The long term goal of this project is to explore the mechanisms and molecular ordering of radiation-induced apoptosis in mammalian cells and the anti-apoptotic signaling systems that constrain this response. We propose the hypothesis that failure to initiate apoptosis in response to radiation may result, in part, from tight regulation of the apoptotic response by constitutively expressed or inducible anti-apoptotic mechanisms. The balance between pro- and anti-apoptotic systems may determine under any set of circumstances the magnitude of the apoptotic response in vitro and in vivo. Our recent studies on the molecular ordering of ceramide-mediated apoptosis in irradiated cells and its modulation by the 1,2-diacylglycerol (DAG) - protein kinase C (PKC) anti-apoptotic signaling system provide an approach to test this hypothesis. Further, our recent discovery that radiation-induced DNA damage signals apoptosis by activating the enzyme ceramide synthase provides an opportunity to study the mechanism of post-mitotic apoptosis. This proposal presents a detailed plan to purify ceramide synthase and clone and sequence its gene, and to study the role of ceramide synthase in radiation-induced apoptosis. Specifically, experiments are planned to explore the role of ceramide synthase in p53-mediated apoptosis induced by radiation, explore the regulation of ceramide synthase at the G2/M checkpoint of irradiated cells, and study the role of ceramide synthase in irradiated cells undergoing post-mitotic apoptosis. We also present a plan to further study the molecular ordering of ceramide-mediated apoptosis in irradiated cells and its anti-apoptotic control via DAG-PKC. Specifically, experiments are presented to explore signals that regulate mitochondrial apoptosis in a cell-free system and to localize mitochondrial damage in the functional ordering of radiation-induced apoptosis in intact cells. In addition, we suggest to study the involvement of ceramidase in operating an anti-apoptotic checkpoint of ceramide-mediated apoptosis in irradiated cells via signaling the activation of the DAG/PKC pathway and the ERK-mediated anti-apoptotic mechanism. The proposed experiments address new and heretofore unknown mechanisms of radiation-induced cell kill and may provide an approach to investigate new hypotheses on mechanisms of radiation resistance. Improved understanding of these pro- and anti-apoptotic signaling systems and their coordinated function may yield opportunities for pharmacological interventions in in vivo models, with important potentials for clinical applications in the radiation management of human cancer.
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