Bcl-2 regulates a caspase-3/caspase-2 apoptotic cascade in cytosolic extracts Eileithyia Swanton 1 , Peter Savory 1 , Sabina Cosulich 1,2 , Paul Clarke 1,3 and Philip Woodman* ,1 1 School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, England, UK Apoptosis is accompanied by the activation of a number of apoptotic proteases (caspases) which selectively cleave speci®c cellular substrates. Caspases themselves are zymogens which are activated by proteolysis. It is widely believed that `initiator' caspases are recruited to and activated within apoptotic signalling complexes, and then cleave and activate downstream `eector' caspases. While activation of the eector caspase, caspase-3, has indeed been observed as distal to activation of several dierent initiator caspases, evidence for a further downstream proteolytic cascade is limited. In particular, there is little evidence that cellular levels of caspase-3 that are activated via one pathway are sucient to cleave and activate other initiator caspases. To address this issue, the ability of caspase-3, activated upon addition to cytosolic extracts of cytochrome c, to cause cleavage of caspase-2 was investigated. It was demonstrated that cleavage of caspase-2 follows, and is dependent upon, activation of caspase-3. Moreover, the activation of both caspases was inhibited by Bcl-2. Together, these data indicate that Bcl-2 can protect cells from apoptosis by acting at a point downstream from release of mitochon- drial cytochrome c, thereby preventing a caspase-3 dependent proteolytic cascade. Keywords: ICH-1; caspase cleavage; cytochrome c; dATP Introduction Apoptosis is a form of cell death activated by the induction of cellular suicide pathways in response to a variety of stimuli. Recent advances have led to a widely accepted model, whereby many of the morphological features of apoptotic cells are a consequence of the action of a conserved family of cysteine proteases (caspases) on speci®c cellular substrates (Nicholson et al., 1995). Caspases themselves are present as proenzymes that are readily cleaved and activated during apoptosis, providing the cell with a means to rapidly amplify its apoptotic response [see Cohen (1997) for review]. In addition, the broadly similar patterns of protein cleavage and consequent morpho- logical changes that occur during apoptosis, irrespec- tive of the stimulus, are consistent with activation of a proteolytic cascade. Evidence for such a cascade has come from examination of apoptotic cells and biochemical studies. Firstly, induction of apoptosis is accompanied by the activation of several caspases within the same cell (Faleiro et al., 1997; Polverino and Patterson, 1997). Secondly, evidence from the differ- ential timing of caspase activation after addition of apoptotic stimuli are consistent with a hierarchy of caspase activities (Faleiro et al., 1997; Li et al., 1997a; Takahashi et al., 1997). In vitro studies are also consistent with a caspase cascade, since puri®ed recombinant caspases will cleave each other eectively [see Cohen (1997)]. However, the relevance of these studies to activation of caspases in vivo are proble- matic, since the concentrations of caspases and incubation conditions may not be physiological. Moreover, additional caspase substrates, and factors that might regulate the speci®city of caspase action, are absent from such studies. Although several recent studies have avoided some of these diculties by adding exogenously-activated caspases to naive extracts (Martin et al., 1996; Muzio et al., 1997; Orth et al., 1996), these still fall short of demonstrating that endogenous levels of caspases are sucient to drive a proteolytic cascade. Evidence for such a dependency when using endogenous proenzymes as the sole source of caspase activity remains limited. The pathway leading to caspase activation varies according to the apoptotic stimulus, though several features are conserved. Thus, cell death caused by activation of the TNFR-1 or CD95/Fas receptors is brought about by the recruitment of the adaptor protein FADD (Fas-Associated protein with Death Domain) (Chinnaiyan et al., 1995). In the case of the TNFR-1, FADD recruitment requires prior binding of TRADD (TNFR-1-Associated Death Domain protein) (Hsu et al., 1995). FADD in turn recruits procaspase- 8 via a homophilic interaction between the death eector domains of these proteins, and this event is closely followed by autocatalytic cleavage and activation of the clustered zymogen (Boldin et al., 1996; Yang et al., 1998). The TNFR-1 receptor can also mediate activation of caspase-2 via the recruit- ment of a death-inducing signalling complex. In this case RIP (Receptor-Interacting Protein) (Stanger et al., 1995) acts as an adaptor for the recruitment of RAIDD (RIP-Associated ICH-1/CED-3-homologous protein with a Death Domain), which subsequently binds to procaspase-2 (Duan and Dixit, 1997). As for caspase-8, it is believed that caspase-2 will autoacti- vate when oligomerized (Butt et al., 1998). In contrast, the activation of caspase-9 by apoptotic stimuli proceeds via recruitment of the zymogen to a signalling complex containing Apaf-1 (Apoptotic protease activating factor-1) and cytochrome c (Li et al., 1997b; Liu et al., 1996; Zou et al., 1997), which is *Correspondence: P Woodman Current addresses: 2 Zeneca Central Toxicology Laboratory, Alderley Park, Maccles®eld, SK10 4TJ, UK; 3 Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK Received 29 July 1998; revised 15 October 1998; accepted 15 October 1998 Oncogene (1999) 18, 1781 ± 1787 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc