Bcl-2 Prevents Caspase-independent Cell Death

Abstract

Nitric oxide (NO) is implicated in apoptosis and has both cytotoxic and cytoprotective effects. Exogenous NO induced the death of PC12 and HeLa cells via a process showing features of both apoptosis and necrosis, with chromatin condensation, nuclear compaction, and mitochondrial swelling. Activation of caspases was not observed during NO-induced cell death. In addition, cell death was not inhibited by peptide caspase inhibitors or by expression of p35, a baculovirus-encoded caspase inhibitor, indicating that NO-induced cell death was independent of caspases. NO-induced cell death was enhanced by Bax expression in a caspase-independent manner and prevented by the anti-cell death protein Bcl-2. Although Bcl-2 has previously been shown to prevent cell death by inhibiting caspase activation, these results indicate that it can also prevent cell death via a caspase-independent mechanism.Nitric oxide (NO) 1 is enzymatically generated from L-arginine by constitutive or inducible NO synthase, and has a number of physiological roles, including smooth muscle relaxation, and neurotransmission (1, 2). NO has also been implicated in a variety of pathological phenomena, such as septic shock, ␤-cell destruction, and transplant rejection (1, 2). Some of these pathological events are closely related to apoptotic cell death (3,4). In many studies, NO has been shown to induce apoptosis, although the precise mechanism involved is still unclear (5-7). In contrast, some investigators have suggested that NO also has the ability to prevent cell death (8 -10), which seems to be mediated by the inhibition of caspases (8), common mediators of apoptosis (11). So far, more than 10 caspases have been identified in mammals. Apoptosis is also regulated by Bcl-2 family proteins, including anti-apoptotic proteins such as Bcl-2 and Bcl-x L , and pro-apoptotic proteins such as Bax and Bak (12). Accumulating evidence suggests that Bcl-2 acts upstream of caspase activation to prevent apoptosis (13,14).In this study, we analyzed NO-induced cell death, particularly focusing on the role of caspases as well as the influence of apoptosis-regulating molecules such as Bcl-2 family proteins. EXPERIMENTAL PROCEDURESReagents-Caspase inhibitors and substrates were purchased from Peptide Inc. (Minoh, Japan). Other chemicals were purchased from Wako Chemical Co. (Tokyo, Japan).Cell Lines and Transfection-HeLa cells, a human cervical carcinoma-derived cell line, and PC12 cells, a rat pheochromocytoma cell line, were maintained in RPMI 1640 culture medium, as described elsewhere (15). A stable transfectant of PC12 cells expressing mouse Bax (designated as PC12-Bax) was obtained by infecting retrovirus that was produced from the packaging cell line ⌿2 transfected with the retroviral vector pBC140 (15) containing mouse bax cDNA. A stable transfectant of PC12 cells expressing human Bcl-2 (designated as PC12-Bcl-2) was obtained by transfecting the pUC-CAGGS vector bearing the human bcl-2 cDNA using electroporation (13). Empty vector-transduced cells were used as the...

Bcl-xS and Bax induce different apoptotic pathways in PC12 cells

“…The ®nding that Bcl-2 and Bcl-x L can inhibit Baxinduced cell death further suggests that these two antiapoptotic proteins can inhibit caspase-independent cell pathways, and is in agreement with previous studies showing that apoptosis induced by a defect in the ubiquitin pathway or by nitric oxide are mediated by a caspase-independent pathway which is blocked by Bcl-2 (Okuno et al, 1998;Monney et al, 1998). The ®nding that Bax induces caspase-independent cell death is in agreement with previous reports (Xiang et al, 1996;Pastorino et al, 1998;Gross et al, 1998), although other studies show that the Bax-induced cell death requires caspase activity (Pan et al, 1998;Martinou et al, 1998;Finucane et al, 1999;Cheng et al, 1997).…”

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

“…Bcl-2 acts by suppressing both caspase-dependent and caspase-independent pathways of apoptosis (Monney et al 1998, Okuno et al 1998, Cosulich et al 1999. Considering that caspase-dependent and caspase-independent pathways could drive the cells to apoptosis, the role of caspase-3 in the apoptosis of P. brasiliensis-infected A549 epithelial cells was investigated.…”

Apoptosis without caspases: an inefficient molecular guillotine?

“…108 However, only a subset of caspases (-1, -3, -6, -7, -8 and -10) have so far been demonstrated to be inhibited by recombinant p35 in vitro, 109 and there are at least three reported cases where p35 cannot rescue cells from apoptosis. 84,101,110 Of course, this could be due to p35 expression levels that are too low for caspase inhibition, as many investigators have problems to highly overexpress p35 due to its viral origin. Alternatively, p35 may not block all caspases.…”

“…84,85 More recent examples include apoptosis induced by the GD3 ganglioside, 86 class I MHC antibodies, 87 puromycin, 88 polyamine analogues, 89 CD-2 and staurosporine, 90,91 intracellular acidification, 92 the retinoid AHPN, 93 E4orf4, a novel adenovirus death factor, 94 irradiation, 95,96 VP-16, dexamethasone and actinomycin D, 97 ± 99 PML, 100 and nitric oxide (NO). 101 Even some aspects of Fas-induced apoptosis (when triggered with the adapter FADD) may occur caspaseindependently although in this case necrotic morphologies were observed as well. 102 In our own hands, we have not seen any apoptotic system in culture (using various physiological and toxic stimuli) where Z-VAD.fmk could save cells from dying.…”