Oncogenes and cell death

Oncogenes Elizabeth A Harrington, Imperial

Cancer

and cell death

Abdallah

Fanidi and Gerard

Research Fund, London,

I Evan

UK

Several recent studies have implicated oncogenes and tumour suppressor genes in the regulation of programmed cell death fapoptosis). Lesions in the cell death pathway appear to be important in both carcinogenesis and the evolution of drug resistance in tumours. They include deregulated expression of genes such as M-2, loss of ~53, and autocrine activation of anti-apoptotic signal transduction pathways. Paradoxically, a number of dominant oncogenes appear to act as potent inducers of apoptosis. This suggests that the pathways of cell proliferation and cell death may be tightly coupled, an idea that may have dramatic implications for models of oncogene co-operation and carcinogenesis.

Current Opinion

in Genetics

and Development

Introduction Although the incidence of cancer is common, affecting one in three persons, it is a clonal disease that arises through expansion of a single affected cell. This implies that such a cell arises only once in every three individuals during the entire course of their lives, out of all the billions of proliferating cells within the body. The cancer cell is, therefore, extremely rare. This rarity is in itself surprising because, in principle, any mutant cell that achieves some growth advantage over its fellows might be expected to undergo clonal expansion and thereby provide an increased target site for yet further carcinogenic mutations. Indeed, most models of carcinogenic progression follow this principle of sequential accumulation of lesions that progressively increase malignant potential. Carcinogenic progression would thus appear to be an inevitable consequence of natural selection within the soma-given enough mutations. The chances of such mutations arising are obviously increased in organisms like man that are physically large (and thus comprise large numbers of target cells), long-lived, and in which substantial cell proliferation continues throughout life. The extreme rarity of the cancer in man must, therefore, imply the existence of powerful mechanisms to suppress neoplasia. One possible anti-neoplastic mechanism involves activation of cell suicide pathways in those cells at risk of neoplastic transformation. This idea has received support from the fact that a number of oncogenes and tumour suppressor genes appear to exert a direct influence on the survival and death of cells. Programmed cell death is an important homeostatic regulator of tissue mass and architecture during development and also in the adult, and has been substantially conserved

1994,

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throughout multicellular evolution 11-61. In this review, we discuss the known relationships between oncogenesis and programmed cell death (apoptosis), and present some ideas as to how the processes of cell proliferation and cell death are linked. We suggest a model in which obligatory coupling of cell proliferative and cell suicide pathways provides a powerful and inbuilt mechanism for suppressing neoplastic transformation. If correct, this model may well provide novel approaches for pharmacological intervention in cancer.

Dominant

oncogenes

and induction

Proto-oncogenes have a well established role as genes encoding components of signal transduction pathways, promoting cell proliferation and differentiation. Recently, some dominant oncogenes have been shown to exhibit a novel and surprising biological property: the ability to trigger programmed cell death. This raises one particularly intriguing question: is the induction of cell death by oncogenes an aspect of some normal function exerted by these signal transduction elements, or is it merely a catastrophic consequence of their inappropriate or deregulated expression?

MYC

The c-myc proto-oncogene encodes a short-lived, sequence-specific DNA-binding protein, whose expression is elevated or deregulated in virtually all tumours 171. Evidence favours the notion that the c-myc protein, c-Myc, is a transcription factor. It possesses an amino-terminal domain with transcriptional

Abbreviations bHLHLZ-basic helix-loop-helix PDCF-platelet-derived

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leucine zipper; ICF-insulin-like growth growth factor; TNF-tumour necrosis factor.

Biology

of cell death

Ltd ISSN 0959-437X

factor;

Oncozenes

activating activity, and a carboxy-terminal DNA-binding/dimerization basic helix-loop-helix leucine zipper (bHLHLZ) domain akin to that present in several known transcription factors 18-121; however, target genes for c-Myc have not yet been well defined (reviewed in 1131). The tight autoregulation normally exhibited by c-myc 114,151 suggests that such altered expression as seen in tumours is unlikely to be a mere consequence of upstream oncogenic mutations, and probably results from lesions within c-myc itself. Expression of c-myc (or of another member of the myc gene family) appears to be necessary and, in some cases, sufficient for cell proliferation (reviewed in 1131). Deregulated c- rnyc expression is associated with inability to withdraw from the cell cycle [12,16,17*1, and suppression of differentiation W-221. In summary, it seems likely that c-myc encodes a transcription factor that promotes cell proliferation and suppresses growth arrest by modulation of appropriate growth-related target genes. Given its growth promoting and oncogenic properties, it is perhaps surprising that c-Myc has for some time been recognized as a potent inducer of cell death. Transgenic animals whose lymphocytes express deregulated c-myc often exhibit increased sensitivity to induction of apoptosis in lymphoid organs 123-251. There is substantial oncogenic synergy in transgenic mice expressing both c-myc and the anti-apoptotic gene bcl-2, suggesting that cell death is an important limitation in the oncogenic development of proto-tumours driven by c-myc alone 1261. Finally, high level expression of c-myc under inducible promoters appears to be toxic for cells 1271. In general, this toxicity associated with c-myc was largely assumed to be a non-specific consequence of its altered expression. However, recent in uifro studies of the induction of cell death by c-Myc have demonstrated that death occurs by the active process of apoptosis, and is aggravated by deprivation of growth factors or application of cytotoxic agents 117,281. Moreover, the cytotoxic attribute of cMyc is inseparable from its mitogenic property. Higher levels of expression of c-Myc correlate both with increased proliferative rate and with increased sensitivity to apoptosis 117’1. Identical regions of the c-Myc protein are required for both growth promotion and induction of apoptosis - specifically, the amino-terminal transactivation domain and the carboxy-terminal DNA-binding and dimerization bHLHLZ domain 117’1 - and dimerization with the heterologous partner protein, Max, is necessary for both transforming and apoptotic functions of c-Myc 1121.These latter two observations strongly suggest that c-Myc induces apoptosis via a transcriptional mechanism, presumably by modulating appropriate target genes. Two simple models have been invoked to explain the induction of apoptosis by c-Myc following serum deprivation or treatment with cytotoxic agents (Fig. 1). The first argues that cell death arises because of a conflict in signals between the growth promoting action of c-Myc and the growth suppressive or cytostatic effects of low serum levels or drugs (for example, see

and cell death

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129,301; Fig. la). Induction of apoptosis is, therefore, a pathological consequence of ‘inappropriate’ c-Myc expression coupled with impeded growth, and is not a normal function of c-Myc. An alternative to this ‘conflict’ model is to propose that the induction of an apoptotic programme is a bonafide and obligate component of c-Myc action that necessarily accompanies proliferation, meaning that proliferation and apoptosis are coupled. In order to proliferate successfully, a cell would require two independent sets of signals, one to trigger mitogenesis and the other to suppress the concomitant apoptotic programme (Fig. lb). According to this ‘dual signal’ model, cells expressing c-Myc die in low serum, not because of a conflict in growth signals, but because they are deprived of specific serum factors that suppress the c-Myc induced apoptotic programme. Moreover, this model offers an alternative explanation as to why expression of c-Myc induces apoptosis in cells exposed to cytotoxic and cytostatic agents. The c-Myc-protein induces apoptosis in drug-treated cells, not because of a conflict between the growth promoting effects of c-Myc and the cytostasis induced by the drug, but because cells expressing c-Myc have a primed apoptotic pathway and are poised to commit suicide in response to genotoxic factors. The attractive biological rationale for the ‘dual signal’ model is that it intrinsically suppresses neoplastic transformation. Any mitogenic lesion in a dominant oncogene necessarily drives both cell growth and cell suicide. Thus, the affected clone will spontaneously delete itself when it outgrows the paracrine environment that supplies it with survival factors - in effect, surveillance against neoplastic transformation is hardwired into the proliferative machinery. Several lines of evidence favour the ‘dual signal’ model. Fibroblasts expressing c-Myc undergo apoptosis in the presence of cycloheximide 117’1, implying that all the machinery necessary for apoptosis pre-exists in cells as a consequence of c-Myc expression. This indicates that the transcriptional apoptotic programme implemented by c-Myc does not arise as a consequence of a conflict in growth signals. Rather, it is already present, although suppressed, in cells that exhibit no overt sign of apoptosis - for example fibroblasts expressing high levels of c-Myc growing in high serum. The clear implication is that serum contains agents that suppress the apoptotic programme. Recently, research in my laboratory has identified the principal elements within serum that suppress c-Myc induced apoptosis in fibroblasts: the insulin-like growth factors (IGFs) and platelet-derived growth factor (PDGF). Other tested mitogens (e.g. epidermal growth factor, fibroblast growth factor, and bombesin) possess no anti-apoptotic activity. Intriguingly, the abilities of IGFs and PDGF to block c-Myc induced apoptosis are not dependent upon the mitogenic activity of either factor, because both suppress apoptosis under conditions in which cell proliferation is profoundly blocked with cytostatic agents and during the post-commitment (growth factor independent) S/G2 phases of the cell cycle (EA Harrington, A Fanidi, M Bennett, GI Evan, unpublished data). In this

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Oncogenes

(a) Conflic;

and cell proliferation

model Apoptosis Low serum/ cytostatic drugs

Mitogens

Proliferation

(b) Dual signal model Proliferation

Mitogens Apoptosis q ‘;u,~~~’

/ Bcl-2

D 1994 Currenl Opinion in Genetics and Development

Fig. 1. Two alternative models to explain Myc-induced apoptosis in serum-deprived fibroblasts. (a) The conflict model. In this model, Myc produces two signals: the first for proliferation, mediated by mitogens, and the second for induction of apoptosis, mediated by low serum or cytostatic drugs. induction of apoptosis arises, therefore, from a conflict in growth signals from mitogens and low serum. (b) The dual signal model. In this model, induction of apoptosis is a normal and obligate function of Myc that is regulated by availability of anti-apoptotic survival factors and Bcl-2.

way, anti-apoptotic cytokines are acting analogously to B&2, which also blocks c-Myc induced apoptosis [31e-33el yet has no detectable mitogenic potential 126,341 (discussed below). The idea that cytokines that inhibit apoptosis do so in a manner unlinked to their mitogenic activity, fits well with the known survivalpotentiating activities of both IGF-1 and PDGF in postmitotic cells, such as neurons [35*,361. Presumably, other cell lineages are dependent upon different survival factors. For example, interleukin-3 appears to be a major anti-apoptotic cytokine for many haematopoietic lineages 137-391.

Other

dominant

oncogenes

The idea that there is an obligate link between proliferation and apoptosis is reinforced by studies with other dominant oncogenes. Adenovirus ElA is the principal early gene responsible for driving host cell cycle progression, which the virus requires for productive infection. ElA encodes a highly evolved multi-functional polypeptide which, like c-Myc, is both oncogenic and a potent inducer of apoptosis 1401.In order to defeat the obligate linkage between proliferation and cell death, and so allow the virus to gain a replicative toe-hold, adenovirus has evolved two independent anti-apoptotic mechanisms, both encoded by the ElB gene. The p55ElB protein sequesters and inactivates ~53 [41,421, a component of some cell suicide pathways (see below), and the pl9ElB protein is a functional homo-

logue of Bcl-2 [40,431. Another example of the apparent obligate growth/death dual function of oncogenes is the chimaeric homeobox oncogene E2A-PBXl, generated during t(1;19) chromosomal translocations in childhood leukaemia. Transgenic mice that constitutively express E2A-PBXl in lymphocytes show high incidence of lymphomas, attesting to the oncogenic nature of the gene. However, they also exhibit evidence of massive lymphocyte apoptosis in the pre-malignant phase [44*1. The c-fos oncogene is also implicated in the control or execution of apoptosis. Recent studies of the topographical expression of a transgenic LacZ marker driven by the c-fos regulatory element in mouse embryogenesis [451 suggest that sustained expression of c-fos coincides with regions that will undergo apoptosis. At present it is unclear whether such c-fos expression is part of a cell-autonomous suicide programme, or whether it merely indicates stimulation of the affected cells by some external suicide signal, such as tumour necrosis factor (TNF) or Fas (see below). As for c-Myc, however, deregulated expression of c-fos induces apoptosis in serum-deprived primary fibroblasts. Thus, substantial evidence is emerging that activated mitogenic proto-oncogenes can be potent triggers of apoptosis. This presents an interesting paradox, because observation tells us that activated forms of the same genes are important elements in carcinogenesis; indeed some, like c-myc, appear to be universally activated in tumours. The clear implication must be that such tumours must have evolved mechanisms to suppress the obligate apoptosis associated with expression of dominant oncogenes. Recently, a variety of such anti-apoptotic lesions have been identified.

Oncogenes cell death

and the suppression

of programmed

Bcl-2

If the proliferative and apoptotic pathways are coupled in vertebrate cells, then mechanisms that suppress apoptosis are likely to be important determinants in promoting carcinogenesis and drug resistance. An example of such a death suppression is the deregulated expression of the hcl-2 proto-oncogene. This gene was first identified as the site of reciprocal translocation on human chromosome 18 in follicular B cell lymphoma, and encodes a membrane associated protein, Bcl-2, that is present in endoplasmic reticulum, nuclear and outer mitochondrial membranes [46,471. Bcl-2 is widely expressed during embryonic development, but in the adult is confmed to immature and stem cell populations, and long-lived cells such as resting B lymphocytes and peripheral sensory neurons 1481. Targeted expression of Bcl-2 to lymphoid cells in transgenic mice leads to an increase in the numbers of mature resting B cells and potentiates their longevity. Affected T cells are markedly resistant to the cytocidal effects of radiation, glucocorticoids and anti-CD3, but thymic

OncoEenes

censorship appears normal. Bcl-2 transgenic mice go on to develop a low incidence of malignant lymphoma. However, co-expression of c-myc with hcl-2 gives rise to a markedly enhanced incidence of tumours, significantly greater than that seen with either hcl-2 or c-myc alone (reviewed in [491>. Similar synergy between bcl-2 and c-myc is observed in vitro [26,31*-33’1, and occurs because expression of bcl-2 specifically blocks the ability of c-myc to induce apoptosis [31*-33.1 without affecting its mitogenic capacity [33’1. Expression of Bcl-2 similarly blocks ElA-induced apoptosis and can functionally replace pl9El~~ 1501. B&2 expression also protects cells from the cytocidal effects of a variety of toxic agents [51-531, and abrogat’es the increased sensitivity to cytotoxic and cytostatic agents elicited by expression of c-Myc [33’1. However, Bcl-2 does not appear to mediate its antiapoptotic effects through enhanced capacity to exclude or metabolize drugs, nor does it confer any direct increased physical resistance to genotoxic agents t541 per se. Rather, it acts to suppress the tendency of damaged cells to commit suicide despite any damage they might sustain. Thus, direct suppression of apoptosis represents a discrete mechanism of resistance to anti-neoplastic drugs that may have important implications in the effective design of future cancer therapies. B&2 is one of a growing family of related proteins that have been conserved throughout multicellular evolution, and which include Ced-9 of C. elegans [31, the mammalian proteins Bcl-Xs and B&XL 155’1, M&l 1561, Al [571 and Bax 158’1, and the viral proteins p30 (baculovirus) [591, BHRFl (Epstein-Barr virus) [6Ol, VGl6 (Herpesvirus saimiri) [611, LMWS-HL (African Swine Fever virus) 1621and p19 E*u (adenovirus) [40,501. Functionally, members of the family fall into two camps. B&2, Bcl-XL, BHRFl, p19Ell’, MCI-1 and Ced-9 all inhibit programmed cell death induced by factor deprivation, deregulated c-Myc or genotoxic damage ([50,63’,641; A Fanidi, EA Harrington, GI Evan, unpublished data). In contrast, both Bax and the smaller splice variant of B&X, Bcl-Xs, antagonize the antiapoptotic activity of Bcl-2. Bax heterodimerizes with Bcl-2 and thereby antagonizes its anti-apoptotic activity 158.1. It is unclear, however, whether Bcl-2 blocks a default Bax-dependent suicide pathway, whether Bax blocks a B&2 mediated survival function, or whether the relative levels of Bcl-2 and Bax simply determine the propensity of an individual cell for programmed cell death, as has been proposed 158’1. In contrast to Bax, Bcl-Xs does not heterodimerize with B&2, but appears to antagonize Bcl-2 by interfering with upstream or downstream effecters of B&2 [55-l. It is not known whether Bcl-Xs can also antagonize Bax, and so suppress cell death. B&2 knockout mice exhibit stunted growth and postnatal mortality. Initially, haematopoietic development is normal, but is followed by complete apoptotic involution of primary lymphoid organs. Animals develop polycystic kidneys with concomitant renal failure, fail to develop pigment during the second hair

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follicle cycle and consequently turn grey at five to six weeks. Nevertheless, the biochemical functions of B&2 remain unknown, although there are some data to support a role in the control of oxidative damage 165,661. Bcl-2 has also been shown to interact with the R-Ras 23 kDa protein believed to be involved in some aspects of signal transduction 167’1.

Other

dominant

anti-apoptotic

lesions

Deregulated expression of Bcl-2 or its homologues currently represents the best-defined class of dominant anti-apoptotic lesion. However, there are hints that other classes of gene might also be involved. The discovery of anti-apoptotic cytokines and their attendant signal transduction pathways raises the real possibility that autocrine loops within such pathways might contribute to the suppression of apoptosis during carcinogenesis. In fibroblasts, for example, activation of the IGF-1 signalling pathway confers similar attributes upon a cell as does deregulated expression of Bcl-2 (EA Harrington, A Fanidi, M Bennett, GI Evan, unpublished data). Affected cells become factor-independent for survival, and become more resistant to the lethal effects of dominant oncogenes or cytotoxic and genotoxic agents. Mutational activation of the IGF-1 receptor and its downstream effecters is, therefore, an example of a further potential class of anti-apoptotic oncogenic lesion. Other cell lineages presumably possess their own characteristic survival pathways which could become subverted in an analogous way; for example, the IL-~ signalling pathway in haematopoietic ceils. The existence of anti-apoptotic signalling pathways and the dependence of cells upon them for survival also underscores the potential importance of the localized cellular environment in determining the survival and evolution of tumour cells. A number of cytokine-mediated pathways for the triggering of apoptosis have been identified, implying that some degree of control over cell death is exerted through killing rather than suicide. The TNF [681, Fas [69-711, and low affinity nerve growth factor receptors 1721 are all examples of related receptors that trigger apoptosis when engaged by appropriate ligands. In all cases, Ile nova protein synthesis is not required for cell death to occur, implying the pre-existence of apoptotic machinery in susceptible cells. However, sub-lethal stimulation of the TNF pathway does cause the rapid induction of a number of genes whose expression can then ameliorate any subsequent suicidal response to TNF. One such gene, A20, encodes a novel zinc finger protein of unknown molecular function, which confers resistance to TNF when expressed in naive cells [73*1. Moreover, a survey of A20 expression in breast carcinomas showed a strong correlation between high levels of expression and resistance to TNF 173.1. At present, it is not known whether A20 acts as a generic anti-apoptotic agent, or whether its action is restricted to TNF-mediated killing. If the latter is the case, then its role in tumorigenesis would presumably be significant only in instances in which TNF/Fas-type

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killing was a restraint to neoplastic transfo.nnation. Intriguingly, A20 [741, like Bcl-2 [751, is induced by the Epstein-Barr virus membrane-associated oncoprotein, latent membrane protein 1 (LMPl).

The role bf tumour death

suppressor

genes in cell

P53

Loss or inactivation of the p53 tumour suppressor gene is the most common single lesion in human neoplasia 176-781. Introduction of wild type ~53 into p53 negative tumour cells inhibits proliferation by implementing a Cl arrest l79-811; attesting to its action as a growth suppressor. Levels of ~53 rapidly increase following DNA damage 1821, mainly through stabilization of the normally short-lived ~5.3 protein, and this appears to be an important component of the Cl arrest that follows DNA damage [831. In part, growth arrest is mediated through p53-dependent transactivation of genes [84’,851; for example GADD45, a growth-arrest and DNA damage-responsive gene deleted in patients suffering from some forms of ataxia-talangiectasia 184’1, and WAFlKIPl, which encodes an inhibitor of Cl @in-dependent kinases [86’,87’1. Tumour cells with spontaneous loss of ,p53, and cells derived from ~53 knockout mice, fail to arrest in Cl following y-irradiation. Consequently, they enter S-phase with unrepaired DNA, and sustain a substantially elevated risk of further mutation [831. The notion that ~53, in addition 10 implementing a cell cycle checkpoint, might also be an important component of the apoptotic pathway, was suggested by observations that the re-introduction of wild-type ~53 into p53-negative tumour cells triggers apoptosis [88*,8Yl, and was subsequently cotirmed by studies with ~53 knockout mice. The p53-negative mice develop apparently normally, but exhibit a markedly increased susceptibility to neoplasia - almost all develop tumours by six months of age 190’1. Thymocytes derived from p53-negative mice are markedly radiation and drug resistant when compared with thymocytes derived from normal isogenic animals [91,92*1. However, not all pathways triggering apoptosis require ~53, because p53nuli thymocytes remain normally susceptible to cytocidal effects of glucocorticoids, a pathway that does not involve DNA damage. The simplest conclusion is, therefore, that ~53 is an essential part of a genotoxic damage pathway leading to apoptosis (referred to by Lane as ‘Guardian of the genonle’ 19311,but is not involved in non-toxicological triggers of apoptosis. However, there are several caveats to this idea. First, although ~53 null mice show a markedly increased incidence of tumours, this in fact represents only a lO-20-fold increase over that seen in normal mice. Such increased ,tumour incidence is not, arguably, so dramatic given that ~53 is absent from all cells, any one of which could give rise to a tumour. This suggests the existence of p53-independent mechanisms for

suppressing tumorigenesis. Second, cells from p53null mice retain their capacity to undergo apoptosis in response to genotoxic damage, it merely requires more extreme damage to initiate it. Thus, ~53 cannot be an obligate component of the apoptotic machinery. Third, induction of apoptosis by c-Myc (A Fanidi, GI Evan, unpublished data) and ElA [941 requires ~53, although neither oncogene directly induces DNA damage. In the case of ElA, induction of apoptosis may arise because ElA induces stabilization and accumulation of ~53 1951. However, c-Myc causes no equivalent stabilization or accumulation of ~53, even under conditions of serum deprivation (A Fanidi, T Littlewood, GI Evan, unpublished data). More generally, if the sole function of ~53 is to respond to DNA damage, it is not immediately obvious why so many DNA hmiour viruses have evolved such elaborate mechanisms to inactivate it [42,961. A real possibility exists, therefore, that the role of ~53 in mediating apoptosis may not be restricted solely to responding to DNA damage. This conclusion is particularly germane to ~53 knockout studies, the underlying assumption of which is that animals and cells so derived are otherwise normal apart from the specifically engineered lesion. If, however, ~53 were involved in physiological processes of apoptosis during development, significant selection might occur even within the soma of p53nu” animals. Cells derived from such animals might therefore differ from their normal counterparts in a variety of arcane and unpredictable ways. Nonetheless, it is clear that ~53 plays a pivotal role in mediating the cellular response to DNA damage, and orchestrates both a Cl growth arrest and the decision to commit suicide. Precisely how it integrates these two functions, however, remains a mystery. One particularly important question is whether the transactivating function of ~53 is required for both growth arrest and induction of cell death.

The retinoblastoma

protein

The retinoblastoma susceptibility locus encodes a 105 kDa nuclear protein, pRb, that has a well described role as a regulator of cell cycle progression (97-1011. Loss of pRb occurs in a number of malignancies (reviewed in [99,1021), and re-introduction of pRb into pRb-negative tumour cells suppresses tumorigenicity and the neoplastic phenotype [103,1041. As with ~53, the retinoblastoma protein is a principal target for functional inactivation by early proteins of the DNA tumour viruses [105,1061. Although there is no evidence of a direct role for pRb in mediating programmed cell death, pRb-knockout mice exhibit both abnormal cell proliferation and massive apoptosis in certain tissues, for example the central nervous system and haematopoietic organs. Inappropriate proliferation in pRbnul1 mice is consistent with the established role of pRb in regulating cell cycle progression, but the substantial apoptosis observed is more surprising and has no immediate explanation. If, however, proliferation and apoptosis are coupled processes, as we have suggested, then apoptosis might occur because cells lacking pRb are

Oncogenes

unable to stop proliferating and, as a consequence, rapidly exhaust from their local environment those factors necessary for their survival.

Control of cell death by oncogenes and tumour suppressor genes: implications for carcinogenesis Suppression

of cell death in carcinogenesis

The propensity of dominant oncogenes to drive apoptosis, the frequent inactivation of ~53 in tumours, and the identification of dominant oncogenes that promote survival rather than cell proliferation, all attest to the importance of suppression of programmed cell death during carcinogenesis. To date, only a very limited number of anti-apoptotic lesions have been identified in turnours. However, this probably has more to do with an historical emphasis on processes driving cell proliferation/cell-cycle progression, rather than any real paucity of anti-apoptotic mechanisms. Indeed, the involvement of cytokines and signalling pathways, anti-apoptotic genes like bcl-2, and tumour suppressor proteins in apoptosis, all suggest that the mechanisms regulating programmed cell death may be as diverse and complex as those regulating proliferation and differentiation. Identification of mechanisms by which tumour cells evade cell death is important, because it may reveal novel targets for therapeutic intervention in cancer and degenerative diseases. Although suppression of cell death appears to be strongly selected during carcinogenesis, it is intriguing that it never appears to involve deletion of the basal apoptotic machinery itself. Cells that have lost ~53, or that have high level expression of Bcl-2 or its homologues, retain the ability to undergo apoptosis, they merely have a raised threshold at which it is triggered. This highlights an interesting aspect of apoptosis, namely the apparent dichotomy between processes that establish and foster the cells’ potential to undergo apoptosis, and those that are actually involved in dismantling the dying cell. All known antiapoptotic lesions in tumours appear to be examples of the former. This may imply that the basal apoptotic machinery cannot be lost because it is entirely comprised of components of some other essential process, for example, cell cycle progression. This would make biological sense, in that any cell that loses the ability to suicide would necessarily ‘fail-safe’ as a cell unable to proliferate and so constitute no neoplastic risk.

Oncogene

co-operation

Suppression of cell death is produced by a type of carcinogenic lesion that is functionally distinct to those that promote cell proliferation. Clearly, the two different types of lesion would be expected to synergize, especially if, as suggested above, the pathways of cell proliferation and cell death are coupled. Such synergy

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is indeed observed - for example between c-myc and bcl-2 - but it differs from the classical form of oncogene co-operation, such as .that observed between cmyc and rus. Fibroblasts co-expressing c-m. and ra.s exhibit morphological transformation, loss of contact inhibition and focus formation [lOfl. However, such cells die in low serum as quickly as those expressing c-myc alone, meaning that activated rras has no antiapoptotic activity in fibroblasts (A Fanidi, GI Evan, unpublished data). In contrast, cells co-expressing cmyc and bcl-2 exhibit a normal flat morphology and do not form foci [33’, 1081. They are, however, resistant to both serum deprivation and cytotoxic drugs (33’1. Thus, oncogene co-operation encompasses at least three independent-types of attribute (exemplified in Fig. 2 by Myc, Ras and Bcl-2). Assessment of the contributions of each in cancer should prove enlightening.

(integration

of signalling

pathways)

18 1994 Current Opinion in Genetics and Lkvelopmn~ J

Fig. 2. Interaction of oncogenes. At least three independent attributes can be ascribed to known dominant oncogenes: Myc promotes proliferation, Bcl-2 suppresses cell death, and Ras serves to integrate signalling pathways. Cooperation can occur between oncogenes of differing attributes, for example between Myc and Ras to give transformed loci.

We have argued th& the obligate dual action of c-Myc in promoting the contradict&y processes of proliferation and programmed cell death represents an important restraint on carcinogenic progression. This is because mutations that activate myc genes are intrinsically unstable and result in the suicide of any affected cell that outgrows its limited supply of survival factors. The intrinsic self-limitation of c-Myc mutations may represent a general evolutionary strategy to suppress neoplasia. For instance, introduction of activated TU.S into many types of naive cell, not only induces morphological changes consistent with transformation, but also produces profound growth arrest [lo!+1121. Similarly, expression of Bcl-2 in naive cells blocks apoptosis, but also suppresses cell proliferation (J Marvel, M Collins, personal communication; A Fanidi, unpublished data). Thus, independent activation of each of the three oncogenes, myc, ras and bcl-2, suppresses the possibility of further mutation and carcinogenic progression, either by killing the affected cell or by preventing its replication. Accordingly, a tumour cell might only arise through the simultaneous acquisition of at least two independent mutations, each of which counteracts the obligate selective disdavantages of the other.

For example, the lethal effects of Myc or ElA would be suppressed by Bcl-2, whilst c-Myc in turn ‘might overcome the growth suppressive effects of B&2 or Ras. This notion raises the intriguing possibility that tumour cells may possess certain combinations of mutations that are obligatorily inter-locked, in which case pharmacological interference with any one of these critical lesions might prove sufficient to unpick the neoplastic knot and trigger involution of the tumour.

Acknowledgements

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1s.

PENN L, BROOKS M, LAUFEHE, Linuzwoon T, MORG~N~IXI~N J, EVAN G, LEE W, LAN0 H: Domains of Human c-myc Protein Required for Autosupprcssion and Co-operation with ras Oncogcncs arc Overlapping. Mol Cell Biol 1990, 10:49614966.

16.

EILERSM, SCHIRMS, BISHOP JM: The IMYC Protein Activates Transcription of the Alpha-Prothymosin Gene. f%fLIOj 1991, 10:133-141.

17. .

We wish to thank the participants of the recent Danbury Centre Meeting on Cell Death (Ott ,12-14th, 1993) for their invaluable comments and discussion.

EVAN G. WYLLIE A. GII.I)ERT C, LITI~.EWO~D T, LANI) H, BROOKS M. WAIFRS C. PENN L, HANCOCK D: Induction of Apoptosis in Fibroblasts by c-myc Protein. Cell 1992, 63:119-125. The c-Myc protein induces apoptosis in serum-deprived or drug treated Iibrobiasts. Induction of apoptosis by c-Myc requires the same functional domains as does induction of cell growth. 18.

COPPOLAJA, COLE MD: Constitutivc c-myc Oncogcnc Expression Blocks Mouse Erythrolcuhcmia CciI Diffcrcntiation But Not Commitment. Nutfrre 1986, 320:760-763.

Papers of particular interest, published within the annual period of review, have been highlighted as: . of Sp&d interest .. of outstanding Interest

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DMITROVSKYE, KUEHL WM, HOLLISGF, KIHSCHIR. BENIXR TP, SEGAL S: Expression of a Transfcctcd Human c-myc Onto gene Inhibits Diffcrcntiation of a ~Mousc Etythrolcukacmia Cell Line. Nature 1986, 322:74&%7SO.

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EA Harrington, A Fanidi and GI Evan, Biochemistry of the CeU Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WCZA 3PX, UK.

129