p53-mediated apoptosis of cells with DNA damage or oncogene overexpression is a major mechanism for its function as a tumor suppressor. Many antitumor treatments such as ionizing radiation and DNA-damaging chemotherapy agents kill cancer cells through the p53-dependent apoptosis pathway. The long-term goal of the proposed studies is to understand the molecular mechanism controlling p53-mediated apoptosis so that better therapeutic strategies can be developed. The specific aims in this proposal represent the beginning of our efforts to define in detail the components of the biochemical pathway of p53-dependent apoptosis and its regulation by employing a cell-free system that recapitulates p53-dependent activation of caspases. First, the gene coding for a recently purified caspase-activating protein CAP110 will be cloned, and its role in p53-dependent apoptosis will be examined in both cell-free and cell-based systems. Second, an affinity-based chromatographic scheme will be used to purify a candidate protein tyrosine phosphatase essential for p53-dependent caspase activation in the cell-free extracts. Its gene will be cloned and its function in p53-dependent apoptosis will be examined in cells. Third, a systematic fractionation scheme will be employed to identify other components in the cell-free extracts that are required for p53-dependent activation of caspases. Forth, an anti-apoptotic activity from transformed p53-/- mouse embryo fibroblasts will be purified and cloned. Northern blot analysis will be performed to determine whether its expression is negatively regulated by p53. Understanding p53-dependent apoptosis and its regulation at the levels of biochemical process will help improve current anticancer therapies for tumors with wild-type p53 and provide targets for the development of drugs which restore the apoptotic response in p53 deficient cancer cells. Dr. David E. Fisher will supervise this project and provide guidance in my transition to an independent investigator.
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