ACTIVATION OF CPP32-LIKE PROTEASE IN TUMOR NECROSIS FACTOR-INDUCED APOPTOSIS IS DEPENDENT ON MITOCHONDRIAL-FUNCTION

Mitochondria have been implicated in apoptosis, however, the precise m echanisms whereby mitochondria exert their effect are not clear. To ga in further insights, we generated a panel of cells from ML-1a cells th at were rendered respiration deficient by ethidium bromide treatment. Two respiration-deficient clones were subsequently reconstituted by fu sion with platelets, Respiration-deficient clones were resistant to TN F-induced apoptosis, whereas ML-1a and reconstituted clones were sensi tive. In contrast, inhibition of proliferation and induction of differ entiation by TNF were still observed in respiration deficient clones, suggesting a selective requirement of respiration in TNF-induced apopt osis, Furthermore the apoptosis machinery is not completely altered in respiration-deficient cells because they underwent apoptosis after st aurosporine treatment. Next, we showed that apoptosis induced by TNF a nd staurosporine were blocked by z-DEVD-CH2F, an inhibitor of CPP32-li ke cysteine protease, suggesting the involvement of CPP32-like proteas e in both apoptosis signaling pathways. Interestingly, TNF activated C PP32-like protease in the parental and reconstituted clones but not in respiration-deficient clones, and staurosporine in all clones. Thus, the apoptosis signaling block in respiration-deficient clones is locat ed at a step before CPP32-like protease activation, which can be bypas sed by staurosporine.