The homozygous inactivation of certain genes, termed anti-oncogenes, has been associated with the pathogenesis of a wide variety of tumors. In particular, inactivation of the retinoblastoma susceptibility gene (RB) has been associated with the etiology of retinoblastomas as well as several other types of human tumors including bladder, breast, small cell lung and prostate carcinomas. Although the function of the nuclear RB protein is unclear, recent observations have suggested that RB can regulate the transcription of at least one proto-oncogene (c-fos) through a specific cis-acting element. This cis-acting element, termed RCE, is also found in the promoters of the c-myc, TGF-beta1, IL-6, JunB, and IGF-2 genes as well as the RB gene itself, suggesting that RB can regulate the expression of a set of growth factors and proto-oncogenes involved in modulating cell growth. The focus of the proposed work is to understand the mechanism through which RB functions to regulate RCE-mediated transcription. At least three factors have been identified as binding to the RCE by gel mobility shift analysis. The proposed experiments will determine the function of these distinct factors in mediating transcriptional regulation by RB in vivo. Preliminary experiments have suggested that RB may be able to inhibit the binding of the factors directly through protein-protein interaction. The proposed experiments will examine how RB regulates binding of the factors and to map the domain of RB responsible for the observed repression of binding. Experiments win involve the introduction of a RB protein, tagged with a specific epitope, into cell lines deficient in RB protein using a vaccinia vector. The effect of the RB protein on binding of the factors in the infected cells will be determined. Similar experiments will also be performed using RB synthesized in vitro in a transcription/translation reaction that will be added to nuclear extracts prepared from retinoblastoma cells. Particular emphasis Will be placed on examining mutant RB proteins similar to those found in human tumors to determine their ability to regulate RCE transcriptional activity in vivo and to regulate binding of factors to the RCE in vitro. Since SV40 T-antigen and EIA bind directly to RB to presumably inactivate its function, the effect of these viral proteins on binding of the factors to the RCE will be addressed both in vitro and in vivo. The successful completion of the project should significantly increase our understanding of the mechanism of action of an anti-oncogene and could potentially lead to a biological treatment for certain cancers.
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