Apoptosis is a morphologically and biochemically distinct form of cell death that has been observed during development, after withdrawal of trophic hormones or treatment with chemotherapeutic agents, and during the course of cell-mediated immunotoxicity. The present proposal seek funds for collaborative studies by two laboratories with a long-standing interest in the mechanism of chemotherapy-induced apoptosis. Previous studies from these laboratories have demonstrated that l) topoisomerase- directed agents and a variety of other clinically useful antineoplastic agents induce apoptosis in a variety of cell types, 2) the morphologic changes of apoptosis are accompanied by the early, quantitative cleavage of a number of nuclear proteins including poly(ADP-ribose polymerase) and the nuclear lamins, and 3) the morphological and biochemical events of apoptosis can be recapitulated with synchrony and high fidelity in a unique cell-free assay developed using the cytosol from cells committed to apoptosis. Using this cell-free system, we have more recently identified two distinct protease activities that appear to play pivotal roles during the course of apoptosis. One, termed priCE (protease resembling interleukin-1-beta converting enzyme), cleaves the nuclear enzyme poly(ADP-ribose) polymerase prior to the earliest detectable cleavage of DNA into nucleosomal fragments. Inhibitors of priCE prevent all of the morphological and biochemical events associated with apoptosis (including cleavage of protein and DNA), suggesting that activation of priCE occurs very early in the process of apoptosis. A second protease with a distinct inhibitor profile is responsible for lamin degradation that occurs later in apoptosis and is required for the nuclear reorganization that results in apoptotic bodies. We now propose a series of experiments designed to characterize these proteases, investigate their structure and regulation, and further evaluate their role in controlling apoptosis. in particular, we propose to l) clone the cDNA for priCE using one of three different strategies, 2) utilize a novel fluorogenic assay and immunological probes against priCE to study the regulation of priCE in tissue culture cells and clinical tumor specimens that differ in their intrinsic ability to undergo apoptosis, 3) identify additional priCE substrates, 4) characterize in greater detail the lamin protease, and 5) isolate and characterize a recently demonstrated naturally-occurring inhibitor of apoptosis. These studies should provide insight into the structure and regulation of biochemical activities that appear to play a pivotal role in the control of chemotherapy-induced cell death.
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