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.

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The “destruction box” (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.