescence in situ hybridization (FISH) using double-labeled probes showed clear and paired hybridization signals at the corresponding regions of both two chromatids in 18 out of 60 metaphases examined. While by single-labeling, only 7 were positive in 60 metaphases observed and the fluorescent spots were seen only at one chromatid.. The modified FISH protocol is useful for mapping cDNA sequences of a few kilobases.

Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 8; DNA, Complementary; Esophageal Neoplasms; Genes, Tumor Suppressor; Humans; In Situ Hybridization, Fluorescence; Tretinoin; Tumor Cells, Cultured

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

The retinoids are reported to reduce second primary aerodigestive tract tumors in patients with prior lung or head and neck carcinomas. Yet, the optimal retinoid useful for chemoprevention and those mechanisms linked to this chemoprevention are not identified. This study reports an in vitro model for carcinogen-induced transformation of immortalized human bronchial epithelial (BEAS-2B) cells that was adapted to study the anti-carcinogenic effects of all-trans-retinoic acid (RA). Following exposure to carcinogens: cigarette smoke condensate (CSC) or N-nitrosamine-4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK), BEAS-2B cells exhibited evidence of transformation. This included an increased anchorage independent growth or acquired ability to form tumors in athymic mice. This transformation was inhibited by RA as demonstrated by a lack of augmented anchorage independent growth or tumor formation in athymic mice for the cells treated with RA. The BEAS-2B cells transformed by NNK exhibited an increase in cyclin E expression which was associated with an increase in the cyclin E-Cdk2 kinase activity. Over-expression of human cyclin E by transfection shows cyclin E enhances the basal clonal growth of BEAS-2B cells. In both the parental and transformed BEAS-2B cells, RA down-regulated cyclin E protein levels which was associated with an inhibition of growth and an accumulation of cells in G1. The data reported here suggest the decline of cyclin E expression represents a potential mechanism for the RA-induced growth suppression which is linked to the anti-carcinogenic effects of RA. Thus, this study reports the adaption of an in vitro model of lung carcinogenesis suitable to test the activity of chemoprevention agents.

Topics: Adenoviridae; Animals; Bronchi; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Drug Evaluation; Enzyme Activation; Epithelial Cells; Humans; Mice; Models, Biological; Nitrosamines; Protein Serine-Threonine Kinases; Rabbits; Simian virus 40; Smoke; Transfection; Tretinoin

The adenocarcinoma cell line derived from an intercalated ductal epithelium of a human salivary gland (HSG) proliferates autonomously mediated by an epidermal growth factor-(EGF)-like molecule with a molecular weight of 46 kDa and an EGF receptor (EGFR). The c-erbB2 protein, a member of EGFR family was also expressed in HSG cells and was involved in the growth signal pathway of HSG cells as well as EGFR. The autocrine growth is regulated by glucocorticoid and retinoic acid (RA) via their receptors. Retinoic acid receptor (RAR) of HSG cells revealed a transcriptional activity in vivo, and the heterodimerization between RAR and 9-cis retinoic acid receptor (RXR) is requisite for the binding with a specific DNA element termed RA response element in vitro. RXR alpha and RXR beta were cloned from HSG cells, and these RXRs, together with RAR, seemed to play a physiological role in RA signaling in vivo.

Topics: Adenocarcinoma; Bucladesine; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Receptors, Retinoic Acid; Salivary Gland Neoplasms; Signal Transduction; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The biological features of HL-60 and all-trans-retinoic acid resistant HL-60 cells (HL-60/ RA) were studied in terms of cell proliferation, cytokinetics, chromosome distribution and G-band karyotypes. Results indicated that the two cell lines exhibited similar morphology and doubling time and similar cytokinetic features, but the cell population in S phase of HL-60/RA is smaller than that of HL-60 cells. Their chromosome number is around 45 +/- 3 for HL-60 and 45 +/- 1 for HL-60/RA cells. G-band karyotype analysis showed that they have a similar diploid karyotype and some abnormal chromosomes. These results demonstrated that HL-60 and HL-60/RA cell lines were originated from the same source.

Topics: Cell Division; Cell Transformation, Neoplastic; Chromosome Banding; Diploidy; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Karyotyping; S Phase; Tretinoin

Vitamin A and its analogus, the retinoids, are agents that are known to induce differentiation and inhibit growth on cell in vitro and in vivo. These agents show promise as prophylactic and therapeutic ones in human cancer and were noted extensively by scholars abroad, but the research has just been developed in recent years in our country. Retinamide (named briefly R II), a kind of new retinoids made in China, can induce differentiation and inhibit growth of cell, but its toxic effect is smaller than its maternal chemical compound (retinoic acid). Using automatic image analysis technology, polyacrylamide gel LDH isoenzyme electrophoresis, radioimmunoassay and condensing reaction of Con A, we have observed induction of differentiation of the R II on SGC-7901 cells. Our experiments indicate that R II can inhibit cell proliferation, decrease obviously average DNA content and nuclear area, reduce CEA secretion and condensing of the cells, and change LDH isozymoraphy of SGC-7901 cell with promoting H-type LDH and reducing M-type LDH. The change of morphology under light microscope by R II showed that the cells spread, flattened and partially lined up, and the alkalophilic quality of cytoplasm became weak. These results suggest that SGC-7901 cell under the action of R II should undergo the changes of reverse differentiation in morphological, physiological and bio-chemical aspects.

Topics: Antineoplastic Agents; Carcinoembryonic Antigen; Cell Transformation, Neoplastic; DNA, Neoplasm; Humans; L-Lactate Dehydrogenase; Stomach Neoplasms; Tretinoin; Tumor Cells, Cultured

To determine the effect of retinoic acid on the development of severe dysplasia or carcinoma in situ from endocervical cells containing human papillomavirus (HPV) type 16.. Two independent lines of HPV 16-immortalized endocervical cells were reconstructed into two squamous epithelial tissues using the organotypic raft culture system to examine the differentiated phenotype. The effect of retinoic acid on dysplastic morphology of differentiation of the epithelia was examined by light microscopy of stained sections and electron microscopy. The endocervical cell type cytokeratin expression pattern was determined by indirect immunofluorescence using specific monoclonal antibodies. Ribonucleic acid expression of the HPV 16 E7 oncogene was examined by in situ hybridization.. Untreated HPV 16-immortalized endocervical cells were reconstructed into squamous dysplastic lesions resembling carcinoma in situ observed in women. Retinoic acid-treated rafts formed epithelia composed of two to three cell layers of columnar-like cells resembling simple epithelium of the endocervix. Electron microscopy and cytokeratin expression patterns confirmed the histology of a differentiated endocervical phenotype after treatment with retinoic acid. Expression of HPV 16 E7 was modestly lower in treated epithelia, preferentially in basal cells.. Retinoic acid prevents the histology and cytokeratin differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells. Because the epithelia closely mimic HPV 16-containing severe dysplasias and native endocervical epithelium in women, this immortalized endocervical cell-raft system may be useful as a model to assess the efficacy of agents such as retinoic acid for preventing progression of these lesions to malignant cervical carcinoma.

Topics: Carcinoma in Situ; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Cervix Uteri; Female; Humans; Keratins; Microscopy, Electron; Papillomaviridae; Tretinoin; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Liarozole has been reported to inhibit P450 enzymes responsible for the catabolism of retinoic acid. This suggests that it may increase the effectiveness of cancer chemopreventive agents, such as retinoic acid, and pro-vitamin A carotenoids, such as beta-carotene, which may yield retinoids. To test this we have utilized the 10T1/2 cell assay system of neoplastic transformation. Simultaneous treatment with Liarozole (10(-5) M) potentiated by a factor of 1000 the ability of low concentrations of retinoic acid (10(-10) M) to inhibit carcinogen-induced neoplastic transformation, to up-regulate gap junctional communication and to increase connexin43 expression. When tested under conventional culture conditions, Liarozole itself partially suppressed neoplastic transformation and up-regulated gap junctional communication; this ability appears due to the presence of retinol in the serum component of cell culture medium. When assays for junctional communication and of connexin43 expression were performed under defined conditions, in the absence of serum, Liarozole was ineffective alone, yet still augmented the effects of retinoic acid. HPLC analysis of cell culture medium demonstrated that Liarozole (10(-5) M) completely protected retinoic acid (10(-6) M) from catabolism over a 48 h period. Potential effects of Liarozole on the activities of carotenoids were also examined. Inhibition of neoplastic transformation by the pro-vitamin A carotenoid beta-carotene, but not by the non-pro-vitamin A carotenoid canthaxanthin, was moderately potentiated by Liarozole. The augmentation of response to beta-carotene was more apparent when tested under defined conditions; here Liarozole strongly increased junctional communication and Cx43 expression. In contrast, Liarozole did not potentiate the activity of canthaxanthin under defined conditions, while increasing the activity of 4-keto retinoic acid, a likely metabolite. Liarozole also elevated connexin43 expression in early passage human fibroblasts. These data indicate that rapid catabolism of retinoic acid limits its in vitro activities, that a portion of the action of beta-carotene as a cancer preventive agent is due to its conversion to retinoic acid and that canthaxathin exerts its chemopreventive action largely independent of conversion to 4-keto retinoic acid.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; beta Carotene; Carotenoids; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Connexin 43; Drug Synergism; Fibroblasts; Gap Junctions; Humans; Imidazoles; Mice; Mice, Inbred C3H; Skin; Tretinoin; Up-Regulation

Retinoids, including all-trans-retinoic acid, its isomers, and fifty synthetic retinoids (retinobenzoic acids), were tested for differentiation-inducing activity on human leukemia cell lines HL-60 and NB4. A good linear correlation, with an r value of 0.91, between the ED50 values for the differentiation-inducing activity towards HL-60 cells and that towards NB4 cells was found.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Drug Screening Assays, Antitumor; Gene Expression Regulation, Leukemic; Granulocytes; Humans; Least-Squares Analysis; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoids; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensi

Topics: Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Female; Humans; Keratinocytes; Male; Models, Biological; Papillomaviridae; RNA, Messenger; Transforming Growth Factor beta; Tretinoin; Uterine Cervical Neoplasms

The present study was directed to characterizing the reversion of neoplastic epidermal JB6 RT101 cells by AP-1 inhibiting drugs. Treatment of tumorigenic JB6 RT101 cells with retinoic acid (RA), fluocinolone acetonide (FA) or forskolin (FN) induced drug dependent (reversible) reversion of transformation. A synergistic effect on reversion was found with the three drugs in combination. Cells reverted by these three drugs also showed reduced levels of AP-1 transcription factor activity. After long term exposure of RT101 cells to FA, enrichment of flat revertants occurred in the population while a few unreverted cells formed foci. These unreverted cells appeared to be FA-resistant. Cloning of cells following RA treatment revealed stable reversion at least 2 months after drug withdrawal. Stable revertants showed lower basal AP-1 activity than RT101 cells (P < 0.01) and unstable revertants returned to transformed phenotype and elevated AP-1 activity within days following drug withdrawal. To our knowledge this is the first demonstration that drug induced reversion co-selects for reduced AP-1 activity. These data suggest that the JB6 RT101 cell line is a useful cell model for studying reversion of transformation and that inhibition of AP-1 activity may be one molecular mechanism of reversion. Considering the development of resistance with FA alone and the relative inefficiency of RA or FN alone, combinations of the three AP-1 activity inhibitors RA, FA and FN may be useful for further animal and clinical studies.

Topics: Animals; Anticarcinogenic Agents; Cell Transformation, Neoplastic; Cells, Cultured; Colforsin; Cyclic AMP; Drug Resistance; Fluocinolone Acetonide; Glucocorticoids; Mice; Phenotype; Sensitivity and Specificity; Skin; Skin Neoplasms; Time Factors; Transcription Factor AP-1; Transcriptional Activation; Tretinoin

Cellular senescence is characterized by a finite proliferative capacity in vitro. Moreover, the proliferative capacity of dermal fibroblasts harvested from humans is inversely proportional to the age of the donor, suggesting that senescence in culture is a manifestation, at the cellular level, of processes that occur during in vivo human aging. As cellular senescence is a program that ultimately decreases cell proliferation, it has been hypothesized that the genetic mechanisms responsible for the negative growth regulation of senescence may also be involved in the suppression of neoplastic transformation. Retinoic acid (RA) and its derivatives are effective negative growth regulators and are known to inhibit tumor growth, in vitro and in vivo. As a first step in examining a role for retinoic acid in the regulation of cellular aging in human fibroblasts, we examined the expression of the nuclear receptors for RA (RAR alpha, RAR beta, and RAR gamma) in human donors of different ages. These studies demonstrate a selective up-regulation of RAR beta, in response to RA, in fibroblasts that manifest a decreased proliferative capacity. We extend these observations to show that this finding is independent of the age of the donor and correlates with the proliferative capacity of the culture as a whole. Nuclear run-on studies show that the increase in RAR beta mRNA accumulation is mediated by a striking increase in the transcription of the RAR beta 2 isoform. Senescent fibroblasts manifesting the transcriptional increase of the RAR beta 2 isoform also demonstrate transcriptional repression of the protooncogene, c-fos. Functional studies demonstrate that RAR beta 2, like the tumor suppressor gene p53, can inhibit oncogene-induced focus formation. These data provide further support for the contention that genetic events important in cellular senescence may also play a significant role in tumor suppression in humans. Moreover, these observations suggest that RA, through transcriptional regulation of RAR beta 2, may mediate aspects of the negative growth control that characterizes both states.

Topics: Actins; Cell Division; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Cellular Senescence; Fibroblasts; Humans; Kinetics; Proto-Oncogene Proteins c-fos; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Skin; Time Factors; Transcription, Genetic; Tretinoin; Up-Regulation

We examined the effect of three RA isomers, including ATRA, 13C-RA and 9C-RA, on the proliferation and differentiation properties of NB4 cells, a cell line established from APL patient and carrying the typical translocation t (15;17). Standard parameters such as cell morphology, cell growth curve, dynamics of cell cycle, expression of clusters of differentiation and reduction of nitro blue tetrazolium (NBT) were used to evaluate the effects of the three isomers. During the first 48 hours of RA treatment, the APL cell maturation was coupled with the cell proliferation. Moreover, significant differences of differentiation-induced effect among RA isomers were observed. ATRA showed better results than 13C-RA while 9C-RA functioned even better than ATRA. These data are helpful to understand the mechanisms, by which RAs induce promyelocytic leukemia cell differentiation and open a new prospect for the clinical use of novel retinoic acid isomers.

Topics: Cell Division; Cell Transformation, Neoplastic; Humans; Isomerism; Isotretinoin; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured

Human neuroblastoma I-type cells isolated from cell lines in vitro are morphologically intermediate between neuroblastic (N) cells, with properties of embryonic sympathoblasts, and substrate-adherent (S) cells having properties of embryonic Schwann/glial/melanocytic cells of the neural crest. I cells have biochemical features of both N and S cells. We propose that the I-type cell represents a malignant neural crest stem cell. The strongest evidence in support of this hypothesis is that: (a) I cells can generate progeny that have neuronal properties, i.e., are committed neuroblasts, or properties of nonneuronal, embryonic neural crest-derived cells; and (b) I-type cells can generate multipotent I-type progeny, indicating their capacity for self-renewal, a feature of stem cells. We report here that I-type cells, derived from four different human neuroblastoma cell lines and experimentally induced to differentiate, give rise to cells with distinct N or S cell phenotypes, indicative of I cell multipotentiality. Experiments with a large panel of I-type subclones, isolated from clonal I-type BE(2)-C cells and exposed to retinoic acid to induce neuronal differentiation or 5-bromo-2'-deoxyuridine to obtain S-type cells, demonstrated that differentiation occurs via induction and selection and not by selection of spontaneously arising variants. The differentiation phenotype was stable. We conclude that human neuroblastoma I-type cells are multipotent embryonic precursor cells of the peripheral nervous system, capable of either neuronal or nonneuronal neural crest cell differentiation.

Topics: Bromodeoxyuridine; Cell Differentiation; Cell Transformation, Neoplastic; Clone Cells; Humans; Neural Crest; Neuroblastoma; Stem Cells; Tretinoin

The differentiation-inducing activity of tanshinone (TAN) and all-trans-retinoic acid (RA) was studied in vitro on a human cervical carcinoma cell line, ME180. The tumor cells were treated with TAN or RA in DMSO (final concentration 0.02%, V/V) on 4 successive days. Cells treated with the same concentration of DMSO alone served as control. Morphologic studies with light and transmission electron microscopy showed that the cells treated with both TAN and RA became well-differentiated. The cell growth, (as revealed by cell counting and 3H-TdR incorporation) was inhibited and the tumorigenicity in nude mice was reduced. No significant difference was observed between the cells treated with TAN and RA.

Topics: Abietanes; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Cell Transformation, Neoplastic; Female; Humans; Mice; Mice, Nude; Phenanthrenes; Tretinoin; Tumor Cells, Cultured; Uterine Cervical Neoplasms

PA-1 cell line is derived from human ovary teratocarcinoma. When it grows in 10% fetal calf serum, it can be induced to differentiate by 10(-5) mol/L retinoic acid (RA). Some morphological changes can be observed after RA induction. By immunostaining of cultured cells, we found that the expression and distribution pattern of desmin and some extracellular matrix molecules, such as fibronectin, laminin and tenascin, had been changed after induction, and these changes were associated with the morphological changes. Cell growth study showed that RA treatment had no effects on growth and autocrine activities of PA-1 cells. These results suggest that some PA-1 cells were induced to differentiate along muscle cells pathway by RA.

Topics: Cell Transformation, Neoplastic; Child; Desmin; Extracellular Matrix; Female; Fibronectins; Humans; Ovarian Neoplasms; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

AP1 is a heterodimeric complex containing products of the Jun and Fos oncogene families. The c-fos and c-jun protooncogenes act as transcriptional activator for numerous cellular genes, and the overexpression of these genes may cause malignant transformation. In this study, to show evidence of a possible inhibition of AP1 transcriptional activity in molecular mechanisms of foodborne molecules, known to be negative modulators of carcinogenesis, we established two rat liver epithelial (REL) cell lines overexpressing either c-fos (43C line) or c-jun (RELcJ1 line) oncoproteins. Contrary to the 43C line, which was spontaneously transformed, the c-jun-transfected REL cells were only transformed in vitro after 12-O-tetra-decanoylphorbol 13-acetate (TPA) exposure. All trans-retinoic acid (RA) abolished the transformation of the 43C line and TPA-treated RELcJ1 cells, suggesting that RA could decrease AP1 activity in these cells despite c-fos or c-jun overexpression. Furthermore, we show for the first time that a flavonoid, quercetin, which is a natural component of vegetables, inhibited only the transformation of the 43C line. The spontaneous transformation of the c-fos-transfected REL cells was associated with the appearance of c-fos/AP1 complexes binding TRE, suggesting that c-fos/AP1 complexes are involved in the antitransforming mechanism of quercetin.

Topics: Animals; Anticarcinogenic Agents; Base Sequence; Blotting, Western; Cell Line; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; Flavonoids; Food; Genes, fos; Genes, jun; Liver; Molecular Sequence Data; Oligodeoxyribonucleotides; Quercetin; Rats; Transcription Factor AP-1; Transfection; Tretinoin

Insulin-like growth factor-binding proteins (IGFBPs) have been shown to both potentiate and inhibit IGF bioactivity in vitro; thus, changes to the type or amount of IGFBPs present in the cellular environment will ultimately affect insulin-like growth factor action. In this study, we have investigated the production of immunoreactive IGFBP-6 by normal human fibroblasts (NHF) and an SV-40-transformed human fibroblast line (AG2804). When analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, IGFBP-6 appeared as a doublet of 32-34-kDa in conditioned medium of both cell lines, with the lower molecular mass band predominating in the NHF cell line. Measured by a specific radioimmunoassay, serum-free NHF, and AG2804 cultures secreted IGFBP-6 at 1.44 +/- 0.09 and 1.23 +/- 0.08 ng/10(4) cells (mean +/- S.E.), respectively. Despite a relatively weak IGFBP-6 signal by ligand blot compared with IGFBP-3, the two proteins were secreted in similar molar concentrations by NHF. Retinoic acid increased IGFBP-6 by 3-fold in NHF and AG2804-conditioned media, maximal at approximately 100 nM retinoic acid. In contrast, IGFBP-6 production was inhibited by transforming growth factor-beta 1 and agents that increase intracellular cAMP concentrations, including dibutyryl cAMP, forskolin, isobutylmethylxanthine, and cholera toxin. This study indicates that IGFBP-6 has a pattern of regulation unique among the IGFBPs, supporting the concept of specific roles for each binding protein in regulating cell growth and metabolism.

Topics: 1-Methyl-3-isobutylxanthine; Bucladesine; Carrier Proteins; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Cholera Toxin; Colforsin; Culture Media, Conditioned; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Humans; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor II; Kinetics; Male; Molecular Weight; Recombinant Proteins; Simian virus 40; Skin; Transforming Growth Factor beta; Tretinoin; Virulence Factors, Bordetella

4-hydroxycarbophenyl retiamide (R II) is a new synthetic analogue of retinol, but with lower toxicity than retinoic acid. We studied its induction effects and its effects on some malignant phenotypes of the MFC cell line. The mechanism of these effects was also explored. MFC cells were grown in complete RPMI 1640 medium supplemented with 10(-5) mol/L R II for five passages. By then the cell growth rate slowed down; the rate of 3H-TdR incorporation and the colony-forming capacity of MFC cells decreased; morphologically, the cells became epithelial rather than fibroblastic with various degrees of polarization. Further investigation about the mechanism of these changes was also undergone. First by flow cytometry, it was shown that the R II-treated cells were retained in G1 phase. Second, dot blot hybridization showed a decrease of more than 61% of c-myc mRNA and an increase of more than 52% of v-fos mRNA. The major chromosome distribution changed from 54-56 to 46-54 with an increase in diploid. Scanning microscopic examination showed that the R II-treated cells were covered by numerous microvilli and pseudopodia with round terminal expansion in contrast to the ruffle protrusions and leaf-like pseudopodia of control cells. All the results suggested that R II could reverse some malignant phenotypes of MFC cells.

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, myc; Mice; Mice, Inbred Strains; Phenotype; RNA, Messenger; Stomach Neoplasms; Tretinoin; Tumor Cells, Cultured

By using MTT and trypan blue exclusion assay, the sensitivity of retinoic acid sensitive HL-60 and resistant HL-60/RA cell to six anti-leukemia drugs such as RA, Ara-c, harringtonine was compared. It was found that all five drugs except RA exhibited an approximately equivalent IC50 to HL-60 and HL-60/RA cells. The results suggest that tumor cells may not develop resistance to differentiation inducer and cytotoxic anti-tumor agent parallel. It also suggests that it is reasonable to use a combination of differentiation and cytotoxic anti-leukemia agents concomitantly or sequentially.

Topics: Antineoplastic Agents; Cell Transformation, Neoplastic; Cytarabine; Drug Resistance; Drug Synergism; Harringtonines; Humans; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured

A full-length cDNA (RA538) was isolated from human esophageal cancer cell line EC8712 after retinoic acid treatment. An expression vector of this cDNA (pRA538) was cotransfected with the neo gene (pDORneo) into parental cancer cell line EC8712. The cell colonies obtained after selection in G418-containing culture medium showed very poor growth, reduced (by 68%-76%) 3H-TdR uptake and morphological changes characteristic of terminal differentiation and programmed cell death (apoptosis). In situ hybridization with RA538 probes revealed expression of mRNA of RA538 in the cytoplasm of the transfected cells. The cells transfected solely with pDORneo after G418 selection showed normal growth pattern and no RA538 expression. However, none of the control cells EC8712 survived the G418 selection. Thus the expression of cDNA RA538 has a similar effect on the esophageal cancer cell EC8712 as the retinoic acid does.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; DNA, Complementary; Esophageal Neoplasms; Gene Expression; Humans; Mice; Mice, Nude; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

We report here the spontaneous in vitro transformation of blood monocytes into fibroblasts in a patient who developed pulmonary fibrosis following ciclosporin-mediated immunosuppression, necessitated by heart transplantation. The blood monocytes with this capacity expressed HLA-DR specificity. Monocytes/macrophages were identified by immunofluorescence using monoclonal antibodies against a specific monocyte/macrophage antigen, while the neo-fibroblasts were identified by electron microscopy and immunofluorescence using monoclonal antibodies against a cytoplasmic enzyme specifically involved in the synthesis of collagen. The secretion of collagen was demonstrated using antibodies against collagen. Both the monocytes/macrophages and the neo-fibroblasts express macrophage and fibroblast markers and are able to synthesize collagen. The all-trans retinoic acid derivative (all-trans RA) inhibits this in vitro transformation of HLA-DR monocytes/macrophages into neo-fibroblasts. Therefore, the possible therapeutic role of all-trans RA in controlling the development of fibrosis remains open to investigation. Until now, no efficient therapy is known for fibrotic diseases which are often lethal when affecting the lungs.

Topics: Aged; Cell Transformation, Neoplastic; Cells, Cultured; Cyclosporine; Fibroblasts; HLA-DR Antigens; Humans; Immunocompromised Host; In Vitro Techniques; Male; Monocytes; Pulmonary Fibrosis; Tretinoin

Genetic alterations in elements of normal signal transduction mechanisms are known to be oncogenic events often resulting in aberrant activation of programs of gene transcription. We have investigated the effect of N-ras oncogene-induced tumorigenic transformation on the transcription factor AP-2. N-ras oncogene-induced transformation of human teratocarcinoma cells PA-1 results in sixfold elevated AP-2 mRNA levels. However, the level of AP-2-mediated trans-activation is dramatically inhibited in these cells. We show here that the high-level expression of AP-2 ultimately results in transcriptional "self-interference". The activation domain of AP-2, when fused to the DNA-binding domain of GAL4, is sufficient for self-interference. Non-N-ras PA-1 cells constitutively expressing AP-2 or GAL4-AP-2 fusion protein from an SV40 promoter exhibit reduced AP-2-mediated transcriptional activation, inhibition of differentiation, and promotion of anchorage-independent growth, properties that are similar to N-ras-transformed PA-1 cells. Thus, AP-2 is placed in the N-ras signal transduction pathway, and many of the biological effects of N-ras can be accomplished by overexpression of AP-2. This is the first evidence that inhibition of the activity of a transcription factor by self-interference contributes to a physiological process.

Topics: Base Sequence; Binding Sites; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Molecular Sequence Data; Peptide Fragments; Protein Binding; Recombinant Fusion Proteins; Teratocarcinoma; Transcription Factor AP-2; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transformation, Genetic; Tretinoin; Tumor Cells, Cultured

In an attempt to define the role of plasminogen activator in invasiveness and differentiation of human melanoma cells, the modulation of these parameters was studied in two melanoma clones characterized by marked differences in their basal features, using 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid, two differentiation inducers, and doxorubicin, a cytotoxic agent. TPA induced only slight reductions, whereas retinoic acid and doxorubicin caused an increase in invasiveness, enzymatic activity and differentiation in the clone showing low invasivity, low urokinase-type plasminogen activator levels and high differentiation. In contrast, in the clone showing high invasivity, high urokinase-type plasminogen activator levels and low differentiation it was found that: TPA was ineffective; retinoic acid induced a reduction of plasminogen activator but no modifications of invasiveness and differentiation; doxorubicin caused a decrease in invasiveness and plasminogen activator activity but no modification of morphological features. The different behaviour of the two clones thus could be related to the basal features of the clones. The results reported here indicate that in the presence of these drugs the associations between invasiveness and urokinase-type plasminogen activator activity and between invasiveness and differentiation are lost. Drug treatment therefore significantly affected the features of the clone characterized by low biological aggressiveness (high differentiation, low invasiveness), whereas the highly aggressive clone did not show a consistent response to drug treatment.

Topics: Cell Survival; Cell Transformation, Neoplastic; Chemotaxis; Doxorubicin; Humans; Melanoma; Neoplasm Invasiveness; Plasminogen Activators; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The inhibitory effect of a third generation retinoid R8923 and all-trans retinoic acid (RA) on malignant transformation of Balb/3T3-A31 cells induced by 3-methylcholanthrene (3-MCA) and 12-0-tetradecanoyl phorbol-13-acetate(TPA) was studied in paper. Malignant transformation of Balb/3T3-A31 cells was evaluated by scoring transformation foci and soft agar assay. Actively growing Balb/3T3-A31 cells (1.5 x 10(4) cells per 60-mm-diameter glass dish) were cultured in 5ml of Eagle's Minimum Essential Medium supplemented with 10 percent fetal bovine serum. Twenty-four hours after plating, the cells were treated with 3-MCA (2 micrograms/ml) for 72 hours. TPA was added into the medium at a concentration of 100 ng/ml for 2 weeks. Thirty-six days after cell plating, the transformation foci were counted and the soft agar assay of the cells isolated from each glass dish was performed. Results showed that there were 16.0 +/- 1.58 transformation foci/dish and the colony forming efficiency in soft agar assay was 138.6 +/- 14.47/10(3) cells in 3-MCA and TPA treated dishes (control group). When the cells were exposed to R8923 or RA (at a concentration of 10(-6) M), the transformation foci were 11.2 +/- 0.84 /dish and 9.2 +/- 1.10/dish respectively, and the corresponding colony forming efficiency values were 66.1 +/- 7.68/10(3) cells and 64.8 +/- 4.46/10(3) cells. They were significantly lower than that of the control group. These results demonstrated that R8923 and RA could effectively inhibit 3-MCA and TPA induced malignant transformation of Balb/3T3-A31 cells and suggested that R8923 is a potential drug for cancer chemoprevention.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Methylcholanthrene; Mice; Mice, Inbred BALB C; Retinoids; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The growth rate and weight of transplanted tumor of SGC7901 gastric cancer cell line, treated with 10 mumol/L retinoic acid (RA) in nude mice, were significantly reduced in comparison with those of control cells. The morphology of RA-treated cells was changed from ellipse shape with meandering margin of a large nucleus to shuttle shape with smooth margin of a small nucleus. With flow cytometry, it was found that G/O Phase markedly increased after the RA treatment, whereas S and G2 + M phase decreased. Especially the S phase fraction was lower than that of control cells. The above results indicated that RA could reverse biological and morphological phenotypes of the human gastric cancer cells in vitro and could induce differentiation of gastric cancer cells to a certain extent.

Topics: Animals; Cell Transformation, Neoplastic; Flow Cytometry; Humans; Interphase; Mice; Mice, Nude; Neoplasm Transplantation; Stomach Neoplasms; Tretinoin; Tumor Cells, Cultured

We have used a model system of normal HKc and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16) to investigate the effect of RA on the growth of HKc/HPV16 and the expression of the HPV16 oncogenes E6 and E7. These studies found that HKc/HPV16 are about 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Northern blot analysis of mRNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 oncogenes E6 and E7 as well as the early ORFs E2 and E5 is substantially reduced following RA treatment. In addition, protein levels of E6 and E7, as measured by immunofluorescence (E6 and E7) and Western blot (E7) are also decreased by RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. The RA treatment (1 nM) of normal HKc, during or immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of dietary retinoids in the chemoprevention of HPV-induced cancers.

Topics: Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Gene Expression; Humans; Keratinocytes; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Protein-Tyrosine Kinases; Repressor Proteins; Tretinoin; Vitamin A

Expression of the soluble lectin L-14 is low in normal and very high in transformed thyroid cells. We show that L-14 gene expression is transiently induced upon thyrotropin stimulation of normal quiescent FRTL-5 rat thyroid cells. Permanent activation of L-14 gene expression is obtained in the same cells infected with a wild-type and a temperature sensitive mutant of Kirsten murine sarcoma virus, both at the permissive and non permissive temperature for transformation. We also find that L-14 mRNA is undetectable in rat brain but is abundant in rat oligodendrocytes precursors transformed by polyoma middle T oncogene. Retinoic acid treatment of these transformed cells leads to acquisition of a differentiated phenotype accompanied by a 30-fold decrease of L-14 mRNA levels. Removal of retinoic acid restores both the transformed undifferentiated phenotype and high L-14 expression. Taken together these results indicate that growth stimulation and induction of cell differentiation are accompanied by strong modulation of L-14 gene expression.

Topics: Animals; Base Sequence; Carrier Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; Fibroblasts; Galectins; Gene Expression; Hemagglutinins; Kinetics; Kirsten murine sarcoma virus; Molecular Sequence Data; Oligodendroglia; Oligonucleotide Probes; Rats; RNA, Messenger; Thymidine; Thyroid Gland; Thyrotropin; Tretinoin

AP-1 transcriptional activity is stimulated by the transformation promoters phorbol 12-myristate 13-acetate ("12-O-tetradecanoylphorbol 13-acetate," TPA) and epidermal growth factor (EGF) in promotion-sensitive (P+) but not in promotion-resistant (P-) JB6 mouse epidermal cell lines. Although TPA stimulates expression of the jun and fos family genes, only c-jun expression shows higher elevation in P+ cells than in P- cells. The present study tests the hypothesis that induced AP-1 activity is required for tumor promoter-induced transformation in JB6 P+ cells. Both retinoic acid and the glucocorticoid fluocinolone acetonide inhibited basal and TPA-induced AP-1 activities that were tested with a stromelysin promoter-chloramphenicol acetyltransferase reporter gene in P+ cells. Since both retinoic acid and fluocinolone acetonide are active in inhibiting TPA-induced anchorage-independent transformation of P+ cells in the dose range that blocks TPA-induced AP-1 activity, their antipromoting effects may occur through inhibition of AP-1 activity. To test the hypothesis with a more specific inhibitor, stable clonal transfectants of P+ cells expressing dominant negative c-jun mutant encoding a transcriptionally inactive product were analyzed. All transfectants showed a block in TPA and EGF induction of AP-1 activity. All transfectants also showed inhibition of TPA-induced transformation, and most transfectants showed a block in EGF-induced transformation. These results indicate that AP-1 activity is required for TPA- or EGF-induced transformation. This work demonstrates that a specific block in induced AP-1 activity inhibits tumor promoter-induced transformation.

Topics: Animals; Base Sequence; Binding Sites; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Fluocinolone Acetonide; Gene Expression; Genes, fos; Genes, jun; Genes, Reporter; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Proto-Oncogene Proteins c-jun; Skin; Tetradecanoylphorbol Acetate; Transfection; Tretinoin

Retinoic acid greatly increases enzyme activity and mRNA expression of the tissue-type transglutaminase enzyme in NIH3T3 cells. This response is blocked in cells transformed with activated H-ras, K-ras or N-ras oncogenes, but not in pSVneo vector transfected cells. Lack of induction by RA of the tissue-type TGase in these ras-transformed fibroblasts suggests intersecting pathways between retinoid action and the ras oncogene.

Topics: 3T3 Cells; Animals; Blotting, Northern; Cell Transformation, Neoplastic; Cloning, Molecular; Enzyme Induction; Gene Expression; Genes, ras; Genetic Vectors; Glyceraldehyde-3-Phosphate Dehydrogenases; Kinetics; Kirsten murine sarcoma virus; Mice; Poly A; RNA; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection; Transglutaminases; Tretinoin

Retinoic acid (RA) has profound effects on cell proliferation and differentiation both in vitro and in vivo. Many human cell lines are known to be sensitive to the growth-inhibitory action of RA. We analyzed established human solid tumor-derived cell lines for their RA sensitivity. Growth inhibition by RA in monolayer was examined by [3H]thymidine incorporation and cell proliferation. Here we report that 11 widely used human cell lines were RA resistant. The majority are carcinoma derived (A-431, BT-20, C-41, ACHN, HCT116, 293, A549, and PA-1); two are sarcoma derived (Saos-2 and A673); and one is a melanoma cell line (A-375). Since nuclear retinoid receptors are implicated in the biological effects of RA, we examined the expression of retinoic acid receptors (RARs) RAR alpha, RAR beta, RAR gamma, and the retinoid X receptors (RXRs) RXR alpha, RXR beta, and RXR gamma in the RA-resistant cell lines by northern blotting and by RNase protection analysis for RAR beta. RAR alpha transcripts were constitutively expressed in all cell lines. By contrast, RAR beta was expressed in only seven RA-resistant cell lines (Saos-2, ACHN, 293, A549, A-375, A673, and PA-1), and its level was enhanced by RA in some cases. In most cell lines, RAR gamma expression was low and was not affected by RA. The RXR genes showed a very distinct expression pattern in the group of selected cell lines. In general, RXR alpha was the most abundantly expressed subtype, RXR beta was expressed at low levels, and RXR gamma could not be detected. In none of the RA-resistant cell lines was RXR expression modulated by RA. The results presented here indicate that the resistance of these human tumor cell lines to RA cannot be simply correlated with expression of RAR or RXR or both.

Topics: Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; Cloning, Molecular; Dose-Response Relationship, Drug; Drug Resistance; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Nuclear Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

F9 embryonal cells can be induced to differentiate and synthesize basement membrane proteins. Perlecan and laminin are two basement membrane constituents that have extensive regions of homology. Expression of perlecan and laminin B1 genes was followed during differentiation of F9 cells by measurements of transcription rate and mRNA abundance using nuclear run on assays and Northern hybridizations, respectively. The rate of precursor protein synthesis was determined by immunoprecipitation from lysates of pulse-labeled F9 cells. The results showed that perlecan gene expression responds more rapidly after induction than does laminin B1 gene expression but is ultimately expressed at a substantially lower level than laminin. Thus, the perlecan and laminin genes appear to be regulated by different mechanisms and their gene products are not made in stoichiometric amounts.

Topics: Animals; Blotting, Northern; Bucladesine; Carcinoma, Embryonal; Cell Transformation, Neoplastic; Gene Expression; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Mice; Proteoglycans; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of G0 arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driven by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions.

Topics: Antibodies, Monoclonal; Antibody Specificity; Blotting, Western; Calcitriol; Cell Transformation, Neoplastic; DNA, Neoplasm; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Humans; Leukemia, Promyelocytic, Acute; Macrophages; Monocytes; Receptor, Macrophage Colony-Stimulating Factor; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

This paper modifies and extends an earlier one on the same subject. It explains why external (but not internal) surface molecules of plasma membrane clusters may be rapidly scattered by any external challenging bioelectrical field. Temporary clusters from challenges may induce mitosis in cells near wounds and in epithelial stem cells. Weak challenges of much longer duration may initiate carcinogenesis by permanent clusters. Basic intracellular ligand/receptors or oncogene products in sufficient concentration at the membrane inner lipid layer may form permanent clusters rapidly. Additive increase of inner surface clusters by initiating agents is equated to promotion; accelerated cluster growth to progression. As a malignant cell grows, its cluster population increases until its membrane becomes permeable enough to stimulate mitosis. A progression mechanism is suggested that is consistent with the known properties of ras p21 proteins. The effect of long term exposure to power transmission line fields on mitosis and carcinogenesis is discussed. An approach to anticancer therapy is suggested, using a hypothesis-based mechanism for the anti-cancer activity of retinoic acid.

Topics: Animals; Anticarcinogenic Agents; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Humans; Lipid Bilayers; Membrane Lipids; Models, Biological; Neoplasms; Phospholipids; Tretinoin

The c-fgr proto-oncogene is expressed in Burkitt's lymphoma (BL) cell and cell lines derived from them. When Epstein-Barr virus (EBV)-negative BL cell lines that contain low levels of c-fgr mRNA are infected with EBV, transcription of the c-fgr gene is further induced. In this paper we show that treatment of EBV-negative and EBV-positive BL cell lines with all-trans retinoic acid also stimulates an increase in c-fgr mRNA levels, varying between 2- and 13-fold depending on the cell line. An increase is detectable 12 to 48 h after treatment, depending on the cell line, suggesting that the c-fgr gene is not regulated directly by retinoic acid but responds to other retinoic acid-induced changes in the cell. We also show that treatment of BL cell lines with all-trans retinoic acid either results in a dose-dependent decrease in growth rate, or has no effect on growth, depending on the cell line. It has previously been suggested that the c-fgr gene product might have a role in regulating the growth of BL cells, since treatment of the EBV-positive BL cell line Daudi with alpha-interferon results in a decrease in c-fgr mRNA levels followed by a decrease in growth rate. Our data indicate that there is no general correlation between c-fgr mRNA levels and growth rate in BL cells and so argue against a role for the c-fgr gene product in growth regulation in these cells.

Topics: Burkitt Lymphoma; Cell Division; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Humans; Proto-Oncogene Mas; Proto-Oncogenes; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. NT2/D1 cells basally express transforming growth factor alpha (TGF-alpha) mRNA and secreted protein. After RA-treatment TGF-alpha expression is markedly reduced. This decline in TGF-alpha expression accompanies the induction of the neuronal phenotype and a marked reduction of tumorigenicity in athymic mice. This suggested a causal link between reduced TGF-alpha expression and the induced differentiation or loss of tumorigenicity of these RA-treated TC cells. To evaluate this possibility, an RA-refractory NT2/D1 subclone was analysed. This subclone, designated NT2/D1-R1, failed to induce differentiation or to decrease TGF-alpha expression despite RA treatment. To further explore the relationship between TGF-alpha expression and RA actions in this human TC cell, a TGF-alpha cDNA was stably transfected and expressed in NT2/D1 cells. RA-treatment of independently obtained TGF-alpha over-expressing clones and a representative control transfectant only expressing the neomycin resistance gene produced a neuronal phenotype similar to parental NT2/D1 cells as assessed by morphologic, immunophenotypic, and gene expression markers of differentiation. RA-treatment of these clones also induced a G1 arrest similar to parental cells. However, only the TGF-alpha over-expressing clones that secreted high levels of TGF-alpha protein into the conditioned media before and after RA treatment still developed tumors in athymic mice despite prior exposure to these cells to RA. This finding demonstrates that TGF-alpha can inhibit the anti-tumorigenic effects of RA in human TCs. Thus, over-expression of a single growth factor that normally declines with RA treatment antagonizes the anti-tumorigenic but not the differentiation actions of RA in this human tumor cell.

Topics: Animals; Blotting, Northern; Cell Transformation, Neoplastic; Culture Media, Conditioned; DNA, Neoplasm; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Mice; Mice, Nude; RNA, Messenger; Teratocarcinoma; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

50 cases were treated with Myelodysplastic Syndrome (MDS) by combined TCM-WM therapy. They were classified into RA 17 cases, RAS 6, RAEB 19, CMML 1 and RAEBT 7. The patients were divided into two groups, one with RA and RAS receiving treatment of hemopoietic and immune drugs plus Chinese medicinal herbs, the other with RAEB, CMML and RAEBT receiving treatment of LD Ara-c and LD Hom chemotherapy plus medicinal herbs. The effective rates were 47.83% and 62.96% respectively, the total effective rate being 56%. 6 cases (RAEB 4, RA 1, RAS 1) were treated with all-trans retinoic acid used as an inducer of differentiation, 2 of them were effective. 11 patients with MDS who had transformed into acute leukemia were treated by LD Ara-c and combined TCM-WM chemotherapy, the remission rate was 54.55% and the survival period was 9-27 months after remission. In some cases low dose chemotherapy resulted in hemocytopenia, bone marrow inhibition, infection, mild nausea and anorexia.

Topics: Adolescent; Adult; Aged; Cell Transformation, Neoplastic; Cytarabine; Drugs, Chinese Herbal; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Pyridoxine; Stanozolol; Tretinoin

We previously reported that human keratinocytes (HKc) immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16) are more sensitive than normal HKc to growth inhibition by retinoic acid (RA), and that RA treatment of HKc/HPV16 inhibits HPV16 E6/E7 mRNA expression (L. Pirisi et al., Cancer Res., 52: 187-193, 1992). We now demonstrate that HPV16 E2 and E5 mRNAs are also decreased by RA treatment of HKc/HPV16, indicating a general inhibition by RA on the expression of HPV16 early genes. In addition, protein levels of E6 and E7, as measured by immunofluorescence, are also decreased in a dose-dependent manner following RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. RA treatment (1 nM) of normal HKc, immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of RA in the chemo-prevention of human papillomavirus-induced cancers.

Topics: Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA-Binding Proteins; Gene Expression Regulation, Viral; Humans; Keratinocytes; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Repressor Proteins; RNA, Messenger; Transfection; Tretinoin

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP-1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP-1 monocytes, PACAP increased basal adenylate cyclase activity 5.3-fold, with an affinity 50-times greater than that of VIP or helodermin (Ka = 3.2 x 10(-11) M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased cAMP generation 2-fold (EC50 = 10(-9) M). Spontaneous and RA-induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP-1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory Gs/Gi subunits of adenylate cyclase.

Topics: Adenylyl Cyclases; Cell Differentiation; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Enzyme Activation; Humans; Hydrogen Peroxide; Isoproterenol; Leukemia; Macrophages; Monocytes; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pulmonary Alveoli; Signal Transduction; Superoxides; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We determined the breakpoints of the RAR-alpha and PML genes in acute promyelocytic leukemia (APL) cells from 40 patients using Southern blot analysis. We also analyzed the relationship between the location of breakpoints, the clinical features of APL and the response to all-trans retinoic acid (ATRA). While the breakpoints of the RAR-alpha gene were consistently within intron 2, we found two major clusters in the breakpoints of the PML gene. The two breakpoint clusters in the PML gene were separated by 10 kb; 5' breakpoints were in intron 3, and 3' breakpoints were around introns 5 and 6. Twenty percent of the patients had 5' breakpoints in the PML gene and 70% had 3' breakpoints. No rearrangement was observed in the remaining 10% of patients in spite of the presence of t(15;17) translocation. There was no relationship between the location of the PML breakpoints, the clinical features at diagnosis and the response to ATRA.

Topics: Adolescent; Adult; Aged; Base Sequence; Blotting, Southern; Carrier Proteins; Cell Transformation, Neoplastic; Child; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; DNA Probes; DNA, Neoplasm; Female; Gene Rearrangement; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Prevalence; Receptors, Retinoic Acid; Restriction Mapping; Translocation, Genetic; Tretinoin

The undifferentiated Y-79 retinoblastoma cell line can be induced by specific agents to express characteristics of mature retinal cells. In the present study, attached Y-79 cell cultures were treated with hexamethylene bis-acetamide (HMBA) and other differentiating agents and examined for "neuronal" and other properties. Immunocytochemical staining was performed with antibodies against neuron- and retina-specific antigens, [synaptophysin, interphotoreceptor retinoid-binding protein (IRBP), neural cell adhesion molecule (N-CAM), and rod- and cone-specific transducin (TR alpha and TC alpha)] and microtubule-associated protein (MAP-1) and tubulin. Enhanced expression of tubulin was observed with cAMP treatment in FBS media. Expression of N-CAM was observed in all groups. Morphological differentiation was pronounced with HMBA and butyrate treatment, with HMBA inducing increased tubulin expression after 2 weeks of treatment. Expression of TR alpha was minimal under all culture conditions, whereas TC alpha was ubiquitously expressed. This supports the concept that Y-79 retinoblastoma is predominantly of cone neuronal origin and that, surprisingly, immunocytochemical differentiation is not correlated with the marked morphological changes induced by the major differentiating agents used.

Topics: Acetamides; Antineoplastic Agents; Butyrates; Cell Adhesion Molecules, Neuronal; Cell Transformation, Neoplastic; Cyclic AMP; Eye Neoplasms; Eye Proteins; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Regression Analysis; Retinoblastoma; Retinol-Binding Proteins; Synaptophysin; Transducin; Tretinoin; Tubulin; Tumor Cells, Cultured

The oncogene jun encodes a transcription factor of the AP-1 family. In mice carrying viral jun (v-jun) as a transgene, wounding is a prerequisite for tumorigenesis, suggesting collaboration between the transgene and a wound-related event. To define possible candidates for this collaborative process, we examined the effect of several wound-related polypeptide growth factors on cells from transgenic mice. Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in embryo fibroblasts and tumor cell revertants from these mice. This effect was specific for the two cytokines and was restricted to cells from v-jun transgenic mice. Anchorage independence required the continued presence of the cytokines. Transfection of transgenic cells with a v-jun expression plasmid also induced anchorage independence and a tumorigenic phenotype in transgenic tumor cell revertants. However, there was no correlation between anchorage independence, expression of Jun, and AP-1 activity. These results suggest that while increased transgene expression can enhance the growth properties of v-jun transgenic cells, there exist other cytokine-dependent mechanisms that have a similar effect. Retinoic acid, dexamethasone, or forskolin inhibits induction of anchorage independence by tumor necrosis factor alpha, interleukin 1 alpha, and transfected v-jun. Although these agents affect both AP-1 transactivation potential and DNA binding in the transgenic cells, the changes are not correlated with the inhibition of growth.

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Colforsin; Dexamethasone; Embryo, Mammalian; Fibroblasts; Gene Expression; Genes, jun; Growth Substances; Interleukin-1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Sarcoma, Experimental; Sensitivity and Specificity; Stimulation, Chemical; Transcription, Genetic; Transforming Growth Factor alpha; Tretinoin; Wounds and Injuries

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

Topics: Cell Transformation, Neoplastic; Humans; Neuroblastoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The control of myogenin (Myf-4), one of the muscle-specific regulatory proteins, is particularly interesting since its expression appears obligatory in myoblasts at the onset of differentiation. We isolated the human Myf-4 (myogenin) gene and determined promoter elements which direct cell type-specific expression and are subject to transactivation by the muscle transcription factors Myf-5 and MyoD1 in fibroblasts. Extrinsic signals such as serum components and purified growth factors or potential intracellular signals such as cAMP down-regulate transcription of the myogenin gene. Constitutive expression of the catalytic subunit of PKA completely suppresses transactivation of the myogenin promoter by Myf-5 or MyoD1 suggesting that cAMP may act via phosphorylation by PKA. In contrast to normal myogenic cell lines in which differentiation and myogenin expression can be induced by the removal of serum components, retinoic acid (RA) is required for differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. This model system was utilized to investigate factors which influence the balance between the transformed state and differentiation. Administration of retinoic acid to BA-Han-1C cells leads to the accumulation of myogenin mRNA approximately 48 h after the addition of RA. This late induction requires ongoing protein- and DNA-synthesis suggesting that trans- and cis-acting factors may be involved in the control. The critical involvement of myogenin in the process of terminal muscle differentiation was also demonstrated in the rat L6 muscle cell line which has been blocked for differentiation by the transforming protein E1a of Ad5 adenovirus. In cells which stably express E1a, myogenin expression is completely suppressed while Myf-5 continues to be synthesized normally. However, E1a inhibits the transactivator function of Myf-5, as demonstrated on GAL4-Myf5 chimeric proteins. A possible interpretation of this result is that Myf-5 or factors activated by Myf-5 are required for the expression of myogenin and myogenin itself is necessary for the terminal differentiation of myoblasts.

Topics: Blood; Blotting, Northern; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; DNA-Binding Proteins; Gene Expression Regulation; Growth Substances; Humans; Muscle Proteins; Muscles; Myogenic Regulatory Factor 5; Myogenic Regulatory Factors; Myogenin; RNA, Messenger; Trans-Activators; Tretinoin; Tumor Cells, Cultured

We analyzed the maintenance of acute myeloid leukemia clonogenic cells (AML CFU-L) in liquid culture in the presence of five potential differentiation inducers: trans-retinoic acid, 1,25-dihydroxy vitamin D3, interferon gamma, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), used singly and with two combined. The culture medium contained either fetal bovine or human serum from normal donors. CFU-L recovery after 7 days was compared to that observed in control cultures. Of AML cases and HL60 cells, 11/15 displayed greater than 50% CFU-L reduction in response to one or more inducers. In 8/9 responsive cases that underwent the nitroblue tetrazolium (NBT) reduction test, an increase in the percentage of functionally mature (NBT-positive) cells was detected. The combination of retinoic acid with interferon gamma was most effective in reducing CFU-L recovering (8 responsive/15 AML cases), G-CSF and M-CSF displayed either inhibitory or stimulatory activity in different AML cases. The type of serum employed generally did not affect the response to inducers of differentiation. No significant inhibition of the recovery of granulocyte-macrophage colony-forming units was determined by the five inducers in experiments with three normal bone marrow samples. Our experiments indicate that biological differentiation inducers can reduce AML CFU-L self-renewal and increase the proportion of differentiated cells at concentrations that do not affect normal myelopoiesis and could be achieved during treatments in vivo.

Topics: Acute Disease; Animals; Calcitriol; Cell Transformation, Neoplastic; Drug Synergism; Granulocyte Colony-Stimulating Factor; Humans; Interferon-gamma; Leukemia, Myeloid; Macrophage Colony-Stimulating Factor; Recombinant Proteins; Tretinoin; Tumor Stem Cell Assay

SV40 large T oncoprotein-transformed murine mesenchymal 3T3 T stem cells (CSV3 cells) can be induced to growth arrest and then differentiate into adipocytes. When differentiation occurs, SV40 T oncoprotein expression is repressed (Estervig et al., J Virol 63:2718, 1989). To determine if repression of T oncoprotein expression can also be induced pharmacologically, the effect of a variety of agents that have been reported to effect differentiation in various cell types but not in 3T3 T or CSV3 cells was tested. This rationale suggests that if any of these agents repress T oncoprotein expression in CSV3 cells, then the results would establish that repression of T oncoprotein expression can be mediated by mechanisms independent of overt differentiation. The results show that dimethylsulfoxide (DMSO) is the only agent tested that represses T oncoprotein expression in CSV3 cells. Repression occurs in a dosage-dependent manner within 24-96 hours after exposure to DMSO. The effect of DMSO on T oncoprotein expression is mediated by posttranslational mechanisms that decrease the stability of the T oncoprotein. DMSO-induced repression of T oncoprotein expression is also associated with reversion of the transformed phenotype in CSV3 cells as demonstrated by the loss of responsiveness to a specific transformation-associated mitogen. These data support the conclusion that the pharmacological repression of T oncoprotein expression represents a form of cancer suppressor activity that can be mediated by a distinct molecular mechanism.

Topics: Acetamides; Animals; Antigens, Polyomavirus Transforming; Azacitidine; Blotting, Northern; Blotting, Western; Butyrates; Butyric Acid; Cell Line, Transformed; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Insulin; Interferons; Mice; Phenotype; Stem Cells; T-Lymphocytes; Tretinoin

To explore the molecular mechanisms by which retinoic acid inhibits oncogenic transformation, we have examined the effects of retinoic acid on the polyomavirus-induced transformation of rat fibroblasts. Treatment of rat F111 fibroblasts with high concentrations of retinoic acid (10(-6) M) partially inhibited the ability of polyomavirus to induce dense focus formation (50-70%). This effect was not secondary to a retinoic acid-dependent block of cellular proliferation. To address the role of the retinoic acid receptor (RAR-alpha) in mediating the transformation-inhibitory effect of retinoic acid, we have overexpressed either RAR-alpha or cellular retinoic acid-binding protein I (CRABP) cDNAs in F111 cells. Introduction of a CRABP I expression vector did not alter the responsiveness of F111 cells to retinoic acid in any detectable fashion. In contrast, overexpression of RAR-alpha increased the sensitivity of F111 cells to the transformation-inhibitory action of retinoic acid by 10- to 100-fold. At high concentrations, retinoic acid inhibited transformation of F111-RAR cells by polyomavirus by about 90%. At near physiological concentrations, retinoic acid inhibited transformation by 25-50% in F111-RAR cells but not in control cells. Retinoic acid did not inhibit either the synthesis of polyoma middle T (mT) or pp60c-src, the cellular target for mT action, or the formation of mT:pp60c-src:PI-3 kinase (phosphatidylinositol-3 kinase) complexes. Therefore, RAR-alpha was not acting as a negative regulator of expression of either the polyomavirus middle T oncogene or the cellular proto-oncogene, c-src. It seems likely that RAR-alpha regulates the expression of cellular genes whose products interact in some way with mT-regulated signaling pathways, leading to a ligand-dependent suppression of polyoma transformation. In addition, RAR-alpha overexpression selectively inhibits the serum-stimulated expression of the c-fos gene, but does not affect the expression of a number of other serum- and polyomavirus-inducible genes including c-jun, junB, c-myc and actin.

Topics: Animals; Antigens, Polyomavirus Transforming; Blood Physiological Phenomena; Carrier Proteins; Cell Cycle; Cell Transformation, Neoplastic; Fibroblasts; Gene Expression; Genes, fos; Polyomavirus; Proto-Oncogene Mas; Proto-Oncogene Proteins pp60(c-src); Rats; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin

According to investigations, it was shown that peripheral blood WBC count increased, percentage of promyelocytes increased, differentiation features emerged, protein kinase C (PK-C) activity elevated, intracellular lysozyme content increased, CFU-F productivity increased and typical t (15; 17) disappeared, when induced differentiation therapy with all trans-retinoic acid (RA) was carried out in patients with acute promyelocytic leukemia. Comparison between RA group and RA+Harringtonin/Ara-C group showed no significant difference in most of the parameters; the curative effect was same in both groups. Complete remission (CR) rate was achieved in 92.6% (25/27 cases) of the patients in the entire group. In order to increase CR rate, prevention and treatment for haemorrhagic complications are critical. Patients with hyperleukocytosis (WBC > or = 100 x 10(9)/L) should be treated with intensive combined therapy as well as aggressive prevention of haemorrhage and pathological changes of lung and brain.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Child; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Remission Induction; Tretinoin

In cells transformed by the highly oncogenic adenovirus type 12 (Ad12), the viral E1A proteins mediate transcriptional repression of the major histocompatibility class I genes. In contrast, class I transcription is not reduced in cells transformed by the nononcogenic Ad5. The decreased rate of class I transcription is, at least in part, the result of a reduced major histocompatibility complex class I enhancer activity in Ad12-transformed cells and correlates with an increase in the levels of a DNA-binding activity to the R2 element of the enhancer (R. Ge, A. Kralli, R. Weinmann, and R. P. Ricciardi, J. Virol. 66:6969-6978, 1992). Employing transient transfection assays, we now provide direct evidence that the R2 element can confer repression in Ad12- but not Ad5-transformed cells. Repression by R2 was observed only in the presence of the positive enhancer element R1 and was dependent on (i) the number of the R2 elements and (ii) the relative arrangement of R2 and R1 elements. The putative R2-binding repressor protein, R2BF, was similar in molecular weight and binding specificity to members of the thyroid hormone/retinoic acid (RA) receptor family. RA treatment abrogated the R2-mediated repression in Ad12-transformed cells and had no effect on the activity of R2/R1-containing promoters in Ad5-transformed cells. These results are consistent with the presence of an R2-binding repressor in Ad12-transformed cells. In the absence of RA, the repressor compromises enhancer activity by interfering with the activity of the positive cis element R1. RA treatment of Ad12-transformed cells may render the repressor inactive.

Topics: Adenovirus E1A Proteins; Adenoviruses, Human; Animals; Base Sequence; Binding Sites; Cell Line, Transformed; Cell Nucleus; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; Enhancer Elements, Genetic; Gene Expression Regulation, Viral; Genes, MHC Class I; H-2 Antigens; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Promoter Regions, Genetic; Recombinant Proteins; Transcription, Genetic; Transfection; Tretinoin

Transcription factor AP-1 is constituted by the various products of the fos and jun proto-oncogene family members, which associate as dimers to bind with variable efficiency to 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive promoter elements (TREs). We have recently shown that DNA binding of AP-1 is regulated by an inhibitory protein, IP-1, whose activity is modulated by phosphorylation. Here it is shown that although AP-1 has a very high affinity for its recognition sequence, its binding to the TRE can be quickly inhibited by the addition of IP-1. IP-1 is more active on AP-1 complexes formed during a shorter period of time. IP-1 activity is blocked by stimulation of the protein kinase C (PKC) signal transduction pathway, achieved by treating HeLa cells with phorbol esters or with a diacylglycerol analog. We observed an increase in AP-1-DNA binding after treatment of the cells with either the calcium ionophore A-23187 or dibutyryl cAMP; this could be ascribed to inhibition of IP-1 activity. A decreased IP-1 activity also correlates with the increase in AP-1-DNA binding after stimulating cells with serum. This suggests that IP-1 is an important target of the various signal transduction pathways. No effect on AP-1 and IP-1 was detected in cells transformed by Ki-ras or v-raf; nor could an effect of inhibition of protein synthesis be observed. We also analysed IP-1 regulation upon differentiation of P19 embryonal carcinoma cells by retinoic acid. We conclude that IP-1 regulation has a pivotal role in the final modulation of Fos-Jun by signal transduction pathways.

Topics: 3T3 Cells; Animals; Blood Physiological Phenomena; Calcium; Cell Transformation, Neoplastic; Cycloheximide; DNA; Genes, fos; Genes, jun; Humans; Mice; Protein Kinase C; Protein Kinases; Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin

The effects of the H-ras oncogene on fibroblast cell tumorigenicity and immunogenicity was studied in transfectants of the BALB/c 3T3 clone A31 fibroblastoid cell-line. Cells that were transfected with MC29-LTR-H-ras (98/6) or MC29-LTR-v-myc + H-ras (98/4v) and were inoculated into syngeneic BALB/c mice were tumorigenic in 100% and 60% of animals respectively. By contrast, transfectants containing the pSV2neo plasmid alone (98/1) displayed normal characteristics both in vitro and in vivo. Inoculation of mice with mitomycin-C-treated 98/1 or 98/4v cells induced an effective protective immunity to a challenge of live 98/4v cells, and a partial immunity against 98/6 cells. Mitomycin-C-treated 98/6 cells failed to render immunity against a challenge of either 98/6 or 98/4v cells. To correlate immunogenicity and tumorigenicity of the different cell types with cell-surface-antigen expression, we prepared MAbs against 98/4v cells in syngeneic mice. Immunohistochemical and immunoblot analysis revealed that MAbs 102 and 104 recognized 2 protein band of 70 and 45 kDa respectively, which were expressed predominantly in 98/1 and 98/4v cells. A third immunoreactive protein band of 44 kDa that reacted with MAb 6 was expressed at a similar cell-surface density on all cell types. Cell-differentiation-inducing agents, such as DMSO, retinoic acid or sodium butyrate, were all found to induce 98/6 cell flattening and morphological changes toward a normal phenotype that were followed by up-regulation of the 70- and 45-kDa antigens. The results suggest that regulation of expression of the 70- and 45-kDa molecules is affected by H-ras, and that expression of these cell-surface molecules may be relevant to tumor cell immunogenicity.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Butyrates; Butyric Acid; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Down-Regulation; Female; Fibroblasts; Genes, myc; Genes, ras; Male; Mice; Mice, Inbred BALB C; Molecular Weight; Phenotype; Tretinoin

We used a series of cell clones from a human teratocarcinoma cell line, PA-1, to study the effect of transformation by an activated N-ras oncogene on the expression of genes involved in retinoic acid (RA)-induced differentiation and growth regulation. Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990). We now report that six different genes of the cluster HOX 1 are sequentially induced by RA in a similar temporal pattern, beginning with genes at the 3' end of the cluster. However, in N-ras-transformed cell clones, RA-induced expression of these homeobox genes is delayed. Hox 1.4 and Hox 1.3, genes abundantly induced in nontransformed clones after 3 days of RA treatment, are expressed in N-ras-transformed cells only after 10 days of RA treatment. At this time, the cells' growth is arrested at very high density, and no differentiated morphologic characteristics are observed. Constitutive expression of a transfected Hox 1.4 gene under the control of a simian virus 40 promotor leads to differentiated cell morphology similar to that of the RA-induced phenotype and restores the growth-inhibitory effects of RA in N-ras-transformed cells. These observations provide evidence that enhanced proliferation in N-ras-transformed cells compromises teratocarcinoma cell differentiation by a mechanism that transiently suppresses homeobox gene induction and implies a central role for homeobox genes in RA-induced cell differentiation. We conclude that stimulation of a putative growth factor signal pathway, associated with ras-induced proliferation, transiently suppresses the induction of transcription factors functionally involved in cell growth and differentiation.

Topics: Amino Acid Sequence; Base Sequence; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Recombinant; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Genes, ras; Humans; Molecular Sequence Data; Multigene Family; Oligonucleotide Probes; Polymerase Chain Reaction; RNA, Neoplasm; Teratoma; Transcriptional Activation; Transfection; Tretinoin

Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 microM retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 microM retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming gene aph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14-20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.

Topics: Blotting, Northern; Blotting, Western; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genes, ras; In Vitro Techniques; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; RNA; Transfection; Tretinoin

Anchorage-independent growth of normal rat kidney (NRK) fibroblast in soft agar depends on both transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). To examine whether c-fos protein is involved in phenotypic transformation of NRK cells, we have transfected and isolated several NRK cell lines that carry the human c-fos gene fused to the metallothionein IIA promoter. A transfectant, Nf-1, had constitutive levels of the human c-fos expression. Anchorage-independent growth of Nf-1 was already stimulated by EGF alone, and the colony sizes of Nf-1 were comparable to those of the parental NRK in the presence of both EGF and TGF beta. Anchorage-independent growth of NRK could be observed in the presence of TGF beta or retinoic acid or platelet derived growth factor (PDGF) and EGF. No growth of NRK in soft agar appeared when basic fibroblast growth factor (bFGF) and EGF were present. By contrast, anchorage-independent growth of Nf-1 was surprisingly enhanced by EGF and TGF beta or retinoic acid or PDGF or bFGF. Expression of the human c-fos gene may compensate the signal to phenotypic transformation induced by TGF beta as well as retinoic acid or PDGF or bFGF.

Topics: Animals; Blotting, Northern; Blotting, Southern; Blotting, Western; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; Fibroblast Growth Factor 2; Genes, fos; Growth Substances; Humans; Kidney; Nuclear Proteins; Phenotype; Plasmids; Platelet-Derived Growth Factor; Rats; Recombinant Proteins; Transfection; Transforming Growth Factor beta; Tretinoin

Transforming growth factor-beta (TGF-beta) plays a complex role as a regulator of proliferation and differentiation of many cell types, including cells of epithelial origin. In this study, we examined whether TGF-beta, alone or in combination with retinoic acid, was able to inhibit the transformation of the murine epidermal cell line JB6. When treated with phorbol myristate acetate (PMA) and other tumor promoters, the nontumorigenic and anchorage-dependent JB6 cells acquired a tumor phenotype, as shown by the acquisition of tumorigenicity and anchorage independence. We found that TGB-beta inhibited the PMA-induced transformation of a subclone of JB6 cells. The effect of TGF-beta was due to an anti-transformation promoting activity, rather than to generalized growth inhibition, since TGF-beta neither inhibited the growth of monolayer cultures of JB6 cells, nor affected the colony-forming efficiency in agar of the JB6-derived permanently transformed RT101 cell line. TGF-beta was synergistic with retinoic acid, a known anti-tumor promoter, in inhibiting the PMA-induced transformation of JB6 cells. Examination of TGF-beta receptor expression on JB6 cells, by both binding and affinity labeling, showed that treatment with PMA significantly decreased TGF-beta receptor expression while retinoic acid counteracted this effect of PMA, thus suggesting that the synergy between retinoic acid and TGF-beta may be due, at least in part, to modulation of TGF-beta receptor expression. TGF-beta, therefore, appears to function as an incomplete antipromoter whose action can be permitted and/or complemented by retinoic acid. Our data demonstrating that TGF-beta has anti-transformation promoting activity suggest that TGF-beta plays a role in maintaining homeostasis of epithelial cells, not only by regulating cell proliferation and differentiation, but also by counteracting events that lead to malignant transformation.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Drug Synergism; Mice; Mice, Inbred BALB C; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tretinoin

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.

Topics: Cell Transformation, Neoplastic; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; NAD+ Nucleosidase; Poly(ADP-ribose) Polymerases; Tretinoin; Tumor Cells, Cultured

The Egr-1 gene (zfp-6) encodes a 'zinc finger'-type transcription factor that is one of the early growth response genes induced, together with c-fos proto-oncogene, in many cell types. Our earlier work indicated that Egr-1 and c-fos may also play roles in differentiation and we now present data to show some features of their regulation. Transcriptional regulation accounts at least partly for the increased steady-state levels of Egr-1 mRNA in differentiating teratocarcinoma cells; this rate increases threefold over the 7-10 days of differentiation of P19 embryonal carcinoma cells with both 0.5% DMSO (to give predominantly cardiac muscle) and 1 microM retinoic acid (to give nerve and glial cells). The stability of Egr-1 transcripts remains the same (T1/2 = 90 min) in undifferentiated EC and differentiated cell products. In contrast, transcripts for c-fos are barely detectable in EC cells and increase 20-fold during differentiation. The basis for this is a marked increase in stability of c-fos mRNA after differentiation. The protein products of both genes parallel the steady-state levels of their mRNAs, but both proteins become more stable in differentiated cells. This is particularly marked for c-Fos protein, which appears as a distinct 58 kDa species in terminally differentiated P19 cells. Both Egr-1 and c-Fos proteins remain at high constitutive levels in differentiated cells indicating a distinct role for these transcription factors, For instance, it appears that this form of Fos protein may not repress the synthesis of the Egr-1 gene as it does during transient expression of serum-stimulated genes.

Topics: Animals; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Gene Expression Regulation, Neoplastic; Mice; Precipitin Tests; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation; Zinc Fingers

The maturation of embryonal neural crest cells is thought to be regulated in part by the milieu into which these cells migrate. Neuroblastoma (NB) is a tumor of very early childhood that is thought to arise in association with the arrested differentiation of embryonal neural crest cells. In culture, neuroblastoma tumor cells differentiate in the presence of retinoic acid, which is also known to influence extracellular matrix protein synthesis. We have cultured neuroblastoma cells on laminin (LN) and fibronectin (FN) substrata to examine the role of extracellular matrix in retinoic acid (RA)-induced differentiation of these tumor cells. These proteins caused morphologic changes in NB cells indistinguishable from those caused by RA. Antiserum to each of these proteins blocked the effects induced by the corresponding protein, but neither antiserum affected the action of RA. Despite the induction of a neuronal morphologic change, matrix proteins did not alter the proliferation of NB cells. These results indicate that LN and FN modulate the differentiation of NB cells without inducing growth arrest and that RA-induced differentiation does not require these matrix proteins.

Topics: Blotting, Northern; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Extracellular Matrix Proteins; Fibronectins; Humans; Laminin; Neural Crest; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

In HL60 cells a nuclear protein of Mr 55,000 is retinoylated, with the formation of a thioester bond. To gain further knowledge on the role of retinoylation we studied it in cell lines with varied responses to retinoic acid (RA). Compared to HL60 the extent of retinoylation (mol/cell) was about fivefold higher in HL60/MRI, a mutant which is more sensitive to RA than HL60. Retinoylation occurred to the same extent and at similar rates in HL60 and in HL60/RA-res, a mutant resistant to differentiation by RA. One-dimensional polyacrylamide gel electrophoresis patterns for the three HL60 cell lines were similar. However, two-dimensional polyacrylamide gel electrophoresis patterns of the three HL60 cell lines were distinct. While we saw the same major retinoylated protein of Mr 55,000 in the three cell lines, the HL60/RA-res cells also contained a high level of a protein with the same Mr and a lower pI. The extent of retinoylation was greater in the RA-sensitive embryonal carcinoma cell line, PCC4.aza1R, than in a RA-resistant cell line, PCC4.(RA)-2. One-dimensional polyacrylamide gel electrophoresis patterns of retinoylated proteins of the embryonal carcinoma cell lines were different from HL60 and from each other. The retinoylation pattern of the normal canine kidney cell line (MDCK) was different from either HL60 or the embryonal carcinoma cells. These results showed the retinoylation was widespread and that the response to RA of different cell types may depend on the retinoylation of specific proteins.

Topics: Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Electrophoresis, Gel, Two-Dimensional; Humans; Kidney; Leukemia; Molecular Weight; Mutation; Neoplasm Proteins; Nuclear Proteins; Teratoma; Tretinoin; Tumor Cells, Cultured

The human teratocarcinoma cell line Tera 2 can be induced to differentiate in vitro after exposure to retinoic acid. We show in this paper that whereas the K-FGF oncogene is expressed in undifferentiated cells, addition of retinoic acid rapidly (less than 60 min) downregulates the expression of this gene. However, when cells are cultured in RA for an extended period of time (greater than 15 days) K-FGF transcripts reappear. We also report that K-FGF is expressed in approximately one-third of primary human germ cell tumours but not in the corresponding normal testicular tissue.

Topics: Cell Differentiation; Cell Transformation, Neoplastic; Down-Regulation; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression; Humans; Kinetics; Proto-Oncogene Proteins; RNA, Messenger; Teratoma; Tretinoin; Tumor Cells, Cultured

It is known that PKC is differently expressed in brain and the peripheral nervous system and is involved in cellular differentiation. We have analyzed 9 human neuronal-derived crest-cell lines for PKC-alpha mRNA. Seven out of nine expressed 9.0 kb and 4.0 kb PKC-alpha mRNAs, but three had high level of 9.0 kb transcription. The different expression of the two messenger RNAs may result from alternative splicing and a different degree of cell maturation. The same cell lines were studied for MYCN gene expression. A possible relation between the two genes is discussed. One cell line expressing high levels of both PKC-alpha mRNA was treated with 10(-5) M retinoic acid (RA). The expression of both messenger RNAs was suppressed when the cells achieved a morphological differentiation and showed neurite-like processes. A decrease of PKC-alpha gene expression was associated to down regulation of MYCN mRNA. These preliminary results suggest that PKC suppression of PKC-alpha mRNA is associated with reversion of the malignant phenotype.

Topics: Cell Transformation, Neoplastic; Gene Expression Regulation, Enzymologic; Humans; Neuroblastoma; Oncogenes; Protein Kinase C; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Balb/3T3 fibroblasts were cultured in type I collagen gel and the effects of tretinoin (all-trans-retinoic acid) were examined on cell growth and the gel contraction produced by cells. Cell proliferation was suppressed and the degree of gel contraction was enhanced by the addition of 10(-7) and 10(-6) M tretinoin. Growth and gel contractility of transformed cells derived from the Balb/3T3 cells were not influenced by this agent. Addition of 12-O-tetradecanoylphorbol ester, which is known to antagonize tretinoin in several biological processes, enhanced gel contraction synergistically with tretinoin. These results suggest that tretinoin influences cell-to-collagen interactions.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Collagen; Fibroblasts; Gels; Mice; Tetradecanoylphorbol Acetate; Tretinoin

The neoplastic Hodgkin's Reed-Sternberg (H-RS) cells in Hodgkin's disease are surrounded in vivo by abundant reactive cells, the function of which may be attributed in part to their elaboration of various cytokines. Thus, a study of the interaction of H-RS cells with exogenous cytokines may provide information as to the mechanism of the clinical and histopathologic changes observed in Hodgkin's disease. This study examined the effect of various cytokines, and of phorbol ester (TPA) and retinoic acid, on the differentiation and proliferation of cultured H-RS cells (cell lines HDLM-1 and KM-H2). In addition, it was determined whether these cells were able to secrete cytokines after being treated with exogenous cytokines. The cytokines used included various types of interleukins (1, 2, and 3), colony-stimulating factors (GM, G, and M), interferons (alpha, beta, and gamma), and tumor necrosis factor (alpha). It was found that these cytokines, used alone or in combination, were not effective in modulating the proliferation and differentiation of cells, or the production of cytokines, in cultured H-RS cells. In contrast, this study revealed that retinoic acid can potentiate TPA-induced growth inhibition in cultured H-RS cells. Retinoic acid, when used alone, exhibited a minimal effect on cell differentiation. No synergistic effects of cytokines and retinoic acid on H-RS cells were observed. The failure of cultured H-RS cells to respond to exogenous cytokines suggests that, during the course of neoplastic transformation, of progression of disease, or of establishment of the cells in culture, H-RS cells lose their dependence on cytokines, although they retain the capacity to produce various cytokines.

Topics: Aged; Biological Factors; Cell Line; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Cytokines; Hodgkin Disease; Humans; Interferons; Interleukins; Lymphoma; Male; Phorbol Esters; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Necrosis Factor-alpha

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR-32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage-independent growth. By Southern blot analysis, BIM cells had both human and rat types of N-myc genes. The human N-myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR-32. The rat N-myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N-myc genes may be involved in the properties of partial reverse-transformation in BIM cells. When treated with various drugs such as db-cAMP, forskolin, and cAMP with isobutyl-methylxanthine, BIM cells expressed a nerve-like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve-like cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Axons; Bucladesine; Calcimycin; Cell Cycle; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Fibroblasts; Humans; Hybrid Cells; Methylcellulose; Neuroblastoma; Neurons; Phenotype; Rats; Thymidine; Tretinoin; Tumor Cells, Cultured

Retinoic acid, hexamethylene bisacetamide, sodium butyrate, and dimethylsulfoxide, four compounds which modulate phenotypic expression in a variety of neoplastic cell lines, all inhibited the induction of tyrosinase activity and melanogenesis by the combination of melanocyte-stimulating hormone and isobutylmethyxanthine in Cloudman S91 melanoma cells. Results were the same in assays of whole cells or in extracts made from them. Only retinoic acid, however, was effective at inhibiting the activation of dopachrome isomerase, another regulatory enzyme in melanogenesis. Despite inhibiting the effects of melanocyte-stimulating hormone (MSH) and isobutylmethylxanthine on tyrosinase activity, all of the agents tested increased the binding of MSH to intact cells. Ultrastructural analysis of treated cells following DOPA cytochemistry revealed that both retinoic acid and hexamethylene bisacetamide arrested melanosomal maturation at stage I-II. Retinoic acid resulted in a derangement of melanosomal structure. The specificity of these agents for preventing the induction of melanogenesis makes them powerful tools for the dissection of this complex cellular process.

Topics: 1-Methyl-3-isobutylxanthine; Acetamides; Animals; Butyrates; Butyric Acid; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Indolequinones; Indoles; Melanins; Melanocyte-Stimulating Hormones; Melanocytes; Melanoma, Experimental; Mice; Microscopy, Electron; Monophenol Monooxygenase; Phenotype; Quinones; Tretinoin; Tumor Cells, Cultured

The regulation of jun family genes and AP1 activity during the course of differentiation of F9 embryonal carcinoma stem cells was investigated. The induction of differentiation by retinoic acid (RA) leads to an accumulation of c-jun mRNA caused by increased c-jun transcription. This induction is an indirect response to RA and requires a functional AP1 binding site within the c-jun promoter. Expression of jun-B mRNA, however, is transiently induced but at a later time point is repressed by RA. The third member of the family, jun-D, is already active in undifferentiated cells and is only slightly induced after differentiation. Differentiation also converts c-jun from being refractory to phorbol esters to a highly inducible state. The development of this response is correlated with increased AP1 activity in RA-treated cells. By contrast, the induction of c-fos by phorbol esters or cAMP is greatly diminished after RA treatment. Transfection experiments indicate that, in the absence of c-Fos, only c-Jun is an effective transactivator. Hence, the major increase in AP1 activity is due to elevated c-jun expression and probably involves positive autoregulation by the c-Jun protein. Furthermore, these results demonstrate that AP1 activity can be stimulated by phorbol ester without concomitant c-fos induction. Forced expression of c-Jun and v-Jun results in activation of at least two differentiation marker genes, EndoB and tissue plasminogen activator, whose regulatory regions contain AP1 binding sites. Thus, the induction of c-jun transcription by RA, although indirect, can have an important role in the differentiation process.

Topics: Base Sequence; Cell Differentiation; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genetic Markers; Humans; Molecular Sequence Data; Multigene Family; Proto-Oncogene Proteins c-jun; RNA, Messenger; Stem Cells; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cholesterol Esters; Depression, Chemical; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Sulfotransferases; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dose-Response Relationship, Drug; Drug Synergism; Guanosine; Humans; IMP Dehydrogenase; Leukemia, Promyelocytic, Acute; Molecular Structure; Phenotype; Ribavirin; Ribonucleosides; Time Factors; Tretinoin; Tumor Cells, Cultured

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Biomarkers, Tumor; Bucladesine; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Glycolipids; Lewis X Antigen; Mice; Phenotype; Teratoma; Tretinoin; Tumor Cells, Cultured

Repeated promotion of 7,12-dimethylbenz[alpha]anthracene-initiated cells in mouse skin with 12-O-tetradecanoylphorbol-13-acetate (TPA) induces them to grow as premalignant skin papillomas and some of these subsequently progress to carcinomas. In this study, we demonstrate that this TPA-induced progression of initiated cells to papillomas and carcinomas could be prevented by exposing them previously to weak promoting regimens or to agents that mimic TPA activity.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzoflavones; Benzyl Alcohols; Cell Transformation, Neoplastic; Cortisone; Dose-Response Relationship, Drug; Indoles; Mice; Mice, Inbred BALB C; Papilloma; Precancerous Conditions; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin; Vitamin E

Twenty-eight compounds were screened for chemopreventive activity by using a rat tracheal epithelial cell transformation inhibition assay. In this new assay, chemicals were tested for their ability to inhibit the formation of transformed rat tracheal epithelial cell colonies which arise following exposure to the carcinogen benzo(a)pyrene. The 15 positive compounds were N-acetylcysteine, bismuththiol, calcium glucarate, (+/-) catechin, diallyl disulfide, glycaric acid, D-glucaro-1,4-lactone, N-(4-hydroxyphenyl)retinamide, D-limonene, mesna, retinoic acid, rutin, quercetin, silymarin, and taurine. In examining the nature of compounds that inhibited rat tracheal epithelial cell transformation, several possible chemopreventive mechanisms appeared to be predominant: compounds that were positive (a) increased glutathione levels or enhanced conjugation; (b) increased cytochrome P-450 activity; (c) displayed nucleophilic activity; or (d) induced differentiation. Thirteen compounds were negative in the rat tracheal epithelial transformation inhibition assay: crocetin, difluoromethylornithine, ellagic acid, esculetin, enoxalone, ibuprofen, levamisole, nordihydroguaiaretic acid, L-2-oxothiazolidine-4-carboxylate, piroxicam, sodium butyrate, D-alpha-tocopherol acetate, and polyethylene glycol 400. It was evident from these results that this assay would not detect compounds that were (a) anti-promoting in nature; (b) glutathione inhibitors; (c) differentiation inhibitors; (d) O6-methylguanine inhibitors; (e) organ specific; or (f) inactive. The rat tracheal epithelial cell transformation inhibition assay appeared to identify chemopreventive compounds that act at early stages of the carcinogenic process.

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; DNA Damage; Epithelium; Fenretinide; Glucaric Acid; In Vitro Techniques; Quercetin; Rats; Trachea; Tretinoin; Vitamin E

Polyclonal antibodies were used to assay human embryonal carcinoma (EC), differentiating EC, yolk sac carcinoma, and teratoma cells for expression of viral early antigen (T-Ag) after infection with simian virus 40 (SV40). Cells of four EC lines were induced to differentiate by cultivation at low density or by exposure to retinoic acid or dimethyl sulfoxide. After infection with SV40, T-Ag was expressed by 1%, or less, of the cells (presumed to be differentiated derivatives) in only some EC cultures whereas the antigen was synthesized by a significant percentage of the yolk sac carcinoma, teratoma, and differentiating EC cells. Also, viral late proteins were produced by EC cells infected with human adenovirus type 7 (Ad7), and SV40 T-Ag was expressed by EC cells after infection with PARA, which is an Ad7-SV40 hybrid virus containing the SV40 T-Ag sequence controlled by Ad7 late regulatory sequences. Thus, T-Ag is not synthesized by the parental EC cells infected with SV40, but it is expressed in cultures of infected differentiated derivatives. The EC cells produce T-Ag, however, when expression of the viral protein is controlled by the Ad7 regulatory sequences in PARA particles. These results demonstrate that expression of T-Ag after infection with SV40 is an indicator of EC cell differentiation and also raise the possibility that, as in mouse EC cells infected with the virus, the SV40 regulatory sequences controlling T-Ag synthesis are not active in human EC cells.

Topics: Adenoviruses, Human; Animals; Antigens, Viral; Capsid; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Embryonal Carcinoma Stem Cells; Humans; Hybridization, Genetic; Male; Mice; Neoplastic Stem Cells; Simian virus 40; Testicular Neoplasms; Tretinoin; Tumor Virus Infections; Virus Replication

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.

Topics: Acetamides; Adenovirus Early Proteins; Cell Transformation, Neoplastic; Chloramphenicol O-Acetyltransferase; DNA, Neoplasm; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasms, Germ Cell and Embryonal; Oncogene Proteins, Viral; Testicular Neoplasms; Transfection; Tretinoin; Tumor Cells, Cultured

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

Two Hodgkin's Reed-Sternberg cell (H-RS) lines, HDLM-1 and KM-H2, have phenotypes and functional properties very similar to those of H-RS cells in tissues. These two types of cells were induced to differentiate with a combination of phorbol ester, retinoic acid, and extracellular matrix. The induced cells displayed the morphology of histiocytes or histiocytelike cells, with a small, round or oval, eccentric nucleus and abundant cytoplasm. In ultrastructural studies, many cytoplasmic projections and rugae were observed. These induced cells exhibited abundant cytoplasmic lysosomal enzymes, such as esterase, acid phosphatase, alpha 1-antitrypsin, or lysozyme. The histiocytic nature of these induced cells was further confirmed by the increased expression of many monocyte/histiocyte markers, including CD11b, CD11c, CD13, CD14, CD15, CD33, CD68, Mac387, and 1E9. In functional tests, the induced cells were shown to produce interleukin-1, tumor necrosis factor, macrophage colony-stimulating factor, and/or prostaglandin E2. Phagocytosis was detected in less than 5% to 10% of the cells when Candida albicans was added to cultures. The results strongly suggest that H-RS cells are related to cells of histiocyte lineage.

Topics: Adult; Antigens, Differentiation; Antigens, Neoplasm; Arachidonic Acids; Biological Factors; Cell Transformation, Neoplastic; Cytokines; Extracellular Matrix; Gene Rearrangement; Histiocytes; Hodgkin Disease; Humans; Ki-1 Antigen; Lymphocytes; Male; Phagocytosis; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

A recombinant N-ras oncogene, under the transcriptional control of a corticosteroid-inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N-ras-induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185-1192, 1988). We have employed the UR61 cell line as a model for ras oncogene-induced neuronal differentiation. In UR61 cells, dexamethasone-induced expression of the recombinant N-ras gene resulted in time-dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146-4149, 1987; Wolfman and Macara: Nature 325: 359-361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782-788, 1987) interactions between oncogene ras-coded p21 and protein kinase C (PK-C; Ca++/phospholipid-dependent protein kinase), we employed the protein kinase inhibitor H-8 (N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride) and phorbol 12,13-dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N-ras action. Treatment of UR61 cells with PDBu depleted cells of PK-C and failed to promote neurite outgrowth but enhanced N-ras-induced neurite outgrowth and ODC activity. H-8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N-ras-induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras-induced neurite outgrowth, suggesting that these two ras-triggered events are mechanistically independent. These findings suggest that certain actions of N-ras can occur in cells depleted of PK-C, and thus, the role of PK-C in ras-induced differentiation differs from its role in ras-induced mitogenesis and transformation.

Topics: Animals; Cell Differentiation; Cell Transformation, Neoplastic; Colforsin; Dexamethasone; Epidermal Growth Factor; Isoquinolines; Nerve Growth Factors; Neurons; Oncogene Protein p21(ras); Ornithine Decarboxylase; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Kinase Inhibitors; Rats; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

We have identified a tumour-related 43 kd cytoplasmic protein (LC/p43) using a monoclonal antibody against the total proteins of human hepatoma cell line PLC/PRF/5. LC/p43 is preferentially expressed in a variety of tumours of human and animal origin, whereas no expression was detected in several normal adult tissues tested. LC/p43 expression was induced in rodent fibroblasts upon transfection with several viral oncogenes. Expression in non-transformed peripheral blood lymphocytes could be induced by treatment with phytohaemagglutinin (PHA) and subsequent culture with interleukin II, whereas retinoic acid treatment of transformed cells caused a drastic reduction of the antigen in the cells. Sequence analysis of three tryptic peptides of LC/p43 revealed 50-70% homology to different domains of the eukaryotic and prokaryotic translation-elongation factors EF1-alpha and EF-Tu, respectively.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Cell Transformation, Neoplastic; Fibroblasts; Fluorescent Antibody Technique; Gene Expression; Humans; Immunoblotting; Interleukin-2; Liver Neoplasms; Liver Neoplasms, Experimental; Lymphocytes; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Peptide Elongation Factors; Phenotype; Phytohemagglutinins; Sequence Homology, Nucleic Acid; Transfection; Tretinoin

All-trans retinoic acid induces leukemic cells from patients with acute promyelocytic leukemia (M3) to differentiate in vitro to mature granulocytes which express the CD15 antigen and are capable of respiratory burst function. Of 35 M3 samples, only one failed to respond. In eight cases, we compared the efficacy of two naturally occurring isomers of retinoic acid, all-trans RA and 13-cis RA. Both isomers induce maximal differentiation at 10(-6) mol/L. The maximal response was maintained at 10(-7) mol/L for the all-trans but not for the 13-cis RA. We also observed that the metabolites 4-oxo-all-trans and 4-oxo-13-cis were effective at 10(-6) mol/L. This 1 order of magnitude difference in the in vitro differentiating potencies of all-trans RA and 13-cis RA in the blasts of promyelocytic leukemias predicts a difference in the clinical efficacy of the two drugs.

Topics: Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Isomerism; Leukemia, Promyelocytic, Acute; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured

The effects of TPA (12-0-tetradecanoylphorbol-13-acetate) and RA (retinoic acid) were investigated on the cell lines HL60 (acute promyelocytic leukemia) and K562 (erythroleukemia) and on cells from patients with several kinds of leukemia. There were 14 cases of acute lymphocytic leukemia (ALL), 2 cases of chronic lymphocytic leukemia (CLL), 23 cases of acute myeloid leukemia (M1-M7), 5 cases of chronic myelocytic leukemia in blast crisis (CML-BC) and 2 mixed leukemias. In almost all of the cases examined, after TPA exposure cells from patients with proven myeloid leukemia became adherent to the substrate, while lymphoid leukemia cells remained in suspension, allowing the differentiation of lymphoid from myeloid blasts. The only exception was in one case of CLL, which had cells that became adherent with long filamental projections. In addition, increased phagocytosis following TPA exposure permitted characterization of M7 as this was the only myeloid leukemia negative for phagocytosis. Further discrimination between the subtypes of myeloid leukemia could be based on the increased lysozyme production seen after TPA in M4 and M5. Esterase positivity allowed the discrimination of M1 cells, which were negative before and after TPA treatment. In agreement with the results of other authors, TPA and RA led to independent ways of differentiation, granulocytic-like lineage and monocytic-like cells being favored by RA and TPA, respectively. The capacity of the same cell to differentiate into more than one lineage, depending on whether RA or TPA was used, was only seen in the present study with M3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Transformation, Neoplastic; Diagnosis, Differential; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tetradecanoylphorbol Acetate; Tretinoin

Experimental carcinogenesis has led to a concept that defines two discrete stages in the development of skin tumors: (i) initiation, which is accomplished by using a mutagen that presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought about most commonly by repeated application of phorbol esters. We have created a transgenic mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas, some of which went on to develop squamous cell carcinomas and, more frequently, underlying sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor promoters and for assessing antitumor and antiproliferative agents.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Genes, ras; Keratinocytes; Mice; Mice, Inbred Strains; Mice, Transgenic; Plasmids; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Levels of epidermal growth factor (EGF) receptor expression vary widely among cell lines derived clonally from a chemically transformed population of rat liver epithelial cells. Retinoic acid (RA), a derivative of vitamin A that stimulates differentiation in a number of embryonal cell lines, increases the level of 125I-EGF binding in several clones of the transformed cell lines. One such cell line, GP6ac, which reverts to a less transformed phenotype when treated with RA, exhibited a 3-4-fold increase in surface EGF receptors with prolonged (2-5-day) RA exposure. The increase persisted as long as the cells were treated with RA. The increase in surface EGF receptors was due to induction of receptor biosynthesis, which occurred within 4 h at both the mRNA and protein levels and persisted until the RA was withdrawn. Paradoxically, the RA response was accompanied by an initial 40-50% decrease in 125I-EGF binding during the first 12 h of RA treatment. The decrease was due primarily to a reduction of receptor affinity. Since the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also decreases 125I-EGF binding and increases EGF receptor biosynthesis in GP6ac cells, we tested the effect of RA in cells depleted of protein kinase C by prolonged treatment (18 h) with 10 microM 12-O-tetradecanoylphorbol-13-acetate. The absence of protein kinase C did not affect the induction of receptor mRNA and protein or the decrease in binding during the early period of RA exposure. This indicates that RA induction of EGF receptor synthesis in GP6ac cells involves signaling pathways distinct from those utilized by phorbol esters.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Cell Line; Cell Transformation, Neoplastic; Cycloheximide; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Gene Expression; In Vitro Techniques; Methylnitronitrosoguanidine; Precipitin Tests; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.

Topics: Calcitriol; Cell Transformation, Neoplastic; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Intermediate Filaments; Lymphoma, Large B-Cell, Diffuse; Phagocytosis; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vimentin

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.

Topics: Animals; Benzimidazoles; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Embryonal Carcinoma Stem Cells; Endoderm; Fibroblasts; Humans; Neoplastic Stem Cells; Teratoma; Tretinoin

Differentiation induction therapy provides an alternative for treatment of patients with acute myeloid leukaemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytarabine (Ara-C) + dimethylformamide (DMF) on the differentiation of blasts from 24 AML patients was studied. Nonadherent mononuclear cells were cultured at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M RA, 10(-6) M Ara-C and/or 100 mM DMF alone and in combination with each other for 6 days in primary culture at 37 degrees C in a humidified incubator under 5% CO2. The triple combination of 10(-6) M RA + 10(-6) M Ara-C + 100 mM DMF induced 90% of blasts from 22 out of 24 AML patients to differentiate. These highly effective results justify a clinical trial of this triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Cytarabine; Dimethylformamide; Humans; Leukemia, Myeloid, Acute; Tretinoin

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transforma

Topics: Actins; Biomarkers, Tumor; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Flow Cytometry; Humans; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The effect of three retinoids, 13-cis-retinoic acid, arotinoid ethyl sulfone and retinal acetate, on methylcholanthrene (MCA) induced transformation of 10T1/2 cells was studied. It appears that 13-cis-retinoic acid and arotinoid ethyl sulfone prevent transformation in a direct way at the last stage of carcinogenesis. Retinal acetate, however, requires cell-to-cell contacts to inhibit the transformation of 10T1/2 cells.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Methylcholanthrene; Mice; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

E-5166 is a newly synthesized polyprenoic acid that has been reported to control epithelial differentiation and to have antiproliferative effects on various tumor cells in vitro. This study examined the effects of E-5166 on the proliferation of keratinocytes. Three kinds of keratinocytes were used: normal human keratinocytes, a human trichilemmoma-derived K-TL-1 cell line, and a Pam 212 cell line. Cell proliferation was measured by 3H-thymidine incorporation and also by determining cell numbers. E-5166 was found to have significant antiproliferative effects on each of the cell lines studied. When Pam 212 cells were cultured in the presence of E-5166, cell proliferation was inhibited in a dose-dependent fashion. The inhibitory effect appeared to be reversible, since removal of E-5166 from the culture medium resulted in a subsequent return of cell proliferation. For normal human keratinocytes and K-TL-1 cells, E-5166 inhibited 3H-thymidine incorporation in a dose-dependent fashion. Furthermore, E-5166 showed significantly stronger antiproliferative capacity than Ro 10-9359, one of the aromatic retinoids, on Pam 212 cells and normal human keratinocytes.

Topics: Animals; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Humans; Keratinocytes; Male; Mice; Tretinoin

The changes of protein kinases (PKs) in the nuclear matrix of HL-60 cells during the differentiation by retinoic acid (RA) were examined by in situ assay after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the nuclear matrix of HL-60, at least seven species of PKs (83, 63, 58, 46, 42, 38, and 31 kDa) were always detected. Among these PKs, 63 kDa PK was increased dramatically during differentiation by RA. Two-dimensional electrophoresis revealed that two species of 63 kDa PKs (pI 5.0 and 5.4) were presented in the nuclear matrix and increased similarly during the differentiation. This increase of 63 kDa PKs may be related to the lobulation of the nucleus and the nuclear matrix accompanying the differentiation by RA.

Topics: Cell Nucleus; Cell Transformation, Neoplastic; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Humans; Protein Kinases; Tretinoin; Tumor Cells, Cultured

Retinoic acid causes a significant inhibition of cell growth of the tumor cell line BA-HAN-1C. This growth inhibition is the same whether the cells are treated with a pulse dose of retinoic acid (RA) or continuously expand to RA. The determination of RA and its degradation products within the culture medium and in the cells showed that after 24 hours 13-cis-RA was the major retinoid in all cells (96 ng/10(6) cells); all-trans-RA represented 56 ng/10(6) cells. After 48 hours 4-hydroxy-RA and a small amount of 5,6-epoxy-RA was found in the cells and also in the culture medium. 4-hydroxy-RA increased up to 96 hours, whereas 13-cis- and all-trans-RA were not detectable in the cells after 96 hours. We conclude that the BA-HAN-1C cells take up and metabolize RA. Nonlinear fit analysis of the time behavior of the RA concentration in medium demonstrates that the RA uptake unexpectedly follows a mono-exponential time function. Discussion of the experimental results in connection with a proper compartment model shows that uptake and metabolism of RA cannot be described really by a first order kinetics. The mathematical analysis leads to a more complicated kinetic model with certain restrictions for the corresponding rate constants.

Topics: Animals; Cell Compartmentation; Cell Transformation, Neoplastic; Kinetics; Mathematics; Models, Theoretical; Rats; Rhabdomyosarcoma; Tretinoin; Tumor Cells, Cultured

We have analyzed the changes in the steady-state levels of ornithine decarboxylase (ODC) mRNA during differentiation of HL60 cells, a human promyelocytic leukemia cell line. Induction of differentiation with either retinoic acid, dimethylsulfoxide, dibutyryl cAMP or dihydroxy-vitamin D3 resulted in a decrease of the cellular content of ODC RNA. Such a decrease occurred late after induction and coincided with the slowing of cell growth activity and with the expression of a cell surface differentiation marker (CD11b antigen). In contrast, the inducers provoked a rapid reduction of c-myc RNA levels, which preceded both the slowing of cell growth and the expression of the differentiation marker. When the cells were treated with a phorbol ester (TPA), the down-regulation of ODC was preceded by a transient increase in the steady-state levels of this RNA. However, such an increase was not observed with other inducers. The possible significance of these results in relation to the control of HL60 cell differentiation is discussed.

Topics: Bucladesine; Calcitriol; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Ornithine Decarboxylase; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin

The present study reports light and electron microscopic observations of hamster cheek pouch epithelium exposed to 25 micrograms DMBA (DMBA = 9,10 Dimethyl, 1,2 Benz(a)-nthracene, RA = Retinoic Acid) and 25 micrograms DMBA along with 25 micrograms, 50 micrograms and 100 micrograms retinoic acid. Significant delay in tumour induction was observed in the animals treated with DMBA + retinoic acid. DMBA + retinoic acid treated cheek pouch developed papillary epidermoid carcinomas which were less invasive and less keratinized than only DMBA treated animals. At cellular level DMBA treated animals showed keratinized cells with thick bundles of tonofilaments, broken basement membrane, wide intercellular spaces and loss of desmosomal attachments, whereas animals treated with DMBA + retinoic acid showed decrease in the intercellular spaces, maintenance of basement membrane and intracellular organelles with suppression of keratinization, indicating the differentiating effect of retinoic acid on DMBA transformed cells of hamster cheek pouch.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Papillary; Carcinoma, Squamous Cell; Cell Nucleus; Cell Transformation, Neoplastic; Cheek; Cricetinae; Cytoplasm; Epithelium; Female; Male; Mesocricetus; Microscopy, Electron; Mouth Mucosa; Mouth Neoplasms; Neoplasm Invasiveness; Tretinoin

Retinoic acid (RA)-induced differentiation of human teratocarcinoma (T2) cells results in a change from a normally non-permissive phenotype for human cytomegalovirus (HCMV) infection to cells which are fully permissive. We have used this system to analyse factors associated with differentiation which may regulate HCMV gene expression. Differentiation of T2 cells results in an increase of c-ras expression. Consequently, we have introduced ras expression vectors into T2 cells. We find that, as with RA induction, transfection of T2 cells with oncogenic human Ha-ras results in cells which are permissive for HCMV infection and gene expression. However, unlike RA which induces a cessation of cell proliferation and terminal differentiation, ras transfection only appears to result in changes associated with early events in RA-induced differentiation of T2 cells.

Topics: Cell Transformation, Neoplastic; Cytomegalovirus; Cytomegalovirus Infections; Gene Expression Regulation; Genes, ras; Humans; Phenotype; Teratoma; Transfection; Tretinoin; Tumor Cells, Cultured

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.

Topics: Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Gene Expression; Humans; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasms, Germ Cell and Embryonal; Phosphotransferases; Receptors, Cell Surface; Receptors, Somatomedin; Teratoma; Tretinoin; Tumor Cells, Cultured

The relationship between plasminogen activator (PA)/plasminogen activator inhibitor (PAI) activity and morphological differentiation was investigated in human neuroblastoma (NB) cells treated with retinoic acid (RA). Conditioned medium from nine NB cell lines and one closely related neuroepithelioma line was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. All NB cell lines were shown to secrete urokinase (UK)-type PA (mol. wt., 52 kDa), and all except two produced tissue PA (mol. wt., 65 kDa). Identification of the PAs was made based on molecular weight and sensitivity to inhibition by anti-UK and anti-tPA antibodies. Several cell lines expressed PA inhibitory molecules; two molecular-weight forms were observed (35 and 40 kDa) in different cell lines. Complex formation with [125]I-labelled proteases revealed specific binding with UK and trypsin but not thrombin, plasmin, or kallikrein. After treatment for 6 days with 1 microM RA, six of the cell lines exhibited an increase in cell-associated and/or secreted tPA activity, corresponding to morphological differentiation of the cells as manifested by extensive neurite outgrowth. A decrease in UK and UK-complex secretion was observed in several of these cell lines. Three cell lines exhibiting no detectable morphological alterations with RA treatment also showed no dramatic changes in PA/PAI activity. These results suggest that morphological differentiation of NB cells may be associated with alterations in the regulation of PA activity.

Topics: Cell Line; Cell Transformation, Neoplastic; DNA, Neoplasm; Drug Screening Assays, Antitumor; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Neuroblastoma; Plasminogen Activators; Plasminogen Inactivators; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We have examined the effects of all-trans retinoic acid (RA) on confluent holding recovery (cell survival) and on the fixation of initial transformational damage expressed as the ultimate yield of transformed foci following X-irradiation of density-inhibited cultures of Balb/3T3 cells. Non-cytotoxic concentrations of RA suppressed both recovery of potentially lethal damage and neoplastic transformation in a dose-dependent manner when added for 24 h during post-irradiation confluent holding after a dose of 5 Gy. At 100 microM, RA inhibited the fixation of initial transformational damage by 80%. These findings are discussed in terms of the hypothesis that retinoids may allow a selective enhancement of the inactivation of certain irradiated tumor cells in vivo while reducing the risk of secondary malignancies in successfully treated patients.

Topics: Animals; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Mice; Mice, Inbred BALB C; Tretinoin; X-Rays

In 1986 we reported on the capacity of cis-diaminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 micrograms/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.

Topics: Antigens, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Cisplatin; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Retinoids that cause inhibition of methylcholanthrene-induced neoplastic transformation of C3H/10T1/2 cells enhance gap-junctional communication in carcinogen-initiated cells. Dose-response studies using retinoids of diverse structures and potency demonstrated a good correlation between these two events. Junctional permeability was enhanced by retinol and tetrahydrotetramethylnaphthalenyl propenylbenzoic acid (TTNPB) at concentrations from 10(-10) to 10(-6) M, and by retinoic acid between 10(-8) and 10(-6) M, the same concentrations that inhibited neoplastic transformation. Retinoic acid inhibited permeability at 10(-10) M, at which concentration transformation was enhanced. Retinoids caused similar alteration sin communication in parental 10T1/2 cells. Communication between initiated and 10T1/2 cells was not influenced by TTNPB. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited junctional communication in initiated cells, in 10T1/2 cells and between these two cell lines. After repeated exposure of 10T1/2 cells to TPA only retinoid-enhanced communication was blocked; in contrast, basal communication became refractory. It is proposed that much of the chemopreventive action of retinoids can be explained by the enhanced junctional communication of growth regulatory signals.

Topics: Animals; Benzoates; Cell Communication; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cells, Cultured; Intercellular Junctions; Kinetics; Methylcholanthrene; Mice; Mice, Inbred C3H; Retinoids; Tretinoin; Vitamin A

Retinoic acid (RA) and butyric acid (BA) were investigated for their effect on in vitro migration of highly metastatic murine B16a melanoma cells. These potential antitumor agents are known to alter the cytoskeleton. Our initial studies determined the 72 h cytostatic/cytotoxic concentrations of RA (1 X 10(-6) M 1 greater than 1 X 10(-5) M) and BA (1.5 mM)/ greater than 2.0 mM). Cytostasis by RA and BA was confirmed by autoradiography and radioisotope incorporation. For migration assays, cells were plated on 3 and 5 microns diameter pore polycarbonate membranes. Complete media was added containing RA or BA at time of plating. For BA pretreatment studies, BA was added to cells for 72 h prior to plating cells in fresh BA on the membranes. Top and bottom surface of the membranes were examined after 72 h of incubation by scanning electron microscopy. Although RA and BA induced cells on top of the membrane to change morphology as shown by phase, transmission and scanning electron microscopy, only BA enhanced the deformability of cells to allow for passage through the 3 micron diameter pores. Butyric acid enhanced migration through 3 micron diameter pore membrane by 511%. For 5 micron diameter pore membranes, 55.2% of the plated number of untreated early passage cells migrated to the bottom surface as compared to 57.3% for BA-treated cells and 14.9% for RA-treated cells. However, if cellular proliferation over the 72 h period was factored in, BA increased migration by 456% over the controls and pretreatment of cells with BA for 72 h prior to plating increased migration by 893%. Without considering proliferation, RA inhibited migration by 75% over controls. The decrease in migration observed in RA-treated cells was due to an inhibitory effect on cellular migration and a decrease in proliferation.

Topics: Animals; Butyrates; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Cytoskeleton; Melanoma; Mice; Microscopy, Electron; Microscopy, Electron, Scanning; Thymidine; Tretinoin; Tumor Cells, Cultured; Uridine

In order to investigate further the role of gap-junctional intercellular communication in the process of cell transformation, we examined the effects of chemicals that modulate gap-junctional communication on the induction and maintenance of transformed foci in BALB/c 3T3 cells. When dibutyryl cyclic AMP, retinoic acid, fluocinolone acetonide, or dexamethasone was added during the induction of cell transformation by standard (3-methylcholanthrene alone) or two-stage (low dose of 3-methylcholanthrene plus phorbol ester) protocols, there was a significant decrease in the number of transformed foci. When BALB/c 3T3 cells are transformed, there is selective intercellular communication between transformed and between surrounding nontransformed cells: transformed cells communicate among themselves but not with surrounding normal cells. Addition of dibutyryl cyclic AMP, retinoic acid, fluocinolone acetonide, or dexamethasone to culture dishes in which transformed foci were present induced communication between transformed cells and surrounding normal cells. In the continuous presence of these chemicals, there was a clear decrease in the number of transformed foci. These chemicals therefore appear capable of reestablishing intercellular communication between transformed and nontransformed cells and of diminishing the number of transformed foci. However, when transformed cells were isolated and placed in culture dishes at clonal density in the presence of these chemicals, there was hardly any decrease in the number of transformed colonies, suggesting that the chemicals cannot revert the phenotype of transformed cells in the absence of normal cells. These results suggest that chemicals that modulate intercellular communication not only inhibit the induction of transformed foci but also revert transformed cells to the normal phenotypes by establishing intercellular communication with surrounding normal cells.

Topics: Animals; Bucladesine; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Dexamethasone; Fluocinolone Acetonide; Intercellular Junctions; Mice; Mice, Inbred BALB C; Tretinoin

Treatment with 10(-5) M retinoic acid causes loss of anchorage-independent growth in src-transformed RR1022 cells but not in ras-transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10(-5) M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10(-5) M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage-independent growth in these two cell lines.

Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Genes, ras; Kirsten murine sarcoma virus; Phosphates; Phospholipids; Protein Kinase C; Tretinoin

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Topics: Acetylcholine; Animals; Antibodies, Monoclonal; Cell Line; Cell Transformation, Neoplastic; gamma-Aminobutyric Acid; Mice; Neurons; Neurotransmitter Agents; Teratoma; Tretinoin

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Dimethylformamide; Dose-Response Relationship, Drug; Drug Interactions; Fibronectins; Mice; Peptides; Time Factors; Transforming Growth Factors; Tretinoin

Topics: Adolescent; Adult; Alkaloids; Cell Transformation, Neoplastic; Child; Female; Harringtonines; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Tumor Cells, Cultured

A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of [3H]thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested (epidermal growth factor, platelet-derived growth factor, transforming growth factor-beta, and retinoic acid) is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; Growth Substances; Insulin; Kidney; Kinetics; Peptides; Platelet-Derived Growth Factor; Rats; Transforming Growth Factors; Tretinoin

The rat embryo fibroblast focus assay is used to evaluate the transforming potential of several oncogenes. The sensitivity of this assay increased fivefold when retinoic acid was added to tissue culture media. Retinoic acid probably acts by selectively inhibiting the proliferation of nontransformed cells.

Topics: Animals; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Embryo, Mammalian; Oncogenes; Plasmids; Rats; Tretinoin

Previous studies have shown that all-trans-retinoic acid fails to inhibit chemically induced transformation in 10T1/2 cells except at toxic levels, whereas retinol and many synthetic retinoids are potent inhibitors. In contrast, in many systems retinoic acid is a more effective modulator of differentiation and carcinogenesis than is retinol. In any attempt to explain this anomaly, we have studied the differential metabolism of retinoic acid and retinol by 10T1/2 cells and by their initiated and transformed derivatives, and have also reexamined these cells for the presence of retinoid-binding proteins. Whereas retinoic acid was depleted from the medium bathing 10T1/2 and initiated 10T1/2 cells within 48 h of treatment, retinol was concentrated 500-fold by these cells, and disappeared from the culture medium no faster than from cell-free medium. Retinoic acid metabolism by a number of transformed cell lines varied widely. There was no apparent correlation between metabolizing ability and transforming agent (methylcholanthrene, X-rays, fission spectrum neutrons, and plasmid oncogene transfection). Uptake of retinoic acid was seen in all cell lines and was not correlated with its metabolism. Retinol was metabolized minimally by all cell lines tested; metabolism of retinol was not correlated with retinoic acid metabolizing ability. Retinoic acid-induced growth inhibition and cytotoxicity were not correlated with metabolizing ability, suggesting that the rate of metabolism of retinoic acid is not a major determinant of its acute biological effects. Using sensitive radioimmunoassays, cellular retinoic acid- (CRABP) and retino-binding proteins (CRBP) were both detected in 10T1/2 and initiated 10T1/2 cells. CRABP levels of about 16 ng/10(6) cells were about 4-fold higher than CRBP levels. Therefore, lack of CRABP does not explain the failure of retinoic acid to inhibit carcinogen-induced transformation in these cells. These studies suggest that the inability of retinoic acid to inhibit transformation in the 10T1/2 cell system may be due to its rapid metabolism and clearance from the medium. On the other hand, the high cellular uptake and stability of retinol in these cells could be an important factor in the inhibition of neoplastic transformation by this retinoid.

Topics: Carrier Proteins; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Receptors, Retinoic Acid; Time Factors; Tretinoin; Vitamin A

The effects of all-trans retinoic acid (RA) on the growth and biochemical properties of five clonal strains of neoplastically transformed rat liver epithelial cells were studied. These cell strains were derived clonally from a single line of normal diploid rat liver epithelial cells that had been transformed by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The results show that RA induces inconsistent alterations in selected phenotypic properties of these five different cell strains. Retinoic acid either depressed, enhanced or produced no effect on the colony-forming ability in soft agar, on the activity of gamma-glutamyl transpeptidase, on the amount of cell-associated fibronectin, and on the binding capacity of 125I-epidermal growth factor (EGF). The only consistent correlation observed among cell strains was between the cellular ability to grow in soft agar and the amount of cell-associated fibronectin. Enhancement of anchorage-independent growth by retinoic acid was not mediated through changes in the number of EGF receptors. Our data demonstrate that the responses to retinoic acid of clonal subpopulations of chemically transformed rat liver epithelial cells are inconsistent, even when the clonal subpopulations are derived from a common precursor.

Topics: Animals; Cell Transformation, Neoplastic; Epithelium; ErbB Receptors; Fibronectins; gamma-Glutamyltransferase; Liver; Methylnitronitrosoguanidine; Rats; Tretinoin

The purpose of the present studies was to determine the influence of the chemopreventive agents selenium, 4-(hydroxyphenyl)-retinamide (4-HPR) and beta-carotene, on functional differentiation (lactogenesis) of the mouse mammary gland cells. The hormone-induced expression of the milk-protein genes, beta-casein, epsilon-casein and the whey acidic protein (WAP) was used as molecular marker of differentiation of the mammary cells in organ culture medium containing insulin, prolactin, aldosterone and hydrocortisone. Quantitative determination of the cellular concentration of the respective mRNA was ascertained by molecular hybridization of RNA to the specific cloned cDNA probes using both the solution and the filter hybridization methods. Selenium at 100 nm concentration caused a pronounced inhibition of accumulation of the respective mRNAs. The retinoid, 4-HPR, also caused a dose-dependent inhibition of expression of these mRNA sequences. In contrast, concentrations of the 3 mRNAs in beta-carotene-treated glands remained similar to those observed in glands cultured in medium containing the hormones and hexane (the latter being the solvent for beta-carotene). The significant antagonistic action of selenium and 4-HPR, however, was reversible after removal of the chemicals from the culture medium. Thus, among the 3 chemicals tested, selenium and retinoid can cause an adverse effect on functional differentiation (lactogenesis) of the mammary cells. This inhibitory effect, however, was reversible. beta-Carotene, on the other hand, caused no apparent antagonistic effect on expression of the milk-protein genes in the isolated whole mammary organ in culture.

Topics: Actins; Animals; beta Carotene; Carotenoids; Cell Transformation, Neoplastic; Female; Fenretinide; Hormones; In Vitro Techniques; Mammary Glands, Animal; Mice; Milk Proteins; RNA, Messenger; Selenium; Tretinoin

Retinoic acid was found to increase the activity of cytidine monophosphosialic acid:lactosylceramide sialyltransferase activity in a nontransformed clonal hamster cell line, NIL 8, and a virally transformed clone, NIL 8-HSV. The potent tumor promoter phorbol-12-myristate-13-acetate (PMA) had no significant effect on sialyltransferase activity in NIL 8 cells but stimulated this activity almost 6-fold when added to NIL 8-HSV cells. There was a synergistically additive effect on sialyltransferase activity when PMA was added to NIL 8 cells in concert with retinoic acid. On the other hand neither PMA nor retinoic acid had an appreciable effect on two other glycosyltransferases measured, uridine diphospho-N-acetylgalactosamine:globotriaosylceramide N-acetylgalactosaminyl-transferase and uridine diphosphogalactose:asialoagalactofetuin galactosyltransferase. Examination of sialyltransferase activity in a human epidermoid carcinoma cell line showed a large increase in enzyme activity in response to retinoic acid administration. Two nontransformed hamster cell lines had less basal sialyltransferase activity but also showed marked elevations after retinoic acid treatment. It is proposed that one of the molecular mechanisms underlying the biological effects of retinoic acid and PMA may be an increase in sialyltransferase activity. Possible regulatory mechanisms are discussed.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Galactosyltransferases; Kinetics; Retroviridae; Sialyltransferases; Tetradecanoylphorbol Acetate; Tretinoin

The present study was designed to determine the effects of dietary 13-cis-retinoic acid and retinyl palmitate on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Female CD-1 mice were initiated with 150 nmol of 7,12-dimethylbenz(a)anthracene and promoted twice weekly with 8 nmol of TPA. Diets supplemented with retinyl palmitate to yield 60,000 or 200,000 IU or 700,000 for 5 wk followed by 350,000 IU per kg of diet (700,000/350,000) fed to mice during tumor promotion resulted in 9%, 37%, and 65% inhibition of the papilloma yield, respectively, at 21 wk of promotion. Although topical applications of 13-cis-retinoic acid have been almost as effective as retinoic acid in preventing the appearance of mouse skin tumors, dietary 13-cis-retinoic acid at 200,000 or 700,000 IU per kg of diet resulted in no reduction in papilloma yield but did result in a dose-dependent decrease in the tumor burden (weight of tumors per mouse). Therefore, dietary retinyl palmitate yielded a dose-dependent inhibition of the number and weight of tumors promoted by TPA, whereas dietary 13-cis-retinoic acid resulted in a decrease in weight but not in number of tumors promoted by TPA.

Topics: Administration, Cutaneous; Animals; Cell Transformation, Neoplastic; Diet; Diterpenes; Dose-Response Relationship, Drug; Female; Isotretinoin; Mice; Papilloma; Retinyl Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Vitamin A

Topics: Animals; Benzo(a)pyrene; Biotransformation; Cell Transformation, Neoplastic; Chromatography, High Pressure Liquid; DNA; Free Radicals; Hydrogen Peroxide; Isotretinoin; Models, Chemical; Oxidation-Reduction; Peroxides; Stereoisomerism; Tretinoin

UV-TDTx cells are cloned from foci arising after C3H10T1/2 cells are sequentially exposed to u.v. irradiation followed by tetradecanoylphorbol acetate (TPA). When grown in pure culture, UV-TDTx cells appear transformed. Co-culture with C3H10T1/2 cells suppresses focus formation by the UV-TDTx cells. In the presence of TPA, however, focus formation by UV-TDTx cells occurs in C3H10T1/2 co-cultures. We now demonstrate that only tumor promoters that activate protein kinase C (TPA, teleocidin) can reverse C3H10T1/2 suppression of UV-TDTx focus formation in co-culture; other promoters (diethyl stilbestrol, dioxin, saccharin, cadmium) are inactive. Retinoic acid, a potent inhibitor of many biological effects of TPA, blocks the action of TPA in UV-TDTx:C3H10T1/2 co-cultures. Focus formation by UV-TDTx cells in co-culture is dependent on the size of the UV-TDTx colony at confluence; if the UV-TDTx colony is below a minimal size when the co-cultures reach density-dependent growth arrest, suppression of focus formation by C3H10T1/2 cells occurs even in the presence of TPA. Finally, TPA must be present prior to confluence to relieve suppression of focus formation. If TPA is added to co-cultures after density arrest, UV-TDTx cells will not subsequently form foci.

Topics: Cell Count; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Humans; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin

Tumor-producing substances that promote lipolysis in vitro may also account for fat mobilization in cachectic cancer patients. Cachexia might improve if this lipolytic action of cancer cells could be halted. This study examined the lipolytic activities of media from four tumor cell lines after treatment with retinoic acid (RA), a cell differentiation inducer. An in vitro adipocyte bioassay measured lipolysis. All four tumor cell lines were intrinsically lipolytic, with elevated baseline lipolytic activities relative to fibroblast-conditioned controls (128% to 287% of control, p less than 0.05). After a 2-week exposure to RA in culture medium followed by 3 days of continued growth in fresh medium, two of four cell lines (both rat prostatic adenocarcinomas) showed significantly reduced lipolytic activities (16% and 61% of corresponding untreated controls, p less than 0.05). These reductions in lipolytic activity after RA treatment were not generalized phenomena; nor were they simply caused by cell differentiation, as the other cell lines (human malignant melanoma and human ovarian teratocarcinoma) showed no reductions despite evidence of cell differentiation. No effect on lipolytic activity was seen after only a 24-hour exposure to RA. We conclude that RA can affect the lipolytic activity of certain tumor cells in vitro, perhaps by influencing tumor-producing lipolytic factor(s).

Topics: Adenocarcinoma; Animals; Cell Line; Cell Transformation, Neoplastic; Female; Fibroblasts; Humans; Lipolysis; Male; Melanoma; Neoplasms; Rats; Teratoma; Time Factors; Tretinoin

The growth of various chemically and virally transformed cell types in culture is inhibited when they are in contact with normal cell types. We show that this growth inhibition is contingent on the presence of junctional communication between the normal and transformed cells (heterologous communication), as probed with a 443 dalton microinjected fluorescent tracer. In cell combinations where heterologous communication is weak or absent there is no detectable growth inhibition; the inhibition appears when communication is induced by cyclic AMP-dependent phosphorylation, and only then. In cell combinations where heterologous communication is spontaneously strong, the growth inhibition is present, but it is abolished when the communication is blocked by retinol or retinoic acid. The cell-to-cell membrane channels of gap junctions are the likely conduits of the signals for this growth control.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Animals; Arvicolinae; Cell Communication; Cell Transformation, Neoplastic; Colforsin; Contact Inhibition; Cyclic AMP; Intercellular Junctions; Ion Channels; Mice; Rats; Tretinoin; Vitamin A; Xanthines

We have studied the regulation of lysosomal glycosidases during morphological differentiation of NB2a neuroblastoma cells. Cells treated with dibutyryl cAMP induced axon-like neuritis and showed a 2-4 fold increase in the activity of 6 lysosomal glycosidases, reaching their highest level after 5 days of treatment. Cells treated with retinoic acid, which induced dendrite-like neurites, did not show significant changes in the glycosidases activity although cell proliferation was also inhibited. There was no change in the pattern of the enzyme secretion during the dibutyryl cAMP treatment and morphological analysis using electron microscopy and cytochemical staining with acid phosphatase indicated the presence of lysosomes in the induced neurites.

Topics: Animals; Bucladesine; Cell Line; Cell Transformation, Neoplastic; Glucuronidase; Glycoside Hydrolases; Lysosomes; Mice; Microscopy, Electron; Neuroblastoma; Staining and Labeling; Tretinoin

By different experimental approaches in culture, we obtained convergent responses to 13 cis-retinoic acid (RA) in rat liver epithelial cell lines. We showed that the degree of transformation of the cells which had already been spontaneously transformed in culture, could be enhanced, since the capacity of these cells both to grow in soft agar and to express gamma-glutamyl transpeptidase increased markedly. We also showed that RA acted synergistically with a promoter, 12-tetradecanoyl-phorbol-13-acetate (TPA), as regards the promoter's property of blocking the metabolic cooperation between cells. RA did not, however, trigger cell transformation in the lines which had not yet been transformed, and unlike TPA, did not by itself inhibit intercellular communications. On the other hand, in our in vivo experiments, it appeared that RA, by slowing down the growth of a transplanted rat hepatoma, might have a slightly protective effect against tumour development.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Drug Synergism; gamma-Glutamyltransferase; Liver; Liver Neoplasms, Experimental; Rats; Rats, Inbred F344; Tetradecanoylphorbol Acetate; Tretinoin

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Insulin; Kidney; Mice; Mitosis; Peptides; Platelet-Derived Growth Factor; Rats; Receptors, Cell Surface; Transforming Growth Factors; Tretinoin

Human teratocarcinoma stem cells are nonpermissive for human cytomegalovirus (HCMV) but become permissive after being induced to differentiate by treatment with retinoic acid. We show that in uninduced teratocarcinoma stem cells, and also in transformed human 293 cells expressing adenovirus E1a gene products, the HCMV immediate-early (IE) 68,000-molecular-weight polypeptide (68K polypeptide) was not expressed, and consequently input viral genomes were not replicated. However, after differentiation of the teratocarcinoma cells, synthesis of the HCMV IE 68K polypeptide was induced, and viral DNA replication occurred. In contrast to our observations for HCMV, simian cytomegalovirus (SCMV) displayed constitutive expression of its analogous IE 94K polypeptide, and the input SCMV genomes were replicated in both uninduced stem cells and 293 cells. Since little, if any, HCMV IE RNA was detectable in human teratocarcinoma or 293 cells after infection under IE conditions, we suggest that a direct transcriptional block to permissivity occurs in these cells. The presence of tandemly repeated sequences which bind nuclear factor I protein in the promoter for the SCMV IE 94K polypeptide gene but not in the promoter for the HCMV IE 68K polypeptide gene may allow the expression of the simian but not of the human IE gene product in transformed cells.

Topics: Adenoviruses, Human; Animals; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cytomegalovirus; DNA Replication; Embryonal Carcinoma Stem Cells; Genes, Viral; Humans; Neoplastic Stem Cells; Promoter Regions, Genetic; Teratoma; Transcription, Genetic; Tretinoin; Viral Proteins; Virus Replication

All-trans retinoic acid (10(-5) M) added at seeding reduces the growth rate and saturation density of normal human embryonic lung fibroblasts of two lines (WI-38 and IMR-90) and similarly inhibits growth of SV40-transformed WI-38 cells (VA13A). The growth inhibitory effects of retinoic acid do not show serum dependency, and the viability of treated cells is 95-99% of controls. Old populations of WI-38 cells (cells at high population doubling levels) are more sensitive to the effects of retinoic acid than are young populations (cells at low population doubling levels), and population life span is reduced by continuous exposure to retinoic acid. When retinoic acid is combined with the glucocorticoid hydrocortisone, inhibition of VA13A cell growth is increased, whereas the retinoic acid-induced inhibition of normal cells is decreased. VA13A cells treated with retinoic acid alone, or in combination with hydrocortisone, exhibit a reversion to a more elongated, fibroblast-like appearance. This paper discusses the clinical implications of the relationship between retinoic acid and hydrocortisone.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Embryo, Mammalian; Fibroblasts; Humans; Hydrocortisone; Microscopy, Electron, Scanning; Simian virus 40; Tretinoin

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1 alpha,25(OH)2D3, like phorbol ester tumor promoters, induces anchorage-independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1 alpha,25(OH)2D3 at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorage-independent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1 alpha,25(OH)2D3 in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1 alpha,25(OH)2D3: other derivatives of vitamin D3 also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1 alpha,25(OH)2D3. JB6 cells were found to contain a receptor specific for 1 alpha,25(OH)2D3 with a Kd of 55.7 pM and Nmax of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1 alpha,25(OH)2D3 also contained the receptor. DNA-cellulose chromatography showed that a 1 alpha,25(OH)2D3-receptor complex interacted with DNA. In contrast to 1 alpha,25(OH)2D3, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1 alpha,25(OH)2D3 when applied with it.

Topics: Animals; Calcitriol; Carcinogens; Cell Adhesion; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; DNA; DNA Replication; DNA-Binding Proteins; Epidermal Cells; Mice; Receptors, Calcitriol; Receptors, Steroid; Tetradecanoylphorbol Acetate; Tretinoin

We found that monolayer cultures of F9 cells induced to differentiate with trans-retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step.

Topics: Acetamides; Bromodeoxyuridine; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Fluorescent Antibody Technique; Laminin; Phenotype; Teratoma; Tretinoin

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.

Topics: Animals; Avian Sarcoma Viruses; Cell Division; Cell Line; Cell Transformation, Neoplastic; Embryo, Mammalian; Fibroblasts; Genes; Genes, Viral; Guinea Pigs; Kinetics; Oncogene Protein p21(ras); Oncogene Protein pp60(v-src); Oncogene Proteins, Viral; Oncogenes; Ornithine Decarboxylase; Retroviridae Proteins; Tetradecanoylphorbol Acetate; Tretinoin

Using C3H/10T-1/2 mouse fibroblasts, we tested whether polyprenoic acid (E-5166) inhibits radiogenic and chemically induced transformation in vitro. Our results show that E-5166 markedly inhibits transformation by X rays and benzo[a]pyrene in a dose-related manner. Maximum inhibition was observed when cells were pretreated with E-5166 prior to carcinogen exposure, and lesser inhibition when E-5166 treatment followed carcinogen exposure. These results indicate that E-5166 can serve as a radio-protective and chemopreventive agent with anticarcinogenic potential.

Topics: Animals; Antineoplastic Agents; Benzo(a)pyrene; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Fibroblasts; Mice; Tretinoin

Retinoic acid has been reported to act as an inhibitor and as an enhancer of mouse skin carcinogenesis in vivo. However, no in vitro cell transformation model has been reported to be sensitive to both effects. In an attempt to provide such a model, the effect of retinoic acid on an early step in carcinogen-induced transformation of mouse epidermal cell line 271c was measured using a recently described assay. The step observed is altered response to extracellular Ca2+ as an epidermal terminal differentiation signal. In six out of twelve experiments retinoic acid increased the frequency of altered colonies resulting from treatment with three chemical carcinogens. The enhancement effect was stronger after DMBA treatment than MNNG or MCA, resulting in up to a 13.7-fold increase in the frequency of colonies exhibiting altered terminal differentiation (TF). On the other hand, up to a 10-fold decrease in TF was observed in other experiments. Both the enhancement and inhibitory effects were greater at the higher doses of retinoic acid tested in the range of 10(-10) - 10(-7) M. Variations in cloning efficiency or surviving colony density did not account for the effects on TF. Enhancement effects tended to be observed at lower doses of carcinogen, or in experiments in which TF resulting from treatment with carcinogen alone was in the lower range observed. However, the factors determining each effect have yet to be defined. The enhancement effect of retinoic acid was not merely suppression of the phenotypic endpoint of the in vitro assays, because treatment of carcinogen-altered cells with retinoic acid or TPA in vitro also enhanced their tumorigenicity in vivo compared to acetone controls. These findings suggest that studies of the determinants of retinoid activity should be a prerequisite to their use in chemoprevention.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Cell Line; Cell Transformation, Neoplastic; Cocarcinogenesis; Dose-Response Relationship, Drug; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

The effects of retinoic acid on a transformed mouse embryo fibroblast cell line (AKR-MCA) were examined. Treatment with retinoic acid restored a non-transformed phenotype to this transformed cell line in a dose dependent manner. Retinoic acid (RA) treated AKR-MCA cells showed a non-transformed morphology, a slower growth rate, and did not grow with anchorage independence. A 38,000 Da protein was phosphorylated to a high degree in the AKR-MCA transformed cell line compared to the non-transformed AKR-2B cell line. RA treatment greatly reduced the level of phosphorylation of this protein in AKR-MCA cells. Growth arrested AKR-MCA cells showed a mitogenic response to nutrient replenishment, but not to epidermal growth factor (EGF). Treatment of AKR-MCA cells with RA restored their ability to respond to EGF while the response to nutrient replenishment was lost. This pattern of growth control was similar to that of the non-transformed AKR-2B cells.

Topics: Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; DNA Replication; Epidermal Growth Factor; Mice; Phosphoproteins; Tretinoin

Retinoic acid (RA) inhibited transformed mouse L-929 and human WISH cell induction of interferon alpha/beta production by nonsensitized mouse spleen cells. The RA effect was both time and dose dependent and acted in near physiologic concentrations. The results suggest that the effect is due to a modulation of a previously described transformed cell surface associated glycoprotein IFN inducer.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Interferon Type I; Kinetics; Mice; Time Factors; Tretinoin

Anchorage-independent growth in vitro is strongly correlated with cellular malignancy in vivo and it has been shown that retinoic acid (RA; a vitamin A analog) inhibits anchorage-independent growth of a wide variety of oncogenically transformed cells (RA-sensitive cells). We report here that decreased or lack of phosphorylation of a group of low molecular weight (20-30 kD) cell surface membrane proteins, particularly one of Mr 28 kD, correlates strongly with RA-induced loss of anchorage-independent growth of RA-sensitive cells. Our studies also show that this group of proteins are not phosphorylated in non-transformed cells which do not grow in an anchorage-independent manner. Analysis of [35S]methionine-labeled proteins revealed that these polypeptides are present in both RA-treated and untreated cell surface membranes. This suggests that modulation of phosphorylation rather than lack of synthesis of these proteins is correlated with anchorage regulation of cells. V8 protease mapping of the 28 kD phosphoprotein from transformed cells, irrespective of their origin or of transforming agents, revealed complete fragment homology. Furthermore, the 28 kD phosphoprotein was found to be phosphorylated exclusively at threonine residues. The data obtained from this study suggest that the ability of cells to grow without anchorage is correlated with the phosphorylation of a group of cell surface membrane proteins and RA inhibits anchorage-independent growth by interfering with the phosphorylation rather than synthesis of these proteins.

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Humans; Membrane Proteins; Molecular Weight; Phosphoproteins; Phosphorylation; Tretinoin

Retinoic acid (RA) treatment of rat tracheal epithelial (RTE) cells, pre-exposed to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), inhibited transformation in a dose-dependent manner. Treatment with RA at concentrations ranging from 3-33 nM reduced the MNNG-induced transformation frequencies by 13-81% and in some experiments by greater than 90%. RA treatment for only 3 days caused 65-75% inhibition of transformation; treatments of longer duration resulted in greater inhibition of transformation. Delaying the onset of RA treatment reduced its effectiveness, but even when RA treatment was delayed for 3 weeks following MNNG exposure, 60% inhibition still occurred. The inhibition of transformation appeared to be irreversible. The colony forming efficiency of cells isolated from transformed colonies 5 weeks after MNNG exposure was drastically reduced when the replated cells were treated with RA either 1 day or 4 days after plating, indicating that RA blocked cell replication. However, cells isolated from transformed colonies at later times after MNNG exposure were increasingly resistant to the antiproliferative effects of RA. The RA concentration causing 50% inhibition (RA-IC50) of colony formation was 0.1-0.3 nM for cells isolated from 3-5 week-old transformed colonies; it increased greater than 100-fold for cells isolated from 12-week-old transformants. Five established RTE cell lines also showed a much increased resistance to the antiproliferative effects of RA; two of these cell lines were even slightly stimulated in their colony forming ability by RA. The RA-IC50 of colony forming efficiency of normal RTE cells was also determined and compared to that of cells isolated from 5-week-old transformed colonies. Since the normal RTE cells require 3T3 feeder layers for clonal growth, both cell types were grown on feeders. For both cell types, the RA-IC50 was similar (150-300 nM); the requirement for relatively high RA concentrations was attributed in part to the rapid RA metabolism by the feeder cells. These experiments show that early RTE cell transformants are growth-inhibited by RA; however, they increasingly lose their sensitivity to the growth controlling effects of RA as they progress to a more advanced stage of transformation. The inhibition of tracheal cell transformation by RA is probably due to the antiproliferative effects of the retinoid.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Trachea; Tretinoin

The inhibitory effects of some antipromoters were studied using a two-stage transformation assay system in vitro with 3-methylcholanthrene (3-MC)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in BALB 3T3 cells. Butylated hydroxyanisole (BHA), a phenolic antioxidant, inhibited TPA-enhanced transformation in a dose-dependent manner, but butylated hydroxytoluene (BHT) did not. Among the three antipromoters tested, retinoic acid (RA) was the most effective inhibitor.

Topics: Butylated Hydroxyanisole; Butylated Hydroxytoluene; Cell Transformation, Neoplastic; Cells, Cultured; Methylcholanthrene; Tetradecanoylphorbol Acetate; Tretinoin

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.

Topics: Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Molecular Weight; Peptide Biosynthesis; Phosphoproteins; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins pp60(c-src); Rats; Time Factors; Transforming Growth Factors; Tretinoin

Present studies in the mammary epithelial cell transformation model in organ culture showed that presence of beta-carotene during the 24 hr treatment (initiation stage) of the glands with the carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA), N-nitrosodiethylamine (DENA) and N-methylnitrosourea (MNU), caused a highly significant (P less than 0.001-0.01) reduction of SCE induced by the same carcinogens. In contrast, 4-hydroxyphenyl retinamide (4-HPR) which is known to act at the promotional stage of carcinogenesis did not show any significant reduction of SCE. Thus findings suggest that beta-carotene can modify the DNA damaging effect of the carcinogens and thereby may also prevent the initiation of the carcinogenic process.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; beta Carotene; Carcinogens; Carotenoids; Cell Transformation, Neoplastic; Diethylnitrosamine; Female; Fenretinide; Mammary Glands, Animal; Methylnitrosourea; Mice; Organ Culture Techniques; Retinoids; Sister Chromatid Exchange; Tretinoin

It has been shown that differentiated derivatives of retinoic acid (RA)-treated F9 embryonal carcinoma cells become non-malignant. In the present study it is asked whether this loss of malignancy is due to cellular differentiation. Because the ability of cells to grow in suspension correlates with in vivo tumorigenicity, we determined the time course of the loss of this property, after RA treatment, with relation to the differentiation to parietal endoderm and the acquisition of normalcy in several common transformation-specific properties of F9 cells. Our results show that pretreatment with RA for 24 h caused 80% inhibition of anchorage-independent growth in F9 cells, and this inhibition reached its highest level (98%) after pretreatment with RA for 48 h and longer. However, all other observed transformation-related properties, and the levels of plasminogen activator (marker for parietal endoderm) remained unaltered at this early post-treatment stage. These observations suggest that the loss of malignancy is a relatively early event in the biochemical pathways involved in the RA-induced differentiation of F9 cells. Furthermore, our data show that the presence of elevated levels of p53 alone may not be sufficient to maintain the anchorage-independent growth and the rapid proliferation of F9 cells.

Topics: Actins; Animals; Bucladesine; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Deoxyglucose; Embryonal Carcinoma Stem Cells; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Mice; Neoplasm Proteins; Neoplastic Stem Cells; Phosphoproteins; Plasminogen Activators; Receptors, Cell Surface; Teratoma; Tretinoin; Tumor Suppressor Protein p53

The effects of N-(4-hydroxyphenyl)retinamide (HPR), a synthetic analogue of vitamin A, on cell morphology, cell cycle kinetics, cytoplasmic matrix, and expression of murine mammary tumor virus (MuMTV) in MuMTV-infected murine mammary tumor cells (GR-3A) were determined. Cellular uptake of HPR was rapid and linear, with zero-order kinetics, during the first 30 minutes of incubation. Flow cytometric analysis of cells treated with nontoxic levels of HPR (10 microM) for 48 hours revealed a reduction in percent cells in the DNA synthetic (S) phase of the cell cycle with a concomitant increase in percent cells in the G1 phase of the cell cycle. Dexamethasone-stimulated MuMTV expression was not affected after 48 hours of HPR exposure, whereas the virus expression was significantly reduced in cells treated with HPR for seven days. The reduction in MuMTV expression was preceded by changes in cell morphology (decreased cell-cell contact and reduced cell flattening) and altered F-actin aggregation. Continuous exposure to HPR (10 microM) for 14 days resulted in reduced cell proliferation rates and decreased cell plating efficiency of GR-3A cells. Taken together, these results indicate that HPR is rapidly incorporated into GR-3A cells and that the effects of HPR on cell profileration, cytoskeletal organization, and cell morphology appear to precede the effects of this retinoid on the expression of the etiological agent of murine mammary tumorigenesis, MuMTV.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Cycle; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Female; Fenretinide; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Microscopy, Electron, Scanning; Tretinoin

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Affinity Labels; Animals; Azides; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Protein Kinases; Subcellular Fractions; Teratoma; Tretinoin

The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

Topics: Animals; Bone Marrow; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Hematopoiesis; Hemin; Humans; Leukemia, Experimental; Lipoproteins; Proteins; Tretinoin

A method for clonal analysis has been developed which allows the characterization of the number and type of progeny cells produced by each single cell arising during clonal evolution. The method is based on a symmetry of self-renewal exhibited by sister cells of the human promyelocytic leukemia cell line -HL60-. This permits the use of one of the sister cells to measure the potential for self renewal of the other. Using a system of sequential daughter cell transfers in semisolid medium, we have analysed self-renewal and differentiation in individual clones exposed to all-trans retinoic acid or dimethylsulfoxide (DMSO). We find that in clones exposed to chemical inducers of differentiation commitment occurs as an all-or-none event which is preceded by coordinated but reversible losses of self-renewal potential. It is concluded that the differentiation pathway of HL60 cells has two distinct portions. These are, first, a predeterministic portion, reflected by coordinated but reversible losses of self-renewal potential, and second, a deterministic portion, reflected by irreversible phenotypic differentiation.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Colony-Forming Units Assay; Dimethyl Sulfoxide; Granulocytes; Humans; Leukemia, Myeloid, Acute; Probability; Tretinoin; Tumor Stem Cell Assay

Topics: Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Humans; Leukemia, Myeloid, Acute; Pseudopodia; Tretinoin

In a case of acute promyelocytic leukemia (APL), the expression of terminal deoxynucleotidyl transferase (TdT), an early lymphoid marker, was detected. Double-fluorescent staining for the myeloid-specific antigens VIM-2 and VIM-D5 in combination with specific antiserum for TdT suggested a mixed leukemic cell population consisting of a morphologically, cytochemically, and immunologically promyelocytic component (80%) and a lymphoid, TdT+ component (20%) that was myelomonocytic in morphology but otherwise without any evidence of nonlymphoid nature. Fluorescent-activated cell analysis revealed that a greater number of cells reacted with monoclonal anti-T antibodies (OKT3, OKT6, and OKT11) than could be identified as lymphoid by TdT expression. As confirmed by double-staining fluorescence microscopy, a large fraction of the promyelocytic leukemia cells were biphenotypic, expressing both myeloid and lymphoid markers (50% positive for VIM-D5 and OKT6, 30% positive for VIM-D5 and OKT3). Subsequently, in vitro differentiation experiments were performed. While treatment of the cells with GCT-conditioned medium favored proliferation, with only a weak and delayed promotion of the cells towards maturation as reflected by enhanced expression of the mature T-marker T3 but persistent expression of the thymocyte antigen, exposure to all-trans and 13-cis retinoic acid resulted in marked differentiation of both the myeloid and the lymphoid cell characteristics. Retinoid treatment resulted in the loss of TdT, a partial disappearance of the T6-antigen, and the expression of the late T cell antigen T3 by almost 70% of the cells. In addition, myeloid maturation was obvious from the morphologic appearance of the cells, as well as from the expression of the OKM1-associated antigen by a majority of the cells. This report concerns a unique case of APL in which, for the first time, a coexistence of promyelocytic and lymphoid elements was detected, with exposure of the cultured leukemic cells to retinoic acid inducing maturation along both the myeloid and the lymphoid lineage.

Topics: Antigens, Surface; Cell Transformation, Neoplastic; Culture Media; DNA Nucleotidylexotransferase; DNA Nucleotidyltransferases; Female; Fibroblasts; Granulocytes; Hematopoietic Stem Cells; Humans; Hybrid Cells; Leukemia, Myeloid, Acute; Middle Aged; Phenotype; T-Lymphocytes; Tretinoin

beta-All-trans-retinoic acid (RA) inhibited the anchorage-independent growth of JB6 cells induced by either mezerein or alpha-epidermal growth factor (alpha-EGF) (a purified fraction of epidermal growth factor). The inhibition was dose dependent for alpha-EGF as well as for RA. Mezerein-induced growth in soft agar was inhibited to a greater extent by RA than was alpha-EGF-induced growth in soft agar, at similar colony yields. The extent of inhibition of anchorage-dependent growth induced by RA was similar for nontransformed JB6 cells and for alpha-EGF-transformed cells, so that transformation was shown not to influence the sensitivity of cells to retinoid inhibition of anchorage-dependent growth. RA was as effective at inhibiting anchorage-independent growth when it was applied after promoter-induced transformation as when it was applied during promoter-induced transformation. Therefore, the antiproliferative effect of RA, without an additional antitransformation effect, was sufficient to account for the reduced colony yield. These results suggest that the antipromoting action of retinoids in JB6 cells may occur by limiting proliferation, the regulation of which may be coupled with the state of differentiation of cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Epidermal Growth Factor; Epidermis; Mice; Skin Neoplasms; Terpenes; Tretinoin

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.

Topics: Animals; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Cytological Techniques; Dimethyl Sulfoxide; Intercellular Junctions; Mice; Mutation; Phenotype; Teratoma; Tretinoin

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Croton Oil; Fluocinolone Acetonide; Mice; Mice, Inbred Strains; Papilloma; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tosylphenylalanyl Chloromethyl Ketone; Tretinoin

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, Thin Layer; Eflornithine; Embryonal Carcinoma Stem Cells; Mice; Neoplastic Stem Cells; Ornithine; Ornithine Decarboxylase; Plasminogen Activators; Polyamines; Putrescine; Teratoma; Tretinoin

By use of indirect immunofluorescence technique and enzyme-linked immunosorbent assay we show that JB 6 mouse epidermal cells have cell surface fibronectin (FN) and release FN into the culture medium. The addition of 10(-8) M 12-O-tetradecanoylphorbol-13-acetate (TPA) to promotable clones caused a 2-fold enhancement of the FN release over solvent control. On the other hand, in non-promotable clones, TPA in concentrations of 10(-8) M or 10(-7) M did not cause increased FN release. Mezerein, a non-phorbol diterpene and second-stage tumor promoter was also found to be active in causing enhanced FN release in promotable but not in non-promotable clones. The vitamin A derivative retinoic acid (RA) antagonized the TPA-caused FN-release in promotable clones. RA had, however, no effect on the basic release patterns, when given alone or given to non-promotable clones together with TPA. These results suggest that the increased release of FN may be a required event for promotion to transformation. Our view is derived from the observation that promotable clones of the JB 6 cell line release increased amounts of FN into their medium upon promoter exposure while non-promotable clones are unaffected.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Epidermis; Fibronectins; Mice; Phorbols; Tetradecanoylphorbol Acetate; Tretinoin

Benzoyl peroxide (BzPo), a free radical generator with tumor promoting activity on mouse skin, is shown to promote neoplastic transformation of JB6 mouse epidermal cells in vitro. Repeated exposures to BzPo are required to readily detect promotion of transformation of JB6 cells. Markedly reduced net synthesis of the major epidermal ganglioside, trisialoganglioside GT1b, (GT) occurs with BzPo treatment as with other tumor promoters active in this system. Addition of ganglioside GT prevents transformation by BzPo while retinoic acid does not.

Topics: Animals; Benzoyl Peroxide; Cell Line; Cell Transformation, Neoplastic; Cocarcinogenesis; Gangliosides; Mice; Peroxides; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

The effect of different retinoids on morphological transformation and anchorage independent growth of Syrian hamster embryo cells has been studied. Retinoic acid and its derivatives were found to induce morphological transformation of hamster embryo cells, and to synergistically increase the transformation frequency when exposed in combination with benzo[a]pyrene. The increase was maintained when the cells were sequentially exposed to benzo[a]pyrene and retinoids in a similar way as observed for tumor promoting phorbol esters. At the same time retinoids were found to strongly decrease anchorage independent growth of a hamster embryo cell line. The present results support previous findings indicating that retinoids may have an enhancing effect on the early stages in carcinogenesis, and an inhibitory effect on the later stages.

Topics: Animals; Benzo(a)pyrene; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Drug Synergism; Embryo, Mammalian; Female; Mesocricetus; Stereoisomerism; Tretinoin

Invasion of chick chorioallantoic membrane (CAM) organ cultures by rat 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 (3Y1/12-10 cells) was inhibited by several retinoids tested. The anti-invasive activity of the retinoids was dependent on retinoid concentration and continuous (4 d) exposure of the CAM. The 50% retinoid dose (dose effective in achieving a response in half of the organ cultures) that inhibited invasion was 0.85 micrograms/ml of retinol palmitate, 0.39 micrograms/ml of retinoic acid, or 0.16 micrograms/ml of retinol acetate. This dose was of the same order of magnitude as that which induced CAM differentiation, and was three- to fourfold less than the dose that caused cytotoxic damage of CAM. In addition, the retinoids inhibited 3Y1/12-10 cell growth by approximately 40% at levels over 10-fold higher than those needed for anti-invasion activity. The findings suggest that the anti-invasive activity of retinoids was at least partly due to direct induction of cell differentiation of the CAM host tissue.

Topics: Adenoviruses, Human; Allantois; Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Chorion; Diterpenes; Extraembryonic Membranes; Neoplasm Invasiveness; Organ Culture Techniques; Rats; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

Single cell clones were isolated from the human teratoma line, Tera-2. The cells of three of these clones were studied. The progressively growing cells were shown to be tumorigenic, and they were characterized by the lack of expression of beta 2-microglobulin and HLA-A,B,C determinants on the cell surface. The majority of the cells expressed Thy-1 antigen and a 90 X 10(3) molecular weight protein recognized by the monoclonal antibody F10.44.2; between a third and half of the cells expressed the sugar specificities detected by the anti-SSEA-1 monoclonal antibody. In response to 5 X 10(-5) M-retinoic acid applied to cells in monolayer culture, the cells differentiated into a population of flat static cells arrested in the G1 phase of the cell cycle. A substantial proportion of these differentiated cells expressed beta 2-microglobulin and 43 X 10(3) molecular weight HLA-A,B,C polypeptides, Thy-1, SSEA-1 sugar determinants, and the 90 X 10(3) Mr protein recognized by F10.44.2. The apparent molecular weight of fibronectin secreted by the cells decreased by about 5 X 10(3) Mr to 235 X 10(3) Mr after differentiation. The progressively growing cells lacked reactivity with reagents that mark cells in the nervous system. Following aggregation and retinoic acid treatment, neuron-like cells were formed. These cells reacted with reagents that also react with human neurons in culture: they reacted with tetanus toxin, the anti-neurofilament antibodies BF10 and RT97, the anti-ganglioside, GQ1c antibody F12 A2B5, and anti-Thy-1. The progressively growing cells of these Tera-2 clones are therefore capable of forming at least two types of cell: the flat cells in monolayer cultures and the neuron-like cells. None of the cell populations reacted with the monoclonal antibody against SSEA-3 and these cloned cells are therefore distinct from previous isolates from Tera-2.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Epitopes; Fibronectins; Humans; Karyotyping; Mice; Mice, Nude; Neoplasm Proteins; Neurons; Phenotype; Teratoma; Tretinoin

We have established conditions to efficiently differentiate embryonic carcinoma stem cells of the line P19 into myogenic cells. As inducers for differentiation, a combination of embryoid body formation in conjunction with treatment with dimethyl sulfoxide and retinoic acid proved to be most efficient. Under these conditions we detected an accumulation of myosin- and actin-specific RNA. Also, large amounts of type IV collagen RNA were produced. Type IV collagen is a component of the muscle basement membrane. In analogy to the F-9 system, we found a drastic decrease in stable p53 mRNA under the differentiation conditions used.

Topics: Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Gene Expression Regulation; Muscles; RNA; RNA, Messenger; Teratoma; Tretinoin

F9 line embryonal carcinoma cells were induced to differentiate into neural direction by long-term treatment of monolayer cultures with retinoic acid and dibutyryl cyclic AMP. Bi- and multi-polar cells appeared, expressing acetylcholinesterase and neurofilament proteins but not markers of glial differentiation including GFA-protein. Nerve growth factor combined with both retinoic acid and dibutyryl cyclic AMP greatly enhanced the development of neuron-like morphology and induced expression of immunoreactivity to tyrosine hydroxylase as well as to Leu-encephalin-like peptides. Similarly, serotonin-like immunofluorescence but not substance P-like immunoreactivity was demonstrable in such cultures. In addition, synaptic-like vesicles were often found in the processes. Analysis of matrix expression in neuronally differentiated F9 cells revealed marked increase in laminin production, as judged by immunofluorescence and immuno-electron microscopy, but no demonstrable intracellular staining for fibronectin or type IV collagen. The results with neuronal cells contrast with the expression of all the three matrix components in endodermally differentiating F9 cells in the same cultures.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Laminin; Mice; Neurons; Teratoma; Tretinoin

The alterations of stimulus-induced membrane potential changes, superoxide (O2-)-producing capacity and phagocytic activity during differentiation of human granulocytes were investigated in the human leukemia cell lines HL-60 and KG-1 differentiating in vitro and in human leukemic granulocytes obtained from chronic myelogenous leukemia patients. HL-60 cells incubated with dimethyl sulfoxide or with retinoic acid showed progressively increasing O2- production as well as membrane potential changes (depolarization) on contact with phorbol myristate acetate or the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, with a concomitant increase in the proportion of mature cells of the granulocytic type. Phagocytosis of latex particles, yeast, and oil droplets appeared 24 h after incubation with dimethyl sulfoxide and anteceded the increment of O2- production and membrane potential changes, both of which appeared concomitantly 3 d after incubation with dimethyl sulfoxide. Similar findings were observed when immature and mature granulocytes obtained from chronic myelogenous leukemia patients were stimulated by phorbol ester, the chemotactic peptide, or calcium ionophore A23187, and the amount of O2- production was parallel to the magnitude of membrane potential changes. HL-60 and KG-1 cells incubated for 1-6 d with phorbol myristate acetate showed neither O2- production nor membrane potential changes on contact with phorbol ester, chemotactic peptide, or A23187, although such cells resembled macrophages morphologically, and their phagocytic activity was significantly increased. O2- production and membrane potential changes in normal granulocytes induced by phorbol ester, chemotactic peptide and A23187 were inhibited by 2-deoxyglucose. These findings indicate that the O2--producing system and the system provoking membrane potential changes may develop concomitantly as human granulocytes mature and differentiate, and that the development of these systems and of phagocytic activity may be independently regulated.

Topics: Adult; Calcimycin; Cell Differentiation; Cell Transformation, Neoplastic; Deoxyglucose; Dimethyl Sulfoxide; Gramicidin; Granulocytes; Humans; Leukemia, Myeloid; Membrane Potentials; Phagocytosis; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Cell Transformation, Neoplastic; Etretinate; Female; Humans; Keratins; Male; Middle Aged; Neoplasm Recurrence, Local; Papilloma; Skin Neoplasms; Tretinoin; Urinary Bladder Neoplasms

To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

Topics: Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cytomegalovirus; Embryonal Carcinoma Stem Cells; Humans; Neoplastic Stem Cells; Stem Cells; Teratoma; Tretinoin; Virus Replication

Retinoic acid (RA) has previously been shown to induce the differentiation of mouse embryonal carcinoma (EC) cells to endoderm-like cells that have a slower rate of proliferation and are nontumorigenic. These cells also acquire the ability to respond to a range of exogenous growth factors. We have analysed the change in growth phenotype for PC13 EC cells using video recordings and autoradiography. We have shown that the endoderm-like cells have a longer cell cycle time than their undifferentiated counterparts (five cell divisions after exposure to RA the differentiated cells had a median cell cycle time of 1800 min compared to 800 min for control cells). The endoderm-like cells also have a progressively decreasing probability of dividing again and this indicates that the differentiation process is accompanied by the acquisition of a limited life-span. The characteristics of mortal cells are well documented, and the endoderm-like cells demonstrate the properties of such cells. In addition, we have confirmed the observation that epidermal growth factor (EGF) can stimulate the proliferation of the endoderm-like cells and have shown, using autoradiography, that 92% of these cells express EGF receptors. Using video recordings, we have demonstrated that the effect of EGF is to shorten the cell cycle of the differentiating cells. We have also shown that EGF can enhance the survival of the endoderm-like cells and thereby prolong their life-span. It is known that EGF and other growth factors can prolong the life-span of mortal cells derived from normal tissues, but we have demonstrated that EGF can have this effect on the differentiated derivatives of a tumour cell.

Topics: Animals; Cell Cycle; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Epidermal Growth Factor; Mice; Neoplasms, Experimental; Teratoma; Tretinoin; Video Recording

A number of mouse and rat cells and their virus-transformed counterparts were tested for sensitivity to Pseudomonas aeruginosa exotoxin A (PEA). In each case, the transformed cells were considerably less sensitive than were the nontransformed cells. In the presence of trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, or retinoic acid, the transformed cells became as sensitive as the nontransformed cells, whereas these drugs had little or no effect on the sensitivity to PEA of the nontransformed cells. Temperature-sensitive virus-transformed normal rabbit kidney cells were sensitized to PEA by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, when these cells were grown as the transformed phenotype, whereas the nontransformed phenotype could not be sensitized. The possibility is discussed that upon malignant transformation a process which is dependent upon calmodulin or protein kinase C strongly decreases the sensitivity of the cells to PEA.

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Calmodulin; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Exotoxins; Kidney; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Protein Biosynthesis; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Rats; Sulfonamides; Tretinoin; Trifluoperazine; Virulence Factors

2',5'-Oligoadenylate (2-5A) synthetase, which polymerizes adenosine triphosphate into 2-5A, is induced upon treatment of cells with interferon (IFN) and is thought to be involved in its antiviral and anticellular action. We report here that retinoic acid (RA) enhanced the level of this enzyme in two human transformed cell lines, WISH and Namalva. Like IFN, RA induced 2-5A synthetase activity in a time- and dose-dependent manner. Addition of anti IFN-alpha, -IFN-beta, or -IFN-gamma antibodies to the medium concomitantly with RA did not prevent such induction; therefore, the effect of RA is clearly not mediated through the induction and externalization of IFN. Pretreatment of cells with actinomycin D inhibited 2-5A synthetase induction by RA, suggesting that RA increased the transcription of the 2-5A synthetase gene. In WISH cells, the growth of encephalomyocarditis virus was inhibited by RA treatment, which is consistent with the hypothesis that 2-5A synthetase plays an important role in the antiviral action of IFN, at least in encephalomyocarditis virus replication. When the anticellular effects of IFN and RA were compared to their ability to induce 2-5A synthetase activity in four human cell lines, there was no strict correlation between the amplitude of the enzyme activity induced and the extent of the antiproliferative effect. It is concluded that the 2-5A system is probably not the only pathway responsible for the antiproliferative effect of both substances. We further suggest that the induction of 2-5A synthetase by IFN and RA might be connected with at least some of the similarities observed between other biological effects of both compounds.

Topics: 2',5'-Oligoadenylate Synthetase; Burkitt Lymphoma; Cell Division; Cell Line; Cell Transformation, Neoplastic; Enzyme Induction; Female; Fibroblasts; Humans; Interferon Type I; Interferon-gamma; Leukemia, Myeloid, Acute; Placenta; Pregnancy; Tretinoin

The effect of retinoic acid (RA) on N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation of primary cultures of rat tracheal epithelial cells was investigated. RA inhibited transformation of rat tracheal epithelial cells by up to 95% at concentrations of 3.3 to 33 nM which did not substantially affect cell survival. The inhibitory effect of RA on transformation was concentration dependent and was also dependent upon timing and duration of treatment. Treatment with RA for only 1 week following N-methyl-N'-nitro-N-nitrosoguanidine exposure diminished the transformation frequency by 30 to 57%, although longer treatment times were more effective. Because RA was able to inhibit transformation effectively at concentrations which were not substantially inhibitory to colony-forming efficiency of rat tracheal epithelial cells, the mechanism of inhibition of cell transformation does not seem to be related to cytotoxic effects of RA known to occur at high RA concentrations.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Epithelium; Male; Methylnitronitrosoguanidine; Rats; Rats, Inbred F344; Time Factors; Trachea; Tretinoin

Utilizing the phenomenon of metabolic cooperation in Chinese hamster V79 cells, we have studied the effects of agents which suppress the 12-tetradecanoyl-phorbol-13-acetate (TPA) enhancement of transformation in vitro, on the TPA suppression of cell-cell communication. None of the agents tested, namely all-trans-retinoic acid, the trimethyl methoxyphenyl analogue of N-ethyl-retinamide, soybean trypsin inhibitor, antipain nor superoxide dismutase, decreased the enhanced recovery effect of TPA on metabolic cooperation. One of the compounds, retinoic acid, significantly increased the % recovery above that observed for TPA alone.

Topics: Animals; Antipain; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Oligopeptides; Phorbols; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Tretinoin; Trypsin Inhibitors

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca2+ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.

Topics: Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Leukemia, Myeloid, Acute; Tretinoin

A 60-kilodalton protein was identified in chromatin digested by micrococcal nuclease during retinoic acid-induced differentiation of human leukemia (HL-60) cells to mature-like granulocytes. The protein was not detected in a retinoic acid-resistant variant of the HL-60 cell line treated with retinoic acid, in HL-60 cells induced with dimethyl sulfoxide, or in normal human granulocytes. This protein may have an important role in the regulation of retinoic acid-induced leukemic cell differentiation.

Topics: Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Granulocytes; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Nucleosomes; Tretinoin

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL-60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation-induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.

Topics: Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; G(M1) Ganglioside; G(M2) Ganglioside; Globosides; Glycosphingolipids; Humans; Leukemia, Myeloid, Acute; Macrophages; Tretinoin

Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin-Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

Topics: Cell Line; Cell Transformation, Neoplastic; Chromatography, Agarose; Drug Stability; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Lymphokines; Nitroblue Tetrazolium; T-Lymphocytes; Tretinoin

The effects of retinoic acid (RA) on cell proliferation, activity of acid phosphatase, protein synthesis and methionine uptake were studied in transformed murine LPA cells. Early inhibition of protein synthesis was demonstrated under experimental conditions in which the rate of cell proliferation was diminished and non-specific effects of vitamin action could be excluded. Measurements of L-methionine uptake revealed a decrease to approximately one-half of that in control cultures after treatment with RA at the concentrations of 5 X 10(-5) M and 10(-4) M.

Topics: Acid Phosphatase; Animals; Biological Transport; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Kinetics; L Cells; Methionine; Mice; Protein Biosynthesis; Tretinoin

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS-polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Antigens, Nuclear; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; Chromatin; Chromosomal Proteins, Non-Histone; Clone Cells; Humans; Leukemia; Nucleoproteins; Tretinoin

A number of characteristics of phorbol ester-mediated mouse skin tumor promotion indicate that cell selection is the underlying biological process. Studies in vivo and in cultured mouse epidermal cells suggest that selection is based on heterogeneous responses of subpopulations of basal cells which can be induced to proliferate or differentiate in response to promoter exposure. Retinoids are effective inhibitors of tumor promotion in mouse skin but do not influence the proliferative response to phorbol esters. Since both retinoids and phorbol esters modify epidermal differentiation, the antipromoter action of retinoids could be related to differentiation responses. Retinoic acid induces transglutaminase activity in cultured mouse epidermal basal cells grown in less than 0.1 mM Ca2+. While this enzyme is associated with terminal differentiation in skin, retinoic acid paradoxically blocks the terminal differentiation of cultured cells. In contrast, phorbol esters and extracellular calcium greater than 0.1 mM induce both transglutaminase activity and terminal differentiation. Enzyme kinetic analyses indicate that the transglutaminases induced by all three inducers are the same enzyme. The increase in activity of transglutaminase by all three inducers requires RNA and protein synthesis. However, the time course of increase and decay of each activity differs for each inducer. A variety of biologically active retinoids induce transglutaminase activity, and their effectiveness correlates with their reported antipromoter activity. Exposures to both retinoic acid and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate are antagonistic resulting in less than additive induction. Induction kinetics with both inducers are more like those of retinoic acid than those of the phorbol ester. Simultaneous exposure to retinoic acid and 12-O-tetradecanoylphorbol-13-acetate protects the epidermal cell population from induced terminal differentiation and cell loss which is observed in response to the promoter alone. These results suggest that the antipromoting action of retinoids could be mediated by modification of phorbol ester-accelerated terminal differentiation through an effect on transglutaminase and cornification. This action of retinoids would block a critical aspect of cell selection involving loss of cells and subsequent regenerative hyperplasia, although simple hyperplasia may still occur.

Topics: Acyltransferases; Animals; Animals, Newborn; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Induction; Kinetics; Mice; Mice, Inbred BALB C; Phorbols; Protein Biosynthesis; Retinoids; Skin; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transglutaminases; Tretinoin

Topics: Animals; Blood Vessels; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chick Embryo; Cricetinae; Gene Expression Regulation; Humans; Macromolecular Substances; Mice; Organ Culture Techniques; Phenotype; Rats; Skin; Staining and Labeling; Tretinoin; Vitamin A; Vitamin A Deficiency

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.

Topics: Cell Line; Cell Transformation, Neoplastic; Decarboxylation; Deoxyglucose; Dimethyl Sulfoxide; Fucosyltransferases; Galactosyltransferases; Hexosyltransferases; Humans; Leukemia, Myeloid, Acute; Sialyltransferases; Substrate Specificity; Tetradecanoylphorbol Acetate; Tretinoin

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.

Topics: Butyrates; Butyric Acid; Cell Count; Cell Cycle; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Macrophages; Tetradecanoylphorbol Acetate; Tretinoin

We describe in vitro studies and a therapeutic trial of retinoic acid (RA) in a patient with acute promyelocytic leukemia (APL) refractory to chemotherapy. Bone marrow promyelocytes from the patient, prior to RA, matured morphologically in liquid culture with RA (97% maturing myeloid cells compared with 26% in control cultures at 7 days). RA-cultured cells displayed leukocyte alkaline phosphatase activity and cytoplasmic maturation (by electron microscopy). Retinoic-acid-treated cells, compared to controls, demonstrated increased functional maturation, with phagocytosis of opsonized zymosan (90% versus 10%) and production of superoxide (measured by nitroblue tetrazolium reduction) in response to phorbol ester, opsonized zymosan, or the chemotaxin F-met-leu-phe. There was no evidence of active proliferation in the cultures. RA-treated cells continued to show 15;17 chromosomal translocation after 7 days in culture. The patient was treated with oral 13-cis-retinoic acid (100 mg/sq m/day) for 13 days. During that time, the peripheral white blood count rose from 300 cu mm to 6,700 cu mm, and the maturing myeloid cell count rose from 54 cu mm to 3,800 cu mm. Bone marrow maturing cells increased from 1.8% to 8.0%. Despite the increasing number of maturing myeloid cells, the patient died on day 13 from disseminated candidiasis. These data confirm that RA induces maturation of leukemic promyelocytes in vitro and suggest that similar maturation is achievable in vivo. We suggest that oral retinoic acid may be a useful adjunct in the treatment of APL.

Topics: Adult; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Leukemia, Myeloid, Acute; Leukocyte Count; Male; Tretinoin

The effects of retinyl acetate and all-trans-retinoic acid on the growth rate, saturation density, and cytoplasmic underlapping of normal and carcinogen-initiated C3H/1OT1/2 cells were examined. Retinyl acetate (a) decreased the saturation density by as much as 45%, (b) had no effect on the growth rate, (c) reduced cytoplasmic underlapping of adjacent cells by 55 to 85%, and (d) inhibited neoplastic transformation by methylcholanthrene. The effects were dose dependent and not significantly affected by the serum concentrations over the range of 2.5 to 10%. In contrast, all-trans-retinoic acid (a) decreased the saturation density as effectively as retinyl acetate, but only in medium containing 10% serum; (b) significantly reduced the growth rate of cells at low density, especially at low serum concentrations; (c) had no effect upon cytoplasmic underlapping; and (d) enhanced transformation at nontoxic concentrations in medium containing 5% serum but had no effect in 10% serum. We conclude that the effects of retinoids on the growth rate and saturation density of cells in culture may not be relevant to their inhibition of neoplastic transformation. Rather, we interpret the results of our experiments on cytoplasmic underlapping as an indication that retinoids inhibit transformation by stabilizing and/or enhancing cell surface receptors involved in cell-cell contact-dependent formation of a stable monolayer.

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Embryo, Mammalian; Kinetics; Methylcholanthrene; Mice; Mice, Inbred C3H; Retinyl Esters; Tretinoin; Vitamin A

Choriocarcinoma cells maintain multiple hormonal functions in culture. We have found that these cells secreted no immunoreactive pregnancy-specific beta 1-glycoprotein (PS beta G), a placental protein. Choriocarcinoma cells can be induced to synthesize low levels of PS beta G by retinoic acid, 8-bromo-cAMP (8BrcAMP), cholera toxin, methyl-isobutylxanthine (MIX), and 5-bromo-2'-deoxyuridine (BrdUrd). The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave synergistic induction of PS beta G. The simultaneous addition of retinoic acid and BrdUrd failed to give even additive induction. In addition to stimulating PS beta G production, retinoic acid increased the production of hCG and its alpha-subunit (hCG alpha) by choriocarcinoma cells. The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave additive induction for hCG and hCG alpha. Passage of choriocarcinoma cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of PS beta G, hCG, and hCG alpha. These retinoid-treated choriocarcinoma cells remained responsive to 8BrcAMP or compounds that increase intracellular cAMP concentrations and to BrdUrd; the production to PS beta G and hCG was greatly stimulated by 8BrcAMP, cholera toxin, or MIX, and the production of hCG and hCG alpha was greatly inhibited by BrdUrd. However, the production of hCG alpha was only slightly induced by these cAMP modulators, and the production of PS beta G was not increased by BrdUrd.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Line; Cell Transformation, Neoplastic; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Cyclic AMP; Deoxyuracil Nucleotides; Female; Fluorodeoxyuridylate; Humans; Pregnancy; Pregnancy-Specific beta 1-Glycoproteins; Tretinoin; Uterine Neoplasms

The ability of N-nitrosodiethylamine (DENA) to induce transformation of the mammary cells was studied in culture of the whole mammary organ from BALB/c female mice. Incidence of nodule-like alveolar lesions (NLAL) in the glands in vitro has been as a measure of transformation. NLALs are analogous to the precancerous hyperplastic alveolar nodules (HAN) of mouse mammary gland in vivo. The mammary glands were treated with graded concentrations (0.1-2.5 microgram/ml) of DENA during lobuloalveolar morphogenesis in medium (Waymouth's MB752/1) containing insulin, prolactin, hydrocortisone and aldosterone. DENA treatment caused a dose-related increased occurrence of NLAL in the glands in vitro and concentration of 1.5 microgram/ml produced the highest incidence of 85%. The high incidence of NLAL was accompanied by a 3-fold increase of DNA repair activity in the DENA treated glands. Incubation of the glands for 6 days after DENA treatment in medium containing N-4-hydroxyphenyl)retinamide and the same hormone mixture caused 61% inhibition of NLAL incidence. The results indicate that DENA is capable of inducing a high level of transformation of the mammary epithelial cells in vitro and that this retinoid can inhibit expression of the transformed cells acting at the promotional level.

Topics: Animals; Cell Transformation, Neoplastic; Diethylnitrosamine; DNA Repair; Female; Fenretinide; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Nitrosamines; Organ Culture Techniques; Tretinoin

Influence of estrogen and progesterone on the inhibitory action of N-(4-hydroxyphenyl) retinamide (4-HPR) was examined during the promotional stage of 7,12-dimethylbenz[alpha]anthracene (DMBA) transformation of the epithelial cells in culture of the whole mammary organs of BALB/c mice. In medium containing insulin, prolactin, hydrocortisone, and aldosterone, 4-HPR caused 68% inhibition of transformation as determined by the presence of nodule-like alveolar structures in the glands exposed to DMBA in vitro. Addition of estrogen and progesterone to the medium reduced this pronounced inhibitory action of 4-HPR to only 15%. While the medium containing insulin, prolactin, growth hormone, estrogen and progesterone was highly conducive to DMBA transformation, 4-HPR inhibition of transformation was limited to only 21%. The antagonistic action of the ovarian steroid hormones was present also at the level of frequency of nodule-like alveolar lesions (NLAL) per gland. Although both ovarian hormones reduced the inhibitory action of 4-HPR, on mammary cell transformation, the antagonistic action of estrogen was noticeably more pronounced.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benz(a)Anthracenes; Castration; Cell Transformation, Neoplastic; Estrogens; Female; Fenretinide; Hormones; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Organ Culture Techniques; Progesterone; Tretinoin

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.

Topics: Animals; Carrier Proteins; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cytosol; Diterpenes; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Mice; Receptors, Retinoic Acid; Retinyl Esters; Tretinoin; Vitamin A

We have examined the effect of physiologic concentrations of dexamethasone on the induction of differentiation of the human promyelocytic leukemia cells (HL-60) and the activity of surface receptors for the f-met-leu-phe chemotactic peptide. Dexamethasone at a concentration of 500 nM was markedly synergistic with dimethylformamide and dimethylsulfoxide, but not with trans-retinoic acid, in inducing f-met-leu-phe binding by HL-60 cells. The increase in peptide binding was due to an increase in the activity of peptide receptors rather than an increase in receptor affinity. The effect of dexamethasone was relatively specific for peptide binding as there was little effect when HL-60 differentiation was assessed by cell morphology or the ability of the cells to reduce nitroblue tetrazolium. Dexamethasone alone had no reproducible effect on HL-60 differentiation as assessed by the three criteria tested. The dexamethasone concentration required for half maximal synergism correlated with the known binding constant of the hormone for steroid receptors in HL-60 cells but the effect of dexamethasone was not blocked with the steroid blocking agents 17 alpha methyltestosterone or progesterone.

Topics: Cell Line; Cell Transformation, Neoplastic; Dexamethasone; Dimethyl Sulfoxide; Dimethylformamide; Dose-Response Relationship, Drug; Drug Synergism; Humans; Indomethacin; Methionine; Methyltestosterone; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Oligopeptides; Progesterone; Receptors, Cell Surface; Receptors, Formyl Peptide; Receptors, Steroid; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Adenosine Triphosphatases; Animals; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Cocarcinogenesis; Cricetinae; Embryo, Mammalian; Mice; Phorbols; Retinol-Binding Proteins; Sister Chromatid Exchange; Tetradecanoylphorbol Acetate; Tretinoin; X-Rays

Topics: Adult; beta Carotene; Canthaxanthin; Carotenoids; Cell Transformation, Neoplastic; Child; Child, Preschool; Electrosurgery; Eye Diseases; Female; Humans; Infant; Male; Neurologic Manifestations; Skin Diseases; Tretinoin; Xeroderma Pigmentosum

Since cell surface gangliosides have been implicated in the regulation of cell growth and differentiation and since tumor-promoting phorbol esters produce a number of membrane changes, the possibility was investigated that ganglioside changes may play a role in promotion of transformation in JB6 mouse epidermal cells. The studies showed that tumor-promoting but not non-promoting phorbol esters produced decreased precursor incorporation into disialoganglioside GD1b and an unknown ganglioside, Gx. These ganglioside responses to promoters were antagonized by the antipromoter retinoic acid, thus suggesting that alterations in ganglioside levels may be involved in promotion of transformation.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epidermis; Gangliosides; Mice; Phorbols; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin

The JB6 mouse epidermal cell line has been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or other tumor promoters resulted in the irreversible acquisition by JB6 cells of tumorigenicity in nude mice and anchorage-independent growth in soft agar. As previously reported, one of the biochemical responses that occurs during TPA treatment is a greater than 75% reduction in a mannose-labeled glycoprotein with an apparent molecular weight of 180,000. This TPA-sensitive glycoprotein has now been identified on the basis of collagenase and pepsin sensitivity as procollagen pro-alpha 1(I) chain. [3H]proline labeling also demonstrated a parallel decrease in the 150,000 procollagen pro-alpha 2(I) component. Two nonphorbol promoters, mezerein and epidermal growth factor, were also active in decreasing procollagen synthesis. Promotion-sensitive and promotion-resistant clonal derivatives of JB6 showed similar basal levels of collagen synthesis as well as similar degrees of TPA-dependent procollagen loss indicating that the collagen decrease may be necessary but is not sufficient to produce the promotion response. Comparison of chemically transformed mouse epidermal cell lines with paried nontransformants suggests the reduced procollagen synthesis is a stably acquired phenotypic change and may therefore be involved in maintenance of the transformed phenotype as well as in its induction.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Epidermis; Mice; Phorbol Esters; Phorbols; Procollagen; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; Embryo, Mammalian; Gamma Rays; Mice; Neoplasms, Experimental; Neoplasms, Radiation-Induced; Radiotherapy; Superoxide Dismutase; Tretinoin; Triiodothyronine; X-Rays

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Contact Inhibition; Epidermal Growth Factor; Mice; Phorbols; Tetradecanoylphorbol Acetate; Tretinoin

The influence of retinoic acid (RA) - a modifier of tumor promotion - on the cell cycle of HeLa cells, and its ability to interfere with the early irradiation-like effects induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been investigated by a variety of different techniques. These include measurement of thymidine incorporation and uptake, of labelling and mitotic indices, and of DNA histograms by flow cytometry. Within 24 h RA (2 X 10-5 M) alone caused a short-lasting inhibition of DNA synthesis; later a decrease of cells in S phase and a steady descending mitotic activity were observed. On combined treatment with RA and TPA (10-8 M), the latter seems to dominate RA in two instances: (1) the transient G2 blockage due to TPA is seen as the earliest effect; however, the cultures do not seem to recover as well if RA is also present; (2) the TPA-induced GI blockage appears to be effective but less pronounced in the presence of both chemicals. Where the third typical TPA-induced effect is concerned, however, both compounds seem to act to a comparable degree in the same time frame; namely by an initial inhibition of DNA synthesis which thus might be a point of critical interference if promoter and modifier age given together.

Topics: Cell Cycle; Cell Division; Cell Transformation, Neoplastic; DNA; Drug Interactions; Flow Cytometry; HeLa Cells; Humans; Mitosis; Phorbols; Tetradecanoylphorbol Acetate; Thymidine; Tretinoin

Topics: Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Epidermis; Mice; Mitogens; Phorbols; Procollagen; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Adult; Basal Cell Nevus Syndrome; Carcinoma, Basal Cell; Cell Transformation, Neoplastic; Etretinate; Facial Neoplasms; Humans; Male; Tretinoin

Retinoic acid and retinol induced functional and morphological differentiation of human promyelocytic leukemia cells (HL-60) into mature granulocytes, but did not induce functional or morphological differentiation of mouse myeloid leukemia cells (M1). Cellular retinoic acid-binding protein, but not retinol-binding protein, was detected on HL-60 cells. Neither binding protein could be detected on M1 cells. These results suggest that retinoic acid-binding protein may be necessary for induction by retinoids of functional and morphological differentiation of myeloid leukemia cells.

Topics: Animals; Carrier Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Induction; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Muramidase; Neoplasm Proteins; Rats; Receptors, Retinoic Acid; Retinol-Binding Proteins; Tretinoin

Topics: Animals; Cell Adhesion; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Methylcholanthrene; Mice; Mice, Inbred C3H; Retinaldehyde; Retinyl Esters; Tretinoin; Vitamin A

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Mice; Phorbols; Skin; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Enzyme Induction; Mice; Ornithine Decarboxylase; Phorbols; Skin; Tetradecanoylphorbol Acetate; Tretinoin

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Blood Cells; Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Rabbits; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Carcinoembryonic Antigen; Cell Line; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Endoderm; Female; Fibroblasts; Lactoperoxidase; Membrane Proteins; Mice; Neoplasms, Experimental; Pregnancy; Surface Properties; Teratoma; Tretinoin

Mammalian cell cultures offer powerful tools for evaluating qualitatively and quantitatively the oncogenic potential of radiation over a wide range of doses with particular importance at the low dose range that is relevant to human exposure and risk. Our studies have shown that early events in the process of radiation induced transformation in both rodent and human cells requires initial replication for fixation of transformation as a hereditary property of cells and further clonal expansion for full expression. Early events (fixation) are inhibited by cell-cell contact and high cell density but can be modified at low temperature where repair processes are slowed. Cell-cell contact and communication in tissue organization may be in part responsible for our findings that radiation oncogenesis induced in utero in hamsters is expressed at a lower frequency than that induced in vitro. Quantitative studies carried out on hamster embryo cells indicate that neutrons are more effective in their carcinogenic potential than x-rays but also more toxic, that splitting the dose of x-rays at low doses leads to enhanced transformation, but that at high doses protracted radiation has a sparing effect. At all dose ranges survival was increased by protracting the radiation dose, thus suggesting that different repair processes must be involved for survival and transformation. Similar observations were seen when the protease inhibitor Antipain was found to enhance transformation in rodent and human cells when present at the time of radiation, but was protective when added after radiation. Survival was not modified under any of those conditions, and Antipain did not affect DNA replication and repair. In our qualitative studies, once cells are transformed by radiation, they exhibit a wide range of structural and functional phenotypic changes, some of which are membrane-associated and are expressed within days after induction. Our current studies on nutritional and hormonal influences on radiation transformation indicate the following: Pyrolysate products from broiled protein foods act in synergism with radiation to produce transformation, whereas vitamin A analogs are powerful, preventive agents. Retinoids inhibit both x-ray-induced transformation and its promotion by TPA; these modifications (enhancement by TPA, inhibition by retinoids) are not reflected in sister chromatid exchanges, but are reflected in the level of membrane associated enzymes Na/K ATPase. Whereas retinoid

Topics: Animals; Antipain; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Clone Cells; Cricetinae; Embryo, Mammalian; Humans; Intercellular Junctions; Liver; Mice; Thyroxine; Tretinoin; Triiodothyronine; Vitamin A

Modification of sister chromatid exchanges and radiation-induced transformation in mouse C3H/10T 1/2 and Syrian hamster embryo cells by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and two retinoids, the trimethylmethoxyphenyl analog of N-ethyl retinamide and beta-all-trans-retinoic acid, has been studied. 12-O-tetradecanoylphorbol-13-acetate alone enhances, and retinoids alone reduce radiation-induced transformation. When both compounds were present, the retinoids not only reduced the oncogenic effects of radiation but completely eliminated the promoting effects of 12-O-tetradecanoylphorbol-13-acetate. These results were not paralleled by changes in sister chromatid exchange frequencies, indicating that, while sister chromatid exchanges may be useful as indicators of primary carcinogen mutagens, they may have little utility when secondary agents after the response of cells to a primary initiator.

Topics: Animals; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cocarcinogenesis; Cricetinae; Crossing Over, Genetic; Mice; Phorbols; Sister Chromatid Exchange; Tetradecanoylphorbol Acetate; Tretinoin

Various natural and synthetic retinoids have been studied for their activity in two biological systems: (a) their activity as inhibitors of methylcholanthrene-induced neoplastic transformation in the C3H/10T1/2 clone 8 mouse fibroblast line (System 1); and (b) their ability to increase the degree of adhesion of C3H/10T1/2 clone 8 cells to a plastic substrate (System 2). These activities were then compared with their known activity in maintaining epithelial differentiation (System 3). With the notable exception of retinoic acid and 13-cis-retinoic acid, which were inactive in Systems 1 and 2, an excellent correlation was observed between activities in Systems 1 and 3 for retinyl acetate, N-(4-hydroxyphenyl)retinamide, retinylidene dimedone, N-ethylretinamide, and N-benzoylretinylamine. Compounds shown to be inactive in System 1 had little or no activity in System 2. However, the ability of retinoids to cause increased adhesion could not be correlated with Systems 1 or 3 in all cases. For instance, retinyl acetate was highly active in Systems 1, 2, and 3, whereas retinylidene dimedone was highly active in Systems 1 and 3 but weakly active in System 2. Conversely, N-(4-hydroxyphenyl)retinylamide was highly active in Systems 1 and 3 but caused a decrease in System 2. The lack of activity of 3 but caused a decrease in System 2. The lack of activity of retinoic acid isomers in the C3H/10T1/2 clone 8 system is paradoxical and may provide important information on requirements for their activation and/or transport.

Topics: Amides; Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Fenretinide; Isotretinoin; Methylcholanthrene; Mice; Neoplasms, Experimental; Retinoids; Retinyl Esters; Structure-Activity Relationship; Tretinoin; Vitamin A

Studies of tumor induction on mouse skin have provided insight into the basis biology of chemical carcinogenesis, but molecular mechanisms have been more difficult to elucidate. Mouse epidermal cell cultures have proven to be a valuable model for performing mechanistic studies. Previous data have indicated that such cultures proliferate and differentiate in a manner highly analogous to epidermis in vivo. In addition, carcinogen metabolism, DNA repair, and responses to tumor promoters are quite similar in mouse skin in vivo and in vitro. Recent data have extended these observations toward defining the biological characteristics of initiated cells and elucidating the mechanism of action of promoters and antipromoters. When mouse epidermis is cultured under conditions of low extracellular Ca++, proliferation is enhanced and terminal differentiation is inhibited. Addition of Ca++ induces terminal differentiation. If cells are treated with carcinogens under low Ca++ conditions and subsequently switched to standard Ca++, cell colonies which do not terminally differentiate evolve. Such colonies continue to synthesize keratin, are subculturable, and may represent preneoplastic cells. In other experiments, epidermal cells derived from mouse skin treated with carcinogens in vivo also demonstrate prolonged in vitro survival and subculturability while controls have a limited lifespan. Such studies suggest that biological alterations can be detected in epidermal cells exposed to carcinogens well before and the phenotypic expression of neoplasia. Exposure of epidermal cells to phorbol-ester tumor promoters induces ornithine decarboxylase (ODC). This induction is enhanced by corticosteroids and markedly inhibited by retinoids. Ultraviolet light also induces ODC in epidermal cells, but kinetic studies suggest that the early pathway of induction (afferent to the nucleus) is different from that of phorbol esters. The later pathways (efferent from the nucleus-i.e., transcription and translation) appear to be similar. Retinoids have only a minor suppressive effect on ODC induction by UV while corticosteroids enhance UV induction to the same extent as seen with phorbol esters These results suggest that the site of retinoids is in the afferent pathway while steroids act on the efferent pathway.

Topics: Animals; Carcinogens; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Environmental Exposure; Enzyme Induction; Epidermal Cells; Epidermis; Methylnitronitrosoguanidine; Mice; Neoplasms, Experimental; Ornithine Decarboxylase; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Cell Adhesion; Cell Membrane; Cell Transformation, Neoplastic; Extracellular Space; Fucose; Glycopeptides; Hexosaminidases; Mannose; Membrane Proteins; Mice; Tretinoin

Topics: Animals; Cell Adhesion; Cell Transformation, Neoplastic; Cells, Cultured; Drug Evaluation, Preclinical; Fibroblasts; Mice; Structure-Activity Relationship; Time Factors; Tretinoin; Vitamin A

Previous studies indicated that the potent tumor promoter 12--0--tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2 degrees RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2 degrees RE cells (clone E11). Both TPA and EGF (approximately 10(-8) M) induced a 3--5 fold increase in agar cloning efficiency of E11 cells. In addition, macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol--12,13--didecanoate (PDD) and ingenol--3,20--dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4 alpha PDD and 4--0--meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol--3,20--bidenzoate failed to induce growth in agar of normal 2 degrees RE cells. Dexamethasone (10(-5)--10(-6) M), trans retinoic acid (10(-5)--10(-6) M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all ten randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells in monolayer culture in the presence of TPA did not enhance their subsequent growth in agar. This system therefore provides an example in which TPA appears to enhance the acquisition of a stable cell property, and thus may be a useful model for studying mechanisms of tumor promotion and progression.

Topics: Adenoviruses, Human; Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dexamethasone; Diterpenes; Embryo, Mammalian; Epidermal Growth Factor; Peptides; Phorbols; Protease Inhibitors; Rats; Tetradecanoylphorbol Acetate; Tretinoin

Animal studies have shown. a) an association between vitamin A and cancers of epithelial origin, and b) that vitamin A and its analogues delay tumour appearance, retard tumour growth and regress tumours induced by carcinogenic polycyclic aromatic hydrocarbons. Human epidemiological and biochemical studies suggest that cancers of epithelial origin may be associated with vitamin A deficiency. Vitamin A and its analogues may have a prophylactic and a therapeutic role in cancers of epithelial origin.

Topics: Animals; Bronchial Neoplasms; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Humans; Lung Neoplasms; Mouth Neoplasms; Neoplasms, Experimental; Pharyngeal Neoplasms; Tretinoin; Vitamin A; Vitamin A Deficiency

Following the well established prevention of chemical carcinogenesis by vitamin A in several species of laboratory animals, we performed in vitro studies with human diploid fibroblasts in culture. Vitamin A-palmitate, alltransretinoic acid and the analogue compound Ro 10-9359 were found to reduce the formation of active intermediates following the administration of G3H Benzo(a)pyrene to the cells. This effect which lead to a considerable decrease of alkylated DNA is not based on a direct inhibition of Benzpyrene metabolizing enzymes by the retinoids but by a preferential inhibition of the de novo synthesis of these enzymes. This caused the well known substrate mediated enzyme induction of benzpyrene metabolizing enzymes to cease. From our data we conclude that the mechanism of the cancer protective effect of vitamin A with respect to certain carcinogens is based on an inhibited activation of procarcinogens. This effect can also be expected in human tissues.

Topics: Benzopyrenes; Biotransformation; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Fibroblasts; Humans; Neoplasms; Tretinoin; Vitamin A

Spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells) displayed an increased adhesion when cultured in the presence of 10(-6) M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [15-(14)C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [(14)C]retinol was taken up by the cells. The hydrocarbon [(14)C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T12 cells was also active in synthesizing [(14)C]anhydroretinol and [(14)C]metabolite-310 from [(14)C]retinol. Moreover incubation of metabolite-310 with the 3T12 microsomes yielded anhydroretinol (40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [(14)C]anhydroretinol incubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.-Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

Topics: Animals; Animals, Newborn; Cell Adhesion; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Microsomes; Models, Biological; Skin; Tretinoin; Vitamin A

Topics: Carcinogens; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Growth Inhibitors; Tretinoin; Vitamin A

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.

Topics: Animals; Avian Sarcoma Viruses; Bucladesine; Cell Differentiation; Cell Fusion; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cholera Toxin; Muscles; Phorbols; Plasminogen Activators; Tetradecanoylphorbol Acetate; Tretinoin; Vitamin A

Extracts prepared from several lines of transformed cells were examined for the presence of cellular binding proteins specific for retinoids. Extracts of human retinoblastoma cell line WERI-Rb1 contained a cellular binding protein specific for retinoic acid, whereas extracts of human retinoblastoma cell line Y-79 contained cellular binding proteins for both retinol and retinoic acid. Upon purification, the latter two binding proteins proved to have properties similar to those of the corresponding proteins obtained from bovine retina. Smaller amounts of these binding proteins were detected in extracts of undifferentiated and differentiated neuroblastoma and McCoy cells. HeLa and rat glioma cells had no detectable amount of binding proteins. The 11-cis-retinal-binding protein, present in extracts of human, rat, and bovine retina, was not found in any of the cell lines examined.

Topics: Animals; Carrier Proteins; Cattle; Cell Line; Cell Transformation, Neoplastic; Eye Neoplasms; Humans; Mice; Molecular Weight; Rats; Retinoblastoma; Retinol-Binding Proteins; Tretinoin; Vitamin A

Topics: Cell Line; Cell Transformation, Neoplastic; Gamma Rays; Tretinoin; Vitamin A

Topics: Carotenoids; Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Dose-Response Relationship, Drug; HeLa Cells; L Cells; Retinaldehyde; Tretinoin; Vitamin A

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diterpenes; DNA, Neoplasm; Mammary Neoplasms, Experimental; Melanoma; Neoplasm Proteins; Neoplasms, Experimental; Retinol-Binding Proteins; Retinyl Esters; Sarcoma, Experimental; Tretinoin; Vitamin A