Cellular Adaptation in the Origin and Development of Cancer1

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(CANCER RESEARCH 51, 2751-2761. June 1. I991| Perspectives in Cancer Research Cellular Adaptation in the Origin and Development of Cancer1

Emmanuel Farber and Harry Rubin Departments of Pathology and of Biochemistry, Medical Sciences Building, University of Toronto, Toronto. Ontario, Canada M5S IA8 [E. F./, and Department of Molecular and Cell Biology and I'irus Laboratory, Stanley Hall, University of California, Berkeley, California 94720 [H. R.]

Introduction Despite the obvious importance of such systems, their study in disease has received little current emphasis, especially with A dominant view today in cancer research assumes that the a mechanistic orientation. Since disease processes are largely major need for our understanding of cancer resides in the expressions of how living organisms react and respond to thorough documentation of the many alterations in gene struc perturbations in the external and internal environments, adap ture, organization, and arrangement that we know are present tive or protective responses and their modulations and mecha in some malignant cell populations and in their immediate nisms would seem to be of the greatest concern in fundamental precursors. While attractive from some points of view, espe studies of disease pathogenesis. Although such considerations cially in the hope of delineating a relatively simple cause-effect have been the subject of occasional deliberations over the years relationship between the genome and cancer, this simple for (e.g., Refs. 2-8), they have received little attention in the mulation largely ignores a fundamental property of all living detailed studies of chronic disease in general and cancer in forms, the ability to respond and adapt to many environmental particular, especially from the viewpoint of mechanism. perturbations. Given the widespread occurrence of cancer in In the face of the many hazards, including some carcinogenic many species and given the unusually prolonged nature of the ones, to which biological systems have been exposed for mil process of cancer development in most instances in humans lions of years, it seems appropriate to ask the question,"What and in animals, often lasting from one-third to two-thirds of mechanisms might have evolved to preserve biological conti the life span of the organism, it would indeed be unlikely and nuity?" Probably many earlier species have been eliminated by unbiological for cancer development to proceed without the exposure to one or more of such hazards. In those that have participation of many active responses, including adaptive ones, survived and endured, what is the nature of the adaptations to the various new cellular phemomena that are part of the that have evolved and could a knowledge of these be useful in carcinogenic process. Immunological surveillance has been a designing new approaches to the prevention of cancer or even popular speculation proposed to account for this prolonged its treatment (9)? What essential roles, if any, do these adaptive process. However, immunological responses are only a small processes play in the development of different cancers? part of this spectrum of reactions, with considerable doubt (1) that they participate actively prior to the relatively late appear Adaptive Responses during Cancer Development ance of unequivocal cancer with its many new biological com ponents such as tissue destruction with cell death, inflamma Cancer development with the different known etiological tion, ulcÃ©ration,etc. agents, chemicals, radiations, DNA viruses, and some RNA All living forms have evolved in a largely unfriendly or even viruses without oncogenes is a very prolonged process as already hostile environment in which protective or adaptive responses mentioned. The only apparent exceptions to this are the special to many different forms of potential injury or harm have been carcinogenic processes induced with oncogene-containing retro- essential for survival and reproduction. In fact, it appears that viruses (10). The long period of development is characteristi the acquisition of mechanisms for many different adaptive cally associated with the presence of various types of focal responses could be considered to be just as important for proliferations of an entirely benign nature (some designated as survival and reproduction as the development of the essential "benign neoplasms") that may show a slow cellular evolution pathways for the basic physiological needs of living organisms to cancer (9). such as for energy and for the syntheses of the small and large During this prolonged process, there appear to be a minimum molecules that are the component parts of the pathways. It is widely recognized that a variety of physiological adaptive re of three general types of cellular adaptive responses: type A, sponses to varying altitudes, other environmental influences, early acute transitory responses, usually of a diffuse, zonal, or and hormonal modulations as well as in aging are fundamental regional nature, largely reversible; type B, clonal constitutive properties of living organisms. In addition, the vast array of adaptation with the development of a new cell population that different xenobiotic chemicals and organisms as well as radia is associated with a resistance of the organ or tissue and of the tions to which all living forms have been exposed since the early organism to environmentally induced cell damage; and type C, forms evolved some 2 to 3 billion years ago would require patterns of adaptation of cells to a variety of environmental highly versatile protective systems to be developed. Such pro modulations during neoplastic transformation. The main sub tective or adaptive mechanisms are known to be present stance of this communication is to discuss briefly each of these throughout the whole spectrum of living forms from single cell three types of adaptations and to suggest how they might be microorganisms to highly differentiated eukaryotes. important as one set of guidelines for cancer prevention and cancer therapy. This overview is limited to cellular responses Received 9/18/90; accepted 3/25/91. that seem to participate actively in the step-by-step sequence of ' The authors' research included in this article was supported by grants from events leading to malignant neoplasia. Adaptive responses of the National Cancer Institute of Canada and the Medical Research Council of Canada (MT-5994) (E. F.): and from National Institutes of Health. CA 15744. the whole organism, especially the late ones in response to fully- and grant 1948. Council for Tobacco Research. USA (H. R.). formed cancers, are omitted. 2751

Acute Transitory Adaptive Responses a major adaptive set of responses of many different organisms from single cell to the human (31-34). The mechanisms include The known responses in this category relate mainly to carci- photoreversal of pyrimidine dimers, mismatch repair acting on nogenesis with radiations and with mutagenic chemicals. Al incorrectly paired bases from unfaithful DNA replication, en though it is our conviction that similar adaptive responses might zymatic alterations with recombinant events and excision re be found in cancer development with cancer-related DNA vi pair. Interesting enzymes are the alkyltransferases which re ruses and some RNA viruses (e.g., Refs. 11 and 12), data in move methyl or other alkyl groups from the O6 position of this area are scanty. guanine, the O* position of thymine, or the phosphate groups Reversible Inductions of Metabolizing Enzymes. One of the of DNA and transfer the alkyl groups to a cysteine S in the most general and basic forms of adaptation to xenobiotic chem transferase itself. In E. coli, the transfer is part of a very icals, including, of course, the majority of drugs, is induction interesting adaptive response to alkylating agents associated of enzyme patterns that relate to the metabolism and possible with the ada gene. detoxification of the chemical (13-17). This type of response is The DNA repair systems are very widespread in nature and seen throughout the living world including the simplest bacte their adaptive protective role is clearly seen in many systems ria. In mammals, this often includes two major aspects of including, of course, humans. The susceptibility to skin cancer detoxification: metabolic activation to more polar derivatives, of humans with xeroderma pigmentosum, with their several such as via the cytochrome P-450 system and other oxidative types of deficiencies in the repair enzyme systems, is a striking as well as reductive pathways (phase I); and conjugation of example of the importance of the DNA repair systems for metabolic products for excretion or other pathways of removal survival. (phase II). The enzymes involved often show transitory in creases in both transcription and translation and usually return Cianai Constitutive Adaptation to their baseline levels after the exposure to the xenobiotic is over. During a systematic study of the sequence of steps in the It is well documented that these acute responses are fre carcinogenic process in the liver (35), it became evident that quently associated with the more rapid excretion of the poten another form of adaptation exists, one that originates in rare tially toxic xenobiotic with an obvious protective result (13- single hepatocytes scattered throughout the liver and that is 20). Apparently, the multiple nature of the responses to so constitutive, rather than diffuse and readily reversible (9, 36). many xenobiotics facilitates both metabolism and excretion. This response pattern has been seen with genotoxic chemical Included in the xenobiotic chemicals are a smaller group of carcinogens. It involves the induction of a new resistance phe- actual or potential mutagenic ones, genotoxic carcinogens, that notype in a rare hepatocyte and the expansion of these rare form a significant percentage of chemical carcinogens. These hepatocytes to form focal proliferations (hepatocyte nod agents also induce a spectrum of enzymes in liver and other ules).The recognition of this new adaptive phenomenon has organs that encompass both phase I and phase II components important potential implications not only for a new conceptual (21, 22). The obvious roles of those components in the genesis framework for the development of some cancers but also in of more toxic mutagenic derivatives, most often electrophilic tumor promotion, in cancer prevention, in the analysis of reactants, are well established. However, any role of these resistance of many primary cancers to chemotherapy, and in reactive derivatives in the survival of the organism is not evident the analysis of how some living forms have adapted, presumably at this time. through evolution, for survival in a hostile environment. Acute Reactive Proteins. Another reaction that is often seen Background. During the first few years of our study of the in the higher organisms is the synthesis and secretion of acute mechanisms of chemical carcinogenesis in the liver, we gradu reactive proteins, especially by the liver (23). These proteins are ally became impressed by five considerations, each of which transitorily elevated in the blood during an episode of injury suggested that some new approach was needed. and return to their baseline values when their injury is over. (a) Cancer development in humans and animals with chem They are considered to play a protective role in the acute injury. icals, DNA viruses and some RNA viruses, radiations, and diet Heat Shock Proteins. A third general adaptive response to is an inordinately long process, requiring from one-third to heat, toxic chemicals and a wide spectrum of other environ two-thirds of the life span of the organism (9, 37). This is mental perturbations is the synthesis of "heat shock proteins" interesting and puzzling, since it can be clearly shown in exper (24-26). This response, like the xenobiotic-induced enzyme imental animals and in some humans that exposure to a carcin induction, is present in Escherichia coli, in yeast, and in a wide ogen began or occurred many years prior to the first appearance spectrum of organisms including insects and mammals. There of cancer and was brief and transitory. Although a dominant is an overall agreement that these responses are often protective role for immunological surveillance was first suggested by and should be considered as adaptive (26). Thomas in 1959 (38, 39), the evidence for this during chemical Thus, at least three different adaptive response patterns can carcinogenesis is unimpressive (1). Alternative hypotheses for be distinguished on exposure to many different xenobiotics and the very prolonged nature of the carcinogenic process are nec at least two, Responses 1 and 3, are present in a wide range of essary if we are to develop rational approaches to cancer organisms, from single cell to highly complex multicellular. prevention. Responses that span such a broad spectrum of living organisms (b) Autonomous or semiautonomous growth of initiated cells probably play an important role in maintaining the integrity is a property acquired late in the carcinogenic process. Initiation and function of the different living forms, especially as to how with one of many different carcinogens is at no time followed they react to and survive environmental disturbances (27-30). by spontaneous or autonomous proliferation of any cells in the DNA Repair Enzymes. There is no doubt that the develop liver or in other organs. Only with large doses of carcinogens ment of a variety of mechanisms for the repair of DNA, altered and periods of exposure much longer than are required for by mutagenic chemicals and radiations and perhaps viruses, is initiation does one see focal lesions with autonomous cell 2752

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. CELLULAR ADAPTATION AND THE ORIGIN OF CANCER proliferation. If the initiated altered cells induced by exposure effects of the agents used in their generation and cross-resist to a carcinogen do not grow spontaneously, how might a ance to other xenobiotics and to a dietary deficiency to which differential stimulation of their growth be created? The growth that rat was not exposed (35, 36). of the rare altered cell leading to focal proliferations ("benign (c) The resistant hepatocytes induced with quite different neoplasms") is a key phenomenon in promotion in virtually all regimens show an unusually common biochemical pattern (35, experimental carcinogenesis and in many human systems of 36). This pattern shows consistent decreases in several phase I cancer development. components including total cytochromes P-450 and several (c) Virtually every chemical carcinogen is an inhibitor of cell mixed function oxygenases, consistent increases in many phase proliferation (mitoinhibition) (see Ref. 40 for references II conjugating systems, characteristic alterations in glucose therein). Haddow (41) suggested in 1938 that inhibition of cell metabolism, and a reproducible altered pattern of iron and proliferation could be an early effect of carcinogens and that, heme metabolism (36). in such an environment, resistant cells might arise and be It is important to emphasize that many different enzymes encouraged to proliferate. Recently, it has been reported that and components contribute to this phenotype. For example, two non-carcinogenic promoting agents for hepatocarcinoge- the decrease in total cytochromes P-450 is so large (60-80%) nesis, phÃ©nobarbital(42-45) and orotic acid (46-48), also show that several different ones must be involved (53, 54). This considerable inhibition of hepatocyte proliferation, not unlike multiplicity of active components in the resistant hepatocytes that seen with the carcinogens. could readily allow for considerable variation in the levels of (infection with v-H-ras and v-ra/(12) and in cultured than are the hepatocytes in the mature normal and surrounding rat liver epithelial cells during transformation induced by ex liver. In contrast to the hepatocytes in the latter which are posures to /V-methyl-yV'-nitro-A'-nitrosoguanidine (50, 51). arranged predominantly as single cell plates, those in the nod The resistance phenotype (52) is manifested at three levels of ules are in the form of double cell plates and acini (60). organization: (a) physiological (behavior), resistance to inhibi However, this difference in organization and architecture is not tion of cell proliferation (mitoinhibition); (b) cellular, resistance an expression of abnormality, since the hepatocytes in the to visible cytological responses to some cytotoxic environments; nodules undergo spontaneously a radical differentiation with and (c) biochemical-molecular, the presence of a biochemical restructuring and remodelling (61, 62). This restructuring, pattern that diminishes or counteracts possible toxic effects of clearly genetically programmed, involves the cell to cell orga xenobiotics. nization and the blood supply as well as the biochemical pattern. (a) At the physiological level, the key manifestation of the This phenomenon is seen in several models of hepatocarcin- resistance phenotype in the rare hepatocyte is the ability to ogenesis. In every respect, this is truly a process of differentia proliferate vigorously in response to a proliferative stimulus in tion, analogous to other examples of differentiation in normal an environment, such as that created by the presence of one of development. many carcinogens and some promoters, in which the vast (b) Rats with hepatocyte nodules are unusually resistant to a majority of hepatocytes, the nonresistant ones, are inhibited. lethal dose of a potent hepatotoxic agent, carbon tetrachloride This is a common basis for the promoting ability of many (63). Rats with nodules show complete resistance to a dose of hepatocarcinogens and may also be important with two non- CC14that is lethal for 100% of normal rats. carcinogenic promoting agents, the drug, phÃ©nobarbitaland the This demonstration of survival value indicates that the ge normal metabolite and obligatory precursor of pyrimidine nu- netic information necessary to develop hepatocyte nodules and cleotides, orotic acid (42-48). to have them remodel by differentiation could have been favored (b) The hepatocyte nodules show resistance to cytotoxic during the long period of evolution. 2753

Another aspect, possibly related to survival, is the resistance Relevance of Clonal Adaptation to Carcinogenesis and Cancer. of the hepatocytes in nodules to the cytotoxic effects of some Tumor promotion, despite its frequent association with an hepatotoxic xenobiotics in vitro and in vivo (see Refs. 35 and element of mystique, is basically clonal expansion of initiated 64). cells. In the liver, this leads to the genesis of hepatocyte nodules. (c) At the biochemical-molecular level, the biochemical pat The concept of clonal adaptation places tumor promotion in tern can readily account for the resistance of the nodules to the liver in quite a different perspective. This part of the cytotoxic effects of xenobiotics and for the more efficient ex carcinogenic process is only in part and perhaps only periph cretion of at least one carcinogen, 2-acetylaminofluorene (65). erally related to the ultimate development of cancer but becomes A marked decrease in activation associated with the large an important way in which the living organism responds in an decrement in phase I components, coupled with the more adaptive fashion to commonly occurring xenobiotics in the efficient conjugation and excretion of active moieties generated, environment. On this basis, scientifically, the neoplastic deriv associated with the several conjugating systems in phase II, ative, the cancer, is perhaps only incidental while the adaptive component is the most important from the perspective of match quite well (65). biology. In this regard, the "turning on" of a new phenotype in The virtual certainty that this pattern is physiologically pro grammed is indicated by its manifold nature with at least 25 to a rare cell during initiation becomes the key to what is seen in 30 different enzymes and other components and by the obser promotion. vations that some agents such as lead nitrate, an interferon, How a mutagenic carcinogen induces a new common con butylated hydroxyanisole, and butylated hydroxytoluene induce stellation of biochemical components that go to make up the a biochemical pattern in normal livers very similar to that seen resistance phenotype is not understood. Although the induction in the hepatocyte nodules but in a reversible or transient manner of mutations in a rare cell by mutagenic carcinogens or metab (36). This special biochemical pattern is not seen in fetal, olites is well known, it remains to be established that this is the neonatal, or regenerating liver but seems to be uniquely asso molecular mechanism for the induction of the resistant hepa ciated with the hepatocyte nodule induced by one of many tocyte. Altered regulatory genes, rare gene rearrangements, gene translocations, or gene amplifications are some possibili different carcinogens. ties. Hypomethylation of some genes, the protein products of These results suggest that hepatocyte nodules represent a new which show considerable elevation in the hepatocyte nodules, state of differentiation, better adapted to survive in a hostile has been found (68-72). Altered metabolic patterns [see Fox environment. The resistance, coupled with the capacity of the and Radicic (73) and Dean and Hinshelwood (5)] coupled with majority of nodules to undergo spontaneous differentiation to "progressive state selection" (see below) offer possible epige- normal-appearing mature liver, indicate that the clonal expan netic alternatives to the mutation hypothesis. sion seen in the early phase of carcinogenesis is genetically It would appear that the study of the control of transcription programmed and is part of a normal physiological pattern of of several of the discrete components of the resistance pheno- adaptation to some types of xenobiotics (9, 35, 36). types, such as glutathione 5-transferase 7-7 (P), 7-glutamyl- Is Clonal Adaptation Also Probable in Other Carcinogenic transpeptidase, D-T diaphorase (quinone reducÃase),and dis Systems? Superficially, there are several similarities in the crete relevant cytochromes P-450, to name but a few, could in overall patterns among many experimental and human systems turn suggest possible underlying mechanisms for clonal adap for cancer development (9, 37). These include: (a) the very long so-called "incubation period" during which the cellular evolu tation including altered methylation. An interesting aspect of clonal adaptation of great practical tion of malignancy from nonmalignant precursor lesions is occurring; (b) the apparent reversibility of many putative pre- importance relates to the resistance of many cancers to chemo therapy (74). It is often implied that resistance to chemotherapy neoplastic or precancerous lesions; (c) the common biological might be a property acquired during the schedule of treatment behavior and appearance of early focal proliferations such as as a mutation or some other genomic alteration including gene polyps, papillomas, and nodules with many different carcino amplification. The experience in the liver and the existence of gens and promoting environments. Interestingly, the colon clonal adaptation suggest the possibility that resistance may be system shows a very large number of changes in the mRNA a primary property of the cancer in some instances. Given the patterns between normal mucosa, polyps, and multiple poly- derivation of cancer by cellular evolution from a nodule with a posis (66). This is quite consistent with the suggestion that resistance phenotype that was used mainly for clonal expansion, polyps may represent a different state of differentiation. It it is certainly conceivable that many cancers in some tissues would seem prudent, therefore, to explore some of these systems may begin with a resistance phenotype. The presence in cancer from the point of view of clonal adaptation. In our opinion, it precursor cell populations of P-glycoprotein, as the product of is quite likely that this type of adaptation, as well as perhaps mdr gene and other components of resistance to xenobiotics others, might well be important in cancer development in including chemotherapeutic agents (52, 57-59), would suggest several systems. that some cancers may begin with a resistance phenotype and The most telling is in the case of malignant melanoma in may have to lose this phenotype as a prerequisite for a positive humans. Clark et al. (67) have shown that the genesis and cytotoxic response to some chemotherapeutic agents. behavior of nevi and their role in the development of melanoma In view of the widespread occurrence of many potential have many similarities to the hepatocyte nodules in rat liver carcinogens in the environment, it remains important to under carcinogenesis. The nevus shows at least two options, differ stand how evolution has enabled organisms to adjust or to entiation to nerve endings as a major pathway and persistence adapt to this ever-present hazard. The long life span of many with further cellular evolution to cancer as a minor one. species in the face of this hazard attests to the efficiency with Whether the resemblances between rat hepatocyte nodules and which evolution has succeeded. The long precancerous period, human nevi extend to biochemical-molecular mechanisms re lasting as long as one-third to one-half or more of the life span, mains a fascinating problem for study. has largely relegated neoplasia to the postreproductive period. 2754

The acquisition of a mechanism to adapt, such as by clonal bryos at cloning densities were the first to be used successfully adaptation, would seem to be an important way in which this in studies of in vitro carcinogenesis by chemicals (80). Up to has been accomplished. If this viewpoint is valid, one would 25% of the clones of these cells underwent morphological have to consider neoplasia as mainly a manifestation of the transformation during a 9-day treatment with carcinogenic imperfection of the adaptive process and cancer as a deviant of polycyclic hydrocarbons, and some of the transformed clones adaptation (75). Such a viewpoint might open several new went on to produce sarcomas when transplanted back into avenues for the development of novel ways to interrupt the Syrian hamsters. The high percentage of clones which became carcinogenesis process and thereby prevent cancer. morphologically transformed indicated to the authors that the Finally, the presence of a major adaptive component during transformation was not the result of "the usual type of randomly carcinogenesis would offer an interesting new perspective to the occurring mutation" (80). However, since the scoring of trans dominant current paradigm of carcinogenesis, the progressive formation was subjective, and only a single stage could be acquisition of abnormal genes and gene products without any discerned, the mechanism(s) of the two stage models could not regard to the adaptive ability and talent of biological systems. be resolved in this system. Our current view would envision the appearance of new phys In 1970, Mondai and Heidelberger (81) carried out a classic iological phenotypes during initiation either by mutation or by series of experiments on chemical carcinogenesis in a line of other mechanisms including epigenetic, and this phenotype mouse prostate cells. They found that every cell exposed to would play a central role in the long adaptive process during methylcholanthrene gave rise upon cloning to a population in carcinogenesis. Once a malignant change supervenes as a rare which a small minority of the cells initiated transformed foci. derivative relatively late in the development of cancer, a se By contrast, only 6% of control cells gave rise to clonal popu quence of altered genes could then begin which might be a lations which had some transformed foci. The fact that 100% reasonable basis for some of the late changes seen in the further of carcinogen-treated cells produced progeny with an increased evolution of cancers. "Progressive state selection" (see below) probability for transformation argued strongly against a muta could play an important role in this progression as well as in tional origin for the transformation. its modulation. A formulation more consistent with modern The work in culture was continued with the C3H10T'/2 line dynamic biology, such as here proposed, not only seems attrac of mouse embryo cells, hereafter referred to as 10T'/2 cells. This tive theoretically but also offers many possible practical ap line was widely used by many investigators to study carcinogen proaches to the ultimate prevention and treatment of cancer. esis induced by a variety of chemical carcinogens (82) and by X-irradiation (83). A finding reported by all who used the system was that the frequency of transformation (the number Neoplastic Transformation of Cells in Culture of transformed foci divided by the number of cells initially seeded) was inversely proportional to the number of cells seeded Initiation or the First Stage of Carcinogenesis in Culture. The (82-88). One particular version of this type of experiment was concepts of initiation and promotion of neoplastic transfor to administer 400 to 600 rads of X-irradiation to a low density mation and the associated two stage model of carcinogenesis of 10T'/2 cells, allow them to grow to confluency, transfer them were developed in the early 1940s from studies of chemically on many dishes over a wide range of concentrations, and score induced tumors in the skin of rabbits and mice (76, 77). In the foci 4 weeks after each cell concentration reached confluency mouse, the first, or initiating, stage was induced by a single (83). A consistent finding was that the number of foci per dish application of carcinogens such as coal tars or purified polycy- (usually less than one per culture) was independent of the clic hydrocarbons and persisted indefinitely thereafter in the number of cells seeded in the transfer. It was concluded that treated area, which generally retained its normal appearance. most, if not all of the cells originally exposed to the X-rays had Repeated application to that area of a promoting agent such as been altered by the treatment in such a manner as to transmit croton oil or TPA,2 which by themselves are not carcinogenic, to their progeny an increased probability for transformation. A resulted in the second stage defined by the appearance of similar conclusion was reached for chemically induced trans tumors. Largely because of the speed of induction of the initi formation of the 10T'/2 cells (87) and of retrovirus-infected rat ated stage, its persistence after removal of the carcinogen, and cells (89). the focal origin of the ensuing tumor, the first stage was The production of heritable change in an entire population generally considered to be mutational in origin (78, 79). The is a radical departure from the quantitative expectation of requirement for repeated application of promoting agents and specific genetic mutation, which usually occurs at a rate of 10~6/ the slowness of onset of the second stage led to the belief that cell division or less. Furthermore, this finding of high frequency, it was nonmutational, or epigenetic, in origin. In fact, however, heritable increase in focus-forming capacity initiated by X-rays whole animal systems were too complex and cumbersome to is consistent with other reports that most or all of the cells provide definitive evidence of these widely accepted explana which survive X-irradiation but receive sublethal hits have tions for the two stages. To add to the complications, it has heritable changes in growth rate (90, 91) and sensitivity to been pointed out that the two-stage model refers only to the mutagenic treatment (92). A correlated finding is that the total number of manipulations used and that many more than two size of the transformation targets of ionizing radiation at high steps occur in the two stage system in animals (49). linear energy transfer is as much as 1000 times higher than that The availability of cell culture systems that could be induced of the targets for specific gene mutations, which are themselves to undergo neoplastic transformation by exposure to carcino larger than that for a single gene (93). Large as it is, the target gens offered a much simpler, easily manipulated, and readily size based on transformation may be an underestimate for the quantified means for settling the question of the nature of two- initiation process, since only a small minority of initiated cells stage carcinogenesis. Primary cultures of Syrian hamster em- undergo transformation (94). 2The abbreviations used are: TPA, IJ-O-tetradecanoylphorbol-IJ-acelate: Several types of experiments with tumor initiators in animals DMBA. 7.12-dimethylbenz[a)anthracene. showed that enduring changes occur in a high proportion of 2755

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. CELLULAR ADAPTATION AND THE ORIGIN OF CANCER carcinogen-treated normal cells just as they do in cell lines. In initiating or first stage, could be considered a high frequency one case, the skin of mice was treated with the carcinogen process, even though its visible manifestation occurs with low DMBA and 5 weeks later almost all the basal cells in the probability. DMBA-treated area responded to croton oil by a faster onset The question of the nature of the second or transforming of DNA synthesis than did the basal cells of mice which had step has since been taken up in the NIH 3T3 cell system, which not been treated with the carcinogen (95). Since epidermis is a can be considered an initiated line, since foci of transformed renewing tissue with continuous turnover of cells, the persistent cells develop if the cultures are left in the confluent state for 10 DMBA-induced responsiveness indicated a heritable increase days or longer in a given range of serum concentrations (101- in the reactivity to promoting agents of all the basal cells in the 103). This capacity for focus formation is highly dependent on area. In another case, DMBA was repeatedly applied to hamster the passage history of the cells. If they are passaged frequently cheek pouches, resulting in the appearance of localized carci at low density in high serum concentration, their capacity for nomas (96). There was a large increase in proteolytic activity transformation steadily diminishes to the point that no trans in the carcinomas and in the surrounding, normal-appearing formed cells arise, even after several successive postconfluent tissues, indicating that a high frequency change had occurred incubations.' Passage of the original cells at low density but in over the entire exposed area. Several studies on tumor induction low serum concentration gives rise to focus-forming cells, just in normal diploid cells show that heritable change occurs in a as prolonged incubation at confluency does. Both conditions high proportion of them after a single dose of X-rays. X- which evoke the transformation constrain the growth and over irradiation of thyroid cells freshly removed from the rat con all metabolism of cells, and it was surmised that the transfor verts 1 of 32 of the clonogenic cells into tumorigenic cells (97). mation represents an adaptive response to that constraint. A similar result was obtained with mammary epithelium re However, growth constraint imposed by the very different moved from rats that had been treated 24 h earlier with a method of limiting the availability of glutamine actually inhibits chemical carcinogen (98). At least 1 in 250 of the cells initiated transformation instead of evoking it (104). The observation that a mammary carcinoma when dispersed and grafted into the fat transformation occurs only under certain specified physiologi pads of isologous recipients. Taken together, these findings cal conditions shows that it is not a spontaneous event in the clearly show that carcinogenic agents produce heritable changes sense that genetic mutations are. The fact that it was favored in a much higher proportion of treated cells than would be by physiological conditions which constrain rather than en expected of conventional mutations. The implication is either courage multiplication was further evidence against a muta- that damage to any one of a very large number of genes can tional origin of the transformation. produce the neoplastic transformation or that the primary Although only a small fraction (usually less than 1%) of the targets are not genetic. An important finding favoring the latter NIH 3T3 cells became focus formers when exposed to growth- interpretation is that X-ray-initiated 10T'/2 cells are irreversibly constraining conditions, the entire population actually under reverted to their original uninitiated condition by treatment went a heritable increase in growth capacity. This was expressed with protease inhibitors begun as late as 10 days, or 13 cell in a substantial increase in saturation density of the entire cell divisions after irradiation (99). There is certainly no reason to population after 3 short passages in low serum concentration expect that protease inhibitors would revert genetic mutations and even greater increases in further passages (102). Clonal in a whole population of cells, particularly at so long a time analysis of a similarly treated population revealed that all the and so many divisions after X-irradiation. cells had acquired an increased capacity for focus formation, Transformation or the Second Stage of Carcinogenesis in Cul although less than 1% of the cells seeded from the uncloned ture. The promotion stage is defined as that which leads to parental population initiated foci in a direct assay (102, 105). tumor formation. Transformation is the cell culture analogue There was an extremely wide range of capacities to produce of tumor formation and can be considered the second stage of foci among the clones, and some of them drifted up or down in carcinogenesis in vitro (94). Although all the X-irradiated 1OT'/2 that capacity as they were repeatedly passaged and assayed for cells appear to be initiated, only a small fraction of them (about focus formation (102). It is apparent that the population does 10~6) actually produce transformed foci (94). It was concluded not fall into two simple classes of transformed and nontrans- that the second step in transformation is a very rare event. formed cells; rather it consists of a mosaic of cells with a very Fluctuation analysis of clone size distribution of transformed large range of capacities for transformation in confluent cul cells agreed with the hypothesis that the second event is a tures. There may be an almost continuous spectrum of poten spontaneous mutation, with a constant small probability of tialities or probabilities for transformation among cells when occurring every time a cell divides (94). This conclusion was at the appropriate conditions are applied. In addition, the expres odds with an earlier finding that no cells capable of producing sion of the transformed state in the development of foci depends foci on transfer appeared in carcinogen-treated lOT'/z cultures on the initial state of the cells, a variety of factors in the assay, until about 3 weeks after they had become confluent and net and the differential in growth rates among the cells at growth had long since stopped (84). It also seems inconsistent confluency. with the transforming effect of the tumor promoter, TPA, on These considerations indicate that there is an optimal degree 10T'/2 cells exposed to 100 rads of X-rays, which is insufficient of constraint for eliciting the adaptive growth response that radiation to significantly increase the frequency of the rare characterizes transformation. If the serum concentration in focus found among control cultures (100). The addition of TPA confluent cultures is too low or too high or if cells are seeded to the lightly X-rayed cells increased focus production to the at such high concentrations that they become crowded as soon extent that almost every culture arising from a single cell had as they attach and spread on the dish, the number of foci per 1 or more foci. The effect of TPA was therefore similar to seeded cell is reduced. It is important to take these factors into increasing the dose of X-rays to 400-600 rads, in that it account when comparing the results obtained in cell culture increased the focus-forming capacity of almost every cell. On with those obtained in the animal. In cell culture, sparse cell this basis, the promoting or second stage in these cells, like the 3 H. Rubin, unpublished observations. 2756

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. CELLULAR ADAPTATION AND THE ORIGIN OF CANCER populations in high serum concentrations are growing and serum concentrations or at high cell densities. We cannot, in metabolizing at a maximal rate and the adaptive response fact, exclude the possibility that the variation in these cellular associated with transformation is brought on when constraints capacities is continuous and therefore distinctive from the dis associated with confluency or low serum concentrations are crete nature of mutations. introduced. In the animal, most cell types are normally under 4. The capacities for focus formation and tumor development the constraint introduced by the 3 dimensional contiguity of are reversible for the entire population of transformed cells. the organized tissue. For example, very few cells of the intact Although adaptation criteria 1 and 4 have been demonstrated liver are synthesizing DNA at any moment, and less than 0.01 % in cells other than the NIH 3T3 line, it is only in this cell that are in mitosis (106). If the liver epithelial cells are dispersed all 4 criteria have been demonstrated. and seeded at low density in culture, 80% of them enter DNA Speculative Mechanism of Carcinogenesis. Given the evidence synthesis and mitosis within 48 h (107). Therefore, in the that the growth properties of a high proportion of cells treated animal, the optimal level of growth and metabolism for neo- with carcinogens are heritably altered, the question arises of plastic transformation of initiated cells may come only when how such pervasive change in whole populations might be they are stimulated to multiply. This appears to be the case in elicited. First, however, it must be noted that heritability among liver carcinogenesis, where nodule formation by carcinogen- somatic cells of the capacity for tumor formation differs in treated cells is brought on by compensatory growth (49). By several respects from that associated with transmission of spe contrast, transformation of rat liver cells in culture is enhanced cific gene mutations in organisms. Not only does transforma by keeping the liver cells in the confluent, relatively inhibited tion in culture occur with high frequency in response to carcin state for 3 weeks at each month passage, instead of serially ogens but the phenotype is heterogeneous, involves the gradual passaging them once a week just as they reach confluence (50). loss of many of the differentiated properties of the treated cells, Thus, growth stimulation in vivo and growth inhibition in vitro and can be prevented in the 10T'/2 line by protease inhibitors, may converge on the optimal condition for transformation. which have no effect on mutation frequency in mutagen-treated Just as reversal was demonstrated for the initiated state, so cells (111). It appears, therefore, that proteases play an essential has it been shown for the transformed state. It was first reported role in the initiation of transformation in lOT'/z cells. that cultivation of transformed Syrian hamster cells at low Protease activity in yeast cells responds to a change in sub density reversed several of their in vitro growth properties and strates. Yeast cells multiply exponentially until they exhaust reduced their capacity for tumor formation (108). Then it was their main substrate, glucose, and remain at a constant number found that the same type of low density growth fully reversed until they start utilizing another substrate (112). The second the morphological and tumor-producing properties of a clone phase of growth is much slower than the first, and it is accom of 10T'/2 cells that had been transformed by X-irradiation (109). panied by a 300-fold increase in protease activity. An inactivat Similar treatment of spontaneously transformed NIH 3T3 cells ing mutation in a key protease results in a failure of other resulted in a loss of their focus-forming capacity and of their proteolytic and hydrolytic enzymes to be processed to an active ability to form tumors in nude mice (102, 110). In both the form. If a mutant cell is exposed to wild type cytoplasm through 10T'/2 and NIH 3T3 cells, the transformed phenotype was mating or heterokaryon formation, the mutant segregants can perpetuated by maintaining the cells at high density. The re maintain indefinitely the wild type content of active proteases versibility of the transformed state of a whole culture considered and hydrolases normally dependent on the now inactive pro alongside its induction under conditions of constraint, and the tease (112). The yeast system demonstrates clearly that an graded response of the entire population leave little room for altered phenotype can be transmitted to progeny by epigenetic doubt that the transforming step, like that of initiation, is in its means without the involvement of cytoplasmic genes (113). The inception a nongenetic process. demonstration of the epigenetic persistence of wild type pro Adaptive Origin of Spontaneous Transformation. The diverse tease content may have particular significance for neoplastic evidence for the adaptive origin of "spontaneous" transforma transformation since: (a) the initiated state in lOT'/z cells can tion of NIH 3T3 cells can be summarized as follows: be reversed by protease inhibitors (99, 111); (b) there is a large 1. No cells are capable of producing dense foci under standard increase in protease content in carcinomas and their surround conditions if the cells had been repeatedly transferred at low ing normal appearing tissue of the carcinogen-treated hamster density in high serum concentrations. Focus-forming cells can cheek pouch (96); (c) the addition of proteases to density- be detected, however, by reassaying cells from the standard inhibited chick embryo cells stimulates them to multiply (114); assay within a few days after the culture becomes confluent and (d) a serine protease that is assayed as an activator of plasmin- net increase in cell number has ceased. Cells capable of forming ogen is elevated in primary tumor cells, in cells transformed by foci in the standard assay also arise even if cultures are passaged viruses, and in a number of tumorigenic cell lines (115); (e) the at low density, if the serum concentration in the passages is induction by TPA of secretion of the protease which activates maintained at a low level. Unlike mutations, therefore, "spon plasminogen has been used to order histologically distinct taneous" transformation is not really spontaneous but occurs classes of human colonie adenomas from the most benign to only when the appropriate inducing conditions obtain. the most advanced premalignant state (116). It seems plausible, 2. Although the capacity to form foci arises in only a fraction therefore, that persistent perturbation of the concentration or (less than 1%) of the population repeatedly passaged at low localization in cells of proteases and other hydrolytic enzymes density in low serum, the bulk of the population gains the might result in a transmissible defect of growth regulation. It capacity to multiply to higher saturation densities. Each cell is not unlikely that such perturbation would increase the genetic also gives rise to a clone with an increased probability to form instability of the cells, introducing genetic changes which might foci in the standard assay. The involvement of the bulk of the contribute to tumor progression. population in these changes in growth capacity is inconsistent It remains a question what the target for carcinogens might with a mutational origin. be that could initiate and propagate such epigenetic changes in 3. There are many degrees of adaptation to multiply in low cells. One possibility is carcinogen-induced damage to the mem- 2757

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. CELLULAR ADAPTATION AND THE ORIGIN OF CANCER branes of lysosomes which contain most of the cellular hydro- increasing efficiency depending on their progression to malig lases. It is noteworthy that UV light, which is a common skin nancy (116). It has also been reported that tumor promoters carcinogen, can in very small doses cause hemolysis of human enhance the proliferation of preneoplastic rat liver cells to a erythrocytes which are virtually without DNA or RNA (117). significantly greater extent than normal unaltered liver cells The hemolysis, which represents the leakage of hemoglobin (124). Finally, there is the finding discussed earlier that TPA from the erythrocyte, is apparently the result of a direct effect has the same transforming effect on 10T'/2 cells that have of UV light on its membrane, and it has been conjectured that received 100 rads of X-rays as does raising the X-ray dose to the mechanism underlying the changes in skin during carcino- 400-600 rads (100). Thus, promoting and initiating agents may genesis by UV light is closely comparable to that which in ultimately produce the same result in cells, although the path creases the permeability of the erythrocyte (117). Mitochondria way through which they act may differ. That only a rare cell in of yeast cells are also disrupted by UV light (118). This effect an initiated field is the actual antecedent of the tumor would is of special interest in cancer where the most commonly follow from the intrinsic heterogeneity of growth capacities in observed biochemical change is increased glycolysis, which was the cell population (102, 103, 125, 126). considered a regulatory response to defective respiration (119). An interpretation similar to that developed here was pro A relation between damaged mitochondrial membranes and posed by Blum to account for epidermal cancer induced in mice defective respiration became quite plausible with the advent of by UV light (117, 127). Results from a large series of carefully the chemiosmotic model of oxidative phosphorylation and its quantitated experiments indicated that successive doses of UV dependence on transmembrane gradients of electrons and pro light progressively accelerate the proliferation of the epidermal tons (120). Nongenetic changes in surface structures can be cells. Blum concluded that the results could not be explained perpetuated for hundreds of cell generations, as has been dem by separable induction and growth phases or by somatic muta onstrated by direct visualization in Paramecium (121). Other tion. Rather, the tumor cells could be considered as differing methods must be developed to demonstrate long term mem from normal cells only in the quantitative sense of proliferating brane effects in metazoan cells; thus the proposal is as yet more rapidly in the regulated context of the epidermis with hypothetical. It is an interesting fact, however, that the carcin each successive exposure to UV light. Although UV light un ogenic polycyclic aromatic hydrocarbons are highly lipophilic doubtedly damages cells, the major response of the epidermis and probably localize to a large extent in membranes (122). is an increase in the rate of mitosis and a consequent increase Although speculative, the suggestion of carcinogenesis through in thickness of the skin (117). If the radiation is not repeated, membrane damage would appear to be testable by studies of the epidermis returns to its normal thickness. The increased the effects of carcinogens on, for example, membrane traffick mitosis can be viewed as an overshoot of the adaptive response ing, cell permeability, erythrocyte hemolysis, protease distri to cell damage. If the radiation is repeated frequently, the skin bution, and mitochondrial function. It is well to keep in mind, remains thickened and carcinomas and sarcomas appear within however, that the definitive evidence in cell culture against a a few months. Blum designated the cellular response to the mutational origin of both the initiation and promotion stages repeated UV irradiation "progressive acceleration of growth." came from quantitative experiments on the growth behavior of He indicated that all the cells exposed to the treatment were living cells. Much remains to be done at that level to obtain a incrementally altered but noted that very slight differences in fuller understanding of neoplastic transformation. their accelerated growth rates would, over a period of months, General Considerations. As noted earlier, the two stage model result in the progeny of one cell outgrowing the others to form of cancer refers to the manipulations used in experimental a tumor. In fact, however, there were signs of the beginning of carcinogenesis, but many more than two steps are involved (49). several tumors on as small an irradiated expanse as the mouse Foulds (123) believed that "initiation establishes an expanse or ear, and sometimes carcinomas and sarcoma tissue were found region of incipient neoplasia that is coextensive with the area in the same tumor mass (117). of exposure to carcinogenic action and has a permanent, repl- Progressive acceleration of growth can account for the pro icable new capacity for neoplastic development which can be gressive increase in number and thickness of transformed foci augmented by repetition of the carcinogenic stimulus." In this of NIH 3T3 cells repeatedly passaged at high density (110) or view, the initiated cells, as observed in the classical skin carci in stepwise decreases in serum concentration (105). To this nogenesis model (17, 78), are not dormant tumor cells but are should be added the selective effect of maintaining cells under cells with an increased reactivity to further applications of conditions of moderate growth constraint. It has been proposed carcinogens or of promoters. The changes effected by initiation that such constraint selects not only for individual cells with are not restricted to the places where tumors emerge at a later increased growth capacity but also for metabolic states within time. Foulds' view coincides with the evidence presented here cells that are subject to continuous phenotypic fluctuation in that the carcinogenic stimulus affects the entire population of growth capacity (102, 105, 110, 126). Successive repetitions of exposed cells, although only a small fraction progress to the this process would lead to relatively stable increases in growth overtly transformed state. If the initiation step is seen as en capacity under conditions of moderate constraint, and the proc ess was called "progressive state selection" (102). Those con dowing cells with an increased reactivity, the promotional treat ment may differ only in degree rather than in kind; i.e., pro straints, which are known to lower metabolic rates as well as moting agents may be effective only on cells previously sensi growth, could lead to an increase in protease, as occurs in yeast tized by a more potent, initiating agent. That this is the case is with the transition from exponential growth to the stationary suggested by the previously mentioned observation that mouse phase, and with the switch from rich to minimal medium (128). epidermis initiated by treatment with DMBA enters DNA Such changes in previously sensitized, i.e., initiated, cells would synthesis more rapidly when treated with croton oil than does lead to increased growth capacity and heterogeneity, just as noninitiated epidermis (95). Similarly, TPA fails to induce the exposure to a carcinogen or promoter would, thereby resulting secretion of plasminogen activator in primary cultures of nor in repeated selection of cells with the greatest growth potential mal human colon but does so in cultures of adenoma cells with and ultimately in neoplasia. The combined effects could be 2758

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2761

Emmanuel Farber and Harry Rubin

Cancer Res 1991;51:2751-2761.

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