Is your initiator really necessary?

J. theor. Biol. (1986) 122, 359-374

Is Your Initiator Really Necessary? R. J. EPSTEIN

University Department and M R C Unit of Clinical Oncoiogy and Radiotherapeutics, Medical Research Council Centre, Hills Rd, Cambridge, U.K. (Received 1 March 1986, and in revised form 6 May 1986) There is little hard evidence for the involvement of specific genotoxic initiators in the pathogenesis of the common carcinoma. Recent findings suggest that sporadic

carcinogenesis is a dynamic and probabilistic process which requires a critical mass of abnormal cells for its expression, and that this requirement may distinguish the evolution of carcinomas from that of paediatric or haematologic malignancies. The proposal that specific carcinogens are neither necessary nor sufficient for tumourigenesis is consistent with the growing realization that aberrant expression of specific oncogenes is neither necessary nor sufficient for cellular transformation. These new perspectives have major implications for basic research strategy and public health policy. Introduction

Cancer is perhaps the most bewildering affliction of modern Western civilization, and as such it is not surprising that many theories have been advanced to explain its occurrence (Comings, 1973; Holliday, 1979; Totter, 1980; Sporn & Todaro, 1980; Cairns, 1981; Rubin, 1985). The most durable of these has been the somatic mutation hypothesis (Boveri, 1929) which states that neoplasms arise following clonal proliferation of a cell that has been transformed by acquired modification of its D N A base-sequence. Central to this hypothesis is the idea that the early stage of transformation is a discrete event, a specific molecular aberration occurring within a single cell. This view of tumour initiation as a highly specific process has gained support from reports linking specific point mutations with oncogene activation (Tabin et al., 1982), specific amino acid substitutions with oncogene product transforming ability (Reddy et al., 1982) and specific subcellular lesions with mutagenicity in vitro (Newbold et al., 1980). Whether such apparent specificity implies the involvement of equally specific environmental carcinogens in the genesis of common human malignancies is far from clear, however. It has long been appreciated that cellular transformation may be induced by genotoxic agents, and that the appearance of tumours in experimental animals may be manipulated by varying the exposure sequence to both mutagenic and non-mutagenic substances (Friedewald & Rous, 1944; Berenblum & Shubik, 1947). To assume by analogy that occult genotoxins are a defining principle of human carcinogenesis is therefore an attractive option from several viewpoints: for the layman, it provides a simple and logical explanation o f a complex and perplexing phenomenon; for the epidemiologist, it offers a constructive approach to cancer 359

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prevention which appears feasible in the medium term; while for the scientist, it suggests a reproducible and quantitative method of analysing malignant transformation in laboratory animals and cell culture. These positive considerations have encouraged the hope that 60-90% of human cancers may ultimately be prevented by improved application of current research strategies 'Perera & Weinstein, 1982). Cases Out o f Control

Yet direct evidence for an obligate initiation-mutation phase in the genesis of sporadic malignancies is lacking (Rubin, 1980), casting doubt on the assumption that environmental genotoxins play a central role in human carcinogenesis. When viewed in context with the slow rate of progress in clinical and preventive oncology (Kolata, 1985a) and the recent rapid advances in basic cell research (Yarbro, 1985), this suggests that a fresh approach to the question of tumour evolution may be timely. Indeed, several such approaches have recently been proposed which eschew the notion of tumourigenesis as a specific process (Weinstein et al., 1984a; Farber, 1984a). Why, then, does the traditional model retain such sway within the scientific community at large? Part of the answer lies in the persuasive evidence of major geographical and occupational differences in the incidence of various tumours. Yet the statistical associations generated by such work--invaluable as they have been in providing testable hypotheses for prospective interventionist trials--have failed to clarify both the quantitative contribution of putative carcinogens and the qualitative mechanisms underlying the observed attributable risks. Dietary factors, for example, have been judged responsible for between 10% and 70% of cancer-related deaths (Doll & Peto, 1981), an estimate which reflects the severely limited availability of hard data in this area. The most obvious reason for this limitation is the difficulty of establishing independent associations between low-level exposures and long-term outcomes, though numerous other problems in interpreting epidemiologic data are well-recognized (Feinstein & Horwitz, 1982; Horwitz et al., 1985; Stewart, 1985; Anonymous, 1985) and include editorial bias towards the publishing of positive associations. In its most extreme form this latter tendency manifests as the "carcinogen of the week" syndrome (Weisburger, 1985) in which preliminary scientific musings become sensationalized in the lay press. The scientific literature, in contrast, is well-known for its rigorous standards of self-criticism; even here, however, data at odds with conventional theory are too often rationalized as being consistent with "multifactorial aetiology", an impressive-sounding but ultimately nebulous phrase which appears with depressing regularity in discussions of sporadic carcinogenesis. For example, the finding of unusually low levels of salivary nitrate in "high-risk" populations for gastric cancer (Forman et al., 1985) would seem surprising given the widely-held view that dietary nitrate ingestion bears a direct quantitative link to gastric carcinogenesis, while the finding that solar exposure is not independently predictive of melanoma incidence when individual pigmentation characteristics are also considered (Elwood et al., 1985) must similarly cast doubt on the assumed quantitative relationship between ultraviolet light exposure and tumourigenesis in oivo. The difficulty of separating the contributions of genetic predisposition and

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environmental exposure is further suggested by the inconsistent conclusions of the numerous migrant studies designed to eliminate these mutually confounding variables. Still another unresolved difficulty in interpreting exposure data relates to the "background" incidence of tumours occurring in unexposed individuals, such as mesotheliomas developing prior to the advent of asbestos (Davies, 1984). Conversely, despite the epidemic carcinogenicity of cigarette smoking, little is known as to which subset of the many carcinogens in cigarette smoke contributes most decisively to cancer risk--or even whether such a subset exists at all. Seen in overview, the debate concerning the role of environmental factors in the pathogenesis of human carcinomas has produced little more than polemics (Higginson & Muir, 1979; Peto, 1980; Epstein & Swartz, 1981) and cannot be expected to obviate the more central issues of tumour biology. From Microbes to Molecules

The idea that certain cancers may be virally initiated has enjoyed popularity among epidemiologists and scientists alike, not least because it provides one of the few biologically respectable models within which events (such as neoplastic transformation) may be regarded as occurring at random. Nonetheless, data relating specific viruses to solid tumours remain circumstantial while hard evidence implicating a definitive role for viruses in molecular carcinogenesis is also lacking. The association of chronic hepatitis B virus (HBV) infection and hepatoma is, on the one hand, unquestionable; yet numerous discrepancies militate against a direct causal connection (Trichopoulos et al., 1982). The heterogeneity of HBV-DNA restriction patterns found in hepatomas (Popper & Mori, 1983) is not consistent with a specific molecular pathogenesis, while the failure to detect genomic HBV-DNA in up to 25% of tumours in HBV carriers tends also to exclude a direct pathogenetic link (Frazer et al., 1984). The recognized association of hepatoma with other causes of chronic active hepatitis (e.g. alpha-l-antitrypsin deficiency) and macronodular cirrhosis (e.g. haemochromatosis) suggests that incorporation of the viral genome is not necessary for tumourigenesis and, conversely, the demonstration of " t u m o u r " HBV-DNA in normal liver parenchymal cells (Arthur et al., 1984) suggests that it is not sufficient. A further report indicating that positive HBV serology is not an independent risk factor for hepatoma among cirrhotic patients (Zaman et al., 1985) further argues against a specific viral pathogenesis. In vitro data also fails to corroborate this view: the mutagenicity of the SV40 virus has been shown to have no locus specificity (Gorbunova, 1982), while maximum induction of gene damage is known to occur within 16 hours of oncogenic virus infection (Lukash, 1981) and to return to normal values subsequently. On balance it would seem more reasonable--even if less n e a t - - t o characterize the sought-after "initial lesion" as an aggregate perturbation of DNA (Becker, 1981; vide infra) occurring within a contiguous population of chronically damaged cells, rather than as a specific genomic event occurring within a single " n o r m a l " (i.e. hitherto uninitiated) ceil. Similarly inconclusive associations abound in the field of viral carcinogenesis. Herpes simplex type 2, for many years the accepted cause of cervical carcinoma by

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dint of serologic evidence, has more recently been shown to play no part in either early (Vonka et al., 1984) or late (Galloway & McDougall, 1983) tumourigenesis; not, however, before another candidate virus emerged (Walker et al., 1983) and a "co-carcinogenic" aetiology had been mooted (zur Hausen, 1982). Claims for the existence of co-carcinogens are remarkably frequent in other virally-related malignancies--aflatoxin and HBV in hepatoma, chronic malarial infection and EpsteinBarr virus (EBV) in Burkitt's lymphoma, regional dietary factors and EBV in nasopharyngeal carcinoma--though the usefulness of these otherwise plausible associations is limited by the fact that they are based on too many variables to be verifiable or, for that matter, falsifiable. It is therefore all the more intriguing to note the recent championing of "preparative co-carcinogens" (Berenblum, 1985), these being defined as non-genotoxic substances which precede and amplify a given mutagenic insult but do not in themselves predispose to malignant transformation. This is an ingenious refinement of classical theory, for it acknowledges the association of non-genotoxic processes with cancer development while preserving the notion of a crucial mutagenic initiating step. Indeed, the only drawback of the theory is the lack of evidence implicating genotoxin exposure as a defining principle of sporadic human carcinogenesis. Such criticisms may seem poorly timed in view of the recent euphoria generated by the newsworthy "oncogene" (Marx, 1982) and the attendant fuelling of the reductionist fire (Maddox, 1983; Erikson, 1984). In addition to their celebrated association with malignant transformation, such genes are transcriptionally active in normal embryogenesis (Muller et al., 1982), growth (Goyette et al., 1983) and differentiation (Matrisian et al., 1985); accordingly, it seems simplistic to equate the transforming potential of these genes when transfected into immortalized monolayer cell cultures with the defining oncogenic capacity of their retroviral homologues. For although dominant (one-step) oncogene-mediated morphologic transformation has indeed been documented in the experimental context described, the prerequisite cellular immortalization has recently been shown to be recessively transmitted (Pereira-Smith & Smith, 1983). This strongly suggests that the crucial initial step in tumourigenesis is stochastic rather than determinative. Such a conclusion is consistent with a view of tumour initiation not as a discrete and irreversible "step" taking place within a prescribed region of the genome (as might be inducible by a mutagen; see Table 1) but as a dynamic and probabilistic process occurring within the genome as a whole. In a qualitative sense this latter process seems likely to overlap considerably with many other processes which we regard as "normal" or physiologic (Farber, 1984a), and it is the implicit denial of this overlap by the initiation-mutation model which most clearly separates the two schools of thought. Oncogene activation has, moreover, been documented in only 15% of human tumours (Farber, 1984b) and the activating mutations relate inconsistently to tumour type, suggesting that the activity of these genes may be consequential rather than causative (Duesberg, 1985). This conclusion is further supported by the finding of widespread amplification of "housekeeping" genes within tumour cells (Nakatani et al., 1985) and of heterogeneity in oncogene expression paralleling other phenotypic traits (Albino et al., 1984). The transforming potential of oncogenes can be increased

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TABLE 1

Theories of cellular transformation Determinative models

Feature Minimum number of "events" Nature of stimulus Dominant or recessive transmission Mediators Influence of intracellular constraints Evidence for frequency in vivot

Carcinogen (initiationmutation )

Oncogene (activation/ aberration)

Probabilistic model

1 specific

2 specific

undefined non-specific

unknown mutagens

dominant retroviruses

recessive mitogens

nil

unknown

crucial

2-3%

< 1%

95%

t Maximum estimated frequency of sporadic human tumours which appear to be "initiated" through these mechanisms.

by mitogenic induction, most likely mediated by interactions between trans-acting factors and promoter sequences, suggesting further that their role in tumourigenesis is not a primary one. While in certain tumour subtypes oncogene activation and/or amplification has been correlated with progression both in vivo (Brudeur et al., 1984) and in vitro (Tainsky et al., 1984), such observations in themselves provide no support for a proximate link between these genes and the initiation of cellular transformation. It is conceivable, of course, that these objections reflect methodologic inadequacy rather than theoretical invalidity, but the point remains that a primary role for oncogene involvement in cell transformation is as yet unproven. W h e n is a C l o n e n o t a C l o n e ?

A key observation favouring the initiation-mutation origin of tumours is the demonstrated monoclonality of some neoplasms, a finding which has been interpreted by many as signifying a general unicellular origin for all malignancies. Most of the evidence for tumour monoclonality, however, has been gleaned from work on paediatric and haematologic malignancies rather than carcinomas. Notwithstanding the obvious similarities between the natural history of carcinoma and that of reticulosis or paediatric tumour, it remains doubtful whether the gross resemblances of end-stage disease necessarily signify common mechanisms of pathogenesis. Indeed, several striking distinctions suggest that they do not: non-cutaneous carcinomas are common and usually fatal epithelial tumours of the post-reproductive age group; paediatric tumours (in which are included, for instance, nephroblastoma, retinoblastoma, and germ-cell tumours) are rare, often characterized by consistent karyotypic anomalies, and not infrequently curable despite advanced-stage disease; while leukaemias, lymphomas and plasma-cell dyscrasias are also comparatively

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uncommon, generally characterized by non-random chromosomal defects, and similarly far more curable than the "log-cell-kill" model of cytotoxic action would predict. Despite their relative rarity, leukaemias remain the commonest malignancy following alkylator therapy and/or irradiation while lymphomas are the commonest following therapeutic immunosuppression. Why should this dichotomy exist between the endemic and the iatrogenic? One explanation would be that these neoplasms--in contrast to carcinomas, for which incidence trends are reversed--are indeed "initiated" by discrete and specific factors such as viruses or iatrogenic mutagens. This hypothesis helps explain the clustering of these tumour types in other clinical contexts: lymphoproliferative malignancies in primary immunodeficiency and DNA repair defects (Merigan, 1981), polyclonal EBV-linked lymphomas reversible on tapering of therapeutic immunosuppression (Starzl et al., 1984) and leukaemia recurrence in transplanted donor marrow (Fialkow et al., 1971). All are compatible with a classical model of malignant transformation in which somatic mutation of a single cell is necessary for transformation, while crucial but poorly-defined systemic constraints play a permissive role in tumourigenesis. Since haemopoietic cells tend to be migratory rather than sessile, these systemic constraints could be expected to be humoral in nature, viz., the familiar theory of "immunologic surveillance". The existence of this mutually constraining humoral interplay is also suggested by numerous clinical observations, including inhibition of haemopoiesis in leukaemia (van Bekkum et al., 1981), "spontaneous" leukaemic remissions (Bernard, 1983) and the anti-leukaemic effect of graft-versus-host disease (Weiden et al., 1981). That such phenomena occur rarely if at all in the context of carcinoma accords with the notion that epithelial cells are subject to different systemic constraints. Conversely, the chemocurability of many haematologic and paediatric malignancies--despite the presumed persistence of several logs of viable tumour cells--argues strongly against the irreversibility of the post-initiation phenotype in these neoplasms, and this contrasts with the acknowledged irreversibility (incurability) of most carcinomas treated with cytotoxic agents. (It is worth noting in passing that not all animal species share the human predisposition to "solid" rather than haematological malignancies. On one level this could be explained by differences in, for instance, DNA repair capacity (diPaolo, 1983) between human and animal cells. Yet some animal species, such as the domestic house-cat, are highly prone to leukaemogenic retroviral infections while relatively resistant to the development of carcinomas; since this species also exhibits low "genetic plasticity" (Warfield, 1984)--a zoological concept manifesting as evolutionary stasis and difficulty in inter-breeding--it is intriguing to ask whether higher structural and functional qualities of the genome may not dictate the relative risks of different neoplasms between species.) There now seems little doubt that most haematologic malignancies are of unicellular origin. Monoclonality of leukaemias has been suspected for decades, originally on the basis of chromosomal analysis (Nowell, 1960); hybridoma technology has made the monoclonal typing of lymphomas a routine undertaking; while more recently, idiotype analysis of immunoglobulin-producing neoplasms has definitively confirmed their clonal nature. "Solid" tumours, in contrast, are characterized by

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phenotypic and antigenic diversity (Ogawa et al., 1980). Multiclonal origins have been demonstrated for malignant (Beutler et al., 1967) and pre-malignant (Hsu et al., 1983) human tumours as well as experimentally-induced animal tumours (Reddy & Fialkow, 1980), while polycentricity of primary human lung (Woolner et al., 1984) and breast (Schwartz et al., 1980) carcinomas is also recognized. Solid tumours of pleoclonal origin may appear to be spuriously monoclonal due to subclone suppression by a single "dominant" clone (Woodruff et aL, 1982), and such confounding may invalidate the conclusions of studies using X-linked markers. The frequent finding of multiclonal origin for carcinomas analysed by this latter technique has, on the other hand, been attributed to inadvertent specimen contamination through normal cellular admixture (Fialkow, 1976). A monoclonal origin for several tumour types has been recently established using restriction fragment length polymorphism analysis (Vogelstein et al., 1985), but it is notable that no carcinomas were included in the study. These anomalies do not support an obligate unicellular origin for carcinomas and, as such, weaken the credibility of an exogenously "initiated" pathogenesis for these tumours. As with haematologic malignancies, somatic mutation seems likely to be involved in the pathogenesis of many paediatric tumours. The proposal that hereditary retinoblastoma evolves through a two-stage process, in which the first stage is a discrete genetic aberration while the second is a critical "spontaneous" (probabilistic) mutation occurring in a genetically predisposed cell (Knudson et al., 1975) has been supported by impeccable evidence (Murphree & Benedict, 1984). There is only the most superficial resemblance between this sequence of events and the two-stage oncogenic process induced in laboratory animals by sequential chemical exposure which forms the model for the initiation/promotion theory of carcinogenesis. Few scientists now doubt that tumour evolution is a "multi-stage" process--the recent demonstration that at least two transfected oncogenes are required to effect the transformation of non-immortalized cells in vitro (Land et al., 1983) provides some objective support for this--yet it seems doubtful whether this conclusion adds much to our understanding of tumour biology. For any process capable of being observed over time will be perceived as consisting of a number of stages, the sole alternative being that no "process' as such will be witnessed at all: moreover, the counterproductive assumption may be made that nothing significant is happening between experimentally observable stages. Hence the true value of the multi-stage model may lie not in what it reveals about tumourigenesis, but in what it reveals about the limits of human perception.

Mutagens, Models and Misconceptions The existence or otherwise of obligate mutagenic initiators is a central issue for public health, since cell transformation in the initiation-mutation model is an event for which no safe mutagen exposure threshold is definable. Nonetheless, the relevance of this model to human carcinogenesis remains controversial: just as its epidemiologic support is inconclusive, so is its experimental support inconsistent. The defining sequentiality of the initiator/promoter hypothesis and the functional

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specificity of the reagents involved have come into question (Iversen & Iversen, 1982; Hennings et al., 1983), while the notion of initiation as a discrete molecular event also seems likely to be simplistic (Scott & Maercklein, 1985). Tumours developing after repeated genotoxin exposure have been proven to arise from significantly more cells than those induced by an initiation/promotion sequence (Reddy & Fialkow, 1983), an observation which defies explanation within a strictly mutational (single- or multi-"hit") model of initiation. The recognition that genetic lesions induced by known mutagens are structurally and functionally heterogeneous (Karran & Williams, 1985) coincides with the realization that mutagenicity cannot be reliably regarded as a predictor of carcinogenicity (Silinskas et al., 1985) and that even classical mutagenic initiators such as nitrosamines are capable of inducing heritable genetic alterations via non-mutational mechanisms (Montesano & Hall, 1983). Further evidence indicates that cell transformation may be "initiated" following damage to any one of many genes (Doniger et al., 1985) and that numerous types of genetic damage may precede transformation following exposure to a specific mutagen (Singer, 1984). Moreover, the failure to demonstrate specific oncogene activation in at least some chemically-induced tumours (Toftgard et al., 1985) tends to exclude this mechanism as a general mode of initiation. These findings support the view that sporadic carcinogenesis is unlikely to arise from a succession of primary point mutations (Weinstein et al., 1984b) irrespective of what part mutations may play in the ultimate expression of the malignant phenotype. What of the predictivity of the model for sporadic human tumourigenesis? The screening of potentially genotoxic substances in short-term carcinogenicity assays has been hampered by inconsistencies (Kroes, 1983 ), and it is difficult not to speculate that these may owe more to a mistaken preoccupation with the experimentallydefined initiation-mutation phase oftumourigenesis (Ray, 1983) than to any intrinsic defect in the tests themselves. Some of the variables contributing to this poor predictivity have been identified, and include differences in genetic stability between human and animal cell lines (DiPaolo, 1983), non-linearity of dose-response relations due to saturable mechanisms of tissue repair (Swenberg et al., 1983), and exaggerated exposure regimens in animal bioassays (Pitot, 1982). Not surprisingly, these same variables confound the applicability of the initiation-mutation model to human carcinogenesis: the problem is circular, constrained by the limitations of the experimental method itself (Smith, 1983). Given that direct alteration of the base sequence of DNA remains the best established molecular mechanism for determining heredity, it is perhaps understandable that the initiation-mutation model should retain popularity in spite of inconclusive epidemiologic support, conflicting experimental data, and limited predictivity of toxicity testing. Base sequence alterations, however, may not prove to be of exclusive importance to carcinogenetic theory (Rubin, 1982). That pathways for cell transformation exist which do not involve the primary binding of an electrophilic reagent to the genetic material is suggested by reports of transformation following cytoskeletal disruption (Iype et al., 1985), free radical formation (Williams, 1983) and interference with DNA polymerase fidelity (Sirover & Loeb, 1976) in the absence of known prior exposure to mutagens. The diversity of these transforming influences

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suggests that they are neither mutually exclusive in their actions nor specific in their targets, yet their indirect genetic sequelae are precisely as heritable as those induced by genotoxins. The association of non-mutagenic stimuli such as diethylstilboestrol, asbestos, foreign bodies and scars with tumourigenesis (Dunkei, 1983) serves also to emphasize the variety of insults which may precede clinically evident neoplasia, and suggests a common non-specific endpoint for the toxicity of these agents. Since most cellular abnormalities seem capable of being induced by either exogenous or endogenous processes (Bridges et al., 1983), the chief importance of these relatively rare associations may lie in the pathogenetic models which they provide for more universal modes of sporadic carcinogenesis. A View from the Cell

Given that the emerging view of tumourigenesis presupposes neither direct nor specific damage to DNA, what can be the nature of the non-specific endpoint(s) suggested by the above? Perhaps the most plausible suggestion for such an endpoint has been the increased DNA synthesis accompanying any stimulus to cellular regeneration (Park & Snee, 1983), the rationale being that cellular capacity for repair and replication of DNA is finite and therefore more prone to error when approaching saturation. The relationship of breast cancer to endogenous oestrogen metabolism, for example, would be compatible with such a mechanism, while remaining unexplained by a genotoxic initiation-mutation model. However, this theory fails to clarify the negligible incidence of small-bowel vis-a-vis colorectal cancer--this being an anomaly which is also unexplained by genotoxic models--and this discrepancy suggests again that a higher order of genome structure may be implicated in tumourigenesis, since such higher-order differences between cells of varying developmental lineage could well underlie the observed organ-specific patterns of sporadic carcinogenesis. Another proposal has been that concerning the hypomethylation of cytosine residues (Boehm & Drahovsky, 1983), a modification which is "epigenetic" (in the sense that the linear base-sequence of DNA remains unchanged, semi-conservatively replicated, and convincingly implicated in the regulation of gene expression and development; Bird, 1978). Hypomethylation is known to lead to the activation of otherwise dormant genes (Ramsden et al., 1985), whereas (hyper)methylation appears to prevent transcription by "locking in" nucleosomal structure (Kolata, 1985b): the former phenomenon can be readily reproduced in vitro with the use of inhibitors (Olsson et al., 1985) and has also been documented in vivo in both carcinomas and pre-malignant lesions (Goelz et al., 1985). Is the link between hypomethylation and tumourigenicity aetiological or epiphenomenal ? Physiological gene activation in eukaryotic cells appears to be a specific process (Brown, 1984), discouraging the notion that generalized hypomethylation plays a primary role in cell transformation; yet it remains an open question as to whether progressive hypomethylation may contribute to the transforming milieu of the pre-neoplastic lesion. Gene expression is also correlated with variations in the higher structure of DNA. Changes in tertiary DNA structure occur with differentiation induction in animal

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cells (Rebouileau & Shapiro, 1983) and with mutagen exposure in plasmids (Lycksell et al., 1985). The affinity of mutagens for DNA seems more specific for tertiary than primary structure (Wilkins, 1984) and, in prokaryotic systems, mutagens may induce primary conformational alterations which correspond to specific patterns of subsequent mutation (Bichara & Fuchs, 1985). These observations serve to emphasize that aberrations of gene regulation--such as have been put forward as models for the initiation of neoplasia (Sachs, 1981)--cannot be assumed to signify primary alterations in the linear structure of DNA, since the higher structure of the molecule may be altered by mutagens and non-mutagens independently of base-sequence alterations. Modulation of higher DNA structure, being more readily reversible than base substitution, could also provide an explanation for the observed dynamic behaviour of neoplasms in vivo (Tatematsu et al., 1983). Many other non-genotoxic pathways for malignant transformation have been proposed, though none conclusively. Genetic recombination has been propounded as one possible mechanism (Caims, 1981) but little firm evidence has emerged to support this as a significant process in human oncogenesis. Anecdotal reports of primary defects in DNA repair associated with carcinoma development (Pero et al., 1983) have similarly not been corroborated despite the potentially coherent model suggested by such observations (Kraemer et al., 1984). Inducible error-prone ("SOS") repair has been well documented to cause specific patterns of mutation in prokaryotic cells (Miller & Low, 1984), though it is only recently that analogous mechanisms have been recognized at all in mammalian systems (Sarker et aI., 1984). The observation that normal excision-repair seems less efficient in transcriptionally inactive chromatin (Kootstra, 1984) has raised the possibility that damage to these regions may predispose to genomic instability and carcinogenesis (Chorazy, 1985). However, no link has emerged which conclusively implicates inapparent deficiencies of DNA repair in spontaneous mutagenesis or sporadic carcinogenesis (Sargentini & Smith, 1985). Chromosomal instability is known to characterize a number of rare syndromes associated with neoplasia (predominantly hematological), and in some cases this instability has been linked to circulating clastogens (Shaham et al., 1980; Emerit & Cerutti, 1981). Less obvious chromosomal instability may occur due to the inheritance of "fragile sites" (Sutherland, 1979), and it has been speculated that subtle degrees of genetic instability may contribute to sporadic carcinogenesis (Hsu, 1983). Efforts to forge a direct aetiological link between ageing and cancer have been similarly fruitless: alterations in DNA synthesis (Collins & Chu, 1985), replication (Linnet al., 1976) and tertiary structure (Thakuv, 1984) have been observed with ageing, but no defined mechanistic link between the general phenomenon of ageing and the probabilistic phenomenon of tumourigenesis has emerged. Tight Junctions and Loose Ends

The technical difficulties in analysing carcinomatous tissue are widely appreciated. In contrast to leukaemias, carcinomas have resisted study for a number of practical reasons: difficulties in mechanically disag'gregating biopsy specimens, in preventing

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normal cellular admixture, in growing epithelial cell lines in oitro, and in analysing growth factor regulatory networks in vivo. These very problems hint at what has been recently referred to as an "old idea" (Rubin, 1985), namely, that the maintenance of normally differentiated phenotype in epithelial tissues--histologically characterized as they are by tight junctions--is critically dependent on intercellular contact. That this is an old idea is chiefly because it is consistent with observations implicating carcinogenesis as a dynamic and probabilistic process (Table 2). Interference with intercellular communication is now established as the primary action of TAaLE 2 Theories o f tumour progression

Determinative models Feature Minimum number of "events" Nature of stimulus Time-course of process Mediators Reversibility Influence of intercellular constraints Evidence of frequency in oioot

Carcinogen (promotion)

Oncogene (activation)

Probabilistic model

1 discrete sequential "'epigenetic carcinogens" sometimes

1 discrete sequential unknown unknown

undefined continuous dynamic growth factors repair enzymes always

minor 30%

unknown 15%

major 60%

t Maximum estimated frequency of these mechanisms operative in multicellular phase of tumour evolution. at least some tumour promoters (Enomoto & Yamasaki, 1985) with potentially reversible cellular deficiencies as their sequelae (DeLuca, 1983): when metabolic co-operation with normal cells is reduced by a "critical mass" of deficient cells, transformation may occur (Trosko et al., 1983). That normal cellular communication inhibits this development is suggested by the far higher frequency of " s p o n t a n e o u s " cellular transformation in vitro than in vivo (Alexander, 1985) and by the well-known clinical and experimental p h e n o m e n o n of foreign body carcinogenesis (Brand, 1982; O p p e n h e i m e r et aL, 1952). Phenotypic alterations in both normal (Levine & Stockdale, 1985) and malignant (Keyner et aL, 1978) cells co-cultivated with non-transformed cells also suggest an informational transfer between cells similar to that seen with hybridized tumour cells (in which the malignant phenotype may be abolished: Klein et al., 1971; Stanbridge et al., 1982) and with blastocyst-implanted teratocarcinoma cells (which yield normal progeny: Mintz & Illmensee, 1975). These p h e n o m e n a indicate that " n e w " , or dominant, information is being imparted to the malignant cell by the normal cell rather than the converse, and this conclusion is confirmed by cell fusion experiments yielding daughter clones of finite division capacity from immortalized parentage (Pereira-Smith, 1983); essentially similar conclusions are hinted at by the postulation of "anti-oncogene" derangements in hereditary neoplasms (K_nudson, 1985). The importance of cell contact in regulating

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gene expression is further stressed by the observation that contact-insensitive cell lines are more susceptible to neoplastic transformation than are normal cells, even though their mutation frequency is unchanged (Nakano et al., 1985). Since the v-src oncogene product is known to inhibit intercellular communication in partly-transformed cells (Chang et al., 1985) and growth to confluence of these same transfected cells increases expression of the gene (Glanville, 1985), it is not difficult to construct a neoplastic positive-feedback loop in which impaired intercellular communication plays a primary role. Constraining of cell behaviour by this intercellular "language" also helps explain the later emergence of the malignant phenotype in vivo. Although the "grammar" regulating cell development remains unformulated (Brenner, 1984), insights into the language's vocabulary are slowly emerging: protein-DNA feedback control has recently been documented in mammalian cells (Cleveland, 1983) while protein-protein interactions may induce genetic aberrations (such as aneuploidy) in the absence of covalent modification of DNA (Zimmerman et al., 1985). These findings heighten the impression of DNA as an exquisitely sensitive microenvironmental interactant rather than as an isolated and autonomous organelle, emphasizing the contribution to cell regulation of an organismic---as distinct from a strictly cytologistic (Smithers, 1962)--"intelligence".

A Modest Proposal If the development of carcinoma is not necessarily contingent upon either environmental (the initiation--mutation hypothesis) or genetic (the oncogene hypothesis) antecedents, how is the perpetuation of this maladaptive tumourigenic potentiality in an otherwise highly successful species to be explained? The mammalian genome is a product of evolution, and it has been claimed that anything produced by evolution is " . . . bound to be a bit of a mess" (Brenner, 1984). As noted previously, carcinomas generally occur in the post-reproductive age group and hence do not exert selection pressure on the species. The eukaryotic genome, being inherently "selfish", has three priorities: it must possess structural irregularity so that it may contain information (Crick, 1982); it must possess functional elasticity so that it may recover from cellular damage (including that due to imperfect.replicative fidelity); and it must possess adaptive plasticity to ensure successful evolution of the species in response to environmental stress. Since evolution will tend to increase the genome's informational content and adaptive capacity at the expense of its repair fidelity, the irregularity and plasticity of the genome must inevitably come to exceed its elasticity. This is simply another way of saying that the survival of the species takes precedence over that of the organism; or that sporadic carcinogenesis may be regarded as the inevitable compromise between genetic stability and organismic adaptability. Cancer is a subject bedevilled by uncertainty and emotion. One thing which is certain, however, is that emotion plays little role in the success of cancer research. Throughout the ages people have sought explanations for tragic and inexplicable occurrences, and in this respect the modern scientific quest for a "cause" of cancer may be little different. Notwithstanding the important advances which have been

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m a d e , h o w e v e r , we r e m a i n c o n f r o n t e d by i r r e d u c i b l e issues o f c h a n c e a n d necessity. This is not to say that any o f the c a r c i n o g e n e t i c m o d e l s p r e s e n t e d are in t h e m s e l v e s valueless or " w r o n g " - - i n the s a m e way that N e w t o n i a n physics is n e i t h e r valueless n o r w r o n g - - n o r is it to say that these m o d e l s need necessarily be m u t u a l l y exclusive. But there is a d a n g e r that o u r p r o f o u n d i g n o r a n c e o f n o r m a l cell b i o l o g y has led to insufficiently critical i n t e r p r e t a t i o n o f the scanty e v i d e n c e a v a i l a b l e , and that by c o n t i n u i n g to s u c c u m b to this t e m p t a t i o n we may be sacrificing the o p p o r t u n i t y to f o r m u l a t e a m o r e m a t u r e u n d e r s t a n d i n g o f t u m o u r biology.

Conclusion T h e r e is n o w suggestive e v i d e n c e that c a r c i n o g e n e s i s is a p r o b a b i l i s t i c p h e n o m e n o n which m a y be i n c r e a s e d in f r e q u e n c y ( " p r o m o t e d " ) by a variety o f e x p o s u r e s and e n d o g e n o u s processes, most o f t h e m n o n - g e n o t o x i c . It also s e e m s likely that the early stages o f t u m o u r g r o w t h in v i v o are d y n a m i c a l l y c o n s t r a i n e d by i n t e r c e l l u l a r i n t e r a c t i o n , a n d that these c o n s t r a i n t s are p a r t i c u l a r l y critical in the e v o l u t i o n o f c a r c i n o m a s . T h e n o t i o n o f an o b l i g a t e m u t a g e n i c initiation p h a s e o f c a r c i n o g e n e s i s , on the o t h e r h a n d , is certainly not p r o v e n a n d m a y well be spurious. REFERENCES ALBINO, A. P., LI- STRANGE, R., OLIEF, A. 1., FURTH, M. E. & OLD, L. J. (1984). Nature 308, 69. ALEXANDER, P. (19851. Br. J. Cancer 51,453. ANONYMOUS (1985). Lancet I, 1311. ARTHUR, M. J. P., HALL, A. J. & WRIGHT, R. (1984). Lancet I, 607. BECKER, F. F. (1981). Am. 3". Pathol. 87, 3. BERENBLUM, I. & SHUBIK, P. (1947). Br. J. Cancer l, 383. BERENBLUM, I. (1985). Cancer Res. 45, 1917. BERNARD, J. & BESSIS, M. (1983). Blood cells 9, 71. BEUTLER, E., COLLINS, Z. & IRX,VIN, L. E. (1967). N. EngL J. Med. 276, 389. BICHARA, M. & FUCHS, P. P. (1985). J. tool. Biol. 183, 341. BIRD, A. P. (1978). J. tool. Biol. 118, 49. BOEHM, T. L. J. & DRAHOVSKY,D. (1983). J N C I 71, 429. BOVERI, T. H. (1929). The origin of malignant tumours. Baltimore: Williams and Wilkins. BRAND, K. G. (1982). In: Cancer--a comprehensive treatise. Becker, F. F. (ed.). New York: Plenum Press. BRENNER, S. (1984). Quoted in: Lewin, R. Science 224, 1327. BRIDGES, J. W., BENEORD, D. J. & HUBBARD,S. A. (1983). Ann. N.Y. Acad. Sci. 407, 42. BROWN, D. D. (1984). Cell 37, 359. BRUDEUR, G. M., SEEGER, R. C., SCHWAB, M., VARMUS, H. E. & BISHOP,J. M. (1984). Science 224, 1121. CAIRNS, J. (1981). Nature 289, 353. CHANG, C., TROSKO, J. E., KUNG, H., BOMBICK, D. & MATSUMARA,F. (1985). Proc. natn. Acad. Sci. U.S.A. 82, 5360. CHORAZY, M. (1985). Cancer Res. clin. Oncol. 109, 159. CLEVELAND,D. W. (1983). Cell 34, 330. COLLINS, J. M. & CHU, A. K. (1985). J- cell. Physiol. 124, 165. COMINGS, D. E. (1973). Proc. natn. Acad. Sci. U.S.A. 70, 3324. CRICK, F. (1982). Life itseIJS London: Macdonald. DAVIES, D. (1984). Br. reed. J. 289, 1164. DELucA, L. M. (1983). J N C I 70, 405. DIPAOLO, J. (1983). J N C I 70, 3. DOLL, R. & PETO, R. (1981). J N C I 66, 1193. DONIGER, J., DAY, R. S. & DIPAOLO, J. A. (1985). Proc. hath. Acad. Sci. U.S.A. 82, 421.

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