title: Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition.
abstract: The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent 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 decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation 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, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: M
authors.initials: M
authors.lastname: Tafani
authors.forename: T G
authors.initials: TG
authors.lastname: Schneider
authors.forename: J G
authors.initials: JG
authors.lastname: Pastorino
authors.forename: J L
authors.initials: JL
authors.lastname: Farber
mesh_terms: D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000502:physiology*; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000502:physiology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D008291:Malate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D009842:Oligopeptides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014409:Tumor Necrosis Factor-alpha; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D016572:Cyclosporine; D008291:Malate Dehydrogenase; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases