Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-a lpha) in the presence of 0.5 mu g/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclospo rin A (CyA) in combination. with the phospholipase A, inhibitor aristolochi c acid (ArA). The MPT is accompanied by the release of cytochrome c from th e mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmenta tion, all of which were inhibited by CyA plus ArA The caspase-3 inhibitor z -Asp-Glu-Val-aspartic acid fluoromethylketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did pre vent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp( OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and d ecreased only slightly the translocation of Bid to the mitochondria, These data indicate that induction of the MTP by TNF-alpha causes a release of cy tochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activa tion of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, t he killing of L929 fibroblasts by TNF-alpha can occur in the absence of eit her caspase-3 or caspase-8 activity. Alternatively, cell death can be preve nted despite an activation of caspase-8.