Apoptosis, a morphologically distinct form of cell death observed during development, withdrawal of trophic hormones, and treatment with chemotherapeutic agents or cytotoxic cytokines, reflects the active participation of endogenous cellular proteases that help diassemble the cell. The present proposal seeks funds for collaborative studies by two laboratories with a long-standing interest in testing the central hypothesis that protease activation and activity plays an important role in the apoptotic process triggered by anticancer agents. Previous studies from these laboratories have demonstrated that 1) antineoplastic agents trigger apoptosis in a variety of cell types; 2) the morphological changes of apoptosis are accompanied by the quantitative cleavage of a number of cellular polypeptides that is mediated by multiple aspartate-directed cysteine proteases (caspases) with differing substrate preferences; and 3) the process of caspase activation induced by cytotoxic cytokines can be inhibited when protein kinase C is activated by phorbol 12-myristate 13-acetate (PMA), which prevents recruitment of the adaptor molecule FADD to ligated death receptors. During the present funding period we demonstrated that the effects of PMA are selectively inhibited by PKC?1 siRNA, identified the death receptors Fas and DR5 as two polypeptides that are phosphorylated in a PMA-dependent manner, mapped phosphorylation sites on Fas, and showed that mutation of one of these abolished the effects of PMA on Fas-mediated apoptosis. In addition, we observed that downregulation of the antiapoptotic Bcl-2 family member Mcl-1 sensitizes cells to the death ligand TRAIL, an agent that was recently introduced into clinical testing. To build on these observations, we now propose to: 1) characterize the sites and biological significance of TRAIL receptor phosphorylation, 2) map the signal transduction pathway leading from PMA treatment to inhibition of death receptor-mediated apoptosis, and 3) perform preclinical studies to determine whether agents that affect death receptor pathway regulation can enhance TRAIL efficacy in tissue culture cell lines and clinical acute leukemia specimens. Collectively, these studies are designed to improve current understanding of the biology of death ligand-induced apoptosis and provide new information that should be helpful in designing more effective cancer treatments. ? ? ?
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