We have recently identified two different pathways of CD95-mediated apoptosis (Scaffidi, C., Fulda, S., Srinivasan, A., Feng, L., Friesen, C., Tomaselli, K. J., Debatin, K.-M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687). CD95-mediated apoptosis in type I cells is initiated by large amounts of active caspase-8 formed at the death-inducing signaling complex (DISC) followed by direct cleavage of caspase-3. In contrast, in type II cells very little DISC and small amounts of active caspase-8 sufficient to induce the apoptogenic activity of mitochondria are formed causing a profound activation of both caspase-8 and caspase-3. Only in type II cells can apoptosis be blocked by overexpressed Bcl-2 or Bclx L . We now show that a number of apoptosis-inhibiting or -inducing stimuli only affect apoptosis in type II cells, indicating that they act on the mitochondrial branch of the CD95 pathway. These stimuli include the activation of protein kinase C, which inhibits CD95-mediated apoptosis resulting in a delayed cleavage of BID, and the induction of apoptosis by the ceramide analog C 2 -ceramide. In addition, we have identified the CD95 high expressing cell line Boe R as a CD95 apoptosis-resistant type II cell that can be sensitized by treatment with cycloheximide without affecting formation of the DISC. This also places the effects of cycloheximide in the mitochondrial branch of the type II CD95 pathway. In contrast, c-FLIP was found to block CD95-mediated apoptosis in both type I and type II cells, because it acts directly at the DISC of both types of cells.
CD95 (APO-1/Fas) is a member of the death receptor family (1). Triggering of this receptor results in the formation of the death-inducing signaling complex (DISC)1 , a complex of signaling proteins recruited by the activated CD95 instantly after the addition of agonistic anti-CD95 antibodies or the CD95 ligand (2).2 Formation of the DISC comprising the adapter molecule FADD/MORT1 (3, 4) and caspase-8 (5, 6) results in the release of active caspase-8 at the DISC and cleavage of various intracellular death substrates (7,8). Both DISC proteins, FADD and caspase-8, have now been demonstrated to be essential components of the CD95 signaling machinery (9 -12).We have recently identified two different CD95 apoptosis signaling cell types (13). Type I cells require activation of caspase-8 at the DISC closely followed by activation of caspase-3. Blocking the release of apoptogenic factors (i.e. cytochrome c and apoptosis-inducing factor) from mitochondria by heterologous expression of Bcl-2 or Bcl-x L had no effect on caspase-8 or caspase-3 cleavage or on CD95 sensitivity of these cells. In type II cells DISC formation was strongly reduced despite similar expression levels of the DISC components CD95, FADD, and caspase-8. In these cells, caspase-8 and caspase-3 were primarily activated downstream of mitochondria, and their activation was blocked by the overexpression of Bcl-2. We now demonstrate that a number of treatments that have been reported to either in...