The Central Executioner of Apoptosis: Multiple Connections between Protease Activation and Mitochondria in Fas/APO-1/CD95- and Ceramide-induced Apoptosis

Abstract

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1β converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (ΔΨm). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the ΔΨm is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the ΔΨm collapse and subsequent apoptosis. Cytosols from anti-Fas–treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their ΔΨm. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + ΔΨm collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Role of caspases and possible involvement of retinoblastoma protein during TGFβ-mediated apoptosis of human B lymphocytes

“…The amount of the active cleaved form of caspase-2 was maximum after 24 h and it disappeared after 48 h. In contrast, the p17 and p19 forms of caspase-3 were apparent 24 h after stimulation with TGFb and the active p17 form, but not the p19 form, was present after 48 h. This di erent kinetics suggested that TGFb could activate a cascade of di erent caspases similar to that described for Fas-mediated apoptosis (Hirata et al, 1998;Kamada et al, 1997;Susin et al, 1997). However, we were not able to detect, by Western blotting, expression of either proforms or active proteolytic fragments of caspases-1, 4, 6 or 7 in BL41 cells after 30 min and 48 h of activation by TGFb (data not shown).…”

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

“…There are numerous studies suggesting that overexpression of Bcl-2 or Bcl-x L can block apoptosis by effects at the mitochondrion (Shimizu et al, 1996;Susin et al, 1997;Yang et al, 1997), whereas Bax and Bak have been shown to exert pro-apoptotic activity (Chittenden et al, 1995;Narita et al, 1998). We examined the effect of STS on the expression levels of Bcl-2, Bcl-x L, Bak and Bax proteins.…”

p27Kip1 induces drug resistance by preventing apoptosis upstream of cytochrome c release and procaspase-3 activation in leukemic cells

“…Apoptosis has been shown to trigger mitochondrial permeability transition (Susin et al, 1997) as well as mitochondrial release of pro-apoptotic factors (Susin et al, 1996;Kluck et al, 1997;Yang et al, 1997). To detect the modi®cations of the mitochondrial membrane potential, we treated mock-transfected and p27 Kip1 -overexpressing cells with 50 mM etoposide for indicated times.…”