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From the Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor B under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Key Words: Fas antigen • apoptosis • necrosis • caspases • oxygen radicals

Research was supported by the Interuniversitaire Attractiepolen, grant 9005097N of the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen, and European Community Biomed Program grant BMH4-CT96-0300. G. Denecker is a research assistant, and P. Vandenabeele a postdoctoral researcher with the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen.

Abbreviations used: Ac-DEVD-amc, acetyl-Asp(OMe)- Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin; Ac-YVAD-amc, acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin; Ac-YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethylketone; BHA, butylated hydroxyanisole; DD, death domain; DR, death receptor; DHR123, dihydrorhodamine, 123; FADD, Fas-associated DD protein; L929hFas, Fas-transfected L929 (cells); MORT, mediator of receptor-induced toxicity; NF-B, nuclear factor B; PI, propidium iodide; TRADD, TNFR-associated DD protein; TRAIL, TNF- related apoptosis–inducing ligand; zAAD-cmk, benzyloxycarbonyl-Ala-Ala-Asp-chloromethylketone; zDEVD-fmk, benzyloxycarbonyl-Asp(OMe)- Glu(OMe)-Val-Asp(OMe)-fluoromethylketone; zD-fmk, benzyloxycarbonyl-Asp(OMe)-fluoromethylketone; zVAD-afc, benzyloxycarbonyl-Val-Ala-Asp(OMe)-aminotrifluoromethylcoumarin; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone.

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