Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression.
Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.
Lüschen
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Institut für Immunologie, Christian-Albrechts-Universität Kiel, Brunswiker Str. 4, 24105 Kiel, Germany.
Ussat
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Scherer
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Kabelitz
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Adam-Klages
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D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019014:fas Receptor / Q000187:drug effects / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C505533:FADD protein, human; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D016328:NF-kappa B; D015842:Serine Proteinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D003513:Cycloheximide; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases