Uncouplers of oxidative phosphorylation can enhance a Fas death signal

Recent work suggests a participation of mitochondria in apoptotic cell deat h. This role includes the release of apoptogenic molecules into the cytosol preceding or after a loss of mitochondrial membrane potential Delta Psi(m) . The two uncouplers of oxidative phosphorylation carbonyl cyanide m-chloro phenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) reduce Delta Psi(m) by d irect attack of the proton gradient across the inner mitochondrial membrane . Here we show that both compounds enhance the apoptosis-inducing capacity of Fas/APO-1/CD95 signaling in Jurkat and CEM cells without causing apoptot ic changes on their own account. This amplification occurred upstream or at the level of caspases and was not inhibited by Bcl-2. The effect could be blocked by the cowpox protein CrmA and is thus likely to require caspase 8 activity. Apoptosis induction by staurosporine in Jurkat cells as well as b y Fas in SKW6 cells was unaffected by CCCP and DNP. The role of cytochrome c during Fas-DNP signaling was investigated. No early cytochrome c release from mitochondria was detected by Western blotting. Functional assays with cytoplasmic preparations from Fas-DNP-treated cells also indicated that the re was no major contribution by cytochrome c or caspase 9 to the activation of effector caspases. Furthermore, an increase of rhodamine-123 uptake int o intact cells, which has been explained by mitochondrial swelling, occurre d considerably later than the caspase activation and was blocked by Z-VAD-f mk. These data show that uncouplers of oxidative phosphorylation can presen sitize some but not all cells for a Fas death signal and provide informatio n about the existence of separate pathways in the induction of apoptosis.