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.

Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline-or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants.

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 κB 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.

The Yin and Yang of Plant Caspases The function of plant metacaspases, identified by limited sequence homology to the animal caspases that control cell death, has remained elusive. Coll et al. (p. 1393 ) have now elucidated the actions of two metacaspases in the small plant Arabidopsis . One metacaspase, AtMC1, promoted cell death, and the other, AtMC2, acted antagonistically to stall cell death. The results help to elucidate the mechanisms by which plants control cell survival during development and defend against pathogen attack.