The death receptor Fas transduces apoptotic death signaling mediated by caspases. In the present study, human hepatoma HepG2 cells showed the Fas-mediated apoptosis mediated by caspase, especially caspase 3, only in the presence of actinomycin D. Interestingly, cytosolic proteins extracted from intact HepG2 cells induced caspase 3 inactivation. Our results reveal that this inactivation was triggered by the direct inhibition of activated caspase 3 by IAP gene family ILP. In addition, a 53 kDa protein was co-immunoprecipitated with antihuman caspase 3 antibody from intact HepG2 cells. This protein was a complex-protein of procaspase 3 and the cell cycle regulator p21 WAF1 (p21). P21 bound to only procaspase 3, but not to activated caspase 3. We also demonstrate that p21 protein-loaded HepG2 cells resist to Fas-mediated apoptosis even in the presence of actinomycin D. Here we report that caspase 3 inactivation for the resistance to Fas-mediated apoptosis is induced by a procaspase 3/p21 complex formation and direct inhibition of activated caspase 3 by ILP.

Caspase 3 is an essential death factor for the Fasmediated cell death, and its inactivation in cells is initiated by an interaction with p21 on mitochondria or with IAP family member ILP. Survivin is also a member of IAP family and is speci®cally expressed during embryogenesis and in tumor cells and suppresses cell death signaling. In our current study, we demonstrated that Survivin translocation into the nucleus is dependent on Fas stimulation and cell proliferation. Survivin also interacts with the cell cycle regulator Cdk4, leading to Cdk2/Cyclin E activation and Rb phosphorylation. As a result of Survivin/Cdk4 complex formation, p21 is released from its complex with Cdk4 and interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death. Here, we propose that Survivin supports procaspase 3/p21 complex formation as a result of interaction with Cdk4 resulting in suppression of cell death signaling.

The death mediator caspase acts as the dominant regulator during cell death induction. The CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain), is essential for the cell death signaling. We recently reported that activation of caspase 3 is regulated by complex formation with p21 or ILP. In the present study, we investigated the binding domain with p21 and ILP to further characterize the caspase 3 inactivation machinery. Our results show that caspase 3 contains p21 binding domain in the N-terminus and ILP binding domain in the active site. Further, the caspase 3 binding domain in p21 was independent of the Cdk-or PCNAbinding domain. We also found caspase 3 protection by p21 from the p3-site cleavage serineproteinase contributes to the suppression machinery. Here, we propose the caspase 3 inactivation system by p21 and ILP as new essential system in the regulation of cell death.

Death receptor Fas transduces cell death signaling upon stimulation by Fas ligand, and this death signaling is mediated by caspase. Recently, we reported that the cell cycle regulator p21 interacts with procaspase 3 to resist Fas-mediated cell death. In the present study, the molecular characterization and functional region of the procaspase 3-p21 complex was further investigated. We observed the p21 expression in the mitochondrial fraction of HepG2 cells and detected Fas-mediated cell death only in the presence of actinomycin D. However, mitochondrial-DNA-lacking HepG2 (MDLH) cells showed this effect even in the absence of actinomycin D. Both p21 and procaspase 3 were expressed in MDLH cells, but the procaspase 3-p21 complex formation was not observed. Interestingly, the resistance to Fas-mediated cell death in the MDLH cells without actinomycin D was recovered after microinjection of HepG2-derived mitochondria into the MDLH cells. We conclude that mitochondria are necessary for procaspase 3-p21 complex formation and propose that the mitochondrial role during cell death is not only death induction but also death suppression.Cell death is an essential phenomenon for cell homeostasis, as well as cell growth, and its occurrence during embryonic and postembryonic development has been well documented (20,39). There are two distinct processes leading to cell death: apoptotic cell death and necrotic cell death (39). Apoptotic cell death is accompanied by the condensation and/or fragmentation of nuclei, as well as apoptotic body formation and chromosomal DNA fragmentation into 180-bp oligomers (39). Multiple studies have demonstrated the important role of apoptotic cell death in various disease states and physiological cell death (21), and many factors involved with the death signaling have been identified.Fas, a transmembrane protein belonging to the tumor necrosis factor/nerve growth factor receptor family (21), transduces the death signaling upon stimulation by Fas ligand or an agonistic Fas antibody, such as the CH-11 clone (41). The molecular mechanism of Fas-mediated apoptosis has been extensively investigated. Caspase is the term used for the interleukin-1␤ converting enzyme (ICE)/CED-3 cysteine proteinase family (1). During death induction, the sequential activation of the ICE and CPP32 subfamilies has been reported (6,27,29,31,33), and this phenomenon is known as the "ICE cascade." At present, 10 genes have been identified as part of the caspase family, and the CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain [7,23]) and caspase 8 (FLICE/MACH [2,19]), in particular acts as the dominant regulator in the death signaling. Therefore, the regulation of CPP32 subfamily activation is an especially important focus for cell death research.Among the members of the CPP32 subfamily, caspase 3 is especially important in the understanding of apoptotic cell death because of its variant substrate specificity. Cytoplasmic serine proteinase (32), caspase 8 (38), and/or cytotoxic-T-lymphocyte-derived granzyme B (4) prote...

Caspase 3 is an essential factor for Fas-mediated cell death and exists endogenously in cells where its activation is suppressed by p21 and ILP. Inside the cell, procaspase 3 interacts with p21 on mitochondria. In the present study, we investigated the molecular basis for procaspase 3/p21 complex formation. During Fas-mediated cell death, mitochondria are damaged, accompanied by decreased mitochondrial membrane-potential and decreased intracellular ATP levels. This mitochondrial damage occurs before an estrangement of the procaspase 3/p21 complex, and we demonstrate that intracellular ATP-deprivation also initiates an estrangement of procaspase 3/p21 complex formation and accelerates Fas-mediated cell death. In addition, our current results revealed that the phosphorylated p21 by PKA interacts with procaspase 3. Here, we report that the mitochondrial role, especially for ATP synthesis, and PKA are necessary for the procaspase 3/p21 complex formation to resist Fas-mediated cell death. Cell Death and Differentiation (2000) 7, 721 ± 728.