Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein.

Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).