Mutational inactivation of the retinoblastoma (Rb) gene predisposes young children to malignant tumors of the eye, and peripheral osteosarcomas as adults. The genesis of a variety of additional human cancers, including small cell lung, bladder and breast carcinomas, is also associated with functional inactivation of the Rb gene. As such, Rb is believed to function in normal cells to limit or constrain cell proliferation and/or facilitate cell differentiation. In recent years, Rb has been shown to physically and functionally interact with a number of cellular transcription factors and to regulate the expression of a handful of growth-related genes. The Rb protein is also subject to a complex series of cell-cycle regulated phosphorylation and dephosphorylation reactions. Is it believed, although not as yet proved, that these cyclic changes in post-translational modification result in alteration of Rb function. Taken together, it is likely that Rb functions as a """"""""molecular switch"""""""", transducing intra- and extracellular signals into differential rates of transcription of key growth-regulating genes. Our long term goals are to precisely define the mechanism(s) by which Rb function is regulated, to identify targets of Rb function, and to relate this knowledge to processes such as cell-cycle progression, cellular senescence, and differentiation. Our experimental approach is to use in vitro assays, such as affinity chromatography and protein-DNA binding, to identify, clone, and characterize cellular proteins that physically interact with the Rb protein or Rb-regulated promoter elements. Thus, in this proposal our Specific Aims are: 1. Identification, cloning, biochemical characterization, and functional analysis of cDNAs encoding proteins that bind specifically to the amino- terminal one-third of the human and murine Rb proteins. 2. Cloning and/or characterization of two nuclear proteins that specifically-bind Rb-regulated elements within the c-fos, c-myc, and TGF- beta1 promoters in vitro and functional analysis of Rb-mediated transcriptional regulation.
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