Larger than life: Mitochondria and the Bcl-2 family

Leukemia Research 31 (2007) 277–286

Invited review

Larger than life: Mitochondria and the Bcl-2 family Joanna Skommer a,∗ , Donald Wlodkowic a , Andrzej Deptala b,c a Department of Clinical Sciences, University of Kuopio, Harjulantie 1 C, 70211 Kuopio, Finland Department of the Prevention of Environmental Hazards, Medical University of Warsaw, Warsaw, Poland Department of Oncology and Haematology, Central Clinical Hospital of the Ministry of Interior and Administration, Warsaw, Poland b

c

Received 3 June 2006; received in revised form 4 June 2006; accepted 16 June 2006 Available online 5 September 2006

Abstract The intrinsic pathway of apoptosis relies on mitochondrial membrane permeabilization, with Bcl-2 proteins serving as its master regulators. They form a complex network of interactions both within the family and with multiple cellular factors outside the family. The understanding of the processes that regulate mitochondrial breach, and mechanisms that direct the pro- and anti-apoptotic functions of Bcl-2 proteins, should assist the development of novel anticancer therapies. Thus, it is of no surprise that research in the field is gaining momentum. In this review we outline the current concepts on regulatory circuits governing mitochondrial rupture and action of Bcl-2 proteins during cell death, and how this burgeoning knowledge is being translated into the clinics with the hope to combat cancer. © 2006 Elsevier Ltd. All rights reserved. Keywords: Mitochondria; Bcl-2; Apoptosis; Cell death; Cancer

1. Introduction Apoptosis is a genetically controlled and complex process central to the development, homeostasis and disease, turned on in response to environmental signals or triggered by intrinsic factors, and designed to kill errant cells in an orderly and clean way. This type of cell death is classically defined by a pattern of molecular events and morphological changes, including condensation of cytoplasm, rounding up, loss of mitochondrial membrane potential, chromatin condensation to compact and simple geometric figures, nuclear fragmentation, blebbing with maintenance of membrane integrity (zeiosis) and finally loss of plasma membrane asymmetry, coupled to the display of phagocytosis markers on the cell Abbreviations: AIF, apoptosis inducing factor; BHdomain, Bcl-2 homology domain; EndoG, endonuclease G; ER, endoplasmic reticulum; MAC, mitochondrial apoptosis-induced channel; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; MOMP, mitochondrial outer membrane permeabilization; PTP, permeability transition pore; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel; ψm , mitochondrial membrane potential ∗ Corresponding author. Tel.: +358 50 923 1982; fax: +358 17 162 705. E-mail address: [email protected] (J. Skommer). 0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.06.027

surface [1]. By these means delinquent cells are marked to be safely removed. Other forms of cell death (e.g., autophagy and necrosis) can also contribute to proper development and control of well-being [2], and any disturbances in the general “to live or not to live” decision-making process may have calamitous consequences for the organism. At least two main pathways lead to apoptosis: (1) extrinsic, consisting of cell surface TNF-related family of receptors, their inhibitory counterparts (decoy receptors) and cytoplasmic adapter or death inhibitory molecules (e.g., FADD or FLIP) and (2) intrinsic, for which mitochondrion is the hub governed by pro- and anti-apoptotic members of the Bcl-2 family. Over the past years, the understanding of the crucial role of mitochondria and their regulators in the control of apoptosis began to emerge, resulting in successful design of mitochondrion targeted anticancer therapies (reviewed, e.g., in refs. [3–7]). Although the myriad of crossroads and multipronged mechanisms ruling the mitochondrial pathway of cell demise is not completely elucidated, recent advances have shed more light on this exciting topic. The major goals of this review are to cover basic mechanisms by which mitochondrial breach leads to cell death, and to outline current concepts on regulatory circuits that direct

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pro- and anti-apoptotic functions of the Bcl-2 family proteins. Finally, the contemporary anticancer treatment approaches based on targeting mitochondrion and Bcl-2 proteins will be summarized.

2. Mitochondrion is calling the shot “We have tried to provide a map of the many roads to cellular ruin through the mitochondrial pathway; the ones most traveled in apoptotic cell death remain to be determined” (Spierings et al. [8]). Mitochondria – “powerhouses”, “gatekeepers” and “gardens of cell death” – have dual function in life and death [9]. Early evidence connecting mitochondria with apoptotic cell demise posited also that pro-apoptotic and pro-survival functions of these organelles are quite distinct [10]. In recent years multiple mechanisms have been anticipated to explain mitochondrial function in cell death, including both mitochondrial physiological processes as well as passive release of apoptogenic proteins into the cytosol upon mitochondrial outer membrane permeabilization (MOMP). In a testimony to the importance of mitochondria in regulating cell fate, a plethora of observations indicate that mitochondrion stands at the nexus of sensing and integrating diverse stress signals, and mitochondrial disturbances often occur long before any discernible signs of apoptosis (e.g., in ATRA-induced death of myelomonocytic cells) [11]. In this regard multiparametric flow cytometry as well as laser scanning cytometry (LSC) assays can prove useful to study the temporal and quantitative relationship between the loss of mitochondrial membrane potential and other apoptotic attributes, as recently described [12,13]. Moreover, in many death scenarios MMP appears to have a better prognostic value for cell demise than other events, e.g., caspase activation, and is induced by any of a number of apoptotic insults, including ligation of death domain receptors, DNA-damaging agents, growth factor withdrawal or irradiation. Mitochondria can act as amplifiers of caspase activity, and vice versa—caspase 3 and caspase 7 can contribute to cytochrome c or AIF release and loss of mitochondrial membrane potential [14]. Should caspase activity be inhibited, caspase-independent processes may still ensure cellular demise. In a further development of this theme, preservation of mitochondrial functions during apoptosis can retard events associated with caspase activation, such as loss of plasma membrane integrity or PS externalization, and several mitochondrial proteins have been identified as rate-limiting for the activation of caspases and nucleases. Formally, MOMP can originate from either mitochondrial outer membrane (MOM) or inner membrane (MIM). Depending on the stimuli and/or cellular context, loss of the mitochondrial membrane potential (ψm ) can occur before, during or after MOMP, indicative of the underlying mechanisms [15]. If the inner membrane partakes in MOMP, a

permeability transition pore (PTP) opens, followed by an osmotically obligatory flux of water and molecules up to 1.5 kDa, and ensuing equilibration of ions between the matrix and the cytoplasm. PTP is a multiprotein complex, suggested to consists of cyclophilin D (CypD) in the matrix, the adenine nucleotide translocator (ANT) in the inner membrane, VDAC and the peripheral benzodiazepine receptor in the outer membrane, and possibly associated with some other proteins (e.g., creatinine kinase from the intermembrane space or cytosolic hexokinase), or to be formed by aggregates of misfolded and otherwise damaged integral membrane proteins [16–18]. Sustained opening of PTP leads to ψm loss and osmotic swelling of the matrix, often sufficient to distort the structure of cristae and to rupture the outer mitochondrial membrane [4,19]. Importantly, many other events apart from PTP opening can induce ψm loss, and its low-conductance opening (rapid flickering of the pore between open and closed states) can still sustain ψm . The universal role of PTP gating as a primary mechanism for MOMP is still a subject of speculation, as swelling of mitochondrial matrix is not always a feature of apoptotic cells, cytochrome c release often precedes loss of ψm , and can even occur in its absence [20,21]. Moreover, inhibition of caspases can protect dying cells from ψm loss in some settings [4,22–24], and caspase 3 and caspase 7 have been recently identified as key mediators of the loss of ψm during apoptosis [14]. Should MOMP be defined as a PTP-associated event, its inhibition by ligands of the PTP constituents is observed. In this vein, loss or inhibition of cyclophilin D does not protect against all apoptotic triggers that employ mitochondrial pathway of cell demise, and thus stimuli and cell type-dependent role of cyclophilin D in modulating cell death is postulated [25]. It also appears that PTP is involved in necrosis or ischemia–reperfusion injury rather than intrinsic apoptosis [26]. Another tenet suggests that MOMP is a process intrinsic to the outer membrane. A fraction of cardiolipin (a lipid restricted to mitochondria) that concentrates at MOM and MIM contact points may be required for the formation of membrane-spanning Bax pores, either alone or acting in concert with MOM proteins. Among MOM proteins suggested to modulate the function of Bcl-2-related proteins are hexokinase (see also below), and mitochondrial fission and fusion-related proteins, such as Drp1, Mfn2 or Fis1 [27,28]. Still, it appears that Bax is activated and recruited to MOM by a signal originating outside the mitochondria. Conceivably, certain proteins of the MOM may act as docking domains for Bax, serving as bona fide regulators, but not initiators, of MOMP. In line, the prevailing model (recently designated as “the innocent bystander scenario”) proposes that the commitment to MOMP rests exclusively within the Bcl-2 family proteins that either promote (BH3-only and multidomain pro-apoptotic members) or prevent (anti-apoptotic members) the formation of membrane-spanning pores [29]. The action of Bcl-2 proteins on mitochondria will be outlined in more details later in this article.

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A hybrid model has been also proposed, asserting that PTP opening does not lead to mitochondrial swelling, but promotes MOMP via the activation of Bax [30,31]. Besides, it has been speculated that mitochondrial hyperpolarization may cause MOMP. Follow-up studies revealed, however, that though hyperpolarization does occur during apoptosis, it is not an absolute requirement for the release of cytochrome c [23]. Finally, the concept that ceramide and sphingosine are able to form channels in mitochondrial membranes has been brought forward [32]. Nonetheless, the diameter of sphingosine channels, unlike that of ceramide ones, is too small to permit the liberation of pro-apoptotic factors residing in the mitochondrial intermembrane space [33]. Regardless of the exact process accounting for MOMP, it can lead to cell death through three discernible and not mutually exclusive mechanisms [8,34,35]: (1) Release of mitochondria-residing factors that promote caspase-dependent (holocytochrome c, Smac/DIABLO and Omi/HtrA2) or caspase-independent (AIF, EndoG and Omi/HtrA2) cell death. It is still under debate whether these proteins are coreleased during apoptosis. In some reports cytochrome c leakage (a well-known trigger of apoptosome assembly) is absent or reduced as compared to that of other mitochondrial proteins. According to another scenario caspase (or other protease) activation is required for the release of AIF and/or EndoG [14,36,37]. Recently, Uren et al. stylishly demonstrated that upon MOMP cytochrome c, Smac/DIABLO and Omi/HtrA2 readily translocate from the mitochondrial intermembrane space, whereas AIF and EndoG remain bound to the inner membrane [38]. It has also been shown that cardiolipinbound cytochrome c catalyzes the oxidation of cardiolipin, and that this event is crucial for the release of pro-apoptotic factors residing in the mitochondrial intermembrane space [39]. Following their liberation, the death-promoting proteins can be further regulated: AIF and EndoG activity can be blocked by heat shock protein 70 (Hsp70) [40], whereas Apollon/BRUCE binds and ubiquitylates Smac/DIABLO and HtrA2, facilitating their proteasomal degradation [41]. Like in a detective story, however, the case is still continuing for mitochondrial proteins. New evidence emerge to actually relief many of them from the charges of homicide, implicating them with apoptosis speed-up rather than initiation (for details see ref. [42]). Maybe mitochondrial factors are not always cats among the pigeons after all? The investigation is likely to go on. (2) Loss of mitochondrial functions imperative for cell survival. Cytochrome c holds a fundamental role in respiration, transferring electrons from complex III to complex IV of the electron transport chain, and hence allowing mitochondrial transmembrane potential (ψm ) to

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be maintained. Although the mitochondrial damage is generally considered as the “point-of-no-return” of the death program, it is an accumulating process and not an all-or-nothing phenomenon. Accordingly, respiratory dysfunctions that occur early during apoptosis can be overcome upon addition of exogenous cytochrome c, becoming irreversible only with a progressive damage to mitochondria over the time [43], and indicative of the intactness of the inner membrane. It also appears that some cells can survive cytochrome c exodus and without respiration (rho◦ cells), providing the metabolic functions of mitochondria (such as amino acid, heme and steroid metabolism, protein import, metabolite transport and ATP production) are maintained. Ordinarily yet ψm dissipation has lethal corollaries, pointing out that the proton-motive force and/or the inner membrane permeability have been distorted, likely leading to: (i) cessation of the import of most proteins synthesized in the cytosol; (ii) Ca2+ and glutathione release from the mitochondrial matrix; (iii) uncoupling of oxidative phosphorylation with cessation of ATP synthesis, oxidation of NAD(P)H2 and glutathione; (iv) hyperproduction of superoxide anion by the uncoupled respiratory chain; (v) a decreased rate of electron transfer that lessens the consumption of mitochondrial pyruvate and its conversion into lactate, which in turn leads to cytoplasmic acidification. Overall, the cell is left to breathe its last. (3) Induction of reactive oxygen species (ROS). Mitochondria are also on the frontline of ROS production, which – if excessive – overpower the cell defence mechanisms and may modify cellular macromolecules and critical cellular targets. Indeed oxidative stress leads to lipid peroxidation, calcium mobilization, mitochondrial permeability transition, ATP depletion, protein oxidation, loss of electron transport and/or DNA damage, and hence promotes cell death [34]. Contradictorily, in some contexts ROS can also stimulate protective mechanisms, such as NF-␬B activation or inhibition of caspases (through the oxidization of cysteine residues at their active sites), hallmarking their regulatory function [34,44].

3. Bcl-2 family proteins The Bcl-2 protein family members are potent regulators of the mitochondrial changes during both apoptosis and necrosis. Their importance is often illustrated by eminent descriptors such as “the lords of death”, “proteins in control”, “gatekeepers and gatecrashers” or “bodyguards and assassins” [45–48]. More than 30 members of the family have been identified over the past years, which based on the functional criteria are classified as pro-survival (Bcl-2, Bcl-XL , Bcl-w, Mcl-1, A1 and Boo) or pro-apoptotic [49]. The latter group is further divided into multidomain pro-apoptotic Bcl2 proteins (Bax, Bak and Bok) that share a high degree of

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structural similarity to pro-survival Bcl-2-like proteins, and so called BH3-only proteins (including Bad, Bim, Bid, Noxa and Puma and among others) that share homology with the Bcl-2 family only in the BH3 region. Only recently, some new members of the family have been characterized, such as Bcl-GL , Bfk Bcl-rambo, Bcl-B or Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) [50–53]. The activity of Bcl-2 family members is a subject to stringent control at transcriptional, post-transcriptional and posttranslational level, pivotal in counterbalancing their pro- and anti-apoptotic functions (extensively reviewed, e.g., in refs. [54] and [55]) and per se will not be the focus of the present review.

4. The network of Bcl-2 family proteins—how does it work? Like rugby-players, Bcl-2 family proteins mostly try to block each other’s moves. The anti-apoptotic family members bind to the multidomain pro-apoptotic members, and thus prevent them from mediating the release of cytochrome c. For example, Bcl-2 can prevent Bax translocation, bind the N-terminal exposed form of Bak and hamper Bax/Bak oligomerisation, inhibiting mitochondrial membrane permeabilization [56,57]. Similarly, BNIP3 binds the activated form of Bax and pro-apoptotic form of Bak preventing their interaction as well as homo-oligomerization [58,59]. The current model of the regulatory network of Bcl-2 family members assumes also that BH3-only proteins come in two flavours, as “de-repressors” or “direct activators” (compare Fig. 1). The BH3-only proteins can bind to the hydrophobic pocket of the anti-apoptotic proteins, freeing activator BH3-only proteins (direct binding model) or the multidomain pro-apoptotic family members (displacement model), to allow their action [49,60–63] (compare Fig. 1 and its description). The activator BH3-only proteins (Bid and Bim) are widely suggested to have toxic effects independent of Bcl-2 inhibition, affecting mitochondria directly or via interaction with Bax (and possibly Bak) [64,65]. For instance, the caspase 8-cleaved Bid (tBid) can be further modified by Nterminal myristoylation, allowing its interaction with Bax or Bak, induction of Bax conformational change and Bax/Bak oligomerization. Moreover, synthetic peptides corresponding to the BH3 domains of Bim and Bid are able to activate Bax. Difficulties in direct demonstration of Bid binding to Bax led to “kiss and run” hypothesis, suggesting a transient nature of Bid/Bax complexes [66]. The pro-apoptotic effects of Bid are blocked upon its binding to the anti-apoptotic family members. The discrepancies exist as to whether the interaction of Bid/Bim with pro-survival Bcl-2-like proteins or that with Bax/Bak-like proteins is of higher affinity and utmost importance for its pro-apoptotic activity [54,60]. The recent evidence favours scenario wherein inhibition of anti-apoptotic Bcl-2 proteins is not sufficient for apoptosis to occur, and requires Bid or Bim-mediated activation of Bax/Bak [62].

Fig. 1. How Bcl-2 proteins act in concert to induce cell death. Constitutively active Bax/Bak is constrained by pro-survival Bcl-2 family members, counteracted by BH3-only proteins (the displacement model). The right set of BH3-only proteins (compare Fig. 2), releasing Bax/Bak from all constraining partners, is required to induce cell death. Alternatively, Bax/Bak may be activated only upon interaction with BH3-only activators (Bid, Bim; the direct binding model) and Bcl-2 anti-apoptotic proteins held this interaction in check until inhibited by de-repressor BH3-only proteins. According to a recent view Bcl-2 antagonists may induce death only in some cells, which survival depends on the action of Bcl-2 anti-apoptotic proteins, e.g., in cells where BH3-only activators have been earlier engaged to activate Bax/Bak, where Bax/Bak have been activated by other factors and/or where Bax/Bak activation by factors other than BH3-only activators is inhibited by Bcl-2 anti-apoptotic proteins [49,60–62]. A more detailed view on the interaction between Bcl-2 family proteins and p53 is depicted in Fig. 3.

The picture is even more complex, as oligomers of tBid are reportedly able to trigger apoptosis without inducing the dimerization of Bax or Bak [65]. Next, it is cardiolipin rather than mitochondrial proteins that has been suggested as a gateway to tBid-induced dysfunctions in mitochondrial bioenergetics [67]. Furthermore, a BH3-like protein modulator of apoptosis-1 (MAP-1) has been identified as a mitochondrial target of Bax [68], interactions between various BH3-only proteins and the anti-apoptotic Bcl-2 family members exhibit a pattern of overlapping selectivity (Fig. 2), and the latter may differently inhibit pro-apoptotic Bcl-2 family proteins [49,60]. Finally, Noxa and the novel BH3-bearing

Fig. 2. Cooperation between different classes of BH3-only proteins, binding promiscuously to their pro-survival counterparts. Note that Puma, Bim and tBid bind avidly to all pro-survival proteins, whereas Noxa and Bad act in concert to bind a broad range of anti-apoptotic proteins. Potentially, antiapoptotic proteins can be individually targeted upon development of selective mimetics of BH3 domains [49,60–62].

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Fig. 3. The impact of p53 on the mitochondrial pathway in apoptosis. The transcription-independent and transcription-dependent (Puma induction) functions of p53 are depicted. Note that the transactivation function of p53 is not limited to the induction of Puma expression; Bax and Bid are among other p53 transcriptional targets from the Bcl-2 family that influence the intrinsic pathway of apoptosis. Moreover, p53 has transrepression functions that affect, e.g., the expression of Bcl-2 and Bcl-XL . Recent evidence indicates also that, apart from Bcl-XL , p53 can also form a complex with Bcl-2 [73].

ubiquitin ligase Mule/ARF-BP1 promote Mcl-1 degradation, whereas Puma can bind to Mcl-1 which is necessary but not sufficient to prevent Mcl-1 degradation [69]. There is also some uncertainty surrounding the action of multidomain pro-apoptotic Bcl-2 proteins. Emerging data indicate that Bax may have a dominant function over Bak in regulating the apoptotic signalling in certain cellular contexts, undermining the widely suggested idea that Bax and Bak serve a completely redundant function during apoptosis [57,70]. Overall, this illustrates the wide scope of functions served by BH3-only proteins to monitor cellular well-being, and the complexity of mutual regulations that occur between Bcl-2 family proteins. A plethora of cellular factors outside the Bcl-2 family may also directly interact with Bcl-2 proteins to modulate their action. For instance, the tumour suppressor p53 can directly activate Bax or Bak, or block Bcl-2 and Bcl-XL , acting as an activator and de-repressor, respectively (Fig. 3) [71,72]. A nuclear protein involved in non-homologous DNA repair, Ku70, can inhibit Bax translocation to the mitochondria [74,75], whereas orphan receptor TR3 reportedly translocates from the nucleus to mitochondria where it binds Bcl-2 and evokes MOMP [76]. In this case Dr. Jekyll and Mr. Hyde character of Bcl-2 is revealed, as it is converted from the protector into the vicious killer, and seemingly analogous effects can be obtained upon cleavage of Bcl-2 and Bcl-XL by caspases, when an MMP-promoting protein fragment is produced [44]. The story carries on as linker histone H1.2, released from the nucleus upon X-ray induced DNA damage, promotes cytochrome c release by activating Bak [77], mitochondriabound hexokinase II (HK-II) can interact with VDAC inhibiting its interaction with Bax [78], and a serine/threonine kinase Raf-1, implicated in the transmission of multiple growth and survival signals, can associate with Bcl-2 and phosphorylate BAD [79]. Bif-1, a member of the endophilin B protein family, has been proposed as anovel Bax/Bak activator [80]. Finally, a recently identified downstream target of p53,

p53AIP1, interacts with Bcl-2 proteins at mitochondria [81]. Thus, Bcl-2 proteins are implicated not only in a complex network of mutual interactions, but also in relations with a wide range of cellular factors outside the family.

5. Effects of Bcl-2 family proteins on mitochondria How Bcl-2 proteins control mitochondrial stability at the level of mitochondrial membrane is another conundrum. According to the basic mechanisms suggested, they form membrane-spanning pores, interact with and regulate preexisting channels such as PTP, and conceivably alter the membrane structure by interactions with membrane lipids. The argument in support for the first hypothesis is the early observation of structural similarity between Bcl-XL or Bid and the pore-forming domains of several bacterial toxins [82], strengthened by subsequent reports on the poration activity of Bax, Bcl-2, Bcl-XL and cleaved Bid in artificial membranes [83]. During apoptosis, the pair of hydrophobic ␣-helices of Bcl-2 has been shown to translocate from the cytoplasm into the lipid bilayer of both ER and mitochondria, forming a channel that may either protect against or propagate apoptosis. Similarly, the ␣-helix 6 of Bid can form channels in planar lipid bilayers [84]. The significance of channelforming activity of BH3-only and anti-apoptotic Bcl-2 family proteins under physiological conditions remains, however, controversial, being detectable only when these proteins are assayed or pre-inserted in the artificial membranes at low pH. Moreover, in cells overexpressing Bcl-2 no Bcl-2 attributable channel activity has been detected. In sharp contrast, it is wellsubstantiated that activation of Bax and Bak upon induction of apoptosis involves their oligomerization (Bax oligomers up to hexamers have been reported), integration into mitochondrial membrane (Bax) and formation of non-selective channels/lipidic pores [56,66] and in the absence of Bax and Bak (double knockouts) MOMP does not occur and cells are

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protected from apoptotic death (whereas necrotic cell death pathway remains intact) [85]. In non-apoptotic cells Bax exists as a monomer either freely in the cytosol or loosely attached to the outer mitochondrial membrane. The activated Bax can be distinguished from its inactive form by exposition of 6A7 epitope (an initial and reversible event that occurs prior to oligomerisation) upon conformational change in the N-terminus, a feature recently employed in an antibody chip technology [66,86]. Moreover, translocation of Bax can be effectively examined by LSC [13]. Some studies indicate, however, that conformational changes in Bax and its subsequent translocation to mitochondria are insufficient for engaging its molecular function. The recent evidence from the Antonsson’s lab supports these observations and demonstrates that it is the Bax channelforming activity, and not conformational change or translocation to mitochondria, which is required for apoptosis [87]. Yet another version of the story suggests that Bcl-2 family members are involved in the formation of mitochondrial apoptosis-induced channel (MAC), the molecular identity of which remains to be determined. Several observations indicate that MAC is responsible for the liberation of cytochrome c early in apoptosis, and based on numerous pharmacological, molecular and immunological studies, Bax is considered as its component [21,88–90]. Alternatively, both Bax and Bak have been shown to interact with some resident mitochondrial proteins, such as previously mentioned components of the permeability transition pore VDAC1 and VDAC2, or adenine nucleotide translocator [91]. Importantly, the Bax–VDAC interaction has promoting, whereas Bak–VDAC1 inhibiting, effects on MOMP [15,91]. As recently demonstrated, Bax can also facilitate calcium-dependent PTP-opening [11]. The changed conductance of existing in mitochondria channels could lead to mitochondrial swelling and the non-specific rupture of the MOM [92,93]. Nevertheless, the action of pro-apoptotic members of Bcl-2 family on VDAC conformation is still controversial, as many groups have failed to validate the requirement of either VDAC or ANT for Bax killing, and no defects in recombinant Bax- or tBid-induced cytochrome c release were observed in cyclopholin D knockouts [25,26,56]. This supports the essential role of Bcl-2 proteins, and not proteins intrinsic to mitochondrial membranes, as MOMP appointers. Another model suggests that Bcl-2 family proteins can alter the composition or curvature of the mitochondrial lipid bilayer. Gong et al. have recently reported that the activity of tBid at mitochondria may be analogous to that of antibiotic polypeptides, which promote the outflow of bacterial cell contents through destabilization of the membrane bilayer structure [94]. Moreover, tBid-mediated lipid and cardiolipin redistribution could induce Bax to bind, intercalate and permeabilize the mitochondrial membrane (an alternative to the “kiss and run” hypothesis mentioned earlier). Indeed, the outer membrane permeabilization can be promoted by BH3/Bax interaction, and the process reportedly requires cardiolipin [39,95].

At this point it is worth noting that apart from controlling mitochondrial membrane permeability directly, Bcl-2 proteins orchestrate several ancillary processes crucial for the regulation of cell fate: (1) Bcl-2 can exert protective effects also when expressed at the endoplasmic reticulum (ER), through regulation of caspase activation, calcium homeostasis or Bax activation [63,96,97]; (2) Bik (a BH3-only protein) has been shown to regulate calcium release from ER upstream of Bak and Bax [98]; (3) ER-targeted Bcl-2 inhibits autophagy and caspase-independent cell death, conceivably through a direct interaction with the autophagy protein Beclin 1 [99,100]; (4) BNIP3 plays a central role in ceramide- and arsenic trioxide-induced autophagic cell death [101,102]; (5) Bax and Bak modulate the unfolded protein response (UPR) and steady-state ER calcium homeostasis [103,104]; (6) Bid is phosphorylated in ATM-dependent manner after DNA damage and translocates into the nucleus. The phosporylated Bid is required for the cell cycle arrest in S phase and thus may play a pro-survival role [105,106]; (7) Bcl-2 protein may modify subcellular localisation of Apaf-1, although this has been observed only in certain cell types [107,108]; (8) the cell cycle regulatory functions of Bcl-2 proteins are also widely recognized [109–111].

6. Boosting the oncologist’s arsenal The seminal discovery that the BCL2 gene inhibits cell death rather than promotes cell proliferation [112,113] gave the foundation for a now widely embraced premise that impaired cell eradication is a crucial step in tumorigenesis. Indeed, disturbances in regulation of cell death underlie many diseases, including cancer, autoimmunity and degenerative disorders and the anti-tumour effects of anticancer drugs are linked to their ability to induce apoptosis (or alternative “back-up” forms of cell death) within tumours. The Achilles’ heel of many tumour cells is that they remain sensitive to some cell death triggers. In fact, proliferationdriving oncogene activation often promotes apoptosis, and thus cancer cells can survive only if additional lesions to circumvent cell death (e.g., inactivation of p53-dependent pathway or Bcl-2 overexpression) are acquired [114]. In this context, overexpression of Bcl-2 and/or Bcl-XL or loss of Bak and/or Bax function, has been suggested to contribute to resistance of tumours to radiation and/or chemotherapy. This has not proven, however, to be a general rule for nonhaematological malignancies, signifying that depending on the cellular context, forms of cell death other than apoptosis (e.g., necrosis, senescence, mitotic catastrophe or autophagy) can also be activated in response to therapy [115]. Still, apoptosis provides the basis for the treatment of tumours arising from apoptosis-sensitive cells/tissues, such as T-cell lymphomas and some other haematological malignancies. In such cases inhibition of apoptosis by virtue of inactivation of p53 pathway or misbalancing of Bcl-2 proteins can change the overall level of cell killing and response to therapy

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[116,117]. Stahnke et al. proposed even a two-parameter flow cytometry approach (concomitant detection of cytochrome c release and cleavage of caspase 3) to predict drug resistance in leukemia patients [118]. Moreover, Bcl-2 up-regulation can inhibit p53-induced apoptosis, and mutant Myc proteins can facilitate lymphomagenesis even in the absence of p53 inactivation by inability to suppress Bcl-2 activity [119]. Accordingly, there is currently a great excitement surrounding the potential of targeting mitochondrial pathway of apoptosis as a strategy to combat malignancy, as summarized in several excellent reviews (e.g., in refs. [3–7]). Expectedly, agents able to restore apoptosis, or stimulate pro-apoptotic proteins and/or signalling pathways that remained functional in malignant cells, are likely to purge of cancer cells while sparing their normal counterparts. It is of no surprise hence that the strategies to overcome the cytoprotective effects of Bcl-2 or related antiapoptotic proteins are emerging. These include shutting off gene transcription, induction of mRNA degradation with antisense oligonucleotides, direct attack on the proteins with peptidic (stabilized ␣-helix of Bcl-2 domains, SAHBs) or small-molecule drugs or bringing into play endogenous antagonists of anti-apoptotic Bcl-2 family proteins. Multiple drugs have been shown to regulate the BCL2 gene expression, including some sythetic retinoids, histone deacetylases inhibitors, peroxisome-proliferator-activated receptor ␥ (PPAR␥)-modulating drugs, or a natural compound curcumin. The most common drug targeting BCL2 mRNA – G3139 (oblimersen sodium) – has shown promising bioactivity in some, but not all studies. Among the major disadvantages of this approach are slow degradation rate of the Bcl-2 protein (which necessitates a prolonged suppression of mRNA accumulation) and G3139-induced inflammatory responses. Drugs attacking directly Bcl-2 proteins are the alternative that is currently being extensively tested in pre- and clinical trials. Pertinent to therapy of follicular lymphoma, a non-Hodgkin’s lymphoma characterized by t(14;18) translocation and constant overexpression of Bcl-2, small molecule “BH3 mimetics” effectively induce apoptosis in FL cells [12,120,121]. Another approach is to disrupt interactions between pro-apoptotic multidomain proteins and their inhibitors from the outside of the family, e.g., Bax–Ku70 interaction [122]. The inhibition of mitochondrial FK-506binding protein 38 (FKBP38) that anchors Bcl-2 and BclXL to mitochondria has also apoptosis-promoting potential [123]. Finally, there are approaches to increase the amount of pro-apoptotic Bcl-2 members within cells, including adenoviral delivery of Bax or Bik [124–126]. In this context, a truncated form of Bax (tBax) is reportedly even more deadly than wild-type Bax, at least in some settings [127–129]. In addition, other mitochondria-directed strategies may be valuable in cancer therapy, and include agents targeting the mitochondrial inner membrane or its proteins, lipophilic cations that preferentially accumulate in mitochondrial matrix of cancer cells (compared to non-transformed cells, tumour cells have generally higher transmembrane

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potential and more mitochondria), toxic peptides that target ψm , agents targeting and depleting mtDNA [3] or compounds acting just down-stream of mitochondria to promote apoptosome assembly (e.g., PETCM, an inhibitor of oncoprotein prothymosin-␣) [130].

7. Closing remarks Elucidating machinery that governs the balance between apoptosis and survival is critical for the understanding of basic pathomechanisms of diverse disorders, such as cancer or autoimmunity. During cancer development and progression pro-survival signals are often hard-wired to pro-apoptotic ones, and thus, in order to survive, neoplastic cells must co-opt additional defects in apoptotic pathways. Repair of such lesions should provide the excellent opportunity to dispose malignant cells while sparing their normal counterparts (widening of therapeutic window). In this regard, triggers of mitochondrial pathway of apoptosis may be exploited with the therapeutic intent in a wide range of malignancies, and inhibitors of Bcl-2-like proteins will hopefully aid conquering bcl-2 linked refractoriness to chemotherapy. Further studies are necessary to develop more potent and selective inhibitors of Bcl-2 and related proteins, and already insights into the binding selectivity of BH3-only proteins to Bcl-2 pro-survival proteins are emerging. The involvement of mitochondria and Bcl-2 family proteins in alternative modes of cell demise also begins to surface, and as a disability to apoptose can be overridden by induction of other types of cell death, this may be a therapeutically important lead. The burgeoning knowledge on the mechanisms regulating cell fate will hopefully advance hand-in-hand with the development of well-tolerated and more effective, if not ultimate, anticancer regimens.

Acknowledgements JS and DW are supported by fellowship from the Centre of International Mobility (CIMO), Helsinki, Finland.

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