The sphingolipid ceramide, a bioeffector lipid, is known to regulate anti-proliferative responses in various human cell lines. In particular, it has been shown that various anti-cancer agents cause the elevation of endogenous ceramide levels accompanied by apoptotic cell death in human cancer cells. Preliminary results from this program demonstrate that: i) Treatment of A549 human lung adenocarcinoma cells with chemotherapeutic agents results in the formation of endogenous long-chain ceramide via either the de novo pathway or the hydrolysis of sphingomyelin through the action of sphingomyelinase (SMase); ii) Agents that cause ceramide generation via these two distinct pathways have synergistic cytotoxic effects; iii) Downstream targets of ceramides that are generated via the two distinct pathways are distinct. These data lead us to the HYPOTHESIS that there are two distinct mechanisms of ceramide formation, namely the de novo pathway and the hydrolysis of sphingomyelin, which function independently and synergistically in mediating apoptosis and growth suppression in human lung cancer cells. As a corollary, we believe that prevention of ceramide clearance should augment chemotherapy action in lung cancer treatment. Therefore, two specific aims are proposed: 1) To determine whether the two distinct mechanisms of ceramide generation act synergistically in mediating apoptotic cell death and/or growth suppression in lung cancer cells. A) Determine which chemotherapeutic agents activate which of the two pathways; B) establish the synergistic effects of anti-cancer agents which activate neutral sphingomyelinase (NSMase) or the de novo pathway, respectively; C) determine the role of ceramide, generated from each pathway, in mediating the action of chemotherapeutic agents by inhibiting each pathway, and D) establish the synergistic effects of activating the two pathways, by overexpression of SMase and serinepalmitoyl transferase (SPT). 2) To identify the mechanisms by which ceramides generated by the two different pathways act synergistically in mediating apoptosis and/or growth suppression. A) Determine the compartmentalization of these pathways, and analyze whether the sub-cellular compartments in which these two pathways are activated are important for their synergistic action; B) test the corollary that clearance of ceramide attenuates the synergistic effects of ceramides in different sub-cellular compartments; and C) determine the down stream targets of ceramides generated via these two pathways. These studies will offer mechanism-based targets for the development of novel cancer therapeutics involving these two distinct ceramide pathways.