APOPTOSIS INDUCTION BY CASPASE-8 IS AMPLIFIED THROUGH THE MITOCHONDRIAL RELEASE OF CYTOCHROME-C

Apoptosis often involves the release of cytochrome c from mitochondria , leading to caspase activation. However, in apoptosis mediated by CD9 5 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated an d, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added activ e caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragm entation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear mem branes intact. Thus, mitochondria were required for complete engagemen t of the apoptotic machinery. In the absence of mitochondria, high con centrations of caspase-8 were required to activate downstream caspases . However, when mitochondria were present, the effects of low concentr ations of caspase-8 were vastly amplified through cytochrome c-depende nt caspase activation. Caspase-8 promoted cytochrome c release indirec tly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apopt osis only at the lowest caspase-8 concentrations, potentially explaini ng why CD95-induced apoptosis can often evade inhibition by Bcl-2.