SEQUENTIAL OPERATION OF CERAMIDE SYNTHESIS AND ICE CASCADE IN CPT-11-INITIATED APOPTOTIC DEATH SIGNALING

The chemotherapeutic agent CPT-11 induced apoptotic cell death in mous e fibroblast 4B1 cells. To examine the intracellular apoptotic death s ignal initiated by CPT-11, ceramide synthesis and the ICE cascade were analyzed. CPT-11-initiated cytolytic activity was prevented by both c aspase inhibitors YVAD-CHO and DEVD-CHO, or ceramide synthesis inhibit or fumonisin B1, and accelerated by sphingomyelin, suggesting the dire ct involvement of ceramide synthesis and the interleukin 1-beta conver ting enzyme (ICE) cascade. In addition, apoptosis was induced by both native and synthesized ceramide and prevented by YVAD-CHO and DEVD-CHO , suggesting the possible involvement of ceramide in ICE cascade opera tion. To directly demonstrate whether ceramide synthesis operates the ICE cascade, proteolytic activity of ICE- or CPP32-like proteinase was analyzed. ICE-like proteinase activity was prevented by fumonisin B1 and YVAD-CHO, but not by DEVD-CHO. In contrast, fumonisin B1, YVAD-CHO , and DEVD-CHO all prevented CPP32-like proteinase activity. These res ults suggest that ceramide synthesis acts as a dominant regulator in C PT-11-initiated death signaling and sequentially operates the ICE casc ade. (C) 1997 Academic Press.