Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.
Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.
Vercammen
D
D
Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium.
Beyaert
R
R
Denecker
G
G
Goossens
V
V
Van Loo
G
G
Declercq
W
W
Grooten
J
J
Fiers
W
W
Vandenabeele
P
P
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009336:Necrosis; D009842:Oligopeptides / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D016166:Free Radical Scavengers; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D016328:NF-kappa B; D009842:Oligopeptides; D017382:Reactive Oxygen Species; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D002083:Butylated Hydroxyanisole; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1