Ceramide is now increasingly appreciated as an endogenous regulator of apoptosis in a number of cell systems. However, the mechanism of action of ceramide, it's relationship to other components of the apoptotic pathway, and its potential use as a chemotherapeutic agents have not been clearly investigated. The applicant's preliminary data show that the chemotherapeutic agent vincristine induces significant and prolonged accumulation of endogenous ceramide, and that ceramide may activate ICE- like proteases as down stream components of its apoptotic mechanism. Therefore, this application aims at: 1) Defining the role of endogenous ceramide in the apoptotic pathway induced in several representative tumor cell lines by multiple chemotherapeutic agents (by determining the ceramide responses to these agents and by determining the role of endogenous ceramide in regulating apoptosis through the modulation of ceramide metabolism in these cells); 2) Determining the mechanism of ceramide induced death. This will concentrate on determining the ability of ceramide to activate ICE-like proteases and the mechanism of this activation in a cell free system; and 3) Developing bacterial sphingomyelinase (bSMase) as a selective intracellular activator of apoptosis in cancer but not in normal cells. These studies will aim and cloning and expressing BSMASE in leukemia cells as well as in normal human diploid fibroblasts (HDF) and determining the effect of this expression on ceramide formation and the apoptotic and growth inhibitory response. Mechanistic studies will aim at defining the intracellular compartment involved in ceramide generation and in mediating apoptosis. Therefore, studies from this application should clarify the role of endogenous ceramide in mediating apoptosis, should provide important mechanistic insight into ceramide-induced death, and finally they should develop a foundation for possible gene-therapy based approaches towards the treatment of cancer.
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