Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.

The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A 2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1␤-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A 2 (cPLA 2 ) and that activated cPLA 2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32͞ caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA 2 and apoptosis. TNF-induced activation of cPLA 2 was accompanied by a cleavage of the 100-kDa cPLA 2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32͞caspase-3. Interestingly, specific inhibition of cPLA 2 enzyme activity by arachidonyl trif luoromethylketone (AACOCF 3 ) partially inhibited TNFinduced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA 2 during apoptosis and identify cPLA 2 as a mediator of TNF-induced cell death acting downstream of caspases.Apoptosis is an active form of cell death controlled by the expression of evolutionarily highly conserved genes, which either activate or suppress the process of cell death (1). The best studied activators of apoptosis include a growing family of caspases, cysteine proteases related to interleukin 1␤-converting enzyme (ICE͞caspase-1), which share sequence homology with ced-3, a gene essential for apoptosis of nematode Caenorhabditis elegans (2). Another family of activators of apoptosis include proteins containing a ''death domain'' originally discovered in the intracellular parts of two receptors capable of mediating apoptosis, type 1 tumor necrosis factor receptor (TNF-R1) and Fas (3, 4). Recently, it was revealed that binding of receptor death domains to each other and to other intracellular proteins containing similar domains initiate the apoptotic signaling: TNF-R1-associated death domain protein (TRADD) binds to TNF-R1 (5), Fas-associated death domain protein (FADD͞MORT1) binds to either Fas or TRADD (6, 7), and FLICE͞MACH͞caspase-8 binds to FADD (8, 9), completing the death-inducing signaling complex. Interestingly, FLICE͞MACH͞caspase-8 contains a caspase-related cysteine protease domain, which possibly couples the receptor crosslinking and activation of apoptotic protease cascade (8, 9). The importance of caspases in TNFand Fas-mediated apoptosis has been clearly demonstrated by studies showing that (i) receptor activation leads to proteolytic activation of at least three family members, FLICE͞MACH͞ caspase-8 (9), CPP32͞caspase-3 (10), and ICE-LAP3͞ caspase-7 (11); (ii) inhibitors of caspases (cow pox virus protein CrmA, baculovirus protein p35, and specific inhibitory peptides) effectively block TNF-and Fas-mediated apoptosis (12-16); and (iii) ectopic expression of caspases in several cells results i...

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO-induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.from the depressive effect of IL-1[~ on glucose-induced insulin secretion [18].These findings led us to hypothesize that among the different stress proteins expressed in heat stressed islet cells, hsp70 may confer protection against NO toxicity. To test this hypothesis we transfected a rat insulinoma cell line, RINm5F, with a vector containing the complete coding region of the human hspTO gene [10]. Analysis of the stably transfected clones revealed that hsp70 confers resistance against the cytotoxicity of NO.