BIOLOGICAL Function of the Retinoblastoma Gene Product

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The long-range goal of our research is to understand the biological function of the retinoblastoma tumor suppressor protein (RB). During the current funding period, we have obtained results to support the idea that RB functions as an inhibitor of cell proliferation and cell death. This dual role of RB allows for terminal differentiation that is coupled to long-term survival, e.g., of muscles and neurons. Inactivation of RB by genetic mutation, by viral oncoproteins, or by phosphorylation, is associated with tumor development. The inactivation of RB, we reason, must be complemented by defects in apoptosis to cause tumor development. The proposed research is designed to identify genetic programs that are regulated by RB to establish terminal growth arrest and the resistance to apoptosis. We are interested in the hypothesis that RB may regulate a single "transcriptome" to block mitogenic and apoptotic stimulation of differentiated cells. The proposed research is based on two interesting RB mutants we have created. The point- mutant N757F can inhibit S-phase entry, but cannot establish mitogen- resistant growth arrest during muscle differentiation. The point-mutant MI is resistant to proteolytic degradation during apoptosis and can protect cells from tumor necrosis factor (TNF)-induced death. We have created a germline MI mutation in the mouse Rb gene and found these Rb-MI mice to be resistant to septic shock. We propose to create a Rb- N757F mice as a model to further study the function of RB in terminal differentiation. We will use proteomics methods to identify the protein- binding defects of RB-N757F. We will use genomics methods to identify genes that are differentially expressed in myocytes that express RB or RB-N757F. We will also identify genes that are differentially expressed in RB versus RB-MI cells under conditions of TNF stimulation. By comparing these two independently derived gene sets, we will be able to determine if RB regulates a common transcriptome to render cells resistant to mitogenic and apoptotic signals. Identification of RB-regulated genetic programs is fundamental to the understanding of RB function in differentiation and apoptosis. These genetics programs are likely to be affected during tumor development. Therefore, the proposed research will result in the identification of important targets for therapeutic intervention in cancer treatment.