THE APOPTOSIS-NECROSIS PARADOX - APOPTOGENIC PROTEASES ACTIVATED AFTER MITOCHONDRIAL PERMEABILITY TRANSITION DETERMINE THE MODE OF CELL-DEATH

Mitochondrial alterations including permeability transition (PT) const itute critical events of the apoptotic cascade and are under the contr ol of Bcl-2 related gene products, Here we show that induction of PT i s sufficient to activate CPP32-like proteases with DEVDase activity an d the associated cleavage of the nuclear DEVDase substrate poly(ADP-ri bose) polymerase (PARP). Thus, direct intervention on mitochondria usi ng a ligand of the mitochondrial benzodiazepin receptor or a protonoph ore causes DEVDase activation. In addition, the DEVDase activation tri ggered by conventional apoptosis inducers (glucocorticoids or topoisom erase inhibitors) is prevented by inhibitors of PT. The protease inhib itor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) com pletely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifes tation of apoptosis-associated changes in cellular redox potentials (h ypergeneration of superoxide anion, oxidation of compounds of the inne r mitochondrial membrane, depletion of non-oxidized glutathione), as w ell as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapab le of preventing disruption of the plasma membrane during protracted c ell culture (12-24 h), even in conditions in which it completely block s nuclear apoptosis (chromatin condensation and DNA fragmentation). El ectron microscopic analysis confirms that cells treated with PT induce rs alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations ar e compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contra st, it would be the availability of apoptogenic proteases that would d etermine the choice between the two death modalities.