Bcl-2, Bcl-x L and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death David CS Huang, Suzanne Cory and Andreas Strasser The Walter and Eliza Hall Institute of Medical Research, Post Oce, Royal Melbourne Hospital, Victoria 3050, Australia Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, is an eective inhibitor of apoptotic cell death induced by many cytotoxic agents. Survival- promoting homologues of Bcl-2 include its close relative, Bcl-x L and the 19 kD protein encoded by the E1B gene of adenoviruses. Whether these proteins are functionally equivalent and whether they can antagonise all or only some pathways to apoptosis is unresolved. We have carried out a systematic comparison of Bcl-2, Bcl-x L and adenovirus E1B19kD activity, using several cell lines and a range of cytotoxic conditions. High levels of expression of each of these proteins inhibited apoptosis induced by growth factor deprivation or treatment with g-radiation, glucocorticoid and various cytotoxic drugs. In contrast, none of them could eectively counter apoptosis induced via the TNF receptor or Fas/APO-1 (CD95). Biochem- ical analysis revealed that all three proteins can associate with Bax and Bak, members of the Bcl-2 protein sub- family that can facilitate apoptosis. The results provide evidence that Bcl-2, Bcl-x L and adenovirus protein E1B19kD are indistinguishable in their ability to regulate the cell death eector machinery. Keywords: apoptosis; Bcl-2; Bcl-x L ; E1B19kD adeno- virus protein; Fas/APO-1 (CD95); TNF Introduction Apoptosis, the physiological cell death process, is characterised morphologically by cell shrinkage, chromatin condensation and DNA degradation (Wyllie et al., 1980). It plays an important role in sculpting tissues during development, in tissue home- ostasis and in host defence systems deployed against invading pathogens (Wyllie et al., 1980; Ellis et al., 1991; Vaux and Strasser, 1996). Since apoptosis triggered by diverse agents can be inhibited by the protein product of the proto-oncogene bcl-2, it seems that several independent biochemical pathways pro- moting cell death converge upon a common ®nal eector mechanism (Hengartner and Horvitz, 1994a; Cory, 1995; Korsmeyer, 1995; Thompson, 1995; Vaux and Strasser, 1996; White, 1996). Studies of cell death in the nematode C. elegans have revealed striking evolutionary conservation of the molecular basis of apoptosis. Two genes, ced-3 and ced-4, are essential for cell death during nematode development while another, ced-9, is essential for cell survival (Ellis et al., 1991; Hengartner et al., 1992; Yuan et al., 1993). No mammalian ced-4 homologue has been discovered thus far, but ced-3 encodes a cysteine protease related to the interleukin-1b convert- ing enzyme (ICE) (Yuan et al., 1993) and ced-9 is a functional and structural homologue of bcl-2 (Vaux et al., 1992a; Hengartner and Horvitz, 1994b). Since apoptosis can be induced by overexpression of cysteine proteases (reviewed by Kumar, 1995) and blocked by pseudo-substrate inhibitors encoded by viruses (Gagliardini et al., 1994; Bump et al., 1995), it appears that cell death is initiated by proteolytic cleavage of one or several critical substrates. Thus, Bcl-2 and Ced-9 may function by blocking a step that leads to the activation or activity of cysteine proteases (Hengartner and Horvitz, 1994a; Cory, 1995; White, 1996). Control of cell death appears to be signi®cantly more complex in mammals than in C. elegans. Whilst the nematode apparently has only one cysteine protease gene, ced-3 (Ellis et al., 1991), there are at least eight in mammals, comprising three distinct subfamilies (Kumar, 1995). In addition, only one survival gene (ced-9) has been identi®ed in C. elegans, whilst there are three closely homologous genes in vertebrates: bcl-2 (Vaux et al., 1988), bcl-x L (Boise et al., 1993) and bcl-w (Gibson et al., 1996), as well as two distantly related genes, A1 (Lin et al., 1996) and Mcl-1 (Reynolds et al., 1994). Furthermore, in mammals, there exists a sub-family of Bcl-2 homo- logues, exempli®ed by Bax (Oltvai et al., 1993), that inhibit the function of the survival proteins by forming heterodimers with them and thereby facilitate cell death when expressed at high levels (Oltvai and Korsmeyer, 1994). It is presently unclear whether Bax sub-family members participate directly in cell death or merely act as antagonists of proteins of the Bcl-2 sub-family. Bax- like proteins have not yet been found in nematodes but it has been postulated that depending on the genetic make-up of a cell, Ced-9 can either block or promote apoptosis (Hengartner and Horvitz, 1994c). Whilst a ced-9 gain of function mutation prevents all developmentally programmed death of somatic cells in C. elegans (Ellis et al., 1991; Hengartner and Horvitz, 1994c), Bcl-2 is not able to block apoptosis triggered by all physiological cell death stimuli in mammalian cells (reviewed by Strasser, 1995a). For example, although B and T lymphocytes from bcl-2 transgenic mice are resistant to numerous cytotoxic agents (McDonnell et al., 1989; Sentman et al., 1991; Strasser et al., 1991a,b, 1994a), they remain sensitive to apoptosis induced by activation of the surface receptor Fas/APO-1 (CD95) (Strasser et al., 1995c). Moreover, although bcl-2 transgene expression enhances the survival of immature B and T cells that have failed Correspondence: A Strasser Received 23 July 1996; revised 30 September 1996; accepted 30 September 1996 Oncogene (1997) 14, 405 ± 414  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00