Altered Apoptotic Responses in Chemically-Resistant Mammalian Cell Variants

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Tissue and organ homeostasis in all multicellular organisms is maintained by a rigid balance between cellular proliferation and cell death. One of the most important mechanisms of cell death is a genetically regulated process of cellular suicide known as apoptosis. Several forms of this process have been shown to involve (a) the apoptotic suppressor, Bcl-2, (b) a factor, Bid, which when activated induces cytochrome c efflux from mitochondria, and (c) several specific proteases, including caspase-3 and caspase-8. The overall purpose of this project is to elucidate the molecular and biochemical roles of these gene products in the model system of apoptosis induced by staurosporine in human HL-60 cultured cells. Experiments will be undertaken to answer three specific questions. (1) What is the staurosporine-activated caspase that processes Bid? The caspase will be purified biochemically. If novel, it will be sequenced, and its gene will be cloned. (2) How does Bcl-2 regulate staurosporine-induced caspase-8 activation? Several possibilities will be considered including the formation of signaling complexes with other components. (2) Does Bcl-2 form heterodimers with Bid in the cytosol and affect the susceptibility of Bid to caspase action? The possible formation of such dimers and their substrate activities will be assayed directly. It is expected that the results will yield fundamental new information about apoptosis applicable to a wide variety of multicellular organisms.