transforming-growth-factor-beta and Retinoblastoma

The product of the retinoblastoma gene (RB1) is believed to function as a negative regulator of cell growth. Recent experimental results suggest that RB1 may exert its growth-suppressing activity by regulating the transcription of a variety of growth-related genes, including FOS, MYC, and TGFBI. A series of biochemical and molecular analyses suggest that RB1 indirectly affects gene expression via cell-cycle-regulated interactions with transcription factors, such as E2F and SPI. Determination of the mechanisms regulating such protein-protein interactions and the identification of additional targets of RB1 function will provide vital insights into the role of this tumor-suppressor gene in mammalian cell proliferation.

Topics: Adenovirus E1A Proteins; Animals; Base Sequence; Consensus Sequence; DNA-Binding Proteins; Eye Neoplasms; Fungal Proteins; Gene Expression Regulation; Genes, fos; Genes, myc; Genes, Retinoblastoma; Humans; Mice; Molecular Sequence Data; Peptide Elongation Factor 2; Peptide Elongation Factors; Promoter Regions, Genetic; Repressor Proteins; Retinoblastoma; Retinoblastoma Protein; Saccharomyces cerevisiae Proteins; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

BIGH3, a secreted protein of the extracellular matrix interacts with collagen and integrins on the cell surface. BIGH3 can have opposing functions in cancer, acting either as tumor suppressor or promoter by enhancing tumor progression and angiogenesis. In the eye, BIGH3 is expressed in the cornea and the retinal pigment epithelium and could impact on the development of retinoblastoma, the most common paediatric intraocular neoplasm. Retinoblastoma initiation requires the inactivation of both alleles of the RB1 tumor suppressor gene in the developing retina and tumor progression involves additional genomic changes. To determine whether BIGH3 affects retinoblastoma development, we generated a retinoblastoma mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing in these mice resulted in enhanced tumor development in the retina. A decrease in apoptosis is involved in the initial events of tumorigenesis, followed by an increased activity of the pro-survival ERK pathway as well as an upregulation of cyclin-dependent kinases (CDKs). Taken together, these data suggest that BIGH3 acts as a tumor suppressor in the retina.

Topics: Animals; Antigens, Polyomavirus Transforming; Apoptosis; Blotting, Western; Cyclin-Dependent Kinases; Disease Models, Animal; Extracellular Matrix Proteins; Humans; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Fluorescence; Retinal Neoplasms; Retinal Pigment Epithelium; Retinoblastoma; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Burden; Tumor Suppressor Proteins

Retinoblastoma is the most common intraocular tumor in children resulting from genetic alterations and transformation of mature retinal cells. The objective of this study was to investigate the effects of SD-208, TGF-β-RI kinase inhibitor, on the expression of some miRNAs including a miR-17/92 cluster in retinoblastoma cells. Prior to initiate this work, the cell proliferation was studied by Methyl Thiazolyl Tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Then, the expression patterns of four miRNAs (18a, 20a, 22, and 34a) were investigated in the treated SD-208 (0.0, 1, 2 and 3 µM) and untreated Y-79 cells. A remarkable inhibition of the cell proliferation was found in Y-79 cells treated with SD-208 versus untreated cells. Also, the expression changes were observed in miRNAs 18a, 20a, 22 and 34a in response to SD-208 treatment (P<0.05). The findings of the present study suggest that the anti-cancer effect of SD-208 may be exerted due to the regulation of specific miRNAs, at least in this particular retinoblastoma cell line. To the best of the researchers' knowledge, this is the first report demonstrating that the SD-208 could alter the expression of tumor suppressive miRNAs as well as oncomiRs in vitro. In conclusion, the present data suggest that SD-208 could be an alternative agent in retinoblastoma treatment.

Topics: Cell Line, Tumor; Cell Proliferation; Child; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Pteridines; Real-Time Polymerase Chain Reaction; Retinal Neoplasms; Retinoblastoma; RNA, Neoplasm; Transforming Growth Factor beta

Retinoblastoma is the most common primary intraocular tumor of childhood and may be heritable or occur sporadically. Anterior diffuse retinoblastoma is an uncommon variant that is thought to be sporadic. We describe a child with anterior diffuse retinoblastoma who presented with a pseudohypopyon. Genetic analysis showed a germline mutation of the RB1 allele that is potentially heritable. Immunofluorescence staining was positive for transforming growth factor beta and for vascular endothelial growth factor and negative for inducible nitric oxide synthase and for hypoxia inducible factor alpha in the tumor seeds, indicating acquisition of nonischemia-mediated survival factors of the tumor seeds in the aqueous humor.

Topics: Anterior Eye Segment; Biomarkers, Tumor; Child; DNA Mutational Analysis; Female; Fluorescent Antibody Technique; Germ-Line Mutation; Humans; Retinal Neoplasms; Retinoblastoma; Retinoblastoma Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To analyze the histopathology and expression of transforming growth factor beta (TGF-beta) in retinoblastoma with and without cataractous changes.. Twenty patients with unilateral retinoblastoma underwent enucleation. None of these patients had received preoperative chemotherapy or radiotherapy. Formalin-fixed, paraffin-embedded tissue sections were examined histologically for the presence of morgagnian globules or liquefaction of lens fibers; TGF-beta was immunolocalized using an anti-TGF-beta antibody.. Two globes showed several morgagnian globules and liquefaction of the lens fibers, representing cataractous changes. One patient had posterior subcapsular cataract; the other, anterior polar cataract. In both cases, prominent cytoplasmic immunoreactivity for TGF-beta was detected in retinoblastoma cells. In contrast, 3 patients showed histologic evidence of minor cataractous changes. The globes with either minor or no cataractous changes revealed minimal to no expression of TGF-beta.. These results suggest that TGF-beta produced by retinoblastoma cells may induce cataract formation. Clinical Relevance The growth factors produced by retinoblastoma cells may lead to associated pathologies, such as cataracts, in the ocular structures. This study implies that when a child presents with a unilateral cataract, retinoblastoma should be excluded as the primary diagnosis.

Topics: Cataract; Child, Preschool; Eye Enucleation; Female; Fluorescent Antibody Technique, Indirect; Humans; Infant; Male; Microscopy, Confocal; Retinal Neoplasms; Retinoblastoma; Transforming Growth Factor beta

N-myc oncogene amplification is frequent in human neuroblastoma and predicts poor prognosis, but the molecular consequences have remained obscure. We report here that enhanced N-myc expression correlates with low or undetectable expression of activin A, but not other closely related members of the transforming growth factor-beta superfamily. N-myc interacts with the activin A promoter, eventually inducing down-regulation of activin A mRNA and protein. This study demonstrates for the first time N-myc-induced down-regulation of a gene implicated in signal transduction. Down-regulation of activin A could deprive neuroblastomas from a signal with growth-inhibitory activities toward the tumor and its stroma and thereby permit neuroblastoma progression.

Topics: Activins; Amino Acid Sequence; Cell Line; Clone Cells; Culture Media, Conditioned; Down-Regulation; Gene Amplification; Genes, Reporter; Humans; Inhibins; Kidney; Molecular Sequence Data; Neuroblastoma; Peptides; Promoter Regions, Genetic; Prostatic Secretory Proteins; Proto-Oncogene Proteins c-myc; Retinoblastoma; Sequence Analysis, Protein; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Retinoblastoma cells are resistant to transforming growth factor-beta (TGF-beta) activity due to the absence of TGF-beta binding. To further elucidate the mechanism of TGF-beta resistance, we studied the expression of the TGF-beta receptors and SMADs by using the Y79 and WERI-Rb-1 retinoblastoma cell lines. Binding of 125I-TGF-beta1 to serine/threonine kinase receptor type II (TbetaR-II) and TbetaR-I was not seen in the retinoblastoma cells. TbetaR-II mRNA was not expressed in these cells, but TbetaR-I mRNA was detected. Mutation analysis revealed no mutation in the coding region of the TbetaR-II gene, and TbetaR-II mRNA could be induced after the differentiation of Y79 cells. Smad2, Smad3, and Smad4, which are involved in TGF-beta signaling, were expressed in the retinoblastoma cells. Transcriptional activation of the TGF-beta-responsive genes was not seen by the transfection of either receptor cDNA alone but could be induced by transfection of both TbetaR-II and TbetaR-I. These data suggest that the defect in the TGF-beta response is caused by the lack of TbetaR-II in the retinoblastoma cells. In addition, TbetaR-I may be functionally inactivated in these cell lines.

Topics: Activin Receptors, Type I; Animals; Cell Line; DNA Mutational Analysis; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Mink; Mutation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retinoblastoma; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Prion diseases are a group of neurodegenerative disorders characterized by intracerebral accumulation of a protease-resistant prion protein (PrP(Sc)) that causes extensive neuronal degeneration and astrogliosis. The regulation of prion protein (PrP) gene expression by a panel of glial and neuronal cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-10, and TGF-beta1) was investigated in human neural cell lines by reverse transcription-polymerase chain reaction and Northern blot analysis. The constitutive expression of PrP mRNA was identified in all human neural cell lines and tissues examined including Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG astrocytoma, KG-1-C glioma, NTera2 teratocarcinoma, NTera2-derived differentiated neurons (NTera2-N), peripheral nerve, and cerebral and cerebellar tissues. In SK-N-SH cells, a 48 hour (h) treatment with 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, or 100 nM phorbol 12-myristate 13-acetate induced a 2.7- to 4.2-fold increase in the level of PrP mRNA, while the exposure to 100 ng/ml IFN-gamma resulted in a 50% decrease. By contrast, none of these cytokines significantly altered the levels of PrP mRNA in IMR-32, NTera2-N, or U-373MG cells. These results indicate that the PrP gene expression is constitutive in a wide range of human neural cell lines and tissues where it is controlled by cell type-specific regulatory mechanisms.

Topics: Astrocytoma; Brain; Brain Neoplasms; Cell Line; Cerebellum; Cytokines; Eye Neoplasms; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-1; Interleukin-10; Neuroblastoma; Neuroglia; Neurons; Polymerase Chain Reaction; Prions; Retinoblastoma; RNA, Messenger; Teratocarcinoma; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

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

Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1.9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor beta 1, an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor beta 1 may be linked to its simultaneous suppression of cell cycle-dependent kinase 4 and cyclin E protein levels.

Topics: Animals; Blotting, Northern; Carcinoma, Hepatocellular; Cell Cycle; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver; Liver Regeneration; Male; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Retinoblastoma; Retinoblastoma Protein; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Tamoxifen has been an effective antiestrogen in suppressing breast cancer growth which is estrogen-responsive or dependent. Early studies have provided circumstantial evidence that transforming growth factor-beta (TGF-beta) may be an autocrine mediator of tamoxifen action. Therefore, it is both fundamentally important and clinically relevant to investigate the relationship between tamoxifen and TGF-beta. In this study, we demonstrated that CAMA-1 cells, which are sensitive to tamoxifen inhibition, did not respond to TGF-beta growth inhibition. The type I and II TGF-beta receptors were undetectable by the radio-ligand affinity labeling technique. Despite the presence of a normal TGF-beta type II receptor gene, the mRNA transcript of the gene was undetectable by the extremely sensitive Intron-differential RNA/PCR method. The possibility that the lack of TGF-beta receptors might be intimately linked to the absence of normal retinoblastoma (Rb) gene products, as suggested by previous studies of retinoblastoma cells, was further investigated. The lack of TGF-beta receptor expression was found due to reasons other than the absence, deletion or abnormality of the Rb gene because a normal Rb gene and its hyper- and hypo-phosphorylated protein products were detected in CAMA-1 cells. In conclusion, our results suggest that the TGF-beta system is not obligatory for antiestrogen growth inhibition of CAMA-1 cells.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Estrogens; Female; Genes, myc; Genes, Retinoblastoma; Humans; Molecular Sequence Data; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Ovarian Neoplasms; Receptors, Transforming Growth Factor beta; Retinoblastoma; Retinoblastoma Protein; Tamoxifen; Transforming Growth Factor beta; Tumor Cells, Cultured