Tumor suppressor genes (or recessive oncogenes) are a class of cellular genes for which the loss of gene function is associated with the formation of human tumors. This is in contrast to oncogenes whose overexpression or abnormal activation of gene function leads to the transformed phenotype. Retinoblastoma is a tumor of the immature retina that occurs in young children, and has served as a prototypic model system for the study of tumor suppressor genes in human cancer. Two lines of genetic evidence support the notion that the retinoblastoma gene (Rb) plays an important role in normal cellular growth processes: 1) the Rb gene is ubiquitously expressed in normal tissues but is inactivated in a subset of human tumors; 2) functional replacement of the Rb gene suppresses the tumorigenic phenotypes, including growth, of proliferating tumor cells. How Rb functions in vivo, however, is not clear. The broad, long-term objectives of this proposal are to understand how Rb acts at the molecular level to bring about its tumor suppression function and to elucidate the role of Rb in normal cellular growth processes. One clue to Rb function was realized with the discovery that viral oncoproteins such as SV40 T antigen, papilloma virus E7, and adenovirus E1A can bind to the Rb protein. Subsequent structure-function analyses by several groups including ours has supported the hypothesis that these, viral proteins may elicit cell transformation by overriding normal growth controls through sequestration of Rb protein, and that Rb may operate via protein-protein interaction. Indeed, we and others have recently identified a number of cellular proteins that can interact with Rb, but the function of many of these proteins remains elusive. To better understand the mechanism of action of Rb in normal cell growth regulation, the function of its cellular targets, namely Rb-binding proteins (RBPs), must be characterized. We will use our expertise in Rb protein structure and function to identify the structural and biochemical properties of RBPs that determine their unique functional interactions with Rb.
The specific aims of the proposed experiments are 1) to further characterize a novel activity that can regulate Rb interaction with a specific RBP protein E2F, 2) to isolate cDNA clones of RBPs, 3) to assess the role of RBPs as potential regulatory elements of cell proliferation, 4) to determine the structure-function relationship of RBPs that govern their binding affinities for Rb, 5) to characterize the spatial and temporal nature of RBP gene expression during development, the cell cycle and cellular differentiation. Since Rb gene inactivation primarily causes tumors in the immature retinas of young children, it is clear that Rb, though itself a ubiquitous protein, operates in a tissue and developmental stagespecific manner. The last aim, in conjunction with aim (2), will also address the issue if specific RBP proteins exist that might mediate the tissue and developmental stage-specific function of Rb. Overall, the information from our studies will provide a better understanding of Rb tumor suppressor gene function and contribute to our understanding of the molecular nature of human cancer.
