Biochemical mechanisms of apoptosis : ordering of the biochemical events in chemical-induced apoptosis.

Abstract

Apoptosis is an important form of cell death induced by anti-cancer drugs and chemicals. The biochemical mechanisms of chemical-induced apoptosis remain, however, largely unknown, and the major biochemical events in this model of apoptosis are poorly characterised. In this study, apoptosis, induced in human monocytic THP.l cells by etoposide and N-tosyl-L-phenylalanyl chloromethyl ketone and in leukaemic U937 cells by etoposide, was accompanied by the release of mitochondrial cytochrome c, activation of caspases, proteolysis of poly(ADP-ribose) polymerase (PARP), DNA fragmentation, externalisation of phosphatidylserine (PS), and reduction in mitochondrial membrane potential (ɅѰm). Time course studies demonstrated that activation of caspases occurred prior to both PS externalisation and reduction in A\|/m. This was further supported by the observation that the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) inhibited all the ultrastructural and biochemical characteristics of apoptosis except the release of cytochrome c. The execution phase of apoptosis may thus be initiated by the translocation of cytochrome c to the cytosol, where it interacts with dATP and apoptotic-protease activating factor-1, resulting in activation of the initiator caspase-9, which in turn activates effector caspases, such as -3 and -7. Cytochrome c release was a later event in non-apoptotic cell death occurring after commitment to cell death and without caspase activation. Thus its release may represent a commitment to cell death in chemical-induced apoptosis. This study also showed that in cells triggered into apoptosis the plasma membrane changes leading to recognition and phagocytosis may be uncoupled from other features of apoptosis by using the mitochondrial inhibitors, antimycin A and oligomycin. These inhibitors blocked increased plasma membrane permeability, PS externalisation and recognition by two classes of phagocytes but not activation of caspases-3 and -7, PAR? cleavage or DNA fragmentation in THP.l cells. PS externalisation was also dissociated from caspase activation in U937 cells. Thus cell surface changes governing safe clearance of apoptotic cells may be regulated by an independent pathway to those mediated by caspases. This finding may have important consequences for attempts to manipulate cell death for therapeutic gain in vivo. Finally, this study demonstrated that in chemical-induced apoptosis caspases functioned solely as executioners and activation of caspases occurred after commitment to cell death, through post-mitochondrial activation pathway(s). Z- VAD.FMK inhibited apoptosis at a stage after this commitment by blocking the post- mitochondrial activation cascade of caspases. This contrasted to its effect in death receptor-mediated apoptosis in U937 cells, where Z.VAD.FMK inhibited apoptosis prior to commitment to cell death induced by tumour necrosis factor-ɑ.