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-1beta converting enzyme (
ICE;
caspase 1) or
ICE-like
enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/
Apopain/
caspase 3 activation, plasma membrane
phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim 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 DeltaPsim 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 DeltaPsim. 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 + DeltaPsim 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.