Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis

Abstract

BelgiumTHE Fas/ APO-l receptor is one of the major regulators of apoptosisl-7. We report here that Fas/ APO-l-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-lJl-converting enzyme (ICE)s--lo, which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/ APO-l rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/ APO-l-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor, CrmA or an antisense ICE construct, substantially suppressed Fas/ APO-l-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/ APO-l-mediated cell death.The signal transduction pathway elicited by Fas/ APO-l is almost completely unknown. Initiation of apoptosis may involve a new class of cysteine proteases, including the product of the C. elegans cell-death gene eed-3, mammalian interleukin-lfJ-converting enzyme (ICE) and the related proteases Nedd-2/Ich-l, prICE and CPP-32 (refs 11-17). Overexpression ofCED-3, ICE or Nedd-2/Ich-1 in Rat-l fibroblasts has been shown to result in apoptotic cell death 12.15. We therefore investigated whether Fas/ APO-I-mediated apoptosis involved an ICE-related proteolytic activity. In L929-APO-l cells 18 or B-lymphoblastoid SKW 6.4 cells, apoptosis triggered by the agonistic monoclonal antibody anti-APO-I was strongly inhibited by the ICE inhibitor YVAD-CHO, a tetrapeptide aldehyde (Ki = 0.76 nM)8 (Fig. la).

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8

“…Furthermore, caspase-8 was potently inhibited by CrmA, which is inhibitory to Fas-stimulated apoptosis of Jurkat cells though not to that induced by staurosporine . This ®nding, in combination with similarities in sensitivity to CrmA of labeled caspases in Fas-stimulated (F22, F20, F19, and F17) and staurosporine-treated (S22, S20, S19, S17) cells, is consistent with the notion that CrmA inhibition of caspase-8 is responsible for the sensitivity of Fasinduced apoptotic cell death to CrmA (Enari et al, 1995;Los et al, 1995;. Thus, caspase-8, a CrmA-sensitive protease, appears to be critical for the initiation of the protease cascade in Fasstimulated cells.…”

“…In particular, we show that the addition of active caspase-8 (MACH/FLICE/Mch5) to extracts from non-apoptotic Jurkat cells can trigger stepwise activation of caspases that is strikingly similar to that induced by Fas stimulation. Finally, we ®nd that caspase-8 is sensitive to CrmA, the inhibitor of Fasinduced apoptotic cell death (Enari et al, 1995;Los et al, 1995;. Taken together, these results support the notion that the activation of multiple caspases is required for the organization of protease cascades as well as for the cleavage of multiple apoptosis-associated substrates.…”

“…It has been reported that Fas-induced cell death is prevented by CrmA (Enari et al, 1995;Los et al, 1995;, in contrast to other cell death events such as staurosporine-induced apoptosis . Interestingly, the protease activity of caspase-8 as assessed by the cleavage of DEVD-MCA (Km *7 mM) was *95% inhibited by preincubation with 100 nM CrmA ( Figure 6a).…”

c-Myc-deficient B lymphocytes are resistant to spontaneous and induced cell death

“…Decreased surface expression of CD95 has been reported to confer resistance to CD95-induced cell death. 34 Engagement of CD95 receptor on the surface leads to the activation of procaspase 8, caspase 3, 35 and ultimately to cleavage of the caspase 3 substrate poly (ADP-ribose) polymerase (PARP). 36 To determine whether higher resistance to anti-CD95-induced cell death in c-Myc-deficient cells correlated with changes in PARP processing, we performed Western blot analysis on these cells.…”

Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway

“…It has been reported that Fas-and tumor necrosis factor (TNF) receptor-mediated apoptosis is driven by caspase family, because it is suppressed by their inhibitors, including cowpox virus CrmA Enari et al, 1995;Los et al, 1995), baculovirus p35 and tetrapeptide caspase-1 and caspase-3 inhibitors (Enari et al, 1995;Los et al, 1995;Schlegel et al, 1996;Chinnaiyan et al, 1996;Hasegawa et al, 1996). It has also been reported that thymocytes from caspase-1-de®cient mice are resistant to Fas-mediated apoptosis, indicating the direct involvement of caspase-1 itself in this process of thymocyte death (Kuida et al, 1995).…”