Bcl-2 PREVENTS TNE- AND Fas-INDUCED CELL DEATH BUT DOES NOT INHIBIT INITIAL PROCESSING OF CASPASE-3

The members of ICE/CED3 family of cysteine proteases (caspases) play important roles in the regulation of programmed cell death. Recent studies have revealed that processing and activation of CPP32 (CASP-3) are crucial for executing cell death in TNF-aand Fasinduced apoptosis. Overexpression of Bcl-2 in HeLa cells inhibits the cell death and CASP-3 like protease activities which are induced by TNF-or or anti-Fas antibody. Proform of CASP-3 disappered after the treatment with TNF-on or anti-Fas antibody in both HeLa cells and HeLa/bcl-2 cells. These results suggest Bcl-2 prevents cell death and activation of CASP-3 but does not inhibit initial processing of CASP-3. Molecular genetic analysis of programmed cell death in Caenorhabditis elegans (C. elegans) revealed that the gene ced-3 executes cell death (8). A ced-3 gene activity is required for all programmed cell death in C. elegans. The analysis of the primary structure of CED-3 revealed that it has a significant homology with that of interleukin-lfl converting enzyme (ICE) that encodes Asp-specific cysteine protease (32). The expression of ice in a number of mammalian cell lines induces apoptosis (20, 31). An increasing number of ICE/CED-3 family members (caspases) has been identified. The mammalian caspase family now includes ICE, ICH-l, ICH-3, CPP32 (Yama/Apopain), Tx (ICH2/ICErelII), ICErelIII, MCH2a, ICE-LAP3 (MCH3/ Abbreviations: Ac-DEVD-CHO, acetyl-DEYD-CHO; Ac-YVAD-CHO, acetyl-YVAD-CHO; CHX, cycloheximide; CrmA, cytokine response modifier A; FLICE, FADD-like ICE; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; Z-Asp-CH2-DCB, benzyloxycarbonyl-AspCHQOC(O)-2,6-dichlorobenzene 5Correspondence to: M. Miura at Osaka University Medical School. Tel: +81-6-879-3581; Fax: +8l-6879-3589; E—mail: mmiura@nana.med.osaka-u.ac.jp CMH-l), MCH4, FLICE (MACHal/MCH5), ICELAP6 and Caspase l2 (ll), and all of the caspase members have been shown to be involved in executing cell death. Apoptotic stimuli lead to the processing of certain members of the caspase family, resulting in the formation of active heterodimeric forms. These active enzymes initiate the consequences of caspase activation and in turn, cells undergo irreversible changes that lead to apoptosis. CASP-3 is the most widely investigated member of the caspase family involved in the execution of cell death in mammals. Recent studies have suggested that proteolytic cleavage and activation of the CASP-3 are functionally important in inducing apoptosis. For example, CASP-3 activity is markedly elevated in cells undergoing apoptosis induced by a variety of reagents, and a tetrapeptide inhibitor that specifically inhibits CASP-3-like activity also blocks apoptosis (23, 28). In contrast to caspase family members which promote cell death, members of the Bcl-2 family inhibit cell death induced by diverse stimuli in many cell types (3, l4, 29). A recent study revealed that Bcl-2 prevents CASP-3 processing and PARP cleavage, which can be observed during the execution of cell death induced by various