Ordering of ceramide formation, capase activation, and mitochondrial changes during CD95 and DNA damage induced apoptosis

To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or gamma-radiation (IR) in relati on to caspase activation and mitochondrial changes in Jurkat T cells. The C er response to all three stimuli was mapped in between caspases sensitive t o benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-alde hyde (DEVD-CHO). Cer production was independent of nuclear fragmentation bu t associated with the occurrence of other aspects of the apoptotic morpholo gy. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by C D95 but did not affect the response to etoposide or IR, placing CD95-induce d Cer formation downstream from caspase-8 and excluding a role for caspase- 8 in the DNA damage pathways. CD95 signaling to the mitochondria required c aspase-8, whereas cytochrome c release in response to DNA damage was caspas e-independent. These results indicate that the caspases required for the Ce r response to etoposide and IR reside at or downstream from the mitochondri a. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Ce r is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution p hase of apoptosis.