Myc pathways provoking cell suicide and cancer

Article metrics

Abstract

A paradox for the cancer biology field has been the revelation that oncogenes, once thought to simply provide advantages to a cancer cell, actually put it at dire risk of cell suicide. Myc is the quintessential oncogene in this respect, as in normal cells it is required for cell cycle traverse, whereas in cancers it is overexpressed and functions as the angiogenic switch. Nonetheless, Myc overexpression kills normal cells dead in their tracks. Here we review Myc-induced pathways that contribute to the apoptotic response. Molecular analysis of Myc-induced tumors has established that some of these apoptotic pathways are essential checkpoints that guard the cell from cancer, as they are selectively bypassed during tumorigenesis. The precise mechanism(s) by which Myc targets these pathways are largely unresolved, but we propose that they involve crosstalk and feedback regulatory loops between arbiters of cell death.

The Myc paradox

The diverse functions and biological effects of Myc oncoproteins have mystified the cancer biology field for over two decades. The pioneering work of Bishop and colleagues established that v-myc was the oncogene captured by the avian MC29 myelocytomatosis transforming virus (Sheiness et al., 1978; Alitalo et al., 1983). On the heels of this revelation were the discoveries that v-myc's cellular homolog c-Myc (Sheiness and Bishop, 1979; Sheiness et al., 1980; Crews et al., 1982; Dalla-Favera et al., 1982), as well as other Myc family members N-Myc and L-Myc (Kohl et al., 1983; Schwab et al., 1984; Nau et al., 1985; Seeger et al., 1985), are activated in many cancers. Indeed, the current tally for Myc activation in human cancer now approaches 70%, suggesting that this event is required for tumorigenesis. Activation of Myc occurs through diverse mechanisms, including translocations, amplifications, or enhanced translation or protein stability (Sears et al., 1999; Stoneley et al., 2000; Noguchi et al., 2001; Alarcon-Vargas et al., 2002; Channavajhala and Seldin, 2002; Popescu and Zimonjic, 2002; Ruggero and Pandolfi, 2003; Tonini and Romani, 2003). However, most often Myc expression is activated indirectly through alterations in signaling pathways that induce or repress Myc transcription. Although gain-of-function mutations in Myc proteins have been described for some tumor types (Bhatia et al., 1993), the real common denominator of Myc activation is the deregulated overexpression of these oncoproteins. This then led to the concept that increasing threshold levels of Myc put the cell at a high risk for transformation.

At first glance, the selection for Myc activation in cancer seemed obvious. First, it was quickly established that enforced Myc expression was sufficient to provoke the entry and continuous, mitogen-independent, proliferation of cells (Cavalieri and Goldfarb, 1987, 1988; Eilers, 1991), and that it effectively blocked terminal cell differentiation (Coppola and Cole, 1986; Maruyama et al., 1987; Freytag, 1988). Subsequently, Myc was shown to be necessary for traverse into S phase of the cell cycle (Heikkila et al., 1987), a finding recently underscored by the conditional knockout of c-Myc (de Alboran et al., 2001; Trumpp et al., 2001). Thus, not surprisingly, both c-Myc and N-Myc are essential for vertebrate development (Stanton et al., 1990; Sawai et al., 1991; Charron et al., 1992; Moens et al., 1992; Davis et al., 1993). In addition, numerous studies showed that Myc activation was sufficient to provoke diverse cancers (Adams et al., 1985; Leder et al., 1986) and, more recently, that Myc is continuously required to maintain the transformed state (Felsher and Bishop, 1999; Pelengaris et al., 1999; Jain et al., 2002). Finally, to round out the story was the revelation that c-Myc functioned as an angiogenic switch (Pelengaris et al., 1999, 2002), and that its expression was in fact essential for proper and coordinate regulation of angiogenic and anti-angiogenic factors in cancer and development (Baudino et al., 2002). This was satisfying – now we know why Myc activation was so pervasive in cancer.

The fly in the ointment was that in the midst of the excitement surrounding the discovery of how Myc functioned as an oncogenic transcription factor (Blackwell et al., 1990; Blackwood and Eisenman, 1991), a paradox confronted the field – Myc was discovered to actually trigger rapid apoptosis (Askew et al., 1991; Evan et al., 1992), an endogenous and conserved program of cell suicide (Ellis et al., 1991). Further, what rapidly became clear was that Myc was the rule, rather than the exception, as other oncogenes such as E1A (Rao et al., 1992; Debbas and White, 1993; Lowe and Ruley, 1993) and E2F-1 (Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994; Kowalik et al., 1995) were then shown to behave in a similar fashion. This raised the hypothesis that apoptotic pathways must be disabled for oncogenes to promote transformation (Askew et al., 1991; Evan et al., 1992) and, as will be told, this hypothesis bore fruit. So then the task became how does Myc kill a cell? This has been a frustrating exercise for the field, but clues have come from understanding how Myc does its job, identifying the targets involved in Myc-induced death, and discovering which pathways are disabled in cancer cells.

The Max network blues

Myc genes are induced as primary response genes of virtually all signal transduction pathways known to be altered in cancer, including, for example, those governed by tyrosine kinase growth factor receptors, NF-κB and β-catenin (Kelly et al., 1983; Renan, 1989; Duyao et al., 1990; Marcu et al., 1997; Zou et al., 1997; He et al., 1998). In turn, Myc proteins take over the chain of command by functioning as master transcriptional regulators of a wide array of ‘Myc target genes’ that execute the cellular response.

Myc family proteins (c-Myc, N-Myc, L-Myc and Myc's ‘second-cousins’ S- and B-Myc) are basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors. Archaic forms of Myc appear to exist even in primitive metazoans, and all Myc proteins bind to the E-box CAYGTG motif that is present in transcription targets induced by Myc (Blackwell et al., 1990; Prendergast and Ziff, 1991; Blackwell et al., 1993). However, the DNA binding and transcriptional activity of Myc, and indeed all Myc's functions, strictly requires its dimerization with Max, a small, and again conserved, bHLH-Zip protein (Blackwood and Eisenman, 1991; Blackwood et al., 1992). Indeed, Max is now recognized as the central and shared dimerization partner of a rather large network of related b-HLH-Zip transcription factors that function, by binding through the same sequence elements, as transcriptional repressors (Figure 1a) (Grandori et al., 2000). These include the Mad family of proteins (Mad1, Mxi1, Mad3 and Mad4) and larger proteins coined Mnt (also known as Max's next tango) and Mga (i.e., the biggest protein of the bunch) (Ayer et al., 1993; Hurlin et al., 1995, 1997, 1999; Meroni et al., 1997). Thus, two potential outcomes result from the binding of Max-containing heterodimers. First, Myc : Max dimers activate transcription through interactions of conserved ‘Myc-boxes’ with transcriptional coactivators (such as TRRAP and BAF53) and their associated histone acetyltransferases (HATs, e.g., GCN5) and/or ATPase/helicases (TIPs, e.g., TIP49) (Figure 1a) (McMahon et al., 1998, 2000; Park et al., 2001; Dugan et al., 2002; Park et al., 2002). Second, Mnt : Max or Mad : Max dimers actively repress transcription through direct protein : protein interactions with the general transcriptional corepressors Sin3a/3b and, in a clear case of guilt by association, with Sin3's tethered corepressors (e.g., N-Cor and the Ski/Sno proteins) and histone deacetylases (HDACs) (Figure 1a) (Ayer et al., 1995, 1996; Heinzel et al., 1997; Hurlin et al., 1997; Nomura et al., 1999). Following binding and recruitment or displacement of the basal transcriptional machinery, these heterodimers then also function to regulate chromatin structure in a general sense, and in fact in some scenarios, in particular in the case of neural crest specification, this function may be required to precisely regulate gene expression (Bellmeyer et al., 2003). Further complexity is provided from the fact that Myc : Max dimers can play the other side of the field, repressing transcription by binding to and disrupting the functions of other transcriptional activators such as Miz-1 (Figure 1c) (Peukert et al., 1997; Seoane et al., 2001, 2002; Staller et al., 2001; Bowen et al., 2002; Herold et al., 2002; Kime and Wright, 2003; Wu et al., 2003). And, as a final blow to convention, there are now reasonable alternatives as to how the network really works, as Myc can effectively displace Mnt : Max complexes, and Mnt loss alone, even in somatic cells that express no forms of Myc, is capable of inducing the ‘Myc’ response (Hurlin et al., 2003; Nilsson et al., 2003) (Figure 1b).

Figure 1
figure1

Transcriptional regulation by the Myc–Max–Mad–Mnt network. (a) Domain structure of Myc, Max, Mad1-4, Mnt and Miz-1. Myc family proteins activated in cancer (c-Myc, N-Myc and L-Myc) dimerize with Max and bind the E-box motif CACGTG to activate transcription through the association of conserved Myc-boxes I and II with the transcriptional coactivators TRAPP and BAF53 (not shown) and their associated histone acetyltransferases (HATs) and ATPase/helicases (TIPs). Dimerization with Max is mediated through the helix–loop–helix (HLH) and juxtaposed leucine zipper (Zip) domains, whereas DNA binding in conferred by a basic (b) region. Domains involved in transactivation (TAD) and transrepression (not shown) are present in regions surrounding the conserved Myc-boxes. The negative partners Mad1, Mxi1, Mad3, Mad4 and Mnt also dimerize with Max and bind to identical response elements but repress transcription through their associations with the general transcriptional corepressors Sin3a/3b through a Sin3-interaction domain (SID) and Sin3-associated histone deacetylases (HDACs). NLS indicates the nuclear localization motif of c-Myc, Max and Mad1 – this is also present in other members of the network. Miz-1 is a POZ-zinc-finger transcriptional activator whose functions are inhibited by association with Myc : Max dimers. (b) Regulation of apoptotic mediators can occur through induction by Myc : Max complexes, and/or by relieving Mnt-mediated repression, which can occur through displacement by Myc : Max complexes, or through Mnt loss. (c) Myc inhibits the transcriptional activation of the cdk inhibitor p21Cip1 by binding to Miz1. In this scenario, the apoptotic response is favored over G1 arrest

Apoptosis induced by Myc could be generally viewed in two ways, and these are not mutually exclusive. Apoptosis is essential for proper embryonic development, tissue morphogenesis and homeostasis (Ellis et al., 1991). Thus, at one level, Myc-induced apoptosis could reflect normal functions of these oncoproteins in regulating cell death programs – in this setting, loss of Myc could either result in the abnormal survival of cells that are supposed to die or, should it be required for survival, in the inappropriate deaths of cells that are supposed to live. On the other hand, Myc apoptotic functions could simply be a pathophysiological response to cells having too much Myc – in this scenario hyperproliferation invoked by Myc induces, in ways that are slowly becoming clearer (see below), a ‘stress’ response that triggers apoptotic programs that eliminate the cell (Sherr, 2001). Gene targeting has revealed that, perhaps with one exception, it is the latter mechanism that is operational. Indeed there is scant evidence from knockouts in mice, or knockdowns of more archaic forms in Drosophila, that loss of c-Myc, N-Myc, Max, or any of the Mads triggers apoptosis in vivo, although effects on cell mass, rates of cell cycle traverse and/or proper differentiation are evident (reviewed in Baudino and Cleveland, 2001). The same cannot be said for Mnt, as loss of Mnt in mouse embryo fibroblasts (MEFs) (Hurlin et al., 2003) or knock down of Mnt using RNA interference (Nilsson et al., 2003) is sufficient to trigger apoptotic programs. Strikingly, since Mnt loss can provoke apoptosis even in somatic cells lacking all forms of Myc (Mateyak et al., 1997), this indicates that Myc may trigger apoptotic programs indirectly, by effectively disrupting Mnt's functions in harnessing the apoptotic response (Nilsson et al., 2003).

Myc response genes in apoptosis – moving targets

Myc-induced apoptosis requires its DNA binding functions and dimerization with Max (Evan et al., 1992; Amati et al., 1993). There has been much debate as to whether Myc's ability to provoke cell death requires its transactivation versus transrepression functions (Xiao et al., 1998; Cole and McMahon, 1999; Conzen et al., 2000), and the bottom line, despite fairly extensive mutagenesis of conserved regions of the N-terminus of the protein, is that the field has failed to identify a mutant defective in transactivation alone. Further, one wonders if this debate is productive, as forms of Myc specifically lacking either function are never found at appreciable levels in vivo, and Myc clearly acts as both an activator and repressor of transcription; thus, it follows that both functions are likely important.

Myc target genes have been identified by a number of approaches, most recently in SAGE and microarray screens designed to capture those targets induced or repressed following conditional activation of the oncoprotein (Coller et al., 2000; Boon et al., 2001; Neiman et al., 2001; Schuldiner and Benvenisty, 2001; Godfried et al., 2002; Iritani et al., 2002; Menssen and Hermeking, 2002; Watson et al., 2002; Yu et al., 2002; Ellwood-Yen et al., 2003; Huang et al., 2003). The cast of targets is ponderous (with at least 647 targets already identified, see http://www.myc-cancer-gene.org). Furthermore, recent genomic screens for Myc binding sites by chromatin immunoprecipitation (ChIP) and other genome-wide assays indicate that this number of targets is underestimated by at least half, and (yet another scary thought) these assays have been biased for only those targets having E-boxes in their promoter-regulatory regions (Zeller et al., 2001, 2003; Fernandez et al., 2003; Haggerty et al., 2003; Mao et al., 2003; O'Connell et al., 2003; Orian et al., 2003).

Given the pervasive genomic response, one blanches at the thought of ever sorting out which target(s) of Myc specifically contribute to triggering apoptotic programs. On top of this, one wonders if the screens that have been performed are truly reflective of the conditions of the premalignant cell that is trying to make the decision to either kill itself or its host. For example, most screens for targets have utilized a chimeric form of c-Myc that is fused to an engineered version of the estrogen receptor (ER) hormone binding domain (Myc-ER) that responds to the ER agonist 4-hydroxytamoxifen (Littlewood et al., 1995). When expressed in cells, this chimera is held in heat shock complexes in the cytoplasm, yet binding of tamoxifen releases Myc-ER and allows nuclear localization and gene regulation. An assumption of the field is that the Myc-ER transgene really behaves as a faithful mimic of native Myc oncoproteins. For example, it is feasible that Myc-ER may recruit other co-factors to transcriptional complexes. In addition, there are concerns about how much Myc one needs to overexpress to mimic the in vivo situation in cancer. In seems quite likely that the thresholds of Myc that can be achieved will drastically influence the outcome. Indeed, this has been underscored in experiments canvassing the genomic binding sites for Myc by ChIP, where at least three classes of genes (high, medium and low affinity) can be recognized depending upon the level of Myc that is expressed by a given cell (Fernandez et al., 2003). Finally, most of the screens that have been performed have addressed scenarios where Myc overexpression is sufficient to induce their expression, whereas comparatively little has been done to evaluate whether Myc is essential for the expression of its identified target genes, other than a survey of a select few in somatic and immortal rat fibroblasts lacking c-myc (Bush et al., 1998).

In truth, most of the insights into which Myc targets play important roles in apoptosis have come from knowledge that has been established on the regulation of apoptosis in general. At its most basic level, apoptosis is controlled by intrinsic survival pathways that are necessary to block the cell death program, and those invoked by extrinsic signals that actively trigger the demise of the cell. Extrinsic apoptotic pathways are direct and efficient signals that provoke cell suicide, and are induced following ligation of the Fas/TNF-α family of death receptors with their ligands (Figure 2). Through the agency of adaptor molecules such as the Fas-associated death domain (FADD) protein, interactions are formed to create the death-inducing-signaling complex (DISC), which attracts and activates ‘initiator’ caspase-8, -2 or -10, which are members of a family of cysteine-directed, aspartate-specific proteases (Ashe and Berry, 2003). These caspases then cleave and activate other ‘effector’ caspases such as caspase-3, -6 and -7, which clip key targets required for cell integrity (Nunez et al., 1998; Earnshaw et al., 1999; Nicholson, 1999; Creagh and Martin, 2001). In contrast, intrinsic cell survival pathways are much more elaborate (Ashe and Berry, 2003). Generally these are pathways that are initiated by ligand binding to cell surface cytokine receptors, which provoke signaling pathways that induce the activities of the Akt/PKB and PKA families of serine/threonine kinases (Datta et al., 1999; Cross et al., 2000). In addition, these signals regulate the activity and/or expression of the NF-κB and Forkhead transcription factors, which generally suppress or induce apoptosis, respectively (Li and Zhu, 2002; Burgering and Medema, 2003).

Figure 2
figure2

Death receptor pathways and points of regulation by Myc. The trimeric death receptors Fas and TNF-R1 interact with their respective ligands, which induces death domain (DD)-mediated associations with the adaptor molecules FADD and TRADD and other components of the death-inducing signaling complex (DISC) (e.g., RIP, RAIDD, TRAF4, and the procaspases-8 and -2). This association triggers cleavage of pro-caspases-8 and –2, leading to cleavage of downstream effector caspases (caspase-3) or to cleavage of the proapoptotic BH3-only Bcl-2 family member Bid, which generates truncated Bid (tBid). tBid activates other proapoptotic Bcl-2 family members Bax and Bak (not shown) and results in the release of proapoptotic factors from mitochondria (MT), most notably cytochrome c, which binds to the scaffold protein Apaf-1. The resulting apoptosome (not shown) that is formed then binds and induces cleavage of procaspase-9, which then cleaves and activates effector caspases, thus amplifying the apoptotic response. TNF-α-mediated pathways also induce signaling cascades that result in the activation of NF-κB, which provides an essential survival function by regulating genes such as the antiapoptotic Bcl-2 family member bcl-X. Myc interfaces with death receptor pathways at several levels: (1) Myc can activate expression of FasL, which could participate in the direct suicide of the cell or, alternatively, could trigger the death of Fas-expressing immune cells that target the cancer cell, a scenario that would explain the immune privilege state of Myc-expressing cancer cells; (2) Myc provokes association of Bid with mitochondria; (3) Myc affects components of the DISC (e.g., RIP) to sensitize cells to TNF-α-mediated apoptosis; (4) Myc compromises the function of TRAF2, effectively disrupting activation of NF-κB; and (5) Myc inhibits the induction of bcl-X. N denotes nucleus

Focal points in the control of intrinsic apoptotic pathways, and which also play a role in amplifying the apoptotic response initiated by cell death receptors, are the functions of key organelles, in particular mitochondria and the endoplasmic reticulum (Ferri and Kroemer, 2001). The integrity of these organelles is regulated by the Bcl-2 family of proteins, which function as dedicated supervisors of cell suicide (Scorrano and Korsmeyer, 2003). Bcl-2 family proteins share regions of homology (so-called Bcl-2 homology (BH) domains) and come in two flavors, those having BH4 domains that are dedicated to protect the cell from apoptosis (e.g., the antiapoptotic proteins Bcl-2, Bcl-XL, and Mcl-1), and those designed to kill it (Figure 3). Further, the assassins come in two varieties. First are the multidomain (BH1-3) proteins Bok, Bax and Bak; and Bax and Bak are together required for most intrinsic and extrinsic forms of cell death (Lindsten et al., 2000; Rathmell et al., 2002; Hahn et al., 2003; Kandasamy et al., 2003; Scorrano et al., 2003). Second is a barrage of BH3-only proteins (e.g., Bim, Bid, Puma and Noxa), which function as signaling molecules that bind to and disrupt the function of their antiapoptotic cousins and/or which can directly activate Bax/Bak (see below) (Figure 3).

Figure 3
figure3

The Bcl-2 family network. Bcl-2 family members share regions of homology, the so-called BH domains and also may contain a domain (TM) that mediates insertion into the outer membrane of the mitochondrion and to that of the endoplasmic reticulum. Antiapoptotic family members contain BH1-4 domains. Proapoptotic family members come in two varieties, BH3-only domain proteins that function to disrupt antiapoptotic family members, or which directly activate other proapoptotic family members having BH1-3 domains (Bax, Bak and Bok)

The functions of Bcl-2 family proteins are regulated at several levels, including changes in their localization, expression and post-translational modifications (phosphorylation, myristylation and deamidation), which modify their functions (Wang et al., 1996; Zha et al., 1996; Datta et al., 1997; del Peso et al., 1997; Breitschopf et al., 2000; Biswas and Greene, 2002; Deverman et al., 2002; Putcha et al., 2003). In addition, some Bcl-2 family proteins are caspase substrates (Cheng et al., 1997; Li et al., 1998; Condorelli et al., 2001). Apoptotic signaling pathways targeting these proteins lead to alterations in mitochondrial functions and to the subsequent release of apoptotic mediators from mitochondria, including SMAC (which compromises the functions of endogenous inhibitors of caspases, the IAP proteins (Vaux and Silke, 2003)), endonuclease G (Li et al., 2001; van Loo et al., 2001), apoptosis-inducing factor (AIF) (Susin et al., 1996) and, most notably, cytochrome c (Liu et al., 1996). Cytochrome c is a key mediator of the mitochondrial cell death response, as it binds to a scaffold protein coined apoptosis activating factor-1 (Apaf-1) that then forms the functional ‘apoptosome’. This higher-order oligomeric complex then activates procaspase-9, which cleaves and turns on effector caspases (Figure 2) (Wang, 2001).

Not surprisingly, the identification of the numerous regulators of apoptosis spurred an immediate response by the Myc field, as it quickly pounced from one mediator to the next, trying (and usually succeeding) to make connections to Myc-induced cell death. One would hope that, at least after the dust settled, the field would agree on pinning the response to one or two key regulators. The distressing fact is that links have been found to most. Here we therefore touch on those that have clear roles in cancer and refer the reader to other sources for other more eclectic mediators of the response (Dang, 1999; Prendergast, 1999).

Dancing with death receptors

A relatively new principle in cancer biology is that apoptosis is an important safeguard that protects the organism from tumor cells. This checkpoint is operational at two levels: an intrinsic response that somehow senses things are amiss in the mutated cell and instructs its suicide; and an immune surveillance mechanism that protects the organism from being overcome by a few bad actors. Both levels of control may be operational in connections of Myc to the death receptor network (Figure 2). First, although essential roles for Myc in cell deaths provoked by death receptor cascades is still lacking in vivo, Myc does sensitize some cell lines to TNF-α- and Fas-induced death (Klefstrom et al., 1994; Hueber et al., 1997; Klefstrom et al., 1997), and in some T-cell hybridomas c-Myc is required for activation-induced death (Shi et al., 1992), which in peripheral T cells is Fas dependent (Green et al., 2003). Exactly at which level Myc affects the Fas/TNF-α response is still unresolved, but some have suggested that this occurs just downstream of these receptor complexes. For example, Myc somehow blocks activation of NF-κB by TNF-α (Klefstrom et al., 1997; You et al., 2002), and loss of the NF-κB family member RelA also sensitizes cells to TNF-α-induced apoptosis (Beg et al., 1995; Doi et al., 1999; Rosenfeld et al., 2000). With regard to Fas, Myc acts independently of FADD (Yeh et al., 1998), and thus must target other regulators or components of the DISC, such as the RIP serine/threonine kinase (Klefstrom et al., 2002) (Figure 2). Second, MEFs and myeloid cells derived from lpr or gld mice, which have inactivating mutations in the Fas and Fas ligand genes, respectively, are resistant to Myc-induced death (Hueber et al., 1997; Amanullah et al., 2002). Third, Myc can also activate, at least in a limited number of cell lines, the transcription of Fas ligand (FasL) (Brunner et al., 2000; Kasibhatla et al., 2000) (Figure 2).

A relatively new but now proven tenet of cancer biology is that apoptotic pathways provoked by oncogenes must be bypassed during tumorigenesis (Askew et al., 1991; Evan et al., 1992; Schmitt et al., 2002). Given these connections of Myc and death receptors, the field was thus poised to accept that these networks and/or their downstream targets would be inactivated in tumors provoked by Myc activity. Indeed, first forays looked promising, as CASPASE-8 was shown to be selectively inactivated by methylation in late-stage and poor-prognosis human neuroblastoma having amplified N-MYC (Teitz et al., 2000). Further, silencing of one of the downstream amplifiers of the response, APAF-1, in malignant melanoma correlates with resistance of Apaf-1-deficient MEFs to Myc-induced apoptosis (Soengas et al., 1999, 2001). Furthermore, loss of Fas functions in lpr mice was shown to accelerate Myc-induced lymphomagenesis in L-myc transgenic mice (Zornig et al., 1995). However, this finding has been challenged by other in vivo studies showing that loss of Fas or FasL does little to Myc-induced apoptosis or transformation (Cameron et al., 2000), and direct links to other regulators and modifiers of the death receptor pathways have been slow in coming.

The failure to find widespread connections of death receptors and Myc does not, however, preclude important roles for these regulators in tumorigenesis. First, Fas/FasL regulate the size and function of the lymphoid compartment, and their loss results in lymphoproliferative autoimmune syndromes that could well cooperate with Myc in transformation (Watanabe-Fukunaga et al., 1992; Takahashi et al., 1994). Even more compelling are the recent revelations suggesting that cancer is an immune privileged condition (Griffith et al., 1995; Strand and Galle, 1998; Abrahams et al., 2003), and that this often occurs through upregulation of FasL in tumor cells, which would target Fas receptor-expressing immune cells for destruction (Shiraki et al., 1997; Peduto Eberl et al., 1999). This may also be relevant in the case of TNF-related apoptosis-inducing ligand (TRAIL) receptor and TRAIL, which are required to suppress tumor development in mice (Cretney et al., 2002; Sedger et al., 2002). This therefore suggests the Myc field would be well-served to look at the other side of the coin, where precancerous Myc-expressing cells would express regulators that specifically target immune cells for destruction. Indeed, MYCN-amplified neuroblastoma has been shown to express high levels of FasL (Shurin et al., 1998; Chen et al., 1999; Takamizawa et al., 2000). Clearly, direct genetic tests of Myc and immune privilege are now warranted.

Myc compromises mitochondrial functions by targeting the Bcl-2 network

A focal point in the control of most apoptotic pathways is the mitochondrion. Convention dictated that this organelle simply functioned as the engine of the cell, by generating the ATP needed for cellular metabolism. However, mitochondria are now also recognized as dangerous time bombs that are poised to release a host of proapoptotic factors that direct cell suicide (Harris and Thompson, 2000) (Figure 2). Most prominent among these is cytochrome c and, like other apoptotic signals, Myc activation provokes the release of cytochrome c (Juin et al., 1999). Rightly so, attention then turned to defining how Myc regulates cytochrome c release.

Key arbiters of the mitochondria are the Bcl-2 family of proteins and Myc affects this network at multiple levels. First is the case of Bax. In the healthy cell, Bax predominantly resides in the cytosol, where its C-terminal mitochondrial binding domain (the α9-helix) is buried within a hydrophobic pocket formed by the BH1-3 domains (Suzuki et al., 2000). However, upon receipt of apoptotic signals, Bax often re-localizes to the outer membrane of the mitochondrion where, along with Bak, it forms higher-order homo-oligomeric structures and both of these events appear to be important regulatory steps that control cell death (Wei et al., 2000; Antonsson et al., 2001; Mikhailov et al., 2003). Bax-deficient (but curiously not Bak-deficient) MEFs are remarkably resistant to Myc-induced apoptosis (Mitchell et al., 2000; Soucie et al., 2001; Juin et al., 2002) and bax has been suggested as a transcription target of Myc in human cells (Mitchell et al., 2000) (Figure 4). However, the latter finding is the exception rather than the rule, and Myc affects Bax in mysterious ways, as Myc is necessary (Soucie et al., 2001) and sufficient (Juin et al., 2002) for Bax activation (Figure 4); yet this can occur without affecting Bax expression, localization or conformation (Juin et al., 2002). Thus attention has turned to other, more indirect, mechanism(s) that could trigger Bax/Bak activation.

Figure 4
figure4

Myc targets the Bcl-2 family. Myc influences the expression and/or activity of Bcl-2 family members at several levels. First, Myc can induce the expression of the BH3-only factor Puma either directly or through the agency of p53. Elevated levels of Puma would then disrupt the functions of Bcl-2 or Bcl-XL, leading to mitochondrial dysfunction and the release of proapoptotic factors cytochrome c, SMAC, endonuclease G and apoptosis-inducing factor (AIF) (see text). Second, Myc can repress the transcription of bcl-2 or bcl-X. Third, Myc may ‘activate’ Bax by other means, as Myc is essential, in at least some cells, for Bax activation. Finally, Myc also provokes the association of the BH3-only factor Bid with mitochondria

Logical mediators of Myc's Bax-dependent response would include the large collection of BH3-only proteins, which activate Bax and Bak by at least two mechanisms. The simplest scenario is that operational for Puma, Bim and Noxa, where apoptotic signaling provokes their association with the antiapoptotic proteins Bcl-2 and Bcl-XL, functionally sequestering these proteins and releasing Bax and Bak to continue the chain of destruction (Coultas and Strasser, 2003). Bad functions in a similar manner, but its associations are disrupted by serine and threonine phosphorylations by the Akt, PKA, and possibly Pim kinases (Datta et al., 1997; del Peso et al., 1997; Harada et al., 1999; Fox et al., 2003), all of which function as important survival signal relays (Cross et al., 2000; Lawlor and Alessi, 2001; Wang et al., 2001) and which can block Myc-induced apoptosis (Kauffmann-Zeh et al., 1997; Weissinger et al., 1997). The second scheme, exemplified by Bid, is much more elaborate, and includes cleavage by caspase-8 following the engagement of death receptors, myristoylation of truncated Bid (tBid) and re-localization, binding and activation of tBid-bound Bak at the mitochondria (Li et al., 1998; Wei et al., 2000; Zha et al., 2000).

Despite the wealth of opportunity, the links of the BH3-only proteins to Myc-induced death are, at least at this juncture, pretty weak. One report suggests that c-Myc somehow activates association of Bid with mitochondria, but in this scenario Bid is not cleaved (Iaccarino et al., 2003) (Figure 4), so here one would argue that it must be acting like other BH3-only proteins to disrupt Bcl-2 and/or Bcl-XL functions. The other link is, however, more straightforward, where Myc activation induces the expression of Puma (Jeffers et al., 2003; Maclean et al., 2003) and Puma does harbor high-affinity binding sites for c-Myc by ChIP analysis (Fernandez et al., 2003). However, PUMA induction may only be direct in human cells (Fernandez et al., 2003), as this response strictly requires Myc's ability to activate p53 in MEFs (see below, Jeffers et al., 2003) (Figure 4).

The most direct connection of Myc and Bcl-2 family proteins has come from studies demonstrating that Myc suppresses Bcl-2 or Bcl-XL expression both in MEFs and primary hematopoietic cells, and in the precancerous B cells of Eμ-myc transgenic mice (Eischen et al., 2001a, 2001c; Maclean et al., 2003) (Figure 4), which overexpress c-myc in B cells by virtue of the immunoglobulin enhancer (Adams et al., 1985). In the hematopoietic compartment, Bcl-XL functions are generally required to support the survival of immature progenitors (Motoyama et al., 1995), whereas this role falls to Bcl-2 in more mature cells (Veis et al., 1993; Nakayama et al., 1994; Matsuzaki et al., 1997). In both scenarios, their transcription is dependent upon cytokine receptor signaling pathways that are activated by the Jak family of tyrosine kinases (Packham et al., 1998; Wen et al., 2001). Myc could in theory disrupt these signaling pathways at many levels but, at least in the case of bcl-X, appears to simply suppress its transcription, as Myc effectively represses bcl-X promoter activity in transient promoter–reporter assays (Nilsson and Cleveland, unpublished). However, this does not imply that this Myc-induced pathway, nor that suppressing bcl-2, is simple. One oddity is that Myc somehow selectively represses the expression of the Bcl-2 family member that is specifically required for the survival of that cell type. For example, in immature B cells, where Bcl-XL functions are required, Myc suppresses bcl-X, whereas in mature B-cells and myeloid cells it is bcl-2 that is suppressed by Myc (Eischen et al., 2001c). Furthermore, exactly how this suppression occurs is not resolved, as Myc appears to require de novo protein synthesis to direct bcl-X suppression (Eischen et al., 2001c), and the reductions of Bcl-XL and Bcl-2 protein observed following Myc activation are more profound than the transcriptional response (Maclean et al., 2003). Regardless of the nuts and bolts of how it all works, these findings do strongly support a model whereby reductions in the steady-state levels of Bcl-2 or Bcl-XL would lead to Bax activation, thus explaining the Bax dependence of Myc's apoptotic response (Figure 4). The importance of this pathway is also underscored by genetic tests of the hypothesis, whereby Bax loss, or Bcl-2 or Bcl-XL overexpression, effectively compromises Myc-induced apoptosis and thus accelerates Myc-induced tumorigenesis (Strasser et al., 1990; Fanidi et al., 1992; Eischen et al., 2001b; Pelengaris et al., 2002). Finally, as proof-in-principle for its relevance as an apoptotic checkpoint, the Myc-to-Bcl-2/Bcl-XL apoptotic pathway is frequently disrupted in lymphomas arising in Eμ-myc transgenic mice (Eischen et al., 2001c), and the expression of these proteins is often elevated in human tumors overexpressing MYC oncoproteins (Coultas and Strasser, 2003).

Myc triggers the Arf–p53 tumor suppressor pathway

The p53 tumor suppressor has been heralded as the ‘guardian of the genome’ (Lane, 1992; White, 1996) and is the most frequently mutated gene in cancer (Zambetti and Levine, 1993). p53 functions as a sequence-specific transcription factor (Zambetti et al., 1992) that is responsive to a wide array of signals that stress the cell, including DNA damage, hypoxia, and hyperproliferative signals emanating from oncogenes such as Myc (Levine, 1997; Giaccia and Kastan, 1998). In these scenarios, p53 functions as a critical checkpoint that either induces cell cycle arrest in the G1 phase of the cell cycle, allowing cells to recover and repair any damage, or provokes apoptosis to eliminate the damaged cell (Giaccia and Kastan, 1998). Over 70 transcription targets have been shown to be directly regulated by p53, and fully half of these have been suggested as mediators of the apoptotic response. These include the induction of proapoptotic genes such as bax, puma, noxa, bim, dr5 and fas, and the repression of bcl-2 (Miyashita et al., 1994a; Miyashita and Reed, 1995; Owen-Schaub et al., 1995; Wu et al., 1997; Oda et al., 2000; Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001; Burns and El-Deiry, 2003). In addition, targets involved in the cell cycle arrest response also have intimate links to the apoptotic program. For example, the ability of Myc to suppress p53-mediated induction of the cyclin-dependent kinase inhibitor p21Cip1, which appears to occur through Myc's ability to disrupt the functions of Miz-1 (Herold et al., 2002; Seoane et al., 2002; Wu et al., 2003), alters the balance of the p53 response in favor of that inducing cell death (Figure 1c).

Given its profound effects in either blocking cell proliferation or provoking apoptosis, the levels of p53 are understandably tightly monitored by the cell. The response is largely harnessed by a feedback loop that involves yet another p53 transcription target, Mdm2 (Wu et al., 1993) (Figure 5). Mdm2 itself functions as an oncogene and is activated in human tumors by amplifications or by alterations in signaling pathways that lead to its overexpression (Momand and Zambetti, 1997). Either scenario leads to high levels of Mdm2 protein, which inhibits p53 at three levels. First, Mdm2 directly binds to p53's N-terminal transactivation domain to disrupt its transcription functions (Momand et al., 1992; Chen et al., 1993). Second, Mdm2 also functions as an E3 ubiquitin ligase that monoubiquitinates p53 (Honda et al., 1997), which then becomes a substrate for polyubiquitination by other E3 ubiquitin ligases (including the p300 coactivator protein; Grossman et al., 1998, 2003). Finally, Mdm2 then shuttles polyubiquitinated p53 to the cytosol for degradation by the proteasome (Freedman and Levine, 1998; Roth et al., 1998). Thus, p53 can be activated indirectly by signals that inhibit Mdm2, such as that which occurs when Mdm2 is phosphorylated by the ataxia telangiectasia serine/threonine kinase (Atm) following γ-irradiation (Khosravi et al., 1999; Maya et al., 2001). Indeed, the N- and C-terminal domains of p53 are highly modified by post-translational modifications, including acetylation and sumoylation of residues in its C-terminus and regulatory phosphorylations at both ends of the protein (Gu and Roeder, 1997; Sakaguchi et al., 1998; Gostissa et al., 1999; Rodriguez et al., 1999; Muller et al., 2000; Kahyo et al., 2001; Schmidt and Muller, 2002). Many of these modifications are important for stabilizing p53, by disrupting its ability to be bound and ubiquitinated by Mdm2, and by regulating its rate of nuclear import and export. The net result of disrupting Mdm2 functions is p53 accumulation, activation and then cell death, a concept underscored by the Mdm2 knockout mouse, which dies early during embryonic development, unless crossed onto a p53-deficient background (Jones et al., 1995; Montes de Oca Luna et al., 1995).

Figure 5
figure5

Myc activates the Arf–p53 tumor suppressor pathway. Myc activation leads to profound increases in the levels of the Arf nucleolar tumor suppressor, which activates p53 indirectly, through blocking the functions of p53's inhibitor Mdm2. Arf-mediated inhibition of Mdm2 occurs through direct binding and inhibition of Mdm2's E3 ubiquitin ligase activity, which normally initiates the program of p53 destruction by the proteasome, and Arf can often also sequester Mdm2 into the nucleolus. p53 also feedback inhibits the transcription of Arf; thus in the absence of p53, or in the presence of p53 mutants, Arf expression is greatly elevated. Myc may also activate p53 by other means as well, as in some cells it can activate p53 transcription, or may activate p53 through the affects on the ATM-DNA repair pathway

Scenarios that lead to p53 activation during transformation include the response of cells to senescence and ‘oncogenic stress’ (Sherr, 1998). Both rely on the function of an essential modifier of the p53 response, the Arf tumor suppressor. Arf is a small basic nucleolar protein encoded by the alternative reading frame of the Ink4a locus (Quelle et al., 1995), which also encodes the p16 cyclin-dependent kinase (Cdk) inhibitor that inhibits D-type cyclin : Cdk4/Cdk6 complexes, which phosphorylate and inactivate the retinoblastoma (Rb) tumor suppressor (Serrano et al., 1993; Xiong et al., 1993). Deletion or silencing of the INK4a/ARF locus is the second most common alteration in human tumors (Ruas and Peters, 1998), and this event is thus a double-whammy, which simultaneously inactivates both the p53 and Rb tumor suppressor pathways (James and Peters, 2000; Sherr, 2001). Arf expression is induced in a protracted manner by replicative crisis that reflects senescence programs and/or ex vivo ‘culture shock’ (Sherr and DePinho, 2000), yet is upregulated in a profound manner following the exposure of cells to activated oncoproteins such as Ras, E1A, E2f1 and Myc (Bates et al., 1998; de Stanchina et al., 1998; Zindy et al., 1998; Dimri et al., 2000; Groth et al., 2000; Lin and Lowe, 2001). When induced, Arf activates p53 indirectly, by binding to Mdm2 and inhibiting its p53-directed E3 ubiquitin ligase activity (Honda and Yasuda, 1999), and by sequestering Mdm2 into the nucleolus (Kamijo et al., 1998; Tao and Levine, 1999; Weber et al., 1999). Like p53-deficient mice (Donehower et al., 1992; Jacks et al., 1994), loss of Arf leads to spontaneous tumor development (Kamijo et al., 1997). The longer latency required for tumor appearance and the altered spectrum of malignancies in Arf-deficient mice suggested that Arf may also operate in a p53-independent manner (Kamijo et al., 1999). Indeed, Arf has been shown to regulate angiogenesis of the eye (McKeller et al., 2002), inhibit ribosomal RNA processing (Sugimoto et al., 2003) and NF-κB signaling (Rocha et al., 2003) in a p53-independent manner, and can, when overexpressed in ArfMdm2p53 triply deficient cells, cause cell cycle arrest (Weber et al., 2000).

The Arf–p53 pathway is an important mediator of Myc-induced apoptosis, as loss of either p53 or Arf impairs Myc-induced apoptosis (Hermeking and Eick, 1994; Wagner et al., 1994; Zindy et al., 1998). Myc activation in primary cells is associated with a profound induction of both Arf and p53 protein levels. This response occurs at several levels. First, in some cell types, p53 is a transcription target of Myc and indeed the gene harbors an E-box in its promoter-regulatory region (Reisman et al., 1993). However, in most cells, p53 protein levels are more profoundly induced following Myc activation (Zindy et al., 1998), usually through the agency of Arf in blocking Mdm2 functions (Honda and Yasuda, 1999; Weber et al., 1999), but perhaps also via effects of Myc on the Atm pathway (Lindstrom and Wiman, 2003).

The conventional notion holds that the Myc-to-Arf-to-p53 response (Figure 5) is largely initiated through transcriptional induction of Arf, as Myc's transcription functions are required for it to induce cell death (Amati et al., 1993). However, how Myc induces Arf remains unclear as the response requires de novo protein synthesis (Zindy et al., 1998). Thus, Myc must work through the agency of other transcription factors or signaling proteins that regulate Arf transcription. Indeed some likely culprits have been identified, including the transcription factors Bmi-1, Twist, Tbx2 and Tbx3, all of which repress Arf transcription and block Myc-induced apoptosis (Jacobs et al., 1999a, 2000; Maestro et al., 1999; Brummelkamp et al., 2002; Carlson et al., 2002; Lingbeek et al., 2002). Furthermore, reductions in Bmi-1 impair Myc-induced tumorigenesis through augmenting an Arf-dependent apoptotic response (Jacobs et al., 1999a). Despite these compelling connections, Myc's effects on the expression and/or activity of these Arf repressors have never been properly evaluated.

Rather than suppress an Arf repressor, the obvious alternative is that Myc induces Arf by activating a positive regulator. One potential target is DMP-1, as DMP-1 is a potent and direct activator of Arf (Inoue et al., 1999) and loss of DMP-1 accelerates Myc-induced lymphomagenesis (Inoue et al., 1999). Another candidate was thought to be E2f1, as Myc induces E2f1 expression in primary cells (Leone et al., 1997; Sears et al., 1997; Baudino et al., 2003; Fernandez et al., 2003) and E2f1 has been suggested to induce Arf directly (Bates et al., 1998). Further, like Myc (Zindy et al., 1998), E2f1 overexpression can trigger p53-dependent apoptosis (Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994; Kowalik et al., 1995; DeGregori et al., 1997). However, Myc's reliance on E2f1 for apoptosis is, at the very least, assay-dependent (Leone et al., 2001; Baudino et al., 2003), as Myc can clearly activate Arf and p53 expression and kill cells in the absence of E2f1 (Baudino et al., 2003). Furthermore, E2f1's role in Myc-induced apoptosis is not observed in transgenic mice programmed to overexpress c-Myc (Rounbehler et al., 2002; Baudino et al., 2003). A third, and perhaps more reasonable candidate as a mediator of the Arf response to Myc is the calcium-regulated serine/threonine kinase DAP (Kimchi, 1998). DAP expression is augmented by Myc, the kinase is sufficient to activate Arf, and loss of DAP impairs Myc's ability to induce Arf and p53 and to trigger cell death (Raveh et al., 2001). However, the physiological role of the DAP kinase as a checkpoint that harnesses Myc's apoptotic and transforming activities has not been addressed in vivo, and Arf is not strictly essential for DAP-induced death, possibly since the TNF-α/Fas pathways are also engaged when this class of kinases are activated (Cohen et al., 1999; Kogel et al., 2003). Finally, another view is that probably too much emphasis is being placed on the Arf transcriptional response, especially when this is compared to the robust induction of the protein (Zindy et al., 1998; Eischen et al., 1999). Thus, evaluating post-translational regulation of Arf may provide further insights into regulators of this response.

Essential tools in establishing which apoptotic pathways may be operational in precancerous cells undergoing the transition to a frank malignancy have been the use of mouse models that faithfully recapitulate human tumors with MYC involvement (Adams et al., 1985; Leder et al., 1986; Stewart et al., 1993; Pelengaris et al., 1999, 2002; Rounbehler et al., 2001). These analyses have underscored the importance of the Arf-p53 and Bcl-2/Bcl-XL pathways as checkpoints that guard the cell against cancer. First, molecular analyses of the lymphomas that arise in Eμ-myc transgenic mice, which are a phenocopy of human Burkitt's lymphoma (BL) that bear translocated MYC-immunoglobulin alleles (Adams et al., 1985), have established that both apoptotic pathways are bypassed during lymphomagenesis. In this model, c-myc overexpression in the B-cell compartment drives high rates of proliferation that are initially offset by increased rates of apoptosis (Jacobsen et al., 1994), and increases in the expression of Arf and p53, and reductions in the expression of Bcl-2 and Bcl-XL, are evident in these precancerous B cells (Eischen et al., 1999, 2001c; Maclean et al., 2003). Strikingly, 80% of the lymphomas that ultimately arise in these mice display hits in the Arf–p53 pathway, by bi-allelic deletion of Arf (25%), inactivating point mutations in p53 (30%) and/or high levels of Mdm2 expression (50% overall) (Eischen et al., 1999). In addition, over half of these tumors also overexpress Bcl-2, regardless of the status of Arf or p53 in these lymphomas (Eischen et al., 2001c). Thus, these apoptotic pathways are bypassed during tumorigenesis in an independent fashion. Further, these events are relevant to the human condition, as BL have a similar frequency in hits in the ARF–Mdm2-p53 pathway (Lindstrom et al., 2001).

Genetic tests have underscored the essential roles of the Arf–p53 and Bcl-2/Bcl-XL pathways in harnessing Myc-driven tumorigenesis. First, crossing Myc transgenics to Arf-, Ink4a/Arf- or p53-deficient mice greatly accelerates the course of disease, and heterozygous transgenics usually display loss of the wild-type allele, proving that they function as classic tumor suppressors in the response (Eischen et al., 1999; Jacobs et al., 1999b; Russell et al., 2002; Schmitt et al., 1999). Inactivation of Arf or p53 in Eμ-Myc lymphomas is mutually exclusive; however, a curiosity is that 50% of all lymphomas, regardless of their p53 or Arf status, also express high levels of Mdm2 (Eischen et al., 1999), suggesting that Mdm2 might have other targets in the tumorigenic response. Furthermore, a key role for Mdm2 in Myc-driven lymphomagenesis has been established by studies demonstrating that Mdm2 overexpression accelerates disease in Eμ-Myc transgenics (Fridman et al., 2003), whereas loss of just one Mdm2 allele markedly impairs their lymphoma development (Alt et al., 2003). Second, enforced expression of Bcl-2 or Bcl-XL, or loss of Bax, also dramatically accelerates tumorigenesis in Myc transgenics (Strasser et al., 1990; Eischen et al., 2001b; Pelengaris et al., 2002), demonstrating that the pathway by which Myc suppresses Bcl-2 or Bcl-XL is also rate limiting for tumor development.

Feedback loops and crosstalk between Myc's apoptotic pathways

The tumor suppressor field has posited that the Arf–p53 pathway is activated by an ill-defined ‘stress’ response to the hyperproliferative signals driven by oncogenes (Sherr, 2001). The profound reductions in Bcl-2 and Bcl-XL proteins that occur following Myc activation could certainly be viewed as a ‘stress’ for the cell, as loss of either protein in mice compromises the survival of select cell types (see above). The downregulation of bcl-X, at least in B cells and MEFs, is independent of Arf and/or p53 status (Eischen et al., 2001c; Maclean et al., 2003), and from a kinetic standpoint this is certainly a rapid response. It is therefore not too much of a stretch to suggest that Myc's ability to downregulate Bcl-2 or Bcl-XL could be the ‘stress’ signal that triggers the Arf–p53 pathway (Figure 6). This hypothesis makes several predictions. First, one would expect that overexpression of Bcl-2 or Bcl-XL would cancel the need for altering Arf and/or p53 when cells are exposed to Myc. Indeed this has been borne out in Bcl-2 retroviral transduction experiments of Eμ-Myc; p53+/− bone marrow, where one no longer observes the selection for loss of the wild-type p53 allele (Schmitt et al., 2002). Furthermore, the lymphomas that arise in Eμ-Myc/Eμ-Bcl-2 double transgenics lack any indication of Arf or p53 involvement (Maclean, Yang and Cleveland, unpublished results), and those arising in Bax-deficient lymphomas lack p53 mutations (but curiously not Arf deletes; Eischen et al., 2001b). Second, one would predict that loss of Bcl-2 or Bcl-X alone might be sufficient to trigger the pathway (Figure 6), and at least in MEFs, knockdown of bcl-2 does appear to trigger the p53 response (Jiang and Milner, 2003). Collectively, this scenario would suggest a model whereby Bcl-2 or Bcl-XL overexpression, and perhaps loss of Bax (or more likely Bax plus Bak), would block Myc's ability to induce Arf and/or p53. In turn, impairing this response would compromise Myc-induced apoptosis, converting Myc into a pure promoter of cell growth and transformation.

Figure 6
figure6

Crosstalk and feedback loops in the Arf–p53 and Bcl-2/Bcl-XL apoptotic pathways provoked by Myc. In Model I, c-Myc activates the two pathways in an independent manner and both contribute to cell death. In Model II, it is proposed that downregulation of Bcl-2 and/or Bcl-XL serves as the ‘stress’ signal that triggers the induction of Arf and p53. Furthermore, at least in some cell types, loss of Arf or p53 results in a marked upregulation in the steady-state levels of Bcl-2, which might then explain why Arf- and/or p53-deficient cells are inherently more resistant to Myc-induced apoptosis

Linear pathways are always convenient, but biology is rarely a straight path, and this holds true when one considers the other side of the coin, where p53 and Arf status could affect the expression of Bcl-2 family members. Some of these connections are indeed set in stone and are direct as, for example, the BH3-only proteins Puma and Noxa are direct targets of p53 (Oda et al., 2000; Han et al., 2001; Nakano and Vousden, 2001), and Myc's ability to trigger Puma expression, at least in MEFs, is strictly p53 dependent (Maclean et al., 2003). Other links are more subtle, but could still affect the response in a major way. For example, p53 has been suggested as a repressor of Bcl-2 transcription (Miyashita et al., 1994b), and indeed in immature B cells of Arf- and/or p53-deficient mice there are substantial increases in the steady-state levels of Bcl-2 protein (Cleveland, unpublished). Thus, perhaps Arf and p53 loss accelerates lymphoma development in Eμ-Myc transgenics simply because there are high levels of Bcl-2 already expressed in these cells (Figure 6). Connections to Bcl-x may come in a more circuitous, but nonetheless effective, route. The expression of bcl-X in B cells has been shown to require the NF-κB family member c-Rel (Owyang et al., 2001), and, at least in some human cell lines, Arf can inhibit NF-κB and bcl-X expression (Rocha et al., 2003); thus, loss of Arf could conceivably lead to indirect upregulation of bcl-X, which would again disable the apoptotic response (Figure 6).

The marvelously (!) complex and multifarious mechanisms of Myc-induced apoptosis certainly represent a continuing challenge, but it is almost assured that they will continue to provide insights into the biology of cancer in a general sense. Clearly, the years ahead will bring more twists and turns, but ultimately it is hoped that this quest will result in novel therapeutic strategies that can be effective in combating cancer.

References

  1. Abrahams VM, Kamsteeg M and Mor G . (2003). Mol. Biotechnol., 25, 19–30.

  2. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S, Palmiter RD and Brinster RL . (1985). Nature, 318, 533–538.

  3. Alarcon-Vargas D, Tansey WP and Ronai Z . (2002). Oncogene, 21, 4384–4391.

  4. Alitalo K, Bishop JM, Smith DH, Chen EY, Colby WW and Levinson AD . (1983). Proc. Natl. Acad. Sci. USA, 80, 100–104.

  5. Alt JR, Greiner TC, Cleveland JL and Eischen CM . (2003). EMBO J., 22, 1442–1450.

  6. Amanullah A, Liebermann DA and Hoffman B . (2002). Oncogene, 21, 1600–1610.

  7. Amati B, Littlewood TD, Evan GI and Land H . (1993). EMBO J., 12, 5083–5087.

  8. Antonsson B, Montessuit S, Sanchez B and Martinou JC . (2001). J. Biol. Chem., 276, 11615–11623.

  9. Ashe PC and Berry MD . (2003). Prog. Neuropsychopharmacol. Biol. Psychiatry, 27, 199–214.

  10. Askew DS, Ashmun RA, Simmons BC and Cleveland JL . (1991). Oncogene, 6, 1915–1922.

  11. Ayer DE, Kretzner L and Eisenman RN . (1993). Cell, 72, 211–222.

  12. Ayer DE, Laherty CD, Lawrence QA, Armstrong AP and Eisenman RN . (1996). Mol. Cell. Biol., 16, 5772–5781.

  13. Ayer DE, Lawrence QA and Eisenman RN . (1995). Cell, 80, 767–776.

  14. Bates S, Phillips AC, Clark PA, Stott F, Peters G, Ludwig RL and Vousden KH . (1998). Nature, 395, 124–125.

  15. Baudino TA and Cleveland JL . (2001). Mol. Cell. Biol., 21, 691–702.

  16. Baudino TA, Maclean KH, Brennan J, Parganas E, Yang C, Aslanian A, Lees JA, Sherr CJ, Roussel MF and Cleveland JL . (2003). Mol. Cell., 11, 905–914.

  17. Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN and Cleveland JL . (2002). Genes Dev., 16, 2530–2543.

  18. Beg AA, Sha WC, Bronson RT, Ghosh S and Baltimore D . (1995). Nature, 376, 167–170.

  19. Bellmeyer A, Krase J, Lindgren J and LaBonne C . (2003). Dev. Cell, 4, 827–839.

  20. Bhatia K, Huppi K, Spangler G, Siwarski D, Iyer R and Magrath I . (1993). Nat. Genet., 5, 56–61.

  21. Biswas SC and Greene LA . (2002). J. Biol. Chem., 277, 49511–49516.

  22. Blackwell TK, Huang J, Ma A, Kretzner L, Alt FW, Eisenman RN and Weintraub H . (1993). Mol. Cell. Biol., 13, 5216–5224.

  23. Blackwell TK, Kretzner L, Blackwood EM, Eisenman RN and Weintraub H . (1990). Science, 250, 1149–1151.

  24. Blackwood EM and Eisenman RN . (1991). Science, 251, 1211–1217.

  25. Blackwood EM, Luscher B and Eisenman RN . (1992). Genes Dev., 6, 71–80.

  26. Boon K, Caron HN, van Asperen R, Valentijn L, Hermus MC, van Sluis P, Roobeek I, Weis I, Voute PA, Schwab M and Versteeg R . (2001). EMBO J., 20, 1383–1393.

  27. Bowen H, Biggs TE, Phillips E, Baker ST, Perry VH, Mann DA and Barton CH . (2002). J. Biol. Chem., 277, 34997–35006.

  28. Breitschopf K, Haendeler J, Malchow P, Zeiher AM and Dimmeler S . (2000). Mol. Cell. Biol., 20, 1886–1896.

  29. Brummelkamp TR, Kortlever RM, Lingbeek M, Trettel F, MacDonald ME, van Lohuizen M and Bernards R . (2002). J. Biol. Chem., 277, 6567–6572.

  30. Brunner T, Kasibhatla S, Pinkoski MJ, Frutschi C, Yoo NJ, Echeverri F, Mahboubi A and Green DR . (2000). J. Biol. Chem., 275, 9767–9772.

  31. Burgering BM and Medema RH . (2003). J. Leukoc. Biol., 73, 689–701.

  32. Burns TF and El-Deiry WS . (2003). Cancer Biol. Ther., 2, 431–443.

  33. Bush A, Mateyak M, Dugan K, Obaya A, Adachi S, Sedivy J and Cole M . (1998). Genes Dev., 12, 3797–3802.

  34. Cameron ER, Morton J, Johnston CJ, Irvine J, Bell M, Onions DE, Neil JC, Campbell M and Blyth K . (2000). Cell Death Differ., 7, 80–88.

  35. Carlson H, Ota S, Song Y, Chen Y and Hurlin PJ . (2002). Oncogene, 21, 3827–3835.

  36. Cavalieri F and Goldfarb M . (1987). Mol. Cell. Biol., 7, 3554–3560.

  37. Cavalieri F and Goldfarb M . (1988). Oncogene, 2, 289–291.

  38. Channavajhala P and Seldin DC . (2002). Oncogene, 21, 5280–5288.

  39. Charron J, Malynn BA, Fisher P, Stewart V, Jeannotte L, Goff SP, Robertson EJ and Alt FW . (1992). Genes Dev., 6, 2248–2257.

  40. Chen J, Marechal V and Levine AJ . (1993). Mol. Cell. Biol., 13, 4107–4114.

  41. Chen MK, Strande LF, Beierle EA, Kain MS, Geldziler BD and Doolin EJ . (1999). J. Surg. Res., 84, 82–87.

  42. Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K and Hardwick JM . (1997). Science, 278, 1966–1968.

  43. Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E and Kimchi A . (1999). J. Cell. Biol., 146, 141–148.

  44. Cole MD and McMahon SB . (1999). Oncogene, 18, 2916–2924.

  45. Coller HA, Grandori C, Tamayo P, Colbert T, Lander ES, Eisenman RN and Golub TR . (2000). Proc. Natl. Acad. Sci. USA, 97, 3260–3265.

  46. Condorelli F, Salomoni P, Cotteret S, Cesi V, Srinivasula SM, Alnemri ES and Calabretta B . (2001). Mol. Cell. Biol., 21, 3025–3036.

  47. Conzen SD, Gottlob K, Kandel ES, Khanduri P, Wagner AJ, O'Leary M and Hay N . (2000). Mol. Cell. Biol., 20, 6008–6018.

  48. Coppola JA and Cole MD . (1986). Nature, 320, 760–763.

  49. Coultas L and Strasser A . (2003). Semin. Cancer Biol., 13, 115–123.

  50. Creagh EM and Martin SJ . (2001). Biochem. Soc. Trans., 29, 696–702.

  51. Cretney E, Takeda K, Yagita H, Glaccum M, Peschon JJ and Smyth MJ . (2002). J. Immunol., 168, 1356–1361.

  52. Crews S, Barth R, Hood L, Prehn J and Calame K . (1982). Science, 218, 1319–1321.

  53. Cross TG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M and Lord JM . (2000). Exp. Cell. Res., 256, 34–41.

  54. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC and Croce CM . (1982). Proc. Natl. Acad. Sci. USA, 79, 7824–7827.

  55. Dang CV . (1999). Mol. Cell. Biol., 19, 1–11.

  56. Datta SR, Brunet A and Greenberg ME . (1999). Genes Dev., 13, 2905–2927.

  57. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y and Greenberg ME . (1997). Cell, 91, 231–241.

  58. Davis AC, Wims M, Spotts GD, Hann SR and Bradley A . (1993). Genes Dev., 7, 671–682.

  59. de Alboran IM, O’Hagan RC, Gartner F, Malynn B, Davidson L, Rickert R, Rajewsky K, DePinho RA and Alt FW . (2001). Immunity, 14, 45–55.

  60. de Stanchina E, McCurrach ME, Zindy F, Shieh SY, Ferbeyre G, Samuelson AV, Prives C, Roussel MF, Sherr CJ and Lowe SW . (1998). Genes Dev., 12, 2434–2442.

  61. Debbas M and White E . (1993). Genes Dev., 7, 546–554.

  62. DeGregori J, Leone G, Miron A, Jakoi L and Nevins JR . (1997). Proc. Natl. Acad. Sci. USA, 94, 7245–7250.

  63. del Peso L, Gonzalez-Garcia M, Page C, Herrera R and Nunez G . (1997). Science, 278, 687–689.

  64. Deverman BE, Cook BL, Manson SR, Niederhoff RA, Langer EM, Rosova I, Kulans LA, Fu X, Weinberg JS, Heinecke JW, Roth KA and Weintraub SJ . (2002). Cell, 111, 51–62.

  65. Dimri GP, Itahana K, Acosta M and Campisi J . (2000). Mol. Cell. Biol., 20, 273–285.

  66. Doi TS, Marino MW, Takahashi T, Yoshida T, Sakakura T, Old LJ and Obata Y . (1999). Proc. Natl. Acad. Sci. USA, 96, 2994–2999.

  67. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery Jr CA, Butel JS and Bradley A . (1992). Nature, 356, 215–221.

  68. Dugan KA, Wood MA and Cole MD . (2002). Oncogene, 21, 5835–5843.

  69. Duyao MP, Buckler AJ and Sonenshein GE . (1990). Proc. Natl. Acad. Sci. USA, 87, 4727–4731.

  70. Earnshaw WC, Martins LM and Kaufmann SH . (1999). Annu. Rev. Biochem., 68, 383–424.

  71. Eilers M, Schim S and Bishop JM . (1991). EMBO J, 10, 133–141.

  72. Eischen CM, Packham G, Nip J, Fee BE, Hiebert SW, Zambetti GP and Cleveland JL . (2001a). Oncogene, 20, 6983–6993.

  73. Eischen CM, Roussel MF, Korsmeyer SJ and Cleveland JL . (2001b). Mol. Cell. Biol., 21, 7653–7662.

  74. Eischen CM, Weber JD, Roussel MF, Sherr CJ and Cleveland JL . (1999). Genes Dev., 13, 2658–2669.

  75. Eischen CM, Woo D, Roussel MF and Cleveland JL . (2001c). Mol. Cell. Biol., 21, 5063–5070.

  76. Ellis RE, Yuan JY and Horvitz HR . (1991). Annu. Rev. Cell. Biol., 7, 663–698.

  77. Ellwood-Yen K, Graeber TG, Wongvipat J, Iruela-Arispe ML, Zhang J, Matusik R, Thomas GV and Sawyers CL . (2003). Cancer Cell, 4, 223–238.

  78. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ and Hancock DC . (1992). Cell, 69, 119–128.

  79. Fanidi A, Harrington EA and Evan GI . (1992). Nature, 359, 554–556.

  80. Felsher DW and Bishop JM . (1999). Mol. Cell., 4, 199–207.

  81. Fernandez PC, Frank SR, Wang L, Schroeder M, Liu S, Greene J, Cocito A and Amati B . (2003). Genes Dev., 17, 1115–1129.

  82. Ferri KF and Kroemer G . (2001). Nat. Cell. Biol., 3, E255–263.

  83. Fox CJ, Hammerman PS, Cinalli RM, Master SR, Chodosh LA and Thompson CB . (2003). Genes Dev., 17, 1841–1854.

  84. Freedman DA and Levine AJ . (1998). Mol. Cell. Biol., 18, 7288–7293.

  85. Freytag SO . (1988). Mol. Cell. Biol., 8, 1614–1624.

  86. Fridman JS, Hernando E, Hemann MT, De Stanchina E, Cordon-Cardo C and Lowe SW . (2003). Cancer Res., 63, 5703–5706.

  87. Giaccia AJ and Kastan MB . (1998). Genes Dev., 12, 2973–2983.

  88. Godfried MB, Veenstra M, v Sluis P, Boon K, v Asperen R, Hermus MC, v Schaik BD, Voute TP, Schwab M, Versteeg R and Caron HN . (2002). Oncogene, 21, 2097–2101.

  89. Gostissa M, Hengstermann A, Fogal V, Sandy P, Schwarz SE, Scheffner M and Del Sal G . (1999). EMBO J., 18, 6462–6471.

  90. Grandori C, Cowley SM, James LP and Eisenman RN . (2000). Annu. Rev. Cell. Dev. Biol., 16, 653–699.

  91. Green DR, Droin N and Pinkoski M . (2003). Immunol. Rev., 193, 70–81.

  92. Griffith TS, Brunner T, Fletcher SM, Green DR and Ferguson TA . (1995). Science, 270, 1189–1192.

  93. Grossman SR, Deato ME, Brignone C, Chan HM, Kung AL, Tagami H, Nakatani Y and Livingston DM . (2003). Science, 300, 342–344.

  94. Grossman SR, Perez M, Kung AL, Joseph M, Mansur C, Xiao ZX, Kumar S, Howley PM and Livingston DM . (1998). Mol. Cell., 2, 405–415.

  95. Groth A, Weber JD, Willumsen BM, Sherr CJ and Roussel MF . (2000). J. Biol. Chem., 275, 27473–27480.

  96. Gu W and Roeder RG . (1997). Cell, 90, 595–606.

  97. Haggerty TJ, Zeller KI, Osthus RC, Wonsey DR and Dang CV . (2003). Proc. Natl. Acad. Sci. USA, 100, 5313–5318.

  98. Hahn P, Lindsten T, Ying GS, Bennett J, Milam AH, Thompson CB and Dunaief JL . (2003). Invest. Ophthalmol. Vis. Sci., 44, 3598–3605.

  99. Han J, Flemington C, Houghton AB, Gu Z, Zambetti GP, Lutz RJ, Zhu L and Chittenden T . (2001). Proc. Natl. Acad. Sci. USA, 98, 11318–11323.

  100. Harada H, Becknell B, Wilm M, Mann M, Huang LJ, Taylor SS, Scott JD and Korsmeyer SJ . (1999). Mol. Cell., 3, 413–422.

  101. Harris MH and Thompson CB . (2000). Cell Death Differ., 7, 1182–1191.

  102. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B and Kinzler KW . (1998). Science, 281, 1509–1512.

  103. Heikkila R, Schwab G, Wickstrom E, Loke SL, Pluznik DH, Watt R and Neckers LM . (1987). Nature, 328, 445–449.

  104. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK and Rosenfeld MG . (1997). Nature, 387, 43–48.

  105. Hermeking H and Eick D . (1994). Science, 265, 2091–2093.

  106. Herold S, Wanzel M, Beuger V, Frohme C, Beul D, Hillukkala T, Syvaoja J, Saluz HP, Haenel F and Eilers M . (2002). Mol. Cell., 10, 509–521.

  107. Honda R, Tanaka H and Yasuda H . (1997). FEBS Lett., 420, 25–27.

  108. Honda R and Yasuda H . (1999). EMBO J., 18, 22–27.

  109. Huang E, Ishida S, Pittman J, Dressman H, Bild A, Kloos M, D'Amico M, Pestell RG, West M and Nevins JR . (2003). Nat. Genet., 34, 226–230.

  110. Hueber AO, Zornig M, Lyon D, Suda T, Nagata S and Evan GI . (1997). Science, 278, 1305–1309.

  111. Hurlin PJ, Queva C and Eisenman RN . (1997). Genes Dev., 11, 44–58.

  112. Hurlin PJ, Queva C, Koskinen PJ, Steingrimsson E, Ayer DE, Copeland NG, Jenkins NA and Eisenman RN . (1995). EMBO J., 14, 5646–5659.

  113. Hurlin PJ, Steingrimsson E, Copeland NG, Jenkins NA and Eisenman RN . (1999). EMBO J., 18, 7019–7028.

  114. Hurlin PJ, Zhou ZQ, Toyo-Oka K, Ota S, Walker WL, Hirotsune S and Wynshaw-Boris A . (2003). EMBO J., 22, 4584–4596.

  115. Iaccarino I, Hancock D, Evan G and Downward J . (2003). Cell Death Differ., 10, 599–608.

  116. Inoue K, Roussel MF and Sherr CJ . (1999). Proc. Natl. Acad. Sci. USA, 96, 3993–3998.

  117. Iritani BM, Delrow J, Grandori C, Gomez I, Klacking M, Carlos LS and Eisenman RN . (2002). EMBO J., 21, 4820–4830.

  118. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT and Weinberg RA . (1994). Curr. Biol., 4, 1–7.

  119. Jacobs JJ, Keblusek P, Robanus-Maandag E, Kristel P, Lingbeek M, Nederlof PM, van Welsem T, van de Vijver MJ, Koh EY, Daley GQ and van Lohuizen M . (2000). Nat. Genet., 26, 291–299.

  120. Jacobs JJ, Kieboom K, Marino S, DePinho RA and van Lohuizen M . (1999a). Nature, 397, 164–168.

  121. Jacobs JJ, Scheijen B, Voncken JW, Kieboom K, Berns A and van Lohuizen M . (1999b). Genes Dev., 13, 2678–2690.

  122. Jacobsen KA, Prasad VS, Sidman CL and Osmond DG . (1994). Blood, 84, 2784–2794.

  123. Jain M, Arvanitis C, Chu K, Dewey W, Leonhardt E, Trinh M, Sundberg CD, Bishop JM and Felsher DW . (2002). Science, 297, 102–104.

  124. James MC and Peters G . (2000). Prog. Cell Cycle Res., 4, 71–81.

  125. Jeffers JR, Parganas E, Lee Y, Yang C, Wang J, Brennan J, MacLean KH, Han J, Chittenden T, Ihle JN, McKinnon PJ, Cleveland JL and Zambetti GP . (2003). Cancer Cell, 4.

  126. Jiang M and Milner J . (2003). Genes Dev., 17, 832–837.

  127. Jones SN, Roe AE, Donehower LA and Bradley A . (1995). Nature, 378, 206–208.

  128. Juin P, Hueber AO, Littlewood T and Evan G . (1999). Genes Dev., 13, 1367–1381.

  129. Juin P, Hunt A, Littlewood T, Griffiths B, Swigart LB, Korsmeyer S and Evan G . (2002). Mol. Cell. Biol., 22, 6158–6169.

  130. Kahyo T, Nishida T and Yasuda H . (2001). Mol. Cell, 8, 713–718.

  131. Kamijo T, Bodner S, van de Kamp E, Randle DH and Sherr CJ . (1999). Cancer Res., 59, 2217–2222.

  132. Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF and Sherr CJ . (1998). Proc. Natl. Acad. Sci. USA, 95, 8292–8297.

  133. Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, Ashmun RA, Grosveld G and Sherr CJ . (1997). Cell, 91, 649–659.

  134. Kandasamy K, Srinivasula SM, Alnemri ES, Thompson CB, Korsmeyer SJ, Bryant JL and Srivastava RK . (2003). Cancer Res., 63, 1712–1721.

  135. Kasibhatla S, Beere HM, Brunner T, Echeverri F and Green DR . (2000). Curr. Biol., 10, 1205–1208.

  136. Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J and Evan G . (1997). Nature, 385, 544–548.

  137. Kelly K, Cochran BH, Stiles CD and Leder P . (1983). Cell, 35, 603–610.

  138. Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y and Shkedy D . (1999). Proc. Natl. Acad. Sci. USA, 96, 14973–14977.

  139. Kimchi A . (1998). Biochim. Biophys. Acta, 1377, F13–33.

  140. Kime L and Wright SC . (2003). Biochem. J., 370, 291–298.

  141. Klefstrom J, Arighi E, Littlewood T, Jaattela M, Saksela E, Evan GI and Alitalo K . (1997). EMBO. J., 16, 7382–7392.

  142. Klefstrom J, Vastrik I, Saksela E, Valle J, Eilers M and Alitalo K . (1994). EMBO J., 13, 5442–5450.

  143. Klefstrom J, Verschuren EW and Evan G . (2002). J. Biol. Chem., 277, 43224–43232.

  144. Kogel D, Reimertz C, Dussmann H, Mech P, Scheidtmann KH and Prehn JH . (2003). Eur. J. Cancer, 39, 249–256.

  145. Kohl NE, Kanda N, Schreck RR, Bruns G, Latt SA, Gilbert F and Alt FW . (1983). Cell, 35, 359–367.

  146. Kowalik TF, DeGregori J, Schwarz JK and Nevins JR . (1995). J. Virol., 69, 2491–2500.

  147. Lane DP . (1992). Nature, 358, 15–16.

  148. Lawlor MA and Alessi DR . (2001). J. Cell. Sci., 114, 2903–2910.

  149. Leder A, Pattengale PK, Kuo A, Stewart TA and Leder P . (1986). Cell, 45, 485–495.

  150. Leone G, DeGregori J, Sears R, Jakoi L and Nevins JR . (1997). Nature, 387, 422–426.

  151. Leone G, Sears R, Huang E, Rempel R, Nuckolls F, Park CH, Giangrande P, Wu L, Saavedra HI, Field SJ, Thompson MA, Yang H, Fujiwara Y, Greenberg ME, Orkin S, Smith C and Nevins JR . (2001). Mol. Cell, 8, 105–113.

  152. Levine AJ . (1997). Cell, 88, 323–331.

  153. Li H, Zhu H, Xu CJ and Yuan J . (1998). Cell, 94, 491–501.

  154. Li LY, Luo X and Wang X . (2001). Nature, 412, 95–99.

  155. Li Q and Zhu GD . (2002). Curr. Top. Med. Chem., 2, 939–971.

  156. Lin AW and Lowe SW . (2001). Proc. Natl. Acad. Sci. USA, 98, 5025–5030.

  157. Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K, Chen Y, Wei M, Eng VM, Adelman DM, Simon MC, Ma A, Golden JA, Evan G, Korsmeyer SJ, MacGregor GR and Thompson CB . (2000). Mol. Cell, 6, 1389–1399.

  158. Lindstrom MS, Klangby U and Wiman KG . (2001). Oncogene, 20, 2171–2177.

  159. Lindstrom MS and Wiman KG . (2003). Oncogene, 22, 4993–5005.

  160. Lingbeek ME, Jacobs JJ and van Lohuizen M . (2002). J. Biol. Chem., 277, 26120–26127.

  161. Littlewood TD, Hancock DC, Danielian PS, Parker MG and Evan GI . (1995). Nucleic Acids Res., 23, 1686–1690.

  162. Liu X, Kim CN, Yang J, Jemmerson R and Wang X . (1996). Cell, 86, 147–157.

  163. Lowe SW and Ruley HE . (1993). Genes Dev., 7, 535–545.

  164. Maclean KH, Keller UB, Rodriguez-Galindo C, Nilsson JA and Cleveland JL . (2003). Mol. Cell Biol., 23, 7256–7270.

  165. Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, Doglioni C, Beach DH and Hannon GJ . (1999). Genes Dev., 13, 2207–2217.

  166. Mao DY, Watson JD, Yan PS, Barsyte-Lovejoy D, Khosravi F, Wong WW, Farnham PJ, Huang TH and Penn LZ . (2003). Curr. Biol., 13, 882–886.

  167. Marcu KB, Patel AJ and Yang Y . (1997). Curr. Top. Microbiol. Immunol., 224, 47–56.

  168. Maruyama K, Schiavi SC, Huse W, Johnson GL and Ruley HE . (1987). Oncogene, 1, 361–367.

  169. Mateyak MK, Obaya AJ, Adachi S and Sedivy JM . (1997). Cell Growth Differ., 8, 1039–1048.

  170. Matsuzaki Y, Nakayama K, Tomita T, Isoda M, Loh DY and Nakauchi H . (1997). Blood, 89, 853–862.

  171. Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, Buschmann T, Ronai Z, Shiloh Y, Kastan MB, Katzir E and Oren M . (2001). Genes Dev., 15, 1067–1077.

  172. McKeller RN, Fowler JL, Cunningham JJ, Warner N, Smeyne RJ, Zindy F and Skapek SX . (2002). Proc. Natl. Acad. Sci. USA, 99, 3848–3853.

  173. McMahon SB, Van Buskirk HA, Dugan KA, Copeland TD and Cole MD . (1998). Cell, 94, 363–374.

  174. McMahon SB, Wood MA and Cole MD . (2000). Mol. Cell. Biol., 20, 556–562.

  175. Menssen A and Hermeking H . (2002). Proc. Natl. Acad. Sci. USA, 99, 6274–6279.

  176. Meroni G, Reymond A, Alcalay M, Borsani G, Tanigami A, Tonlorenzi R, Nigro CL, Messali S, Zollo M, Ledbetter DH, Brent R, Ballabio A and Carrozzo R . (1997). EMBO J., 16, 2892–2906.

  177. Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White E and Saikumar P . (2003). J. Biol. Chem., 278, 5367–5376.

  178. Mitchell KO, Ricci MS, Miyashita T, Dicker DT, Jin Z, Reed JC and El-Deiry WS . (2000). Cancer Res., 60, 6318–6325.

  179. Miyashita T, Harigai M, Hanada M and Reed JC . (1994a). Cancer Res., 54, 3131–3135.

  180. Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B and Reed JC . (1994b). Oncogene, 9, 1799–1805.

  181. Miyashita T and Reed JC . (1995). Cell, 80, 293–299.

  182. Moens CB, Auerbach AB, Conlon RA, Joyner AL and Rossant J . (1992). Genes Dev., 6, 691–704.

  183. Momand J and Zambetti GP . (1997). J. Cell. Biochem., 64, 343–352.

  184. Momand J, Zambetti GP, Olson DC, George D and Levine AJ . (1992). Cell, 69, 1237–1245.

  185. Montes de Oca Luna R, Wagner DS and Lozano G . (1995). Nature, 378, 203–206.

  186. Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Negishi I, Senju S, Zhang Q and Fujii S et al. (1995). Science, 267, 1506–1510.

  187. Muller S, Berger M, Lehembre F, Seeler JS, Haupt Y and Dejean A . (2000). J. Biol. Chem., 275, 13321–13329.

  188. Nakano K and Vousden KH . (2001). Mol. Cell., 7, 683–694.

  189. Nakayama K, Negishi I, Kuida K, Sawa H and Loh DY . (1994). Proc. Natl. Acad. Sci. USA, 91, 3700–3704.

  190. Nau MM, Brooks BJ, Battey J, Sausville E, Gazdar AF, Kirsch IR, McBride OW, Bertness V, Hollis GF and Minna JD . (1985). Nature, 318, 69–73.

  191. Neiman PE, Ruddell A, Jasoni C, Loring G, Thomas SJ, Brandvold KA, Lee R, Burnside J and Delrow J . (2001). Proc. Natl. Acad. Sci. USA, 98, 6378–6383.

  192. Nicholson DW . (1999). Cell Death Differ., 6, 1028–1042.

  193. Nilsson JA, Maclean KH, Keller UB, Pendeville H, Baudino TA and Cleveland JL . (2003). Mol. Cell. Biol., in press.

  194. Noguchi K, Kokubu A, Kitanaka C, Ichijo H and Kuchino Y . (2001). Biochem. Biophys. Res. Commun., 281, 1313–1320.

  195. Nomura T, Khan MM, Kaul SC, Dong HD, Wadhwa R, Colmenares C, Kohno I and Ishii S . (1999). Genes Dev., 13, 412–423.

  196. Nunez G, Benedict MA, Hu Y and Inohara N . (1998). Oncogene, 17, 3237–3245.

  197. O’Connell BC, Cheung AF, Simkevich CP, Tam W, Ren X, Mateyak MK and Sedivy JM . (2003). J. Biol. Chem., 278, 12563–12573.

  198. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T and Tanaka N . (2000). Science, 288, 1053–1058.

  199. Orian A, van Steensel B, Delrow J, Bussemaker HJ, Li L, Sawado T, Williams E, Loo LW, Cowley SM, Yost C, Pierce S, Edgar BA, Parkhurst SM and Eisenman RN . (2003). Genes Dev., 17, 1101–1114.

  200. Owen-Schaub LB, Zhang W, Cusack JC, Angelo LS, Santee SM, Fujiwara T, Roth JA, Deisseroth AB, Zhang WW and Kruzel E et al. (1995). Mol. Cell. Biol., 15, 3032–3040.

  201. Owyang AM, Tumang JR, Schram BR, Hsia CY, Behrens TW, Rothstein TL and Liou HC . (2001). J. Immunol., 167, 4948–4956.

  202. Packham G, White EL, Eischen CM, Yang H, Parganas E, Ihle JN, Grillot DA, Zambetti GP, Nunez G and Cleveland JL . (1998). Genes Dev., 12, 2475–2487.

  203. Park J, Kunjibettu S, McMahon SB and Cole MD . (2001). Genes Dev., 15, 1619–1624.

  204. Park J, Wood MA and Cole MD . (2002). Mol. Cell. Biol., 22, 1307–1316.

  205. Peduto Eberl L, Guillou L, Saraga E, Schroter M, French LE, Tschopp J and Juillerat-Jeanneret L . (1999). Int. J. Cancer, 81, 772–778.

  206. Pelengaris S, Khan M and Evan GI . (2002). Cell, 109, 321–334.

  207. Pelengaris S, Littlewood T, Khan M, Elia G and Evan G . (1999). Mol. Cell, 3, 565–577.

  208. Peukert K, Staller P, Schneider A, Carmichael G, Hanel F and Eilers M . (1997). EMBO J., 16, 5672–5686.

  209. Popescu NC and Zimonjic DB . (2002). J. Cell. Mol. Med., 6, 151–159.

  210. Prendergast GC . (1999). Oncogene, 18, 2967–2987.

  211. Prendergast GC and Ziff EB . (1991). Science, 251, 186–189.

  212. Putcha GV, Le S, Frank S, Besirli CG, Clark K, Chu B, Alix S, Youle RJ, LaMarche A, Maroney AC and Johnson Jr EM . (2003). Neuron, 38, 899–914.

  213. Qin XQ, Livingston DM, Kaelin WG, Jr and Adams PD . (1994). Proc. Natl. Acad. Sci. USA, 91, 10918–10922.

  214. Quelle DE, Zindy F, Ashmun RA and Sherr CJ . (1995). Cell, 83, 993–1000.

  215. Rao L, Debbas M, Sabbatini P, Hockenbery D, Korsmeyer S and White E . (1992). Proc. Natl. Acad. Sci. USA, 89, 7742–7746.

  216. Rathmell JC, Lindsten T, Zong WX, Cinalli RM and Thompson CB . (2002). Nat. Immunol., 3, 932–939.

  217. Raveh T, Droguett G, Horwitz MS, DePinho RA and Kimchi A . (2001). Nat. Cell. Biol., 3, 1–7.

  218. Reisman D, Elkind NB, Roy B, Beamon J and Rotter V . (1993). Cell Growth Differ., 4, 57–65.

  219. Renan MJ . (1989). Cancer Lett., 47, 1–9.

  220. Rocha S, Campbell KJ and Perkins ND . (2003). Mol. Cell, 12, 15–25.

  221. Rodriguez MS, Desterro JM, Lain S, Midgley CA, Lane DP and Hay RT . (1999). EMBO J., 18, 6455–6461.

  222. Rosenfeld ME, Prichard L, Shiojiri N and Fausto N. . (2000). Am. J. Pathol., 156, 997–1007.

  223. Roth J, Dobbelstein M, Freedman DA, Shenk T and Levine AJ . (1998). EMBO J., 17, 554–564.

  224. Rounbehler RJ, Rogers PM, Conti CJ and Johnson DG . (2002). Cancer Res., 62, 3276–3281.

  225. Rounbehler RJ, Schneider-Broussard R, Conti CJ and Johnson DG . (2001). Oncogene, 20, 5341–5349.

  226. Ruas M and Peters G . (1998). Biochim. Biophys. Acta, 1378, F115–177.

  227. Ruggero D and Pandolfi PP . (2003). Nat. Rev. Cancer, 3, 179–192.

  228. Russell JL, Powers JT, Rounbehler RJ, Rogers PM, Conti CJ and Johnson DG . (2002). Mol. Cell. Biol., 22, 1360–1368.

  229. Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW and Appella E . (1998). Genes Dev., 12, 2831–2841.

  230. Sawai S, Shimono A, Hanaoka K and Kondoh H . (1991). New Biol., 3, 861–869.

  231. Schmitt CA, Fridman JS, Yang M, Baranov E, Hoffman RM and Lowe SW . (2002). Cancer Cell, 1, 289–298.

  232. Schmitt CA, McCurrach ME, de Stanchina E, Wallace-Brodeur RR and Lowe SW . (1999). Genes Dev., 13, 2670–2677.

  233. Schmidt D and Muller S . (2002). Proc. Natl. Acad. Sci. USA, 99, 2872–2877.

  234. Schuldiner O and Benvenisty N . (2001). Oncogene, 20, 4984–4994.

  235. Schwab M, Varmus HE, Bishop JM, Grzeschik KH, Naylor SL, Sakaguchi AY, Brodeur G and Trent J . (1984). Nature, 308, 288–291.

  236. Scorrano L and Korsmeyer SJ . (2003). Biochem. Biophys. Res. Commun., 304, 437–444.

  237. Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T and Korsmeyer SJ . (2003). Science, 300, 135–139.

  238. Sears R, Leone G, DeGregori J and Nevins JR . (1999). Mol. Cell, 3, 169–179.

  239. Sears R, Ohtani K and Nevins JR . (1997). Mol. Cell. Biol., 17, 5227–5235.

  240. Sedger LM, Glaccum MB, Schuh JC, Kanaly ST, Williamson E, Kayagaki N, Yun T, Smolak P, Le T, Goodwin R and Gliniak B . (2002). Eur. J. Immunol., 32, 2246–2254.

  241. Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY and Hammond D . (1985). N. Engl. J. Med., 313, 1111–1116.

  242. Seoane J, Le HV and Massague J . (2002). Nature, 419, 729–734.

  243. Seoane J, Pouponnot C, Staller P, Schader M, Eilers M and Massague J . (2001). Nat. Cell. Biol., 3, 400–408.

  244. Serrano M, Hannon GJ and Beach D . (1993). Nature, 366, 704–707.

  245. Shan B and Lee WH . (1994). Mol. Cell. Biol., 14, 8166–8173.

  246. Sheiness D and Bishop JM . (1979). J. Virol., 31, 514–521.

  247. Sheiness D, Fanshier L and Bishop JM . (1978). J. Virol., 28, 600–610.

  248. Sheiness DK, Hughes SH, Varmus HE, Stubblefield E and Bishop JM . (1980). Virology, 105, 415–424.

  249. Sherr CJ . (1998). Genes Dev., 12, 2984–2991.

  250. Sherr CJ . (2001). Nat. Rev. Mol. Cell. Biol., 2, 731–737.

  251. Sherr CJ and DePinho RA . (2000). Cell, 102, 407–410.

  252. Shi Y, Glynn JM, Guilbert LJ, Cotter TG, Bissonnette RP and Green DR . (1992). Science, 257, 212–214.

  253. Shiraki K, Tsuji N, Shioda T, Isselbacher KJ and Takahashi H . (1997). Proc. Natl. Acad. Sci. USA, 94, 6420–6425.

  254. Shurin GV, Gerein V, Lotze MT and Barksdale Jr EM . (1998). Nat. Immun., 16, 263–274.

  255. Soengas MS, Alarcon RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW and Lowe SW . (1999). Science, 284, 156–159.

  256. Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald WL, Lazebnik YA, Cordon-Cardo C and Lowe SW . (2001). Nature, 409, 207–211.

  257. Soucie EL, Annis MG, Sedivy J, Filmus J, Leber B, Andrews DW and Penn LZ . (2001). Mol. Cell. Biol., 21, 4725–4736.

  258. Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, Moroy T, Bartek J, Massague J, Hanel F and Eilers M . (2001). Nat. Cell. Biol., 3, 392–399.

  259. Stanton BR, Reid SW and Parada LF . (1990). Mol. Cell. Biol., 10, 6755–6758.

  260. Stewart M, Cameron E, Campbell M, McFarlane R, Toth S, Lang K, Onions D and Neil JC . (1993). Int. J. Cancer, 53, 1023–1030.

  261. Stoneley M, Chappell SA, Jopling CL, Dickens M, MacFarlane M and Willis AE . (2000). Mol. Cell. Biol., 20, 1162–1169.

  262. Strand S and Galle PR . (1998). Mol. Med. Today, 4, 63–68.

  263. Strasser A, Harris AW, Bath ML and Cory S . (1990). Nature, 348, 331–333.

  264. Sugimoto M, Kuo ML, Roussel MF and Sherr CJ . (2003). Mol. Cell, 11, 415–424.

  265. Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M and Kroemer G . (1996). J. Exp. Med., 184, 1331–1341.

  266. Suzuki M, Youle RJ and Tjandra N . (2000). Cell, 103, 645–654.

  267. Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T and Nagata S . (1994). Cell, 76, 969–976.

  268. Takamizawa S, Okamoto S, Wen J, Bishop W, Kimura K and Sandler A . (2000). J. Pediatr. Surg., 35, 375–379.

  269. Tao W and Levine AJ . (1999). Proc. Natl. Acad. Sci. USA, 96, 6937–6941.

  270. Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG, Look AT, Lahti JM and Kidd VJ . (2000). Nat. Med., 6, 529–535.

  271. Tonini GP and Romani M . (2003). Cancer Lett., 197, 69–73.

  272. Trumpp A, Refaeli Y, Oskarsson T, Gasser S, Murphy M, Martin GR and Bishop JM . (2001). Nature, 414, 768–773.

  273. van Loo G, Schotte P, van Gurp M, Demol H, Hoorelbeke B, Gevaert K, Rodriguez I, Ruiz-Carrillo A, Vandekerckhove J, Declercq W, Beyaert R and Vandenabeele P . (2001). Cell Death Differ., 8, 1136–1142.

  274. Vaux DL and Silke J . (2003). Biochem. Biophys. Res. Commun., 304, 499–504.

  275. Veis DJ, Sorenson CM, Shutter JR and Korsmeyer SJ . (1993). Cell, 75, 229–240.

  276. Wagner AJ, Kokontis JM and Hay N . (1994). Genes Dev., 8, 2817–2830.

  277. Wang HG, Rapp UR and Reed JC . (1996). Cell, 87, 629–638.

  278. Wang X . (2001). Genes Dev., 15, 2922–2933.

  279. Wang Z, Bhattacharya N, Weaver M, Petersen K, Meyer M, Gapter L and Magnuson NS . (2001). J. Vet. Sci., 2, 167–179.

  280. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA and Nagata S . (1992). Nature, 356, 314–317.

  281. Watson JD, Oster SK, Shago M, Khosravi F and Penn LZ . (2002). J. Biol. Chem., 277, 36921–36930.

  282. Weber JD, Jeffers JR, Rehg JE, Randle DH, Lozano G, Roussel MF, Sherr CJ and Zambetti GP . (2000). Genes Dev., 14, 2358–2365.

  283. Weber JD, Taylor LJ, Roussel MF, Sherr CJ and Bar-Sagi D . (1999). Nat. Cell. Biol., 1, 20–26.

  284. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB and Korsmeyer SJ . (2000). Genes Dev., 14, 2060–2071.

  285. Weissinger EM, Eissner G, Grammer C, Fackler S, Haefner B, Yoon LS, Lu KS, Bazarov A, Sedivy JM, Mischak H and Kolch W . (1997). Mol. Cell. Biol., 17, 3229–3241.

  286. Wen R, Wang D, McKay C, Bunting KD, Marine JC, Vanin EF, Zambetti GP, Korsmeyer SJ, Ihle JN and Cleveland JL . (2001). Mol. Cell. Biol., 21, 678–689.

  287. White E . (1996). Genes Dev., 10, 1–15.

  288. Wu GS, Burns TF, McDonald 3rd ER, Jiang W, Meng R, Krantz ID, Kao G, Gan DD, Zhou JY, Muschel R, Hamilton SR, Spinner NB, Markowitz S, Wu G and el-Deiry WS . (1997). Nat. Genet., 17, 141–143.

  289. Wu S, Cetinkaya C, Munoz-Alonso MJ, von der Lehr N, Bahram F, Beuger V, Eilers M, Leon J and Larsson LG . (2003). Oncogene, 22, 351–360.

  290. Wu X, Bayle JH, Olson D and Levine AJ . (1993). Genes Dev., 7, 1126–1132.

  291. Wu X and Levine AJ . (1994). Proc. Natl. Acad. Sci. USA, 91, 3602–3606.

  292. Xiao Q, Claassen G, Shi J, Adachi S, Sedivy J and Hann SR . (1998). Genes Dev., 12, 3803–3808.

  293. Xiong Y, Zhang H and Beach D . (1993). Genes Dev., 7, 1572–1583.

  294. Yeh WC, Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shahinian A, Ng M, Wakeham A, Khoo W, Mitchell K, El-Deiry WS, Lowe SW, Goeddel DV and Mak TW . (1998). Science, 279, 1954–1958.

  295. You Z, Madrid LV, Saims D, Sedivy J and Wang CY . (2002). J. Biol. Chem., 277, 36671–366777.

  296. Yu J, Zhang L, Hwang PM, Kinzler KW and Vogelstein B . (2001). Mol. Cell, 7, 673–682.

  297. Yu Q, He M, Lee NH and Liu ET . (2002). J. Biol. Chem., 277, 13059–13066.

  298. Zambetti GP, Bargonetti J, Walker K, Prives C and Levine AJ . (1992). Genes Dev., 6, 1143–1152.

  299. Zambetti GP and Levine AJ . (1993). FASEB J., 7, 855–865.

  300. Zeller KI, Haggerty TJ, Barrett JF, Guo Q, Wonsey DR and Dang CV . (2001). J. Biol. Chem., 276, 48285–48291.

  301. Zeller KI, Jegga AG, Aronow BJ, O'Donnell KA and Dang CV . (2003). Genome Biol., 4, R69.

  302. Zha J, Harada H, Yang E, Jockel J and Korsmeyer SJ . (1996). Cell, 87, 619–628.

  303. Zha J, Weiler S, Oh KJ, Wei MC and Korsmeyer SJ . (2000). Science, 290, 1761–1765.

  304. Zindy F, Eischen CM, Randle DH, Kamijo T, Cleveland JL, Sherr CJ and Roussel MF . (1998). Genes Dev., 12, 2424–2433.

  305. Zornig M, Grzeschiczek A, Kowalski MB, Hartmann KU and Moroy T . (1995). Oncogene, 10, 2397–2401.

  306. Zou X, Lin Y, Rudchenko S and Calame K . (1997). Curr. Top. Microbiol. Immunol., 224, 57–66.

Download references

Acknowledgements

We thank current and past members of the laboratory for their efforts, dedication and insights into Myc, cell suicide and cancer. We are also deeply indebted to the Roussel-Sherr, Zambetti and Ihle laboratories for our exciting and productive collaborations over the past decade. We apologize for any omissions of work from our colleagues relevant to the problem, as tackling Myc and apoptosis certainly ‘takes a village’. It was simply our intent to focus the review on those pathways shown to be most relevant to cancer. Our research is supported by grants from the NIH and by the American Lebanese Syrian Associated Charities (ALSAC). JA Nilsson is a George J. Mitchell Endowed Postdoctoral Fellow of St Jude Children's Research Hospital.

Author information

Correspondence to John L Cleveland.

Rights and permissions

Reprints and Permissions

About this article

Keywords

  • Myc
  • apoptosis
  • Arf
  • p53
  • Bcl-2
  • death receptors
  • cancer

Further reading