tretinoin and Retinoblastoma

Retinoblastoma (RB), an intraocular tumor of childhood, contains small subpopulation(s) of stem-like cells expressing the ABCG2 drug transporter that can efflux standard chemotherapies. Since chemo-resistant stem-like cells appear to be a driving force in tumor progression and metastasis for a variety of cancers, innovative treatment strategies are necessary to eradicate these rare cell populations. Terminal differentiation, as a means to deplete the pool of stem-like cells in RB, is an intriguing approach to cancer therapeutics. However, the full extent of RB differentiation remains unknown. Differentiation of RB cells has been examined in response to a variety of different agents, including retinoic acid/sodium butyrate, Pigment Epithelial-Derived Factor, as well as Succinylated Concanavalin A. RB cells exhibit morphologic and phenotypic responses to these differentiating agents, although the permanence of these effects is questionable due to reversibility. Further study of differentiation programs may lead to new approaches in the design of strategies to combat the initiation and progression of RB in vivo.

Topics: Antineoplastic Agents; Butyrates; Cell Differentiation; Concanavalin A; Eye Proteins; Humans; Neoplastic Stem Cells; Nerve Growth Factors; Retinoblastoma; Serpins; Tretinoin; Tumor Cells, Cultured

Retinoblastoma (Rb) is the most prevalent intraocular pediatric malignancy of the retina. Significant genetic factors are known to have a role in the development of Rb.. Here, we report the mutation status of 4813 clinically significant genes in six patients with noncarrier of RB1 gene mutation and having normal RB1 promoter methylation from three families having higher risk for developing Rb in the study.. A total of 27 variants were detected in the study. Heterozygous missense variants c.1162G > A (p.Gly388Arg) in the FGFR4 gene; c.559C > T (p.Pro187Ser) in the NQO1 gene were identified. The family based evaluation of the variants showed that the variant, c.714T > G (p.Tyr238Ter), in the CLEC7A gene in first family; the variant, c.55C > T (p.Arg19Ter), in the APOC3 gene and the variant, c.1171C > T (p.Gln391Ter), in the MUTYH gene in second family; and the variant, c.211G > A (p.Gly71Arg), in the UGT1A1 gene in the third family, were found statistically significant (p < 0.05).. This study might be an important report on emphazing the mutational status of other genes in patients without RB1 gene mutations and having high risk for developing Rb. The study also indicates the interaction between the retinoic acid pathway and Rb oncogenesis for the first time.

Topics: Adult; Child; Child, Preschool; DNA Glycosylases; Exome; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Glucuronosyltransferase; Heterozygote; High-Throughput Nucleotide Sequencing; Humans; Male; Mutation, Missense; NAD(P)H Dehydrogenase (Quinone); Pedigree; Promoter Regions, Genetic; Protein Interaction Maps; Receptor, Fibroblast Growth Factor, Type 4; Retina; Retinal Neoplasms; Retinoblastoma; Retinoblastoma Binding Proteins; Tretinoin; Ubiquitin-Protein Ligases

Retinoids have been shown to serve promising therapeutic agents for human cancers, e.g. the treatment of neuroblastoma. Synthetic retinoids, specific for particular retinoic acid (RA) receptors, are tested as new therapy strategies. In the present study, application of recombinant retinoic acid (RA) lowers retinoblastoma (RB) cell viability and induces apoptosis in RB cell lines. Combined treatment of RA and bone morphogenetic protein 4 (BMP-4) increases the pro-apoptotic effect of RA in the RB cells lines WERI-Rb1, Y-79, RB355, RBL-30 and RBL-15, indicating an additive effect. We could show that in WERI-Rb1 cells RA/BMP-4 mediated cell death is at least partially caspase-dependent, whereby RA and BMP-4 additively increased (i) Apaf-1 mRNA levels, (ii) caspase-9 cleavage activity and (iii) the number of activated, cleaved caspase-3 positive cells. Compared to single application of RA and BMP-4, combined RA/BMP-4 treatment significantly augments mRNA levels of the retinoic acid receptors (RARs) RARα and RARß and the retinoic X receptor (RXR) RXRγ suggesting an interaction in the induction of these RA receptor subtypes in WERI-Rb1 cells. Agonist studies revealed that both, RARs and RXRs are involved in RA/BMP-4 mediated apoptosis in WERI-Rb1 retinoblastoma cells. Employing specific RAR subtype antagonists and a RXRß and RXRγ knockdown, we proved that RA/BMP-4 apoptosis signaling in WERI-Rb1 cells requires the RA receptor subtypes RARα, RARß, RXRß and RXRγ. Deciphering signaling mechanisms underlying apoptosis induction of RA and BMP-4 in WERI-Rb1 cells, our study provides useful starting-points for future retinoid-based therapy strategies in retinoblastoma.

Topics: Adult; Apoptosis; Apoptotic Protease-Activating Factor 1; Bone Morphogenetic Protein 4; Caspases; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Receptors, Retinoic Acid; Retinoblastoma; Retinoid X Receptors; Signal Transduction; Tretinoin; Up-Regulation

The homeobox transcription factor orthodenticle homeobox 2 (OTX2) plays a critical role in very early neurogenesis, but can become oncogenic when aberrantly expressed later in life. We previously discovered its novel oncogenic role in the malignant childhood brain tumor medulloblastoma and hypothesize an oncogenic role in retinoblastoma. Primary retinoblastoma tumors and cell lines were analyzed by quantitative-PCR, immunoblotting and immunohistochemistry for OTX2. The effect of modulating OTX2 expression on tumorigenesis was tested pharmacologically and by siRNA. A lentiviral shRNA-engineered vector was used for conditional knockdown studies on tumor growth in vivo. A luciferase reporter assay was used to analyze ATRA's effect on OTX2's promoter. In this study on retinoblastoma, OTX2 was frequently amplified and/or overexpressed in primary tumors and cell lines. Knockdown of OTX2 expression by siRNA or pharmacologic inhibition by all-trans retinoic acid (ATRA) repressed OTX2 expression and cell proliferation and significantly decreased tumor growth in vivo. Loss of OTX2 expression also resulted in decreased expression of C-MYC and CRX, genes previously implicated in retinoblastoma tumorigenesis. Loss of OTX2 expression increased the phosphorylation of RB, a potential mechanism of modulating cell proliferation. Aberrant expression of OTX2 may contribute to the development of retinoblastoma. OTX2 may serve as a common transcription factor that interlinks multiple tumor-driving pathways. These results also show that OTX2 can be genetically and pharmacologically targeted, providing an exciting new therapeutic option that may be less toxic and more efficacious than current treatments.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Otx Transcription Factors; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Retinoblastoma; Retinoblastoma Protein; Signal Transduction; Trans-Activators; Tretinoin

Recombinant adeno-associated virus type 2 (AAV2) vectors have shown great promise in current ophthalmology clinical trials targeting gene delivery to the retinal pigment epithelium (RPE). To treat the majority of retinal diseases, however, gene delivery would need to be targeted to photoreceptor neurons of the outer retina. AAV2 pseudotyped with the AAV5 capsid (AAV2/5) has shown far greater transduction efficiency in photoreceptors compared to standard AAV2 vectors. For clinical trial applications using gene therapy, it is helpful to generate pre-clinical data in human cells wherever possible. There is however very little data, indeed some controversy, as to whether AAV2/5 can be used effectively in differentiated neurons in culture. In this study we show that transduction of the human neuroblastoma cell line SH-SY5Y with recombinant AAV2/5 expressing GFP is well tolerated. Furthermore, we explore the mechanism whereby exposure to retinoic acid (RA) and the phorbol ester 12-O-Tetradecanoylphorbol-13- acetate (TPA) can induce this cell line to differentiate into a stable population of human neurons, with significantly increased levels of AAV2/5 transduction. These observations may be helpful for assessing AAV2/5 vectors in vitro, particularly where it is necessary to generate pre-clinical data for clinical trials of gene therapy to the human central nervous system.

Topics: Ataxia Telangiectasia Mutated Proteins; Carcinogens; Cell Cycle Proteins; Cell Differentiation; Cell Division; Dependovirus; DNA-Binding Proteins; Gene Expression Regulation; Genetic Vectors; Green Fluorescent Proteins; HEK293 Cells; Humans; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Receptors, Platelet-Derived Growth Factor; Retinal Neoplasms; Retinoblastoma; RNA, Messenger; Tacrolimus Binding Proteins; Tetradecanoylphorbol Acetate; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Retinoblastoma is the most common primary intraocular malignancy in children. With the progression of retinoblastoma, retinoblastoma cells lose their ability to differentiate. Regardless of many attempts to identify prognostic factors in retinoblastoma, further investigation for prognostic factors of retinoblastoma progression is still required because of the lack of sensitivity and specificity of these prognostic factors in predicting disease progression. We demonstrated that the differential expression of the neurotrophin receptors TrkA and TrkB is closely related to the differentiation of retinoblastoma cells. While retinoblastoma cells expressed TrkA as well as TrkB, their growth rates were not influenced by the addition of nerve growth factor to the culture medium. In experimental animal models of retinoblastoma, TrkA expression was primarily detected in more differentiated areas with high nm23 immunoreactivity whereas TrkB expression was apparent in more proliferative areas with high Ki67 immunoreactivity. With retinoic-acid-induced differentiation of retinoblastoma cells, TrkA expression significantly increased whereas TrkB significantly decreased. The differential expression of TrkA and TrkB with differentiation of retinoblastoma cells was mediated by extracellular-signal-regulated kinase 1/2 activation, which was confirmed by immunocytochemistry of TrkA. Therefore, our results suggest that the differential expression of TrkA and TrkB could be valuable as a therapeutic target, for instance using specific inhibitors.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Gene Expression Profiling; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasms, Experimental; Nerve Growth Factor; Receptor, trkA; Receptor, trkB; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Tretinoin

To investigate the inhibition of proliferation of retinoblastoma cells (SO-Rb50) and the differentiation of tumor cells into normal cell types by all trans retinoic acid (ATRA).. This was an experimental study. SO-Rb 50 cells were treated with ATRA at different concentrations and growth curves were plotted. IC50 was analyzed by MTI method. Cell cycles before and after drug treatment were analyzed using flow cytometry. Morphologic characters of living cells and hematoxylin and eosin (HE) stained cells were observed microscopically before and 20 days after drug treatment The cell markers of NSE, vimentin, GFAP were detected with immunohistochemical method before and 20 days after drug treatment.. Cell growth was inhibited by ATRA treatment dose-dependently from 0 to 1 x 10(-5) mol/L and the cell viability was decreased. The IC50 of ATRA measured by MTT was approximately 14.05 microm/L. Treated with 1 x 10(-5) mol/L ATRA, the G0/G1 stage cells increased from 56.5% before treatment to 66.6%-81.0% after treatment, whilst S stage cells decreased from 33.1% to 22.3%-15.9%. Cells could attach to poly-lysine coated cover slides and formed small colonies. Some cells changed from round in shape to oval or fusiform shapes. The ratio of nuclear to cytoplasm decreased after the treatment of ATRA. Some cells extended long or short processes 20 days after treatment. Immunohistochemical studies showed that cells were stained moderately positive, strongly positive and weakly positive by NSE, vimentin and GFAP antibodies, respectively before treatment. After the ATRA treatment, cells were stained strongly positive, strongly positive and moderately positive by NSE, vimentin and GFAP antibodies, respectively.. ATRA significantly inhibits the proliferation of SO-Rb 50 cells and cells arrested at G0/G1 stage of cell cycle. ATRA induces differentiation of retinoblastoma cells towards the neurons and gliocytes.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Retinoblastoma; Tretinoin

We previously reported that N-(4-hydroxyphenyl)retinamide (4HPR) inhibits retinoblastoma tumor growth in a murine model in vivo and kills Y79 retinoblastoma cells in vitro. In this work, we assayed different cell death-related parameters, including mitochondrial damage and caspase activation, in Y79 cells exposed to 4HPR. 4HPR induced cytochrome c release from mitochondria, caspase-3 activation, and oligonucleosomal DNA fragmentation. However, pharmacologic inactivation of caspases by the pan-caspase inhibitor BOC-D-fmk, or specific caspase-3 inhibition by Z-DEVD-fmk, was not sufficient to prevent cell death, as assessed by loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, disruption of mitochondrial transmembrane potential (Deltapsi(m)), and ATP depletion. We found that 4HPR causes lysosomal membrane permeabilization and cytosolic relocation of cathepsin D. Pepstatin A partially rescued cell viability and reduced DNA fragmentation and cytosolic cytochrome c. The antioxidant N-acetylcysteine attenuated cathepsin D relocation into the cytosol, suggesting that lysosomal destabilization is dependent on elevation of reactive oxygen species and precedes mitochondrial dysfunction. Activation of AKT, which regulates energy level in the cell, by the retinal survival facto]r insulin-like growth factor I was impaired and insulin-like growth factor I was ineffective against ATP and Deltapsi(m) loss in the presence of 4HPR. Lysosomal destabilization, associated with mitochondrial dysfunction, was induced by 4HPR also in other cancer cell lines, including PC3 prostate adenocarcinoma and the vascular tumor Kaposi sarcoma KS-Imm cells. The novel finding of a lysosome-mediated cell death pathway activated by 4HPR could have implications at clinical level for the development of combination chemoprevention and therapy of cancer.

Topics: Acetylcysteine; Adenosine Triphosphate; Antineoplastic Agents; Benzyl Compounds; Caspase Inhibitors; Cathepsin D; Cell Death; Cell Survival; Cytochromes c; Cytosol; Enzyme Activation; Flow Cytometry; Humans; Hydrocarbons, Fluorinated; Insulin-Like Growth Factor I; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Retinoblastoma; Time Factors; Tretinoin

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.

Topics: Acitretin; Antineoplastic Agents; Benzothiazoles; Blotting, Western; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Humans; Imidazoles; Isotretinoin; Retinoblastoma; Retinoic Acid 4-Hydroxylase; Retinol-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; Thiazoles; Tretinoin

Glutamate is the major excitatory neurotransmitter in the retina and glutamate uptake is essential for normal glutamate signalling. Retinal diseases may induce neurochemical changes which affect retinal cells including retinal pigment epithelium (RPE). The aim of the study was to investigate the expression of glutamate transporter subtypes in RPE and retinoblastoma cells and to clarify the effect of proliferation modulators on the levels of the expressed transporter in the RPE cell line.. Cultured pig RPE cells and two human RPE cell lines, D407 and ARPE-19, as well as the human retinoblastoma cell line Y79 were used. Glutamate transporter expression was evaluated with Western blot analysis and immunocytochemistry.. The study revealed unexpected expression of neuronal glutamate transporter/chloride channel EAAT4 in these three cell lines, but not in cultured pig RPE cells, whereas another glutamate carrier, EAAC1, was present in all cell types utilized. Other transporter subtypes, GLT1, GLAST and EAAT5 were not found. Neither tamoxifen, known to inhibit both proliferation and glutamate uptake in RPE cells, nor retinoic acid nor insulin, also known to affect cell proliferation rates, were capable of changing the total levels of EAAT4 in APRE-19 cells.. Neuronal glutamate transporter EAAC1 is expressed in RPE cells. The robust expression of EAAT4 in cell lines may reflect a role of EAAT4 in cell proliferation and migration. Unaltered steady-state expression of this carrier and chloride-channel protein hints at posttranslational mechanisms of regulation of EAAT4.

Topics: Amino Acid Transport System X-AG; Amino Acid Transport Systems; Animals; Blotting, Western; Carrier Proteins; Cell Division; Cell Line, Transformed; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Excitatory Amino Acid Transporter 3; Excitatory Amino Acid Transporter 4; Excitatory Amino Acid Transporter 5; Glutamate Plasma Membrane Transport Proteins; Humans; Immunoenzyme Techniques; Insulin; Photoreceptor Cells; Pigment Epithelium of Eye; Protein Isoforms; Retinal Neoplasms; Retinoblastoma; Swine; Symporters; Tamoxifen; Tretinoin; Tumor Cells, Cultured

Both all-trans-retinoic acid (ATRA) and pigment epithelial-derived factor (PEDF) regulate cell proliferation and differentiation. Treatment of human Y-79 retinoblastoma and A-RPE 19 pigment epithelial cells with ATRA increased the levels of PEDF protein and RNA. Endothelial cells from bovine retina and human umbilical cord expressed PEDF and the levels were also increased by ATRA. Mouse Müller glial cells and rat C6 glioma cells showed at least a 2.5 fold increase in PEDF RNA levels after ATRA treatment, as measured by quantitative PCR. The PEDF promoter contains a retinoic acid receptor element (RARE). Plasmids containing a PEDF promoter regulating a luciferase gene were transfected into D407 and C6 cells and the luciferase activity measured after incubation in the presence or absence of ATRA. In both cell types ATRA increased the level of luciferase activity suggesting the RARE is functional. Dexamethasone was also effective at increasing PEDF RNA levels in both mouse Muller glial cells and C6 rat glioma cells. To test the effects of PEDF on retinoic acid function, expression of retinoic acid receptors in Y-79 and A-RPE 19 cells was measured by PCR. In Y79 cells, PEDF treatment increased the expression levels of RARalpha and RXRgamma receptors and in the A-RPE 19 cells it resulted in a decrease in expression of the RARbeta and RXRbeta receptors. This study clearly indicates an interaction between PEDF and ATRA. The cell differentiation activities of PEDF may operate through mechanisms orchestrated by retinoids, and the converse may also be true. The differentiation, anti-mitotic, and apoptotic actions of PEDF and ATRA may utilize parallel pathways that converge at key junctional transduction molecules to coordinate cellular quiescence and maintain tissue mass in the presence of signals that stimulate abnormal cell proliferation. It will be an interesting therapeutic strategy to co-administer PEDF and retinoic acid in developing protocols for neovascular diseases in the eye and in cancer.

Topics: Animals; Cattle; Dexamethasone; Endothelial Cells; Eye Proteins; Gene Expression Regulation; Glucocorticoids; Humans; Mice; Nerve Growth Factors; Pigment Epithelium of Eye; Promoter Regions, Genetic; Proteins; Rats; Receptors, Retinoic Acid; Retina; Retinoblastoma; RNA; Serpins; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Cell Cycle; Cell Division; Cell Line, Tumor; Flow Cytometry; Humans; Proliferating Cell Nuclear Antigen; Retinal Neoplasms; Retinoblastoma; Tretinoin

To understand the genetic regulatory pathways underlying the retinoic acid (RA) induction of cone arrestin, gene array technology and other molecular tools were used to profile global gene expression changes in human retinoblastoma cells.. Weri-Rb-1 retinoblastoma cells were cultured in the absence or presence of RA for various periods. DNA microarray analysis profiled gene expression followed by real-time PCR and Northern and immunoblot analyses to confirm the change in expression of selected retinal genes and their gene products. Additional methodology included flow cytometry analysis, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.. DNA microarray analysis of approximately 6800 genes revealed RA-induced upregulation of cone-specific genes and downregulation of rod-specific genes in Weri-Rb-1 cells. Other significantly upregulated mRNAs included chicken ovalbumin upstream promoter-transcription factor (COUP-TF1), retinoid X receptor (RXR)-gamma, thyroid hormone receptor (TR)-beta2, and guanylyl cyclase-activating protein (GCAP)-1. Real-time PCR and/or Northern blot analysis confirmed the expression changes of a subset of genes including the upregulation of a pineal- and retina-specific transcription factor, CRX. RA treatment also led to G(0)/G(1) cell cycle arrest and increased both the intensity of human cone arrestin (hCAR)-immunoreactivity and the number of apoptotic cells. The cell-cycle-arrest stage correlated with the observed microarray results in which the RA treatment downregulated critical genes such as cyclins (cyclin E, cyclin D3) and cyclin-dependent kinases (CDK5, CDK10).. These data suggest that RA induces a subpopulation of retinoblastoma cells to differentiate toward a cone cell lineage while selectively leading other cells into apoptosis.

Topics: Antineoplastic Agents; Blotting, Northern; Cell Cycle; Cell Differentiation; DNA, Neoplasm; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

To investigate the mechanism of all trans retinoid acid (ATRA) inhibition of cell growth and induction of apoptosis in human retinoblastoma Y79 cells.. Antiproliferating effects of ATRA on Y79 cells were studied by (3)H-thymidine incorporation. Cell cycle analysis was performed by flow cytometry, apoptosis of the ATRA-treated cells was determined by DNA fragmentation analysis and JNK phosphorylation analyzed by Western blot.. After 36h treatment of 1 micro mol/L ATRA, (3)H-thymidine incorporation decreased to 40% with Y79 cells arrested in G(0)/G(1) and Sub-G(1) peak appeared. DNA ladder was observed in DNA fragmentation analysis after 36h treatment of ATRA. Curcumin, a JNK blocker, blocked the apoptosis and the growth inhibition induced by ATRA. JNK was phosphorylated in 10 to 20 min.. ATRA can induce the apoptosis in Y79 cells by phosphorylation of JNK, which suggests that ATRA may have clinical application prospects for treatment of retinoblastoma.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Phosphorylation; Retinoblastoma; Thymidine; Tretinoin

Topics: Arrestin; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation; Humans; Melatonin; Nuclear Receptor Subfamily 1, Group F, Member 1; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Retinal Cone Photoreceptor Cells; Retinal Neoplasms; Retinoblastoma; RNA, Messenger; Time Factors; Trans-Activators; Tretinoin

We sought to determine whether differentiation agents such as retinoids and butyrate regulate transcript levels of interphotoreceptor retinoid binding protein (IRBP) and cone rod homeobox (CRX), a homeodomain transcription factor that regulates IRBP promoter activity. WERI-Rb1 retinoblastoma cells were treated with all-trans retinol, all-trans retinoic acid, or butyrate. IRBP and CRX mRNA levels were determined by quantitative RT-PCR. Butyrate at low concentrations increased both mRNA levels but suppressed them at higher concentrations. Retinoic acid had minimal effects. Retinol increased CRX mRNA over four fold. IRBP and CRX transcript levels are sensitive to butyrate and CRX expression is sensitive to retinol.

Topics: Butyrates; Dose-Response Relationship, Drug; Eye Proteins; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Retinoblastoma; Retinoids; Retinol-Binding Proteins; RNA, Messenger; RNA, Neoplasm; Trans-Activators; Tretinoin; Tumor Cells, Cultured; Vitamin A

This study was initiated to investigate the molecular mechanisms of activation of expression of the human cone arrestin (hCAR) gene by retinoic acid (RA), in an in vitro model of retinoblastoma cells.. Human retinoblastoma Weri-Rb-1 or Y79 cell lines were cultured in the absence or presence of RA analogues with transcription or translation inhibitors for various periods. The mRNAs encoding hCAR, retinoic acid receptor (RAR), and retinoid X receptor (RXR) subtypes were analyzed by Northern blot. Immunoblot analysis of hCAR protein was also performed. The hCAR promoter's activity and its responsiveness to RA treatment was evaluated by transient transfection of the hCAR promotor-luciferase reporter constructs, followed by promoter deletion analysis to map the specific regions responsible for the RA response.. In our in vitro model, both all-trans RA and 9-cis RA induced hCAR mRNA in a time- and dose-dependent manner. RA's effect was blocked by either RNA or protein synthesis inhibitors; however, hCAR mRNA's stability was not affected by RA, as determined by RNA decay experiments. Although all RAR and RXR subtypes were detected, only RXRgamma and RARalpha increased dramatically after treatment with RA. An RXR-specific agonist, but not an RAR-specific agonist, also increased hCAR mRNA and protein expression in both Weri-Rb-1 and Y79 cells. RA stimulated hCAR promoter-luciferase activity in transient transfection studies. Subsequently, a region between -852 and -702 of the hCAR promoter, with RA-responsive elements (RAREs), was discovered to be responsible for the RA response.. The hCAR gene is transcriptionally upregulated by RA acting through cis elements within -852 to -702 of the hCAR 5' flanking region. Based on the cumulative data, RXRgamma is most likely the RA receptor subtype involved in hCAR regulation by RA.

Topics: Arrestins; Base Sequence; Blotting, Northern; Dose-Response Relationship, Drug; Gene Expression; Humans; Immunoblotting; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Response Elements; Retinal Cone Photoreceptor Cells; Retinal Neoplasms; Retinoblastoma; Retinoid X Receptors; RNA, Messenger; Time Factors; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Vascular endothelial growth factor (VEGF) is an angiogenic peptide that has been implicated in many retinopathies. Although all trans-retinoic acid (atRA) has long been known as an essential factor in the visual cycle, the role of atRA in the pathogenesis of retinal disease remains elusive. In this study, we investigated the effects of atRA on expression of the VEGF gene in retinoblastoma Y79 cells.. Total RNA prepared from Y79 cells, with or without atRA, was subjected to Northern blot analyses. Reporter constructs consisting of the VEGF promoter-luciferase gene were transfected into Y79 cells. Nuclear factors binding to the VEGF promoter were analyzed by electrophoretic mobility shift assays (EMSAs).. The levels of VEGF transcripts were increased by atRA in a time- and dose-dependent manner. Progressive deletion and site-specific mutation analyses indicated that atRA increased VEGF promoter activity through a G+C-rich sequence that was shown to be an Sp1-binding site by supershift assays. EMSAs showed that Sp1 binding was increased by atRA stimulation. Although no measurable change was observed in Sp1 mRNA levels, Western blot analysis showed an increase in Sp1 protein levels in the atRA-treated cells. These data suggest that atRA increases Sp1 protein levels by posttranscriptional mechanisms, and elevated levels of Sp1 protein induce the expression of VEGF at the transcriptional level.. atRA induced transcription of the VEGF gene through Sp1-binding sites in Y79 cells. Pharmacologic intervention that inhibits the signals elicited by atRA may be effective in treating VEGF-mediated retinopathies.

Topics: Antineoplastic Agents; Binding Sites; Blotting, Northern; Blotting, Western; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Luciferases; Lymphokines; Recombinant Fusion Proteins; Retinal Neoplasms; Retinoblastoma; RNA, Messenger; Sp1 Transcription Factor; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Tyrosinase-related protein 2 (TRP-2), also known as DOPAchrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/DOPAchrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.

Topics: Colforsin; Gene Expression; Genes, Reporter; Humans; Melanoma; Monophenol Monooxygenase; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) activated the extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein kinase (MAPK) of HL-60 human myeloblastic leukemia cells before causing myeloid differentiation and cell cycle arrest associated with hypophosphorylation of the retinoblastoma (RB) tumor suppressor protein. ERK2 activation by mitogen-activated protein/ERK kinase (MEK) was necessary for RA-induced differentiation in studies using PD98059 to block MEK phosphorylation. G0 growth arrest and RB tumor suppressor protein hypophosphorylation (which is typically associated with induced differentiation and G0 arrest), two putatively RB-regulated processes, also depended on ERK2 activation by MEK. Activation of ERK2 by RA occurred within hours and persisted until the onset of RB hypophosphorylation, differentiation, and arrest. ERK2 activation was probably needed early, because delaying the addition of PD98059 relative to that of RA restored most of the RA-induced cellular response. In contrast to RA (which activates RA receptors (RARs) and retinoid X receptors in HL-60 cells with its metabolite retinoids), a retinoid that selectively binds RAR-gamma, which is not expressed in HL-60 cells, was relatively ineffective in causing ERK2 activation. This is consistent with the need for a nuclear retinoid receptor function in RA-induced ERK2 activation. RA reduced the amount of unphosphorylated RAR-alpha, whose activation is necessary for RA-induced differentiation and arrest. This shifted the ratio of phosphorylated:unphosphorylated RAR-alpha to predominantly the phosphorylated form. Unlike other steroid thyroid hormone receptors susceptible to phosphorylation and activation by MAPKs, RAR-alpha was not phosphorylated by the activated ERK2 MAPK. The results thus show that RA augments MEK-dependent ERK2 activation that is needed for subsequent RB hypophosphorylation, cell differentiation, and G0 arrest. The process seems to be nuclear receptor dependent and an early seminal component of RA signaling causing differentiation and growth arrest.

Topics: Antineoplastic Agents; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Enzyme Activation; HL-60 Cells; Humans; Mitogen-Activated Protein Kinase 1; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Resting Phase, Cell Cycle; Retinoblastoma; Retinoic Acid Receptor alpha; Tretinoin

We investigated the effect of two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration-dependent manner. The IC50 for this inhibition by all-trans RA and 9-cis RA was 1.50 and 0.15 microM, respectively. The inhibitory effect of 9-cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all-trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9-cis RA and all-trans RA was more than 90% and 95%, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9-cis RA on the inhibition of Y79 cell growth is much greater than that of all-trans RA.

Topics: Alitretinoin; Antineoplastic Agents; Cell Division; Cell Survival; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Retinoblastoma; Time Factors; Tretinoin; Trypan Blue; Tumor Cells, Cultured

Both 1,25-dihydroxyvitamin D3 (VD) and retinoids have potent effects on keratinocyte proliferation. Parallelism in their action as steroid hormones, which involves interaction of their receptors, and in their therapeutic efficacy for hyper-proliferative skin diseases provides a rationale to investigate their combined action on proliferation in pre-confluent human epidermal keratinocyte cultures. As shown by [3H]thymidine incorporation, all-trans retinoic acid (atRA) at subpharmacologic concentrations and 9-cis retinoic acid (9cRA) diminished the anti-proliferative effect of VD. Pre-incubation of the cells with the retinoids clearly enhanced this effect. Cell-cycle analysis revealed G1 arrest upon VD treatment that was attenuated by retinoic acid (RA). Moreover, Northern and Western blot analysis demonstrated that retinoic acid opposed VD-induced accumulation of transforming growth factor-beta1, p21WAF1, and p27KIP1. Finally, retinoic acid reduced VD-elicited hypophosphorylation of the retinoblastoma protein. AtRA at micromolar concentrations conversely potentiated most of the aforementioned VD-dependent actions. In addition, atRA and 9cRA (but not VD) caused a rapid, sustained reduction of RXR alpha protein. VD receptor protein was induced by VD regardless of the presence of RA. In conclusion, RA modulates VD-dependent effects at different levels of keratinocyte proliferation. This could have implications for the use of combinations of both drugs for skin diseases.

Topics: Calcitriol; Cell Cycle Proteins; Cell Division; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; G1 Phase; Humans; Keratinocytes; Microtubule-Associated Proteins; Phosphorylation; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoblastoma; Retinoid X Receptors; Retinoids; Thymidine; Time Factors; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Tritium; Tumor Suppressor Proteins

Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 microM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 microM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 microM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours.

Topics: Adjuvants, Immunologic; Antigens, Surface; Antineoplastic Agents; Cell Differentiation; Dose-Response Relationship, Drug; Eye Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Immunoglobulins; Immunohistochemistry; Intercellular Adhesion Molecule-1; Neural Cell Adhesion Molecules; Retinoblastoma; Thy-1 Antigens; Time Factors; Tretinoin; Tumor Cells, Cultured

Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all-trans-, 13-cis-, and 9-cis-RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.

Topics: Acetylserotonin O-Methyltransferase; Base Sequence; Blotting, Northern; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Retinoblastoma; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

The induction of apoptosis in cultured retinoblastoma cells by diverse drugs was examined by analyzing DNA fragmentation, a hallmark of apoptosis. First, the ability of six retinoblastoma cell lines to undergo apoptosis was surveyed using etoposide (30 micrograms/ml, 20 h exposure). The NCC-RbC-60 cell line, established in this laboratory showed DNA fragmentation clearly, whereas the other cell lines tested, including the representative retinoblastoma cell line, Y-79, did not show distinct DNA fragmentation. Biochemical modulators, such as A23187, forskolin, retinoic acid, phorbol 12-myristate 13-acetate and okadaic acid, were examined to ascertain whether they could induce apoptosis in NCC-RbC-60 and Y-79 cells after exposure for 20 h. Only okadaic acid induced DNA fragmentation in all the retinoblastoma cell lines tested and it induced DNA fragmentation in Y-79 cells in a time- and concentration-dependent manner. Flow-cytometric analysis and microscopic examination revealed that Y-79 cells treated with okadaic acid for 24-48 h accumulated at the G2/M, especially M, phases, before undergoing DNA fragmentation. Other mitotic poisons, nocodazole, colcemid and taxol, also induced apoptosis in Y-79 cells. In the K1034 cell line, established from non-malignant retinal pigmented epithelium, okadaic acid failed to induce both G2/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.

Topics: Apoptosis; Calcimycin; Cell Cycle; Cell Survival; Child, Preschool; Cholera Toxin; Colforsin; DNA, Neoplasm; Enzyme Inhibitors; Ethers, Cyclic; Etoposide; Flow Cytometry; HL-60 Cells; Humans; Male; Mitosis; Nocodazole; Okadaic Acid; Paclitaxel; Phosphoprotein Phosphatases; Retinoblastoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

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

Topics: Blotting, Northern; Blotting, Western; Cell Line; Chromatography, High Pressure Liquid; Eye Neoplasms; Humans; Radioligand Assay; Retinoblastoma; Retinol-Binding Proteins; RNA, Messenger; Tretinoin; Tritium; Vitamin A

The human retinoblastoma gene (RB1) encodes a protein (Rb) of 105 kilodaltons that can be phosphorylated. Analysis of Rb metabolism has shown that the protein has a half-life of more than 10 hours and is synthesized at all phases of the cell cycle. Newly synthesized Rb is not extensively phosphorylated (it is "underphosphorylated") in cells in the G0 and G1 phases but is phosphorylated at multiple sites at the G1/S boundary and in S phase. HL-60 cells that were induced to terminally differentiate by various chemicals lost their ability to phosphorylate newly synthesized Rb at multiple sites when cell growth was arrested. These findings suggest that underphosphorylated Rb may restrict cell proliferation.

Topics: Cell Cycle; Cell Division; Eye Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Interphase; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Retinoblastoma; Tretinoin; Tumor Cells, Cultured

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Cell Line; Cells, Cultured; Cytosol; Drug Resistance; Eye Neoplasms; Humans; Kinetics; Male; Mice; Rats; Rats, Inbred Strains; Retinoblastoma; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Testis; Tretinoin

Cellular retinoic acid-binding proteins (CRABP) and cellular retinol-binding protein (CRBP) can be found in cells and nuclei. They function in the same way as receptors. CRABP and CRBP were studied in 9 cases of choroidal melanoma and in 3 of retinoblastoma. CRABP was found in 2 cases of melanoma and in 3 cases of retinoblastoma. CRBP was found in 1 melanoma.

Topics: Binding Sites; Eye Neoplasms; Humans; Melanoma; Osmolar Concentration; Retinoblastoma; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin

Human retinoblastoma cells were grown in tissue culture and their differentiation into Flexner-Wintersteiner rosettes was investigated. This process of photoreceptor cell differentiation was only observed in primary cultures and subsequent cell passages of tumors which showed these structures in vivo. Rosettes formed spontaneously within 5-9 d after plating of the tumor cells. Under optimal conditions in certain tumor cell strains, up to 80% of the cultured cell aggregates contained one or more differentiated Flexner-Wintersteiner rosettes. Exposure of retinoblastoma cells to RA or dcAMP did not alter the number of rosettes in culture. Retinoblastoma cells within rosettes continued to synthesize DNA, and mitotic figures were frequently observed in histological sections. Ultrastructural analysis of rosettes formed in vitro showed many of the characteristics described in those found in vivo including a polarized shape, established cell junctions (Zonula adherens), extensive accumulation of mitochondria and microtubules in the apical part of the cells, numerous basal bodies and centrioles as well as cilia typical of the mature photoreceptor cell. Lamellated stacks of membranes were also found and their nature is discussed.

Topics: Bucladesine; Cell Differentiation; Cells, Cultured; DNA; Eye Neoplasms; Humans; Retinoblastoma; Rosette Formation; Tretinoin

The effects of butyrate, retinol, and retinoic acid were tested on growth and differentiation of human Y-79 retinoblastoma cells in monolayer cultures. Treatment with 4 mM butyrate resulted in marked growth inhibition of cells, due mostly to increased death rate. The effect was greater in serum-supported cultures in which the proliferation rate was high and the effect was less in the serum-free, defined medium in which the cells were differentiated and the growth rate was slow, suggesting a cell-cycle-specific action of this substance. Moreover, butyrate induced morphologic changes in the viable cells; these changes consisted of an elongated appearance of the cells and of retraction of long processes formed in the serum-free-supported cultures. Retinol at 20 microM also affected the cell viability both in serum-containing and in serum-free culture medium. Retinoic acid at 50 microM induced reversible growth inhibition of cells growing in serum-containing medium and cell death in the defined medium-supported cultures. Combination of 0.5 mM butyrate with 50 microM retinoic acid resulted in an enhanced inhibition of growth in an apparently synergistic fashion. These results demonstrated that butyrate, retinol, and retinoic acid suppressed Y-79 cell growth in vitro and could be useful in future studies of these compounds in retinoblastoma tumors in vivo.

Topics: Butyrates; Butyric Acid; Cell Division; Cell Line; Cell Survival; Eye Neoplasms; Humans; Kinetics; Retinoblastoma; Tretinoin; Vitamin A

The full expression of Class 1 antigens of the major histocompatibility complex (HLA A,B,C,) on ocular cell surfaces appears to be developmentally regulated; HLA is undetectable in retina from 16-week-old fetal eyes but is present in adult retina. Cultured retinoblastoma (RB) may serve as a useful model of retinal cell development. SF81 is an undifferentiated subculture of in vitro retinoblastoma with little evidence of spontaneous differentiation and lack of expression of HLA, attesting to the "undifferentiated" character of this cell line. RB SF81 cells were treated with retinoic acid, a known stimulator of differentiation (optimal concentration 5 X 10(-8) M). HLA expression was evaluated with immunofluorescence, immunoperoxidase, and radioimmunoassay, using monoclonal antibodies. RB SF81 treated with retinoic acid showed a significant, persistent increase in HLA levels by 24 hours compared with controls. Thus, retinoic acid has been documented to induce the surface expression of HLA in undifferentiated retinoblastoma. Developmental parallels between embryonic retina and in vitro retinoblastoma treated with retinoic acid exist.

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Line; Culture Media; Eye Neoplasms; Fluorescent Antibody Technique; HLA Antigens; Humans; Immunoenzyme Techniques; Radioimmunoassay; Retinoblastoma; Tretinoin

Topics: Autoradiography; Biological Transport; Cell Line; Cell Nucleus; Eye Neoplasms; Humans; Retinoblastoma; Tretinoin; Vitamin A; Vitamin E

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

Receptor proteins for [3H]retinol and [3H]retinoic acid in cultured human retinoblastoma cells have been separated rapidly and reproducibility by two different methods. By isoelectric focusing, the isoelectric point of the retinol receptor is at pH 4.0; the retinoic acid receptor has a higher isoelectric point of 4.3. Polyacrylamide slab gel electrophoresis revealed a slower migration rate for the [3H]retinoic acid receptor compared to the [3H]retinol receptor. The separate nature of the two proteins has thus been established in this unique human cell line.

Topics: Carrier Proteins; Cell Line; Neoplasms, Experimental; Receptors, Drug; Retinoblastoma; Tretinoin; Vitamin A