Introduction

Genetically programmed cell suicide is vital for the proper development and functioning of multicellular organisms. By eliminating redundant, damaged or effete cells, apoptosis sculpts the embryo and maintains tissue homeostasis within the adult. Tight control is essential, since too much cell death can contribute to degenerative conditions but too little sets the stage for cancer and autoimmune disease.

Although the distinctive morphology of dying cells had been recorded from the earliest days of microscopy (Lockshin and Zakeri, 2001), it was Kerr, Wyllie and Currie who first appreciated that the ordered stereotypic changes within the cell during its convulsive death throes, which they coined apoptosis, reflected an underlying genetic programme (Kerr, 1969; Kerr et al., 1972). Over a decade elapsed, however, before the first molecularly defined regulator of the cell suicide programme was identified. The hallmark 14; 18 chromosome translocation in human follicular lymphoma was found to link the immunoglobulin heavy chain locus to a novel gene denoted Bcl-2 (Tsujimoto et al., 1984). Unexpectedly, unlike all previously identified oncogenes, Bcl-2 was shown to promote cellular survival rather than proliferation (Vaux et al., 1988). This discovery engendered the concept that impaired apoptosis is central to tumour development, a view that is now widely embraced (Cory et al., 1999; Hanahan and Weinberg, 2000; Green and Evan, 2002).

Important insights regarding the genetic basis of programmed cell death in the nematode C. elegans were then emerging from the Nobel-prize winning studies of Horvitz and colleagues (Horvitz, 1999). They had determined that the demise of the 131 somatic cells fated to die during worm development required two genes, CED-3 and CED-4, whereas another, CED-9, ensured the survival of all others (Ellis and Horvitz, 1991; Hengartner et al., 1992). In a telling convergence of two disparate fields, human Bcl-2 was shown to convey cell survival in the worm (Vaux et al., 1992) and CED-9 proved to be its structural as well as functional counterpart (Hengartner and Horvitz, 1994). Moreover, the protein encoded by CED-3 was found to be related to a mammalian cysteine protease that activates the cytokine interleukin-l (Yuan et al., 1993).

These landmark discoveries established the strong evolutionary conservation of the core cell death machinery and indicated that Bcl-2 and CED-9 must prevent the activation of a proteolytic demolition programme. Indeed, we now know that the cell is dismantled by a group of proteases termed caspases because of the cysteine in their active site and their specificity for cleavage after aspartate residues (Thornberry and Lazebnik, 1998; Earnshaw et al., 1999; Shi, 2002; Yuan, 2003). Mammalian cells possess about a dozen caspases, maintained as inactive zymogens to preclude untimely cell death. Initiator caspases have a long prodomain that allows their recruitment to scaffold proteins on which they are activated. Once active, they can process and activate downstream effector caspases, which in turn cleave several hundred proteins.

Over the past decade it has become evident that Bcl-2 belongs to an extended family of proteins and the interplay between opposing family members integrates intracellular cues to arbitrate whether initiator caspases are unleashed. Despite much progress, many questions remain about how the Bcl-2 family governs this vital process. This review summarizes current understanding of this vexed central issue and discusses how perturbed regulation of Bcl-2 and its relatives contribute to cancer development and thwarts cancer therapy. We also explore the prospect that drugs targeting these vital regulators could create more effective treatments, particularly for cancers. As the literature in this field is now vast, we also refer the reader to the relevant accompanying reviews and several other recent overviews (Gross et al., 1999a; Strasser et al., 2000; Cory and Adams, 2002; Borner, 2003; Adams, in press).

Cell death circuitry

Opposing factions of Bcl-2-related proteins

There are at least 20 Bcl-2-related proteins in mammalian cells (Figure 1) (Adams and Cory, 1998; Gross et al., 1999a; Cory and Adams, 2002). Bcl-2 and its closest relatives, Bcl-x_L and Bcl-w, protect cells from a wide range of cytotoxic insults, including cytokine deprivation, UV- and -irradiation, and chemotherapeutic drugs (see, for example, Huang et al., 1997a). The more divergent Al and Mcl-1 are also protective. Other Bcl-2 relatives, mainly identified as Bcl-2-binding proteins, promote rather than antagonize apoptosis and fall into two distinct groups. One trio – Bax, Bak and Bok – are very similar to Bcl-2 in sequence and structure, and three conserved segments known as BH1, BH2 and BH3 (Bcl-2 homology) regions form a hydrophobic groove on both the anti- and proapoptotic proteins (Muchmore et al., 1996; Suzuki et al., 2000; Petros et al., 2001; Hinds et al., 2003). Bax and its relatives probably act at least in part by perturbing intracellular membranes, as discussed further below.

Figure 1.

Three subfamilies of Bcl-2-related proteins. The Bcl-2 subfamily promotes cell survival, whereas the Bax and BH3-only subfamilies promote apoptosis. BH1 to BH4 (Bcl-2 homology regions 1–4) are sequences having relatively high homology with Bcl-2. Known -helical regions are indicated. The BH1/2/3 region of the Bcl-2-like proteins, and perhaps in the Bax group, is like a receptor for the BH3 domain of their proapoptotic ligands. Many family members have a carboxy-terminal hydrophobic transmembrane domain (TM, hatched) that aids targeting to intracellular membranes. Other Bcl-2 relatives (Boo/Diva, Bcl-Rambo, Bcl-G, Bcl-B, BNIP3 and Bfk) are not shown here because their function is not yet clear. C. elegans proteins CED-9 and EGL-1 are compared with their mammalian counterparts. Mouse Noxa has a second BH3 domain

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The other proapoptotic relatives, exemplified by Bik, Bad and Bim, are largely unrelated in sequence to either Bcl-2 or each other, apart from the signature BH3 domain, which is obligatory for their killing function. These disparate 'BH3-only proteins' (Huang and Strasser, 2000; Puthalakath and Strasser, 2002) cannot kill in the absence of Bax and Bak (Cheng et al., 2001; Zong et al., 2001) and function upstream, sensing developmental death cues and intracellular damage. Once activated, most bind to Bcl-2 and other antiapoptotic homologues, neutralizing their prosurvival function: the amphipathic BH3 -helix docks within the BH1/2/3 hydrophobic groove of the antiapoptotic protein (Sattler et al., 1997; Petros et al., 2000; Liu et al., in press) (Figure 2a). One unusual BH3-only protein, Bid, may act in addition by interacting transiently with Bax (or Bak) (see below). In the nematode, the single identified BH3-only protein, EGL-1 (Conradt and Horvitz, 1998), is responsible, like CED-3 and CED-4, for all developmental death of somatic cells, but the worm apparently has no Bax-like homologue (Horvitz, 1999).

Figure 2.

3D structures of Bcl-2-related proteins. (a) Bcl-x_L with the BH3 peptide of Bad (blue) bound to the surface groove formed by residues within the BH1, BH2 and BH3 domains (Petros et al., 2000). (b) Bax, showing its C-terminal tail (blue) tucked into the surface grove, but running in the opposite orientation to the BH3 ligand (Suzuki et al., 2000). (c) Bcl-w, showing its C-terminal tail (blue) tucked into the surface groove, but somewhat differently than in Bax (Hinds et al., 2003). The figure was kindly provided by Dr Mark Hinds, Walter and Eliza Hall Institute

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Abutting the C-terminus of most Bcl-2 and Bax homologues and certain BH3-only proteins is a hydrophobic sequence of 16 – 19 residues, which has the features of a helical transmembrane domain (TM in Figure 1) and is important for their targeting to intracellular membranes. Imaging studies of healthy cells place Bcl-2 on the nuclear envelope, endoplasmic reticulum and outer mitochondrial membrane (Monaghan et al., 1992; Krajewski et al., 1993; Lithgow et al., 1994), whereas Bcl-x_L and Bcl-w reside primarily on mitochondria (O'Reilly et al., 2001; Kaufmann et al., 2003; Wilson-Annan et al., 2003). Fewer basic residues flank the TM domain of Bcl-2 than that of other prosurvival proteins, and mutagenesis suggests that this drives the broader membrane distribution of Bcl-2 (Kaufmann et al., 2003).

The relationship of the family members with membranes is complex. Although Bcl-2 is thought from in vitro studies to be an integral membrane protein (Nguyen et al., 1993; Janiak et al., 1994), the avidity of individual antiapoptotic proteins for membranes differs markedly. Whereas Bcl-2 is largely membrane-associated after subcellular fractionation, most of the Bcl-w and a substantial proportion of Bcl-x_L are found in the cytosolic fraction (Hsu et al., 1997; Hausmann et al., 2000; O'Reilly et al., 2001; Wilson-Annan et al., 2003). Proapoptotic Bax and Bak also differ in their location in healthy cells: Bak is associated with the mitochondrial and ER membranes, but Bax is largely cytosolic (Wolter et al., 1997; Hsu and Youle, 1998; Griffiths et al., 1999).

Recent structural studies have provided insights regarding these differences. Surprisingly, in both Bax and Bcl-w, the hydrophobic C-terminal helix occludes the BH 1/2/3 hydrophobic groove (Suzuki et al., 2000; Hinds et al., 2003) (Figure 2b, c) and hence presumably must flip out to interact with intracellular membranes. A corollary is that the binding of proapoptotic BH3-only proteins to Bcl-w, and probably also to at least Bcl-x_L and perhaps to all the prosurvival relatives, requires competitive displacement of the C-terminal domain (Wilson-Annan et al., 2003). Although a structure is not yet available for full-length Bcl-2, its stronger membrane association may mean that its TM domain is not bound to the groove, which would therefore be more readily accessible to BH3 'ligands'. Alternatively, all Bcl-2-like and Bax-like family members might initially be attracted to intracellular membranes by a membrane-bound anchor protein, and stable integration via the TM domain might represent a subsequent event. If so, the putative membrane-anchoring protein might be an activator of Bax/Bak (Wilson-Annan et al., 2003).

Pathways for caspase activation

In considering how Bcl-2, CED-9 and their relatives regulate caspase activation, it is instructive to first outline known pathways for caspase activation. In C. elegans, activation of the caspase CED-3 requires CED-4, and CED-9 is thought to block CED-3 activation by sequestering CED-4 on mitochondria (Chen et al., 2000; del Peso et al., 2000). Cells receiving a developmental death cue respond by producing EGL-1, which binds CED-9, allowing release of CED-4 to activate CED-3 (Conradt and Horvitz, 1998) (Figure 3, left panel). The scaffold protein CED-4 has an N-terminal CARD (caspase recruitment domain) that mediates homotypic interaction with a comparable CARD in CED-3, and a nucleotide-binding domain (NBD) that permits the oligomerization required to promote CED-3 processing (Yang et al., 1998).

Figure 3.

Pathways to apoptosis in C. elegans and mammalian cells. The pathway for programmed cell death in C. elegans (left panel) is compared with the two distinct pathways in mammals (right panel): the stress pathway (A), triggered by diverse cytotoxic conditions such as cytokine deprivation, DNA damage and anoikis, leads to activation of initiator caspase-9, whereas the death receptor pathway (B), triggered by aggregation on the plasma membrane of receptors of the tumour necrosis factor (TNF) family (here typified by Fas), leads to activation of initiator caspase-8. The stress and death receptor pathways are largely independent but may be linked via activation of the BH3-only protein Bid in certain cell types (see text). This model of the stress pathway now appears to be too simplistic (see text and Figure 4)

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The mammalian CED-4 homologue Apaf-l was discovered as a cofactor required for activation of caspase-3 by caspase-9 (Liu et al., 1996; Li et al., 1997; Zou et al., 1997). In contrast to CED-4, Apaf-1 is cytosolic (Hausmann et al., 2000), does not bind Bcl-2 or its close relatives (Moriishi et al., 1999) and, in addition to the CARD and NBD motifs, has a long inhibitory C-terminal domain of WD40 repeats (Zou et al., 1997; Wang, 2003). In cells undergoing apoptosis, cytochrome c released from leaky mitochondria (see below) binds to the WD40 repeats, generating a more open conformation (Hu et al., 1998; Srinivasula et al., 1998). Apaf-1 can then multimerize and associate with pro-caspase-9 to form a heptameric 1 MDa complex termed the 'apoptosome' (Acehan et al., 2002; Wang, 2003).

Diverse types of cellular stress, all inhibitable by the Bcl-2-like proteins (see, for example, Strasser et al., 1991a; Huang et al., 1997a), can contribute via mitochondrial disruption to the activation of caspase-9. But is the Apaf-1/caspase-9 apoptosome the unique counterpart of the CED-4/CED-3 death engine of the nematode, as has been widely believed (Figure 3)? If so, loss of Apaf-1 or caspase-9 should be as effective as gain of Bcl-2 in protecting cells against diverse types of intracellular stress. Consistent with that notion, most mice lacking Apaf-1 (Cecconi et al., 1998; Yoshida et al., 1998) or caspase-9 (Hakem et al., 1998; Kuida et al., 1998) died before birth with enlarged brains due to impaired apoptosis during neuronal development. Recent studies have shown, however, that their lymphocytes, myeloid cells and fibroblasts remain highly sensitive to cytotoxic conditions against which Bcl-2 protects (Marsden et al., 2002). Furthermore, some Apaf-1-null mice develop into healthy adults (Honarpour et al., 2000), Apaf-1 is not required for apoptosis of postmitotic neurons (Honarpour et al., 2001) or deletion of autoreactive thymocytes (Hara et al., 2002), and Bcl-2 can protect embryonic stem cells lacking Apaf-1 from stress-imposed death (Haraguchi et al., 2000). Thus, neither Apaf-1 nor caspase-9 is essential for stress-induced apoptosis. Rather, the Apaf-1/cytochrome c/caspase-9/caspase-3 apoptosome appears to be an amplifier of caspase activity, and death of some cell types (such as developing neurons), but not others (such as lymphocytes) requires the higher levels of activity it generates (Marsden et al., 2002).

The novel route to cell death inferred from these findings still requires caspases: lymphocytes lacking Apaf-l or caspase-9 died with all the hallmarks of apoptosis; the dying cells retained discernable caspase activity, including that of effector caspase-7; and their death was delayed by a broad-spectrum caspase inhibitor (Marsden et al., 2002). Presumably, therefore, stress-induced apoptosis is set in train by caspase(s) acting upstream or independently of caspase-9 (Figure 4). Candidates include other caspases having CARD-containing prodomains (1, 2, 4 and 5 in humans and their mouse counterparts 1, 2, 11 and 12), and even caspase-8 (and -10) cannot be entirely ruled out. As discussed further elsewhere (Adams, 2003), the initiators implicated in different situations include caspase-1 (Marsden et al., 2002), caspase-2 (Lassus et al., 2002) and caspase-12 (Nakagawa et al., 2000), but none can be the sole initiator because no general defects in apoptosis have appeared in mice lacking any one of these caspases (Ranger et al., 2001; Yuan, 2003). Hence, it has been proposed that several initiator caspases act redundantly to initiate stress-induced apoptosis (Marsden et al., 2002) (Adams, 2003). Presumably, their activation requires other scaffold/adaptor proteins, and several candidates bearing CARD and NBD are now known (see Adams and Cory, 2002). It will be intriguing to learn whether any such putative caspase activators are constrained (directly or indirectly) in healthy cells by Bcl-2 and its prosurvival homologs, as the C. elegans model would predict.

Figure 4.

Speculative model for mammalian stress pathways. This model attempts to reconcile recent data indicating that neither caspase-9 nor Apaf-1 is essential for stress-induced apoptosis and that other initiator caspases appear to act upstream (Lassus et al., 2002; Marsden et al., 2002). The model postulates that, in addition to the post-mitochondrial apoptosome pathway, there are initiator caspases (here caspases-1 and 2), perhaps controlled fairly directly by Bcl-2, which act upstream of organelle damage to activate Bax and Bak, perhaps via cleavage of a Bid-like protein that interacts with them. Bax and Bak are presumed to not only damage the mitochondria membrane but also to perturb the ER membrane, leading via an unknown protein ('Activator') to the activation of caspase-12. Once the effector caspase-3, -6 and-7 have been processed, they presumably feed back to create more damage to the organelles, perhaps through cleavage of Bid-like molecules or via unknown membrane proteins. For further discussion, see text and (Adams, 2003)

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Vertebrates have evolved an additional route to cell death that is triggered by cell surface receptors and leads to activation of pro-caspase-8, or its close homologue pro-caspase-10 (found in humans but not mice) (B in Figure 3). Upon ligation of the so-called 'death receptors' of the TNF receptor family (such as Fas/APO-1 and TNF-R1), by their cognate ligands, the aggregated receptors recruit the adaptor protein FADD, either directly or indirectly (Ashkenazi and Dixit, 1998; Strasser et al., 2000; Ashkenazi, 2003). In turn, FADD recruits pro-caspase-8 via homotypic interaction between the 'death effector domain' (DED) of FADD and the related DED within the caspase prodomain. The high local concentration in the resulting 'death-inducing signalling complex' (DISC) permits activation and autocatalysis of caspase-8, which then activates caspase-3.

The death receptor/caspase-8 and stress-induced pathways appear to be largely independent. For example, neither over-expression of Bcl-2 (Strasser et al., 1995; Huang et al., 1997a, 1999; Newton et al., 1998) nor loss of Bim, Apaf-1 or caspase-9 (Hakem et al., 1998; Kuida et al., 1998; Bouillet et al., 1999) protects lymphocytes from apoptosis induced by death receptor ligands. In certain other cell types, however, the two pathways may intersect, because caspase-8 can activate the BH3-only protein Bid, which acts by facilitating the permeabilization of mitochondria (see below) (Li et al., 1998; Luo et al., 1998; Gross et al., 1999b). Furthermore, mice lacking Bid can survive the fatal liver damage provoked by injected anti-Fas antibodies (Yin et al., 1999). This cross-talk is also proposed to operate in certain cell lines, designated type II (Scaffidi et al., 1998), but its physiological relevance remains controversial (Schmitz et al., 1999; Huang et al., 2000).

Role of Bcl-2 relatives in the life or death decision

In response to stress, the Bcl-2 family proteins congregate at intracellular membranes to adjudicate whether the cell should die. Their best-studied location is the outer mitochondrial membrane, but the ER/nuclear envelope is receiving increasing attention (Ferri and Kroemer, 2001; Scorrano et al., 2003; Shore, 2003; Zong et al., 2003). The BH3-only proteins now dock to their 'death socket', the BH1/2/3 groove on their pro-survival relatives (Huang and Strasser, 2000). Stress signals also cause proapoptotic Bax and Bak to undergo conformational changes, whereupon cytosolic Bax translocates and both proteins undergo homo-oligomerization on the outer mitochondrial membrane (Hsu and Youle, 1998; Griffiths et al., 1999, 2001; Antonsson et al., 2001; Nechushtan et al., 2001) and probably also the ER membrane (Zong et al., 2003). The role of each faction and their interactions will be considered in turn.

BH3-only proteins: sensing damage, galvanizing action

The multiplicity of mammalian BH3-only proteins seems to have evolved to allow more sophisticated control over the initiation of cell death (Huang and Strasser, 2000; Puthalakath and Strasser, 2002). Individual BH3-only proteins are expressed only in certain cell types, and some appear to monitor particular subcellular compartments for stress or damage, and/or to respond to specific sets of cytotoxic signals (Figure 5). For example, Bim is required for deletion of autoreactive lymphocytes in vivo (Bouillet et al., 2002; Hildeman et al., 2002) and for apoptosis of T cells in vitro following cytokine deprivation, calcium flux or treatment with Taxol (paclitaxel) but not markedly for apoptosis induced by -irradiation (Bouillet et al., 1999). Bad is required for the death that follows deprivation of glucese (Danial et al., 2003) or (to some extent) of epidermal growth factor (Ranger et al., 2003). Bmf may be required for anoikis, the apoptosis that epithelial cells undergo following their detachment from the extracellular matrix (Puthalakath et al., 2001; Frisch, 2003); pertinently, anoikis is thought to limit metastasis. In another link to tumorigenesis, Noxa (Oda et al., 2000) and Puma/Bbc3 (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 200la) are both induced by the tumour suppressor p53. Importantly, they have now been shown to be critical for apoptosis following genotoxic damage and Puma also for the death induced by several drugs (Villunger et al., 2003) (see below).

Figure 5.

Activation of BH3-only proteins. Individual BH3-only proteins appear to have the principal responsibility for different types of intracellular damage, as shown, albeit with partly overlapping activities. The 'beak' on them represents the BH3 domain. Activation can either be largely transcriptional (Hrk, Noxa, Puma) or primarily post-translational (Bim, Bmf, Bad, Bik, Bid) (see text). Although all BH3-only proteins can bind to and inactivate Bcl-2 and other antiapoptotic homologues, Bid after proteolysis (tBid) seemingly can also interact with and activate Bax (see text)

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Individual BH3-only proteins are also recruited for their death duties in different ways (Puthalakath and Strasser, 2002). In C. elegans, transcriptional regulation predominates: EGL-1 is induced both in all somatic cells fated to die (Conradt and Horvitz, 1998) and, in response to the worm p53 homologue CEP-1, in germline cells that have suffered DNA damage (Hofmann et al., 2002). Mammalian proteins regulated primarily at the transcriptional level include Hrk/DP5 (Imaizumi et al., 1999; Harris and Johnson, 2001), as well as Noxa and Puma/Bbc3.

The BH3-only proteins that are produced constitutively are maintained in a latent form until unshackled by diverse mechanisms. For example, Bad is sequestered by 14-3-3 scaffold proteins after phosphorylation by kinases such as Akt/PKB and protein kinase A (Zha et al., 1996), and its activation requires dephosphorylation, for example by calcineurin (Wang et al., 1999). Conversely, Bik/Nbk activation requires phosphorylation, possibly by casein kinase II (Verma et al., 2001). Bid instead undergoes cleavage by caspases or granzyme B, perhaps regulated by phosphorylation (Desagher et al., 2001), to expose its buried BH3 domain (Li et al., 1998; Luo et al., 1998). The cleaved p7/p15 complex is then prepared for membrane association by N-terminal myristoylation of p15 (tBid) (Zha et al., 2000).

Bim and Bmf seem to be sentinels assigned to check the cytoskeleton (Puthalakath et al., 1999, 2001). In healthy cells, both predominant forms of Bim (the splice variants Bim_EL and Bim_L) are sequestered to the dynein motor complex on microtubules via the dynein light-chain DLC1 (also known as LC8) (Puthalakath et al., 1999). In an analogous way, Bmf is bound to the myosin V motor complex through interaction with DLC2 (Puthalakath et al., 2001). Intriguingly, Taxol, which affects microtubules, promotes release of Bim but not Bmf, whereas anoikis frees Bmf but not Bim. In contrast, UV irradiation of cells releases both Bim and Bmf, and their release is mediated via phosphorylation of the conserved DLC binding motif by JNK (Lei and Davis, 2003). Presumably, another mechanism must operate when the DLC is released in association with Bim, as it is after cytokine withdrawal (Puthalakath et al., 1999).

Bim is also modulated transcriptionally. In cytokine-deprived haematopoietic cells, upregulation of bim expression is mediated in part by the forkhead transcription factor FKHR-L1 (Dijkers et al., 2000; Shinjyo et al., 2001), which is inactive in cytokine-supported cells due to its phosphorylation by PKB/Akt (Dijkers et al., 2002). In neuronal cells, induction of bim following cytokine deprivation instead requires JNK activation (Harris and Johnson, 2001; Putcha et al., 2001; Whitfield et al., 2001). Certain bim transcripts, generated by alternative splicing, encode smaller proteins that lack the restraining DCL1-binding motif and are therefore very potent death inducers (O'Connor et al., 1998; U et al., 2001; Marani et al., 2002), but their low abundance makes their physiological relevance unclear.

For several BH3-only proteins, optimal docking on their prosurvival relatives probably requires not only their BH3 domain but also a membrane targeting function, typically mediated by their hydrophobic C-terminal domain (Figure 1). By ensuring colocalization of the two proteins, the tail enhances their interaction. With Bim, for example, that domain is required both for mitochondrial targeting and proapoptotic activity (Yamaguchi and Wang, 2002). Bid lacks a hydrophobic tail, but its targeting to membranes is promoted after its cleavage by the N-terminal myristoylation of p15 (tBid) (Zha et al., 2000) and by interaction with the cardiolipin present in the mitochondrial membrane (Lutter et al., 2000).

It has been thought that all the BH3-only proteins bind promiscuously to all their pro-survival relatives, but more quantitative analysis may well reveal significant preferences. Consequently, there may be more specialisation among the pro-survival relatives than presently envisioned (Nijhawan et al., 2003).

Bid appears to be exceptional among the BH3-only proteins. Although it can bind to both Bcl-2- and Bax-like proteins in vitro, mutagenesis studies have suggested that the latter are the functionally relevant targets (Wang et al., 1996b). Incubation of activated (cleaved) Bid with mitochondria was shown to promote oligomerization of membrane-bound Bak and rapid cytochrome c release but, curiously, Bid was not detectable within cross-linked Bak oligomers, leading to the suggestion that Bid activates Bax and Bak by a 'hit-and-run' mechanism (Wei et al., 2000). Bid and Bax exhibit synergy in membrane permeabilization (Kuwana et al., 2002), and Bid may interact weakly with Bak (Ruffolo and Shore, 2003). However, since direct interactions have not been proven to occur under physiological conditions, Bid may instead activate Bax and Bak indirectly. For example, since Bid is reported to form homo-trimers in the membrane (Grinberg et al., 2002), it could nucleate Bax/Bak-mediated membrane porosity (see below).

Bcl-2-like proteins: proactive or passive protectors?

Mouse genetic studies (Ranger et al., 2001) suggest that the survival of every cell type requires protection by at least one Bcl-2 homolog. Despite overlapping expression patterns, inactivation of individual genes leads to diverse phenotypes, presumably because the different proteins are more abundant in particular tissues. Bcl-2 is essential for the survival of kidney and melanocyte stem cells, as well as mature lymphoid cells (Nakayama et al., 1993, 1994; Veis et al., 1993; Kamada et al., 1995); Bcl-x_L for neuronal and erythroid precursor cells (Motoyama et al., 1995, 1999; Wagner et al., 2000); Bcl-w for sperm cell progenitors in adults (but not juveniles) (Print et al., 1998; Ross et al., 1998; Meehan et al., 2001); Al for neutrophils (Hamasaki et al., 1998); and Mcl-1 for successful implantation of the zygote (Rinkenberger et al., 2000).

Other genetic studies clearly indicate that homeostasis requires an appropriate balance between the level of prosurvival proteins and that of their BH3-only antagonists. Overexpression of the former provokes an abnormal accumulation of cells within the haematopoietic compartment (McDonnell et al., 1989; Strasser et al., 1991b; Ogilvy et al., 1999) and neuronal lineage (Martinou et al., 1994; Farlie et al., 1995), presumably by thwarting physiologically important death signals delivered through upregulation of BH3-only proteins. Conversely, the devastating consequences of inadequate levels of prosurvival proteins can be precluded by also reducing the level of BH3-only killers: remarkably, loss of just a single allele of bim was sufficient to prevent the kidney failure and immune collapse that otherwise ensues in Bcl-2-null mice (Bouillet et al., 2001a).

How do Bcl-2 and its homologues promote cell survival and how do BH3-only proteins negate this function? Importantly, since none of the BH3-only proteins can kill in the absence of Bax and Bak (Cheng et al., 2001; Zong et al., 2001), the consequence of inactivation of the Bcl-2 homologues must presumably be activation of the Bax-like proteins. How this is achieved and whether it is direct or indirect is the subject of vigorous ongoing debate, and several models are discussed further below and elsewhere (Adams, 2003).

Bax-like proteins: triggers for death

The Bax-like proteins are critical for apoptosis in many cells, although whether this holds for all types remains to be established. While inactivation of either bax or bak alone has little consequence in mice, presumably because of functional redundancy, elimination of both genes dramatically impairs developmental apoptosis in many tissues, typically resulting in perinatal death (Lindsten et al., 2000). Simultaneous lack of Bax and Bak, like loss of Bim (Bouillet et al., 1999, 2002), also perturbs thymic selection and lymphoid homeostasis (Rathmell et al., 2002).

In response to cytotoxic signals, Bax and Bak undergo conformational change and form membrane-associated homo-oligomers (Hsu and Youle, 1998; Griffiths et al., 1999, 2001; Antonsson et al., 2001; Nechushtan et al., 2001), which can also coalesce into huge clusters (Nechushtan et al., 2001). All these events can be blocked by Bcl-2 overexpression, re-enforcing the view that it acts upstream of Bax/Bak. How the oligomers form is unclear. Perhaps some Bax/Bak molecules assume a BH3 donor-like conformation while others retain their BH1/2/3 cleft and behave as 'receptors'. Alternatively, the association may involve intermolecular interactions between the grooves and extruded hydrophobic tails. Recent evidence suggests that the reorganization of Bak requires Bax and that their separate homo-oligomers may interact (Mikhailov et al., 2003). Other evidence also suggests that the functions of Bax and Bak may not be entirely equivalent (Zhang et al., 2000; Gillissen et al., 2003).

The signals activating Bax and Bak are not known. Although tBid has been proposed to activate both proteins (see above), some recent work has suggested that in healthy cells Bax might be sequestered in the cytosol by another protein: a 14-3-3 protein (Nomura et al., 2003); the Ku70 protein, otherwise involved in DNA repair in the nucleus (Sawada et al., 2003); or the Humanin peptide (Guo et al., 2003). Similarly, in healthy but not apoptotic cells, a small proportion of Bak is found in association with VDAC2 in the mitochondrial outer membrane (Chen et al., 2003). If any of these associations is physiologically relevant, an apoptotic signal presumably must free Bax to allow its translocation to organelle membranes.

Once activated, Bax-like proteins appear to cause membrane damage. In particular, they are thought to disrupt the outer membrane of mitochondria, thereby releasing apoptotic mediators: cytochrome c, which activates Apaf-1 as discussed above (Liu et al., 1996); Smac/Diablo and Omi/HtrA2, which antagonize the ability of lAPs (Inhibitors of Apoptosis Proteins) to inhibit caspases (Du et al., 2000; Verhagen et al., 2000, 2002; Suzuki et al., 2001); endonuclease G, which may aid CAD (caspase-activated DNAse) in DNA fragmentation (Li et al., 2001; Parrish et al., 2001); and the contentious flavoprotein AIF, which on the one hand has been implicated in chromatin condensation and large-scale DNA degradation (Joza et al., 2001; Yu et al., 2002), but on the other in protecting neurons from peroxide-mediated apoptosis (Klein et al., 2002). Bax and Bak are also involved in apoptosis initiated from the ER in response to the stress imposed by disrupted calcium homeostasis or excess unfolded proteins (Nakagawa et al., 2000; Wei et al., 2001; Zong et al., 2001; Scorrano et al., 2003), and they may function directly on the ER as well as on mitochondria (Zong et al., 2003).

The mechanism of Bax/Bak-induced membrane permeabilization is highly controversial (see, for example, Harris and Thompson, 2000; Tsujimoto and Shimizu, 2000; Martinou and Green, 2001; Hardwick and Polster, 2002). A model involving mitochondrial swelling and rupture of the outer mitochondrial membrane (Vander Heiden et al., 2000) has been challenged (Kluck et al., 1999). Similarly, the possibility that Bax interacts with a preexisting mitochondrial channel, the 'permeability transition pore' (Tsujimoto and Shimizu, 2000), has not been supported (Martinou and Green, 2001; Mikhailov et al., 2001). Another model, based on the structural resemblance of Bax-like (and Bcl-2-like) proteins to pore-forming proteins such as diphtheria toxin (Muchmore et al., 1996), is that Bax and Bak form membrane channels de novo. Indeed, a novel type of channel has been detected in mitochondria from apoptotic cells (Pavlov et al., 2001), and Bax oligomers can form pores in liposomes that are large enough to release cytochrome c (Antonsson et al., 2000; Saito et al., 2000; Roucou et al., 2002) and even far larger molecules (Kuwana et al., 2002). Both Bid and the lipid composition of the membrane appear to be important in facilitating Bax-induced porosity, although the nature of the pores remains in dispute (Kuwana et al., 2002; Roucou et al., 2002). It has also been suggested that Bax might disrupt the membrane through interaction with the proteins that drive mitochondrial fission and fusion (Karbowski et al., 2002).

Models for the functional interplay of family members

Figure 6 displays several models for the relationship of the different types of Bcl-2 family members. One view (Model 1) is that Bcl-2 is merely a passive receptor for BH3-only ligands and that once all such prosurvival receptors are occupied, Bax/Bak-mediated apoptosis ensues (Cheng et al., 2001). If so, the 'excess' BH3-only proteins must then trigger activation of Bax (Bak). However, with the possible exception of Bid, there is no convincing evidence for binding of BH3-only proteins to Bax or Bak. Hence, a refinement of this notion (Model 2) postulates that most BH3-only proteins (e.g. Bad) interact only with Bcl-2 and titrate it, to prevent Bcl-2 from sequestering tBid, which is postulated to bind via its BH3 domain to Bax and Bak and activate them (Letai et al., 2002). In support of this model, in vitro competition experiments using peptides suggest that the Bad and Bik BH3 domains can displace a Bid BH3 domain from Bcl-2, presumably freeing the Bid BH3 peptide to interact with Bax-like molecules (Letai et al., 2002), but quantitative evidence that the Bid BH3 interacts with Bax or Bak has not been reported.

Figure 6.

Models for the functional interplay of Bcl-2 family members. Here 'BH3' denotes a BH3-only protein; 'Bcl-2', any prosurvival family member; 'Bax', either Bax or Bak; and 'caspase', an initiator caspase. In Model 2, 'Bad' represents any BH3-only protein that can interact only with a prosurvival relative, whereas 'Bid' represents one that can activate Bax/Bak. In Model 3, 'mCED-4' denotes a putative mammalian scaffold protein, other than Apaf-1, that can activate an initiator caspase when released by Bcl-2 (see C. elegans pathway in Figure 3); a second initiator caspase (e.g. caspase-9) is presumed to be activated downstream of Bax and organelle damage (see Figure 4). In Model 4, 'direct' denotes heterodimerization of Bcl-2 and Bax; 'signal', a small molecule that can keep Bax from becoming activated; and 'X', a putative membrane protein that can facilitate Bax activation but is sequestered by Bcl-2

Full figure and legend (33K)

This model must be reconciled with the observation that the absence of Bid has minimal physiological impact (Yin et al., 1999), in contrast to the profound effect of losing both Bax and Bak (Lindsten et al., 2000). Perhaps other Bid-like molecules with redundant function remain to be discovered. It has recently been suggested that Bim (or perhaps certain isoforms of it) actually represents such a molecule, based on in vitro evidence that a Bim BH3 peptide can induce oligomerization of Bax and Bak and cytochrome c release from mitochondria (Letai et al., 2002). This effect might be indirect, however, because no binding of Bim to Bax has been detected in dying cells (Liu et al., in press) and direct addition of full-length Bim (Bim_L) to mitochondria did not provoke cytochrome c release, whereas addition of Bid does (Terradillos et al., 2002).

Analogy with the C. elegans model (Figure 3) would argue that Bcl-2 acts by sequestering a CED-4-like molecule, to prevent it from activating initiator caspases (Model 3 in Figure 6) (Hausmann et al., 2000; Strasser et al., 2000). This would have evolutionary appeal, although further evidence is required even for C. elegans, where recent data indicate that CED-4 release from mitochondria-associated CED-9 and translocation to the nuclear membrane is not sufficient to cause cell death (H R Horvitz, personal communication). Furthermore, no candidates for the putative 'bona fide mammalian CED-4' have yet emerged from the numerous screens for Bcl-2-binding molecules. In Model 3, an initiator caspase is presumed to activate Bax, perhaps via cleavage of a Bid-like protein, but other caspases would be activated after Bax-mediated membrane damage (Figure 4).

Finally, Model 4 (Figure 6) groups three models in which the effector role of Bcl-2 is to prevent Bax activation. The 'direct' version, based on the ability of Bcl-2 to heterodimerize with Bax (Oltvai et al., 1993), envisages that this association precludes the oligomerization of Bax and Bak. Although this model cannot be entirely excluded, Bax heterodimerization with Bcl-2 or Bcl-x_L requires a conformational change, such as that induced by certain nonionic detergents (e.g. NP40, Triton X-100 or octylglucoside) (Hsu and Youle, 1997). Whether Bax and Bak actually assume this configuration within membranes after cells receive a death stimulus remains problematic, because Bcl-2 is not found in the oligomeric complexes of Bax (Mikhailov et al., 2001; Nechushtan et al., 2001). Therefore, Bcl-2 may instead control Bax/Bak activation indirectly. As Bcl-2 can prevent the intracellular calcium flux, and pH and ionic changes that occur early during apoptosis (Matsuyama and Reed, 2000; Yu et al., 2001b), Bcl-2 might prevent the activation of Bax and Bak by a signal through such small molecules. An alternative indirect mechanism is that Bcl-2 sequesters a membrane-bound protein X that is needed to drive Bax/Bak activation (Hsu and Youle, 1997; Wilson-Annan et al., 2003).

In summary, while the mechanism is clearly far from resolved, it does seem likely to us that Bcl-2 and its close colleagues normally exert an active rather than a passive role in promoting cell survival and that this function is compromised by the binding of a BH3-only protein.

Role of the Bcl-2 family in oncogenesis

Antiapoptotic Bcl-2 family members are oncoproteins

As indicated above, the oncogenic capacity of Bcl-2 was first realized through its involvement in the 14;18 chromosome translocation in human follicular lymphoma. That translocation activates constitutive expression of Bcl-2 in the B-cell clone and its progeny by linking the gene to the immunoglobulin (Ig) heavy chain gene locus (Tsujimoto et al., 1984; Bakhshi et al., 1985; Cleary et al., 1986; Yunis et al., 1987), whereas in some cases of diffuse large cell lymphoma and chronic lymphocytic leukaemia, variant translocations activate Bcl-2 by fusion to Ig light-chain loci (see, for example, Adachi et al., 1990; Tashiro et al., 1992).

Presumably, all antiapoptotic Bcl-2 family members have oncogenic potential and indeed the mouse bcl-x_L gene has been shown to be activated by retroviral insertion in 10% of cytokine-independent murine myeloid and T-cell lines (Packham et al., 1998). In the totality of malignancies, mutations that directly affect antiapoptotic Bcl-2 family members appear to be surprisingly rare. However, certain oncogenic mutations probably act indirectly to increase their expression levels. For example, the NF-B pathway is activated in many human malignancies, and NF-B can induce expression of the bcl-X_L and A1 genes (Grumont et al., 1999). High levels of expression in tumours must be interpreted with caution, however, because a tumour is often less differentiated than the surrounding normal tissue and expression levels of Bcl-2 family members often change markedly during differentiation (see, for example, Cory, 1995).

Dangerous oncogenic liaisons: insights from mouse models and human tumours

Mice bearing a transgene mimicking the BCL-2 translocation have an increased incidence of spontaneous B lymphoid tumours. The lymphomas take many months to develop, however, and the penetrance of disease is low (McDonnell and Korsmeyer, 1991; Strasser et al., 1993; Linette et al., 1995), arguing that Bcl-2 overexpression on its own is not highly oncogenic and that progression to malignancy requires synergistic mutation(s). Consistent with this conclusion, BCL-2 translocations can be detected in a small proportion of circulating lymphocytes in some healthy individuals (Liu et al., 1994; Ji et al., 1995; Limpens et al., 1995). Many follicular lymphomas have a relatively indolent progression, and the very early stage may well simply represent a benign overproduction of cells resulting from chronic antigen stimulation of a B-cell clone protected by BCL-2 translocation (Zelenetz et al., 1992; Liu et al., 1994). In keeping with that view, the generation of follicular lymphomal mice has been found to rely upon T cell help (Egle et al., 2003).

Follicular lymphoma often transforms into a more aggressive disease and in certain cases (8%), progression is associated with a myc translocation (Yano et al., 1992). Many tumours from E-bcl-2 mice also exhibit myc rearrangement (McDonnell and Korsmeyer, 1991; Strasser et al., 1993). The inferred collaboration between bcl-2 and myc, first suggested by in vitro studies (Vaux et al., 1988), was verified in mice expressing both a bcl-2 and a myc transgene (Strasser et al., 1990). It is now clear that coexpression of bcl-2 and myc can transform diverse cell types: haematopoietic progenitor cells (Strasser et al., 1990, 1996), breast epithelial cells (Jäger et al., 1997) and pancreatic -cells (Naik et al., 1996; Pelengaris et al., 2002).

The marked synergy between bcl-2 and myc probably mainly reflects inhibition by Bcl-2 of the apoptosis induced by Myc when cytokines or other survival factors become limiting (Askew et al., 1991; Bissonnette et al., 1992; Evan et al., 1992; Strasser et al., 1996), but may also reflect the ability of Myc to over-ride the inhibition of cell-cycle entry by Bcl-2 described below (Evan and Littlewood, 1998; Cory et al., 1999; Evan, 2003) (Figure 7a).

Figure 7.

Synergistic action of Myc and Bcl-2 in oncogenesis. (a) While Myc promotes cellular proliferation, it also activates apoptosis when cytokines become limiting. Bcl-2, by contrast, inhibits apoptosis but also restrains entry into the cell cycle. (b) Apoptotic targets of Myc include the tumour suppressor p53, via the Arf/Mdm-2/p53 axis (Sherr, 2001), and the BH3-only protein Bim (Egle, SC et al., 2003). The tumour suppressor Arf sequesters Mdm2, a negative regulator of p53, and the resulting raised levels of wt p53 can promote apoptosis through targets such as the BH3-only proteins Noxa and Puma (see text). Raised p53 levels can also lead to growth arrest, typically by inducing the cell-cycle inhibitor p21. Inhibition of cell-cycle entry by Bcl-2 is associated with maintenance of repressive complexes of p130 and E2F4, via increased levels of p130 and the cyclin-dependent kinase inhibitor p27, but how it does so is unknown (Vairo et al., 2000)

Full figure and legend (54K)

The rapid tumorigenesis in pancreatic -cells elicited by Myc plus Bcl-x_L (Naik et al., 1996; Pelengaris et al., 2002) led to the suggestion that the combination of enhanced proliferation and survival suffices for full-fledged malignancy (Green and Evan, 2002 #10051). Contrary to that proposal, although expression of both in the B-lymphoid compartment provoked a florid overproliferation of pre-B lymphocytes, the cells were not transplantable and therefore not fully malignant, in marked contrast to the progenitor cell tumours that subsequently developed (Strasser et al., 1990). Therefore, although mutations that enforce cell proliferation and inhibit apoptosis are a potent oncogenic combination, they are not sufficient to fully transform cells. Malignancy probably also requires, as a minimum, bypassing growth arrest and senescence – typically by elimination of p53 function (Hanahan and Weinberg, 2000) (Figure 7b; see further below). Consistent with this view, in addition to their elevated levels of Bcl-2 and/or Bcl-x_L, most spontaneous B lymphoid tumours arising in myc transgenic mice carried mutations that eliminate p53 function (Eischen et al., 1999, 2001b).

There appear to be multiple routes from Bcl-2 overexpression to lymphoid malignancy. In addition to myc translocations (Lee et al., 1989; Yano et al., 1992) and p53 mutations (Sander et al., 1993), histologically transformed human follicular lymphomas bear diverse genetic abnormalities, including an extra copy of chromosome 7; deletion at 6q; putative regulatory mutations of the BCL-6 gene, which encodes a transcriptional repressor; somatic mutation of the translocated BCL-2 allele (see below); and inactivation of the p16 and p15 tumour suppressors (see Lossos and Levy, 2000 and references therein).

Mouse models have also explored the oncogenic impact of bcl-2 in the myeloid lineage. Coexpression of bcl-2 with PML-RARa, the chimaeric gene produced by the t(15;17) translocation associated with human acute promyelocytic leukaemia (APL), enhanced accumulation of immature myeloid cells in the bone marrow and accelerated development of APL (Kogan et al., 2001). Introduction of a bcl-2 transgene into mice homozygous for lpr, an inactivating mutation of Fas (CD95), provoked acute myeloblastic leukaemia in some animals (Traver et al., 1998). Thus, tumour development may sometimes require inactivation of both the 'death receptor' and the intracellular stress pathways to apoptosis (Figure 3).

Bcl-2 overexpression has also been found to facilitate leukaemogenesis induced by ionizing radiation (Gibbons et al., 1999), presumably because mutated cells fail to die. Interestingly, young mice exposed to radiation were more at risk of developing tumours than older mice, perhaps reflecting their higher level of lymphocyte production. Similarly, children exposed to radiation in Hiroshima and Nagasaki had a greater incidence of lymphoid malignancy than adults (see Gibbons et al., 1999).

Do proapoptotic family members act as tumour suppressors?

The oncogenic potential of antiapoptotic Bcl-2 family members suggests that proapoptotic relatives could be tumour suppressors. As there is likely to be substantial redundancy within both the BH3-only group and the Bax-like family, their tumour suppressor function may only arise in specific cell types or in specific situations.

The BH3-only protein Bim seemed a prime candidate, since it is a major regulator of lymphoid homeostasis (Bouillet et al., 1999, 2002). Moreover, the human BIM gene is located at chromosome 2q12 or q13 (Bouillet et al., 2001b), a region where alterations (primarily deletions) have been reported for 14 cases of human malignancy, mostly haemopoietic in origin. Indeed, although loss of Bim does not in itself elevate tumour incidence in mice within the first 12 months of life, we have found that loss of even a single allele of Bim dramatically accelerates leukemogenesis in mice expressing a myc transgene during B lymphopoiesis (Egle et al., 2003). Thus, Bim is indeed a potent tumour suppressor, at least in B cells.

BH3-only proteins may also contribute to tumorigenesis in other lineages. Although inactivation of Bid does not perturb homeostasis during development (Yin et al., 1999), 50% of Bid-deficient mice develop chronic myelomonocytic leukaemia by two years of age (Zinkel et al., 2003). In view of the role of Bmf in anoikis (Puthalakath et al., 2001), it will be interesting to determine whether loss of Bmf contributes to metastasis. Noxa and Puma are particularly attractive candidate tumour suppressors, because both are transcriptionally induced by the tumour suppressor p53 (Oda et al., 2000; Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 200la). Indeed, multiple gene-targeting in a human colorectal cell line has recently demonstrated that Puma is required for apoptosis induced by p53 in p21^-/- cells (Yu et al., 2003). Importantly, disruption of the mouse genes has shown that loss of Noxa modestly impairs p53-induced apoptosis (Shibue et al., 2003; Villunger et al., 2003) whereas loss of Puma markedly enhances cell survival (Villunger et al., 2003). For cell types where both Noxa and Puma are expressed, a tumour suppressor function might require inactivation of both the genes.

Since Bax and Bak have largely redundant function (Lindsten et al., 2000), tumour promotion would be expected to require inactivation of both the genes (and perhaps also Bok in tissues expressing this Bax-like protein). Consistent with this prediction, bax-null mice acquire few spontaneous tumours (Knudson et al., 2001). Nonetheless, loss of Bax has enhanced transformation by potent oncogenes in several cell types: adenovirus ElA in primary mouse embryonic fibroblasts (McCurrach et al., 1997); SV40 T antigen in brain choroid plexus (Yin et al., 1997) and mammary epithelium (Shibata et al., 1999); and myc in B-lymphoid cells (Eischen et al., 2001a). Furthermore, some human colorectal and haematopoietic tumours exhibit mutated Bax or Bak (Rampino et al., 1997; Meijerink et al., 1998; Kondo et al., 2000). Finally, loss of Bax in the HCT116 colorectal cell line abolished the (p53-independent) apoptotic response to sulindac and other nonsteroidal anti-inflammatory drugs but only partially reduced the p53-dependent response to the chemotherapeutic agent 5-fluorouracil (Zhang et al., 2000). These observations suggest that certain cytotoxic signals are Bax-specific in their action and/or that Bak expression in certain cell types is insufficient to mediate apoptosis in the absence of Bax.

Bcl-2 and the cell cycle

Intriguingly, in addition to its central role in regulating apoptosis, the Bcl-2 family influences the cell cycle – or more specifically, the transit between quiescence and proliferation. High levels of Bcl-2 do not affect the proliferation rate of continuously cycling cells (O'Reilly et al., 1996), although Gl may be prolonged under suboptimal conditions (Borner, 1996; Linette et al., 1996; Simpson et al., 1999). Significantly, however, G0 cells overexpressing Bcl-2 (or Bcl-x_L, Bcl-w, E1B 19 kDa) are slow to enter the S phase when stimulated with growth factors (Marvel, 1994, #2047; Mazel et al., 1996, #2805; O'Reilly et al., 1996, #4888). Furthermore, in colon carcinoma cells, elevated Bcl-2 can lead to senescence (Crescenzi et al., 2003). Consistent with these in vitro studies, B and T cells in Bcl-2 transgenic mice turn over more slowly (Linette et al., 1996; Mazel et al., 1996; O'Reilly et al., 1997a,1997b). Conversely, overexpression of Bax (Borner, 1996; O'Reilly et al., 1996) and Bad (Chattopadhyay et al., 2001) neutralizes the cell-cycle barrier imposed by Bcl-2, and T cells from bax transgenic mice enter into cycle more rapidly in response to IL-2 stimulation than normal T cells (Brady et al., 1996).

Importantly, the antiapoptotic and cell-cycle aspects of Bcl-2 function are genetically separable, since mutation of a conserved tyrosine just after the BH4 region (Tyr 28 in Bcl-2 itself) abrogates the cell-cycle constraint but not the survival benefit (Huang et al., 1997b). Evidence that the Drosophila Bcl-2 ('Buffy') can also inhibit cell cycle entry indicates that this function has been evolutionarily conserved (Quinn et al., 2003).

The mechanism of the retarded entry into cycle remains poorly understood. In quiescent cells, retinoblastoma protein family members (pRb, p107, p130) bind to and inhibit E2F transcription factors, and progression through the cell cycle requires their phosphorylation by cyclin-dependent kinases (CDKs) to release E2F for transcriptional activity. Bcl-2 apparently interferes with Gl events downstream of Myc induction but prior to E2F-1 activation (Greider et al., 2002). Inhibition by Bcl-2 correlated with retarded degradation of the CDK inhibitor p27Kipl (Brady et al., 1996; Linette et al., 1996) and therefore decreased activity of its target, CDK2 (Gil-Gómez et al., 1998). Furthermore, Rb remained hypophosphorylated (Mazel et al., 1996) and the level of p130 was elevated (Lind et al., 1999; Vairo et al., 2000). Analysis of Bcl-2 action in cells lacking pRB, p130 or p27 indicated that both p27 and p130 are essential for its inhibitory effect but pRB is dispensable (Vairo et al., 2000; Greider et al., 2002; Crescenzi et al., 2003). These findings suggest that Bcl-2 fosters maintenance of repressive complexes of p130 and E2F4 not only by increasing p130 levels but also that of p27, thereby precluding p130 phosphorylation by CDK2 (Lind et al., 1999; Vairo et al., 2000).

How Bcl-2 mediates the increase in p130 and p27 is unknown. However, since the elevation in p27 did not occur in cells expressing Y28A Bcl-2, p27 levels may be modulated via a protein that binds to Bcl-2 near the conserved Tyr residue. Although overexpressed Raf and the Ca⁺-activated protein phosphatase calcineurin have been reported to bind to Bcl-2 via the adjacent BH4 domain (Wang et al., 1996a; Shibasaki et al., 1997), these interactions remain to be substantiated. Since p27 levels in G0 are regulated by proteins that bind to the U-rich sequence in the 5' untranslated region of p27 mRNA (Millard et al., 2000), Bcl-2 may influence the availability or activity of these proteins, either directly or indirectly.

The ability of antiapoptotic Bcl-2 proteins to foster quiescence may have evolved to restrain their pathogenic potential. Indeed, we have recently found that spontaneous tumours and autoimmune disease are more frequent in Y28A bcl-2 transgenic mice than in those bearing a wt bcl-2 transgene (O'Reilly, DH et al., in preparation). Intriguingly, in some morphologically transformed human follicular lymphomas, the translocated Bcl-2 gene has acquired mutations in residues near this conserved Tyr (Tanaka et al., 1992; Matolcsy et al., 1996). Since p27 is haplo-insufficient as a tumour suppressor (Fero et al., 1998), any reduced capacity of Bcl-2 to upregulate p27 may be critical. Suppression of Bcl-2 capacity to inhibit entry into cycle may account for the counter-intuitive finding that overexpression of Bax in T cells enhanced lymphomagenesis in p53-null mice (Knudson et al., 2001).

Many cell types withdraw from the cell cycle in order to embark into the differentiation mode. Intriguingly, when HL60 cells were exposed to differentiation-promoting agents, Bcl-2 overexpression accelerated cell cycle exit in addition to extending the lifespan of the mature cells (Vairo et al., 1996). Hence, simultaneous promotion of quiescence and survival by Bcl-2 and its homologues may also be a mechanism to facilitate differentiation.

Prospects for novel therapeutic modulators of apoptosis

The striking success of STI571 (Gleevec, Imatinib Mesylate; Novartis) (Druker, 2002), a specific inhibitor of the Bcr-Abl tyrosine kinase that hallmarks chronic myelocytic leukaemia, has greatly stimulated the search for therapeutics that are selective for specific molecular targets and overcome the limited efficacy and drastic side effects of conventional therapies.

As impaired apoptosis plays a central role in the pathogenesis of many diseases, the search is accelerating for novel agents that engage the cell death machinery (Nicholson, 2000; Johnstone et al., 2002; Reed, 2002, 2003). The goals are either to preserve cell viability after acute injury (e.g. to limit tissue damage from sepsis or myocardial infarction) or to delete malignant or autoreactive cells. Promising attempts to minimize cell death are focused on caspase inhibitors (Nicholson, 2000). Strategies to enhance cell death include targeting either Bcl-2 or the IAPs, or engaging the death receptor pathway (Figure 3), by, for example, ligating the receptors for TRAIL (Ashkenazi, 2002). We will restrict discussion here to attempts to modulate Bcl-2 function in the treatment of malignancies (Huang, 2000; Baell and Huang, 2002; Rutledge et al., 2002).

Why does targeting the apoptotic machinery have promise? At least for haematopoietic malignancies, it is now thought that conventional therapies, such as those that damage DNA or perturb microtubules, work mainly by indirectly activating the cell death programme (Fisher, 1994; Brown and Wouters, 1999). Hence, direct activation of the cell suicide machinery should be advantageous (Reed, 2003), and several considerations make Bcl-2 a prime target. First, as discussed above, overexpression of Bcl-2 contributes to oncogenesis in a number of mouse tumour models. Second, experimentally imposed Bcl-2 overexpression renders many cultured cell lines refractory to chemotherapeutic drugs and radiation (e.g. Tsujimoto, 1989; Miyashita and Reed, 1992; Strasser et al., 1994; Huang et al., 1997a), as it does in the cells overexpressing Bcl-2 in transgenic mice, (e.g. Sentman et al., 1991; Strasser et al., 1991a). Finally, Bcl-2 is known to function downstream of the p53 tumour suppressor (Wang et al., 1993; Chiou et al., 1994; Strasser et al., 1994) (Figure 7b), the function of which is lost in most tumours (Hollstein et al., 1991; Levine et al., 1991; Sherr, 2001). Hence, the diminished apoptosis in the tumour cell due to that loss might be overcome by directly targeting Bcl-2.

Diverse strategies for limiting Bcl-2 function are under study. The most advanced studies target Bcl-2 or Bcl-x_L expression with antisense oligonucleotides (Genasense; jointly developed by Genta and Aventis), and early promising results (Jansen et al., 1998; Konopleva et al., 2000; Waters et al., 2000; Banerjee, 2001) have recently prompted the US Food and Drug Administration to designate Genasense as a "Fast Track" product for the treatment of patients with CLL. If follow-up studies confirm this early promise, they will establish proof-of-principle that targeting Bcl-2 in tumours is beneficial and may prompt the development of more effective ways of downregulating Bcl-2 expression. An exciting possibility is the use of RNA interference (RNAi), if effective, safe and acceptable delivery mechanisms can be developed (Hannon, 2002). The use of replication-defective adenoviral vectors for delivery of Bax is well advanced in the clinic (Reed, 2003), and adenoviral vectors that replicate only in the tumour cells are coming on line (Berns, 2002). Delivery of BH3-only proteins might also prove efficacious, since they switch on the cell death machine directly.

Eventually, the most effective way to ablate Bcl-2 function may well be to identify small molecules that mimic the interaction of a BH3 domain with its target groove (Figure 8) (Baell and Huang, 2002; Rutledge et al., 2002). The concept that this domain alone is sufficient to inactivate Bcl-2 and provoke cell death has been supported by studies delivering BH3 peptides (Cosulich et al., 1997; Holinger et al., 1999; Wang et al., 2000b; Polster et al., 2001; Letai et al., 2002; Vieira et al., 2002). Nonspecific effects have not always been ruled out, however, and a particular concern is that their cytotoxicity might be due to the tendency of many helical peptides to penetrate membranes (Schimmer et al., 2001).

Figure 8.

Therapeutic potential of BH3 mimetics. In a normal cell, p53 is upregulated in response to genotoxic damage, such as that elicited by certain chemotherapeutic drugs. The p53 then induces the BH3-only proteins Noxa and Puma, which inactivate Bcl-2 and trigger apoptosis (see Figure 7b). The mutations in tumour cells that inactivate p53 function preclude the apoptotic signals from p53, rendering the tumour cell refractory to the chemotherapeutic drug. However, Bcl-2 will remain functional in the tumour cell despite the p53 mutation. Hence, a small organic molecule or peptide (a 'BH3 mimetic') should bind to the groove of Bcl-2 in a fashion similar to the BH3 domain of a BH3-only protein and directly trigger apoptosis

Full figure and legend (58K)

Although the discovery and design of drugs that mimic or block protein–protein interactions are often challenging (Fairlie et al., 1998; Schepartz and Kim, 1998; Cochran, 2001), the deep groove targeted by BH3 peptides should be amenable to attack. One promising approach is to use rational design (Kuntz, 1992) to create BH3 mimetics, based on the known 3D structures of Bcl-x_L, Bcl-2 and Bcl-w (Muchmore et al., 1996; Petros et al., 2000, 2001; Hinds et al., 2003) and insights about how BH3 domains bind (Sattler et al., 1997). Small organic molecules can be designed to target the key interactions between a BH3 domain and the prosurvival molecule (Hajduk et al., 1997; Fesik, 2000). Alternatively, peptidomimetics can be constructed on a scaffold that mimics the helicity of the BH3 domain (Chin and Schepartz, 2001; Ernst et al., 2002; Kutzki et al., 2002). Abbott Laboratories have an advanced programme based on rational design and lead optimization (Hajduk, 1999, #8914; Fesik, 2000, #8834). Other groups have also identified leads by in silico screening of compound libraries (Wang et al., 2000a; Enyedy et al., 2001), by screening a synthetic library of 16 000 compounds (Degterev et al., 2001), by natural product screening (Nakashima et al., 2000) or by serendipity (Tzung et al., 2001). All the reported lead compounds, or subsequent derivatives with enhanced activity (for example, Degterev et al., 2001; Lugovskoy et al., 2002), bind to Bcl-x_L (and Bcl-2) and many are cytotoxic with cultured cell lines.

A potential difficulty is that, to date, binding experiments testing small compounds and modified peptides have used prosurvival molecules that lack the hydrophobic C-terminal region (e.g. truncated Bcl-x_L) (Muchmore et al., 1996; Sattler et al., 1997). As indicated earlier, where structures are available for longer molecules (Bax and Bcl-w), the C-terminal region appears to restrict access to the BH3 binding groove (Hinds et al., 2003; Suzuki et al., 2000) (Figure 2). It is therefore unclear whether the identified compounds will bind well to the full-length proteins, especially since they typically target a smaller surface area than that occupied by a BH3 domain.

The ability of compounds to bind Bcl-x_L or Bcl-2 in vitro does not establish that this is the basis for their cytotoxicity, and verifying their mechanism of action may well be challenging (Baell and Huang, 2002; Rutledge et al., 2002). Many drugs promote cell death indirectly rather than by binding to Bcl-2 as proposed for Antimycin A (Tzung et al., 2001), and the relatively low affinity (micromolar rather than nanomolar) of many leads for their targets suggests that nonspecific activities are likely to dominate in cell-based assays. Specificity can best be proven by genetic tests. For example, compounds that show selectivity for a particular prosurvival protein should have reduced activity in cells that lack that protein due to disruption of its gene or silencing of its expression. Moreover, compounds that target any of the prosurvival proteins should be inactive on cells lacking both Bax and Bak, which are required for killing by BH3-only proteins (Cheng et al., 2001; Zong et al., 2001).

Will BH3 mimetics be too cytotoxic for normal cells to be useful? Several considerations suggest this need not be the case. Firstly, most tumour cells have acquired multiple defects in cell cycle and other checkpoints and they should therefore be more susceptible than their normal counterparts to the action of BH3 mimetics (Figure 8). Secondly, because the grooves of individual prosurvival proteins vary, it is likely that drugs with selective specificity can be identified, opening the possibility of targeting only the dominant guardian of the tumour.

Clearly, the search for effective BH3 mimetics remains in its infancy, but some of the leads now emerging should serve as templates for improvement in affinities and pharmacological properties. Undoubtedly, the knowledge emerging about the cell death machinery will stimulate their development and, conversely, the drug discovery process should generate novel reagents for illuminating apoptotic signalling pathways. In spite of the challenges ahead, the critical role of Bcl-2 in setting the threshold for cell death endows BH3 mimetics with enormous potential as drugs. In the clinic, the need for less toxic and better targeted anticancer therapies is pressing, and directly activating the cell death machinery by inhibiting Bcl-2 offers great promise.

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Acknowledgements

We warmly thank our colleagues, particularly A Strasser, A Harris, H Puthalakath, P Bouillet, C Day, M Hinds, J Baell, P Colman, K Watson, A Roberts and A Wei for many discussions that influenced this review; G Filby for editorial assistance and P Maltezos for figure preparation. This work was supported in part by grants from the National Health and Research Council of Australia (ID No 257502), the US National Institutes of Health (CA43540 and CA80188) and the Leukaemia and Lymphoma Society (SCOR grant). DCSH is a Viertel Fellow.