The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

Abstract

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is su cient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-¯uoromethylketone (Z-VAD.fmk) completely 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 manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane lea¯et. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12 ± 24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis con®rms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

“…However, Z-VAD.fmk does block nuclear DNA fragmentation ( Figure 2b) and PARP cleavage ( Figure 3d) induced by diamide or t-BHP. Of note, Z-VAD.fmk only partially prevents cytolysis induced by t-BHP and diamide (Figure 3c), thus con®rming the previously reported strong association between mitochondrial dysfunction and loss of cell viability (Boise and Thompson, 1997;Hirsch et al, 1997). Bcl-2 protects against t-BHP-induced disruption of the DC m , yet fails to interfere with the diamide-triggered DC m collapse (Figures 1b and 2c).…”

Redundant cell death mechanisms as relics and backups

“…Depending on cells, conditions and stimuli, inhibition of caspase-8 can truly interrupt lethal signaling, favor a switch from apoptotic to necrotic cell death, [17][18][19] which depends on RIP1, 20,21 or induce (again RIP1-dependent) autophagic cell death. 22 Caspase inhibition has also been shown to lead to a switch from apoptosis to necrosis in, for example, thymocytes responding to DNA damage 23 or from apoptosis to autophagic cell death in, for example, growth factor-deprived neurons. 24 Furthermore, conditions that normally induce apoptosis through cellular stress (e.g.…”

“…59 One of the few diseases in which caspase inhibitors may yield convincing preclinical effects is septic shock, 60 which involves the contribution of caspases, not only as cellular demolition enzymes but also for the biosynthesis of proinflammatory cytokines. Thus, although caspase inhibition can successfully prevent the phenotypic manifestation of apoptosis, it often does not prevent cell death, which is likely to be executed through different mechanisms (including apoptosis-like cell death, autophagy and necrosis) 5,23,24,34,61 and perhaps through phagocytic recognition and destruction. 62,63 As mentioned above, it is possible that different cell types present in the same organism differ in their potential to execute cell death through distinct mechanisms.…”

Signal transduction pathways that regulate cell survival and cell death

“…Cells which sustain traumatic damage or are deprived of growth-factors can die even in the presence of caspase inhibitors (Rathmell and Thompson, 1998). However, in the presence of such inhibitors the morphology of dying cell lacks many morphologic features of apoptosis, including typical nuclear changes (Hirsch et al, 1997). Outside of such experimental systems, it appears that caspase activation and cellular autodigestion may invariably accompany apoptosis in vivo.…”