INDUCTION OF THE MITOCHONDRIAL-PERMEABILITY-TRANSITION CAUSES RELEASEOF THE APOPTOGENIC FACTOR CYTOCHROME-C

It was recently reported that the mitochondrial protein cytochrome c i s required for the induction of apoptosis, and that the overexpression of Bcl-2 caused increased retention of this apoptogenic factor by mit ochondria. Several cellular toxins, including H2O2, tBOOH and Ca++, in duce the Mitochondrial Permeability Transition (MPT); we tested the po ssibility that MPT is an intracellular sensor of toxicity that results in the release of cytochrome c. Ne observe char the release of cytoch rome c from purified mitochondria is stimulated by the classical induc ers of MPT, and is inhibited by the classical inhibitor of MPT, cyclos porin A (CsA). After induction of MPT, mitochondrial supernatants gain ed the activity to induce cleavage of caspase 3 (CPP32) in cytosolic e xtracts, and this gain of activity was inhibited by CsA pretreatment o f mitochondria, and was cancelled by immunodepletion of cytochrome c f rom the supernatants. After induction of MPT, mitochondrial supernatan ts mixed with or without cytosolic extract gained the activity to ladd er nuclei, and this gain of activity was inhibited by CsA pretreatment of mitochondria, and cancelled by immunodepletion of cytochrome c fro m the supernatants. These results demonstrate that the induction of MP T causes release of cytochrome c from mitochondria, which is required for the hallmarks of cytosolic and nuclear apoptosis, caspase 3 activa tion and nuclear laddering, and identify the MPT as a potential intrac ellular sensor of oxidants and other toxins, and as a target for the p harmacological inhibition of apoptosis. (C) 1998 Elsevier Science Inc.