Apoptosis, a morphologically distinct form of cell death observed during development, after withdrawal of trophic hormones, and after treatment with ionizing radiation or chemotherapeutic agents, is characterized by active participation of endogenous cellular enzymes in the disassembly of 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) topoisomerase-directed drugs and other clinically useful antineoplastic agents trigger apoptosis in a variety of cell types; 2) the morphological changes of apoptosis are accompanied by the early, quantitative cleavage of a number of nuclear polypeptides; 3) this cleavage is mediated by multiple aspartate-directed cysteine proteases (caspases) with differing substrate preferences; 4) the pathways leading to activation of the caspases after treatment with etopside, topotecan, paclitaxel or ionizing radiation are distinguishable from the pathways of activation after death receptor ligation; 5) activation of protein kinase C by phorbol 12-myristate 13-acetatae (PMA) has no effect on drug-induced apoptosis but inhibits death receptor-induced apoptosis, at least in part, by preventing recruitment of the adaptor molecule FADD to ligated death receptors; 6) several of the procaspases are phosphoproteins; and 7) one key enzyme activated by caspase-3 is CAD, a unique caspase-activated deoxyribonuclease that cooperates with DNA topoisomerase II in playing an important role in nuclear disassembly downstream of caspases during drug-induced apoptosis. We now propose to 1) more fully characterize the nature and physiological significance of posttranslational modifications detected in procaspses and active caspases; 2) identify the protein kinase C isoform responsible for PMA-induced inhibition of death receptor signaling and examine the signal transduction pathway leading to this inhibition; 3) evaluate the roles of survivin and a novel lymphoma-associated cIAP2/MALT fusion protein in caspase activation and apoptosis; and 4) perform a series of pilot experiments to determine whether disruption of the ICAD/CAD complex will result in CAD activation and cell death independent of prior caspase activation. These studies, which are designed to provide improved understanding of the biochemistry of chemotherapy-induced apoptosis, should be helpful in designing more effective cancer chemotherapeutic treatments in the future.
Showing the most recent 10 out of 84 publications
