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 mouse fibroblast 4B1 cells. To examine the intracellular apoptotic death signal initiated by CPT-11, ceramide synthesis and the ICE cascade were analyzed. CPT-11-initiated cytolytic activity was prevented by both caspase inhibitors YVAD-CHO and DEVD-CHO, or ceramide synthesis inhibitor fumonisin B1, and accelerated by sphingomyelin, suggesting the direct involvement of ceramide synthesis and the interleukin 1-beta converting 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 operation. 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 results suggest that ceramide synthesis acts as a dominant regulator in CPT-11-initiated death signaling and sequentially operates the ICE cascade.