The overall goal of our proposal is to characterize the role of radiation activated second messengers, ceramide and protein kinase C (PKC) in the survival response of cells to ionizing radiation. Our preliminary data suggest that radiation mediated ceramide production enhances radiation apoptosis whereas radiation activation of PKC blocks apoptosis. The three specific aims to be investigated are: l) to define the kinetics of ceramide production and its relationship to apoptosis following irradiation in mammalian cells. We will accomplish these experimental goals by measuring ceramide production following x-irradiation employing the DAG kinase assay and the tritiated palmitate labeling method followed by thin layer chromatography. We will measure apoptosis by the terminal transferase assay. To confirm that radiation mediated ceramide production and apoptosis are linked, we will develop cell lines that are incapable of ceramide production from cells that produce ceramide following irradiation and undergo apoptosis. We will study radiation apoptosis in these cell lines compared to the parent ceramide producing cell lines. We will investigate whether a signal transduction pathway exists whereby radiation activates a membrane associated neutral sphingomyelinase which hydrolyzes sphingomyelin to ceramide following the production of reactive oxygen intermediates. 2) to characterize the kinetics of PKC activation following x- irradiation and the radiobiological effects of PKC inhibition using specific PKC inhibitors and cell models which are deficient in the PKC isoforms. To accomplish these experimental goals, we will employ substrates activated by PKC following x-irradiation and Western immunoblots that indicate which specific PKC isoforms are activated following x-irradiation. We will employ specific inhibitors of PKC activation (Chelerythrine and CGP 41251) to study cell survival and apoptosis following radiation and PKC inhibition. To corroborate these findings without the use of inhibitors, we will employ cells deficient in the PKC isoforms alpha and beta which are more radiosensitive than the parent cell lines. We propose to return the radiosensitive PKC deficient cells to parental radiosensitivity by transfecting these cells with expression vectors for PKC alpha and beta. 3) To investigate the effects of ceramide and the inhibition of PKC activity on the ratios of Bcl2 gene family members as the molecular basis for the radiointeractive killing by these agents. Our preliminary data suggest that increases in intracellular ceramide and the inhibition of PKC modulate mRNA levels of the Bcl2 gene family members. We propose that increasing the intracellular concentration of ceramide and/or inhibiting PKC will increase radiation mediated apoptosis. These data provide the basis for developing novel radiotherapeutic strategies to increase tumor cell killing.
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