Evolving perspectives on the biology and mechanisms of carcinogenesis

Leukemia Research Vol. 10, No. 7, pp. 727-734. 1986. Printed in Great Britain.

0145-2126/86$3.00 + .00 PergamonJournalsLtd.

EVOLVING PERSPECTIVES ON THE BIOLOGY A N D MECHANISMS OF CARCINOGENESIS* ARTHUR C. UPTON Institute of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. (Accepted 17 December 1985)

Abstract--From experimental and epidemiological evidence, radiation-induced cancers appear to arise as multistage, monoclonal growths, which are elicited through various mechanisms, depending on the neoplasm in question and the conditions of exposure. At the molecular level, the process of carcinogenesis may involve the activation of oncogenes and/or the inactivation or loss of anti-oncogenes, through chromosomal rearrangements, point mutations, and other effects of radiation on DNA. In contrast to these mechanisms of carcinogenesis, which result from the absorption of radiation by the tumor-forming cells themselves, abscopal effects resulting from irradiation of other cells may contribute to carcinogenesis under certain conditions, e.g. in the induction of tumors of endocrine target cells through radiation-induced disturbances of hormonal balance. Effects of the latter type, which require the killing of substantial numbers of cells, are not elicited at low doses, thus contrasting with effects of the former type, which may be presumed to have no thresholds. Because radiation carcinogenesis may be mediated through a diversity of effects, the relationship between incidence and dose can vary accordingly. The relationship between the incidence of radiation-induced tumors and the time elapsing after irradiation also varies, depending on the type of tumor in question, species, age at irradiation, exposure conditions, and other factors. Although the variations with dose and time are consistent with multistage models of tumor initiation, tumor promotion, and tumor progression, the precise nature of the successive steps that are involved remains to be determined. The tendency for the tumors to resemble their spontaneous counterparts in age-distribution points to interactions between radiation and other carcinogenic risk factors which are as yet poorly understood. Also poorly understood are species- and organ-differences in susceptibility to radiation carcinogenesis, which bear no consistent relationship to corresponding 'spontaneous' cancer rates. Key words: Carcinogenesis, oncogenes, anti-oncogenes, initiation, promotion, progression.

INTRODUCTION DATING f r o m t h e t u r n of t h e c e n t u r y , t h e carcinogenic effects of ionizing r a d i a t i o n h a v e b e e n i n v e s t i g a t e d m o r e t h o r o u g h l y t h a n t h o s e of any o t h e r physical or c h e m i c a l a g e n t [1-3]. T h e r e l e v a n t r e s e a r c h has i n v o l v e d virtually e v e r y species of l a b o r a t o r y a n i m a l a n d every level of biological o r g a n i z a t i o n . A l t h o u g h the biology of radiation c a r c i n o g e n e s i s r e m a i n s to b e u n d e r s t o o d in detail, certain cellular a n d m o l e c u l a r m e c h a n i s m s can n o w b e implicated, at least in a g e n e r a l way.

* Supported in part by Grants ES 00260 and CA 13343 from the U.S. Public Health Service and Grant SIG-9 from the American Cancer Society. Correspondence to: Dr A. C. Upton at the above address.

MULTISTAGE DEVELOPMENT OF NEOPLASIA: INITIATION, PROMOTION, PROGRESSION P r e s e n t t h e o r i e s of carcinogenesis view c a n c e r as a m o n o c l o n a l g r o w t h t h a t arises t h r o u g h a succession of cellular a l t e r a t i o n s , or stages [4]. Studies of the genetics of c a n c e r cells imply t h a t carcinogenesis can result from: (1) the h o m o z y g o u s i n a c t i v a t i o n or d e l e t i o n of certain genes; (2) the a b e r r a n t activation of o t h e r w i s e n o r m a l genes, as m a y o c c u r t h r o u g h trisomy or c h r o m o s o m a l r e a r r a n g e m e n t s ; or (3) the activation of p o i n t m u t a n t alleles of o t h e r w i s e n o r m a l genes [5]. C h a n g e s of any or all of t h e s e types h a v e b e e n s h o w n r e p e a t e d l y to be inducible by ionizing r a d i a t i o n [3]. T h e a g e - d i s t r i b u t i o n of cancers in vivo [6] a n d the biology of m a l i g n a n t t r a n s f o r m a t i o n in vitro [7, 8] imply t h a t m o r e t h a n o n e a l t e r a t i o n , genetic or otherwise, is necessary to cause cancer. F o r e x a m p l e , the relation 727

728

A.C.

between cancer incidence and age--which can be represented by the expression: I = crt"

(1)

where the cancer incidence I increases as a power function of age (or time after irradiation) t, o~is a constant, and n denotes the number of stages involved--suggests that carcinogenesis evolves through 4-6 successive steps or stages [6]. This range exceeds the number of activated oncogenes that have been thought to be necessary for the transformation of immortalized cells in vitro [8], but it is not inconsistent with the number of steps that are apparently involved in the in vitro transformation of primary hamster embryo fibroblasts into fully malignant cells [9]. 'Initiation', 'promotion', and 'progression' are, of course, operational definitions [4, 10-12] which do not specify the precise nature of the changes underlying the successive stages in carcinogenesis. Nevertheless, methods are becoming available for analysing such changes at the molecular level, which may soon make it possible to define them in detail. EFFECTS ON GENES AND CHROMOSOMES The evidence that most cancers arise from a single transformed cell [13], the heritable nature of the transformed phenotype, the transforming ability of oncogenes [14-16], the association between neoplastic transformation and specific mutations or chromosome aberrations [5, 17], the potency of ionizing radiation as a mutagenic and clastogenic agent [3, 18], and the increased risk of cancer associated with inherited defects

UPTON TABLE I. COMPARISON OFTHE PROPERTIES OFONCOGENES AND ANTI-ONCOGENES

Oncogenes Gene active Specific translocations Translocations not hereditary Dominant Tissue specificity may be broad Especially leukemias and lymphomas

Anti-oncogenes Gene inactive Deletions or invisible mutations Mutations hereditary and non-hereditary Recessive Considerable tissue specificity Solid tumors

From [5].

in D N A repair [5] imply that damage to genes and chromosomes plays an important role in radiation carcinogenesis. Various effects on genes and chromosomes (Table 1) may be conceived to activate oncogenes; these include point mutations [19-21] as well as chromosome rearrangements [17, 22]. Point mutations, deletions, and chromosome rearrangements can, likewise, be implicated as mechanisms for inactivating anti-oncogenes (Table 1). The identity of the specific oncogenes and/or antioncogenes that may be involved in radiation carcinogenesis is still largely unexplored. The K-ras oncogene, however, has been observed to be activated in 4 of 7 radiation-induced thymic lymphomas of C57B1 mice, whereas the N-ras oncogene was activated in 5 of 6

TABLE2. ESTIMATEDRATESOFFORMATIONOFDNA DAMAGEPRODUCTSIN MAN Damage Base loss Depurination Depyrimidination Base deamination Cytosine Base alkylation N-7-Me-G N-3-Me Base dimerization Cyclobutane type Pyr(6-4)Pyo type Base oxidation Thymine Glycol 5-Hydroxymethyl uracil Other oxidized products Single strand breaks Direct From [59].

Events/ cell/day 25,000 1300

Primary source of damage

Experimental system analysed

Spontaneous Spontaneous

Chemical model Chemical model

Spontaneous

Chemical model

84,000 840

S-adenosyl-met. S-adenosyl-met.

Chemical model Chemical model

up to 37,500 up to 12,500

U.V. light

Bacteria

U.V. light

Bacteria

400

Ox. metabolism

Human urine

600

Ox. metabolism

Human urine

2000

Ox. metabolism

Estimated

Spontaneous

Chemical model

350

100,000

Biology and mechanisms of carcinogenesis similar neoplasms induced by 4-nitrosourea [23]. This apparent carcinogen-dependent specificity in oncogene activation is intriguing and, obviously, calls for further investigation. The K-ras oncogene has also been found to be activated in 3 of 6 rat skin carcinomas induced by ionizing irradiation, along with activation, gene amplification, and enhanced expression of the m y c oncogene [24, 25]. In view of evidence that more than one oncogene must be activated to transform a cell [8], it is not clear whether or how the aforementioned oncogenes may have contributed to the pathogenesis of the tumors in question, nor is it clear whether they were activated early or late in carcinogenesis. Although the oncogenes could well have been activated early, through mutagenic or clastogenic effects of radiation, other possibilities remain to be excluded, e.g. thousands of lesions are estimated to occur 'spontaneously' each day in the D N A molecules of every cell, through the effects of natural background radiation, free radicals, and other degradative mechanisms (Table 2). Until more is known about the identity, regulation, and function of oncogenes in radiation-induced tumor cells, their role in the carcinogenic process will remain uncertain. Furthermore, while activation of more than one oncogene may normally be required to cause transformation, all but one of the necessary activation steps may already have occurred before irradiation in some cells, through inheritance of abnormalities via the germ line, previous somatic mutations, or both. Inactivation of anti-oncogenes has been implicated in the heightened susceptibility to radiation carcinogenesis that is associated with certain hereditary traits, e.g. in persons with familial retinoblastoma, the inheritance of a deletion or inactivation of one of two paired homologous anti-oncogenes at the Rb-1 locus has been postulated to result in a situation in which the subsequent elimination or inactivation of the remaining normal allele in a suitable somatic cell may lead to its transformation [5, 26]. A similar mechanism has been implicated in Wilm's tumor [5].

EPIGENETIC

AND ABSCOPAL

EFFECTS

Epigenetic effects of radiation on gene regulation also may conceivably contribute to transformation. Effects on the cell membrane have been invoked to explain the carcinogenesis-enhancing action of phorbol esters and other promoting agents [27]. It is noteworthy, therefore, that a dose of less than 10 mSv suffices to inhibit the uptake of iodoeoxyuridine into the D N A of mouse bone marrow cells, presumably through effects on the nuclear membrane [28]. Tumor-enhancing effects of phorbol ester on cell membranes have also been attributed to the blocking of intercellular metabolic cooperation between contiguous cells [11, 29, 30]. Whether comparable mechanisms are involved in the cell density-dependent suppression of transformation that is postulated to occur in rat mammary gland cells irradiated in vivo [31] remains to be determined.

729

Evidence that phorbol ester can cause D N A strand breaks in mouse skin cells, through the release of superoxide radicals from leukocytes [32], suggests that its promoting effects may also be mediated through 'radiomimetic' influences [33], which have been little explored as yet. Radiation-induced changes in the homeostatic relationships among cells also may profoundly influence the probability of transformation and subsequent neoplasia. The importance of these relationships is exemplified in tumorigenic effects on endocrine organs, where radiation-induced disturbances of hormonal feedback mechanisms have been observed to play an important role [3, 34, 35]. In the induction of ovarian tumors in the mouse, likewise, both ovaries must be sterilized by radiation in order to elicit tumor formation in either one [34]. The induction of pituitary tumors in the mouse also may depend on disturbances of hormonal feed-back regulation resulting from radiation damage to distant target organs [34]. Similarly, in the induction of thymic lymphomas in the mouse, the thymus itself need not be irradiated, but damage to the entire hemopoietic system is required for maximal tumorigenesis [36]. Also, in the pathogenesis of osteosarcomas, the killing of osteoblasts by locally deposited radium has been postulated to promote carcinogenesis through stimulation of regenerative proliferation in surviving cells [37]. The above examples are but a few of the many instances in which homeostatic relationships among cells have been observed to influence the probability of neoplasia. Other noteworthy instances include the effects of immunological impairment on susceptibility to carcinogenesis [38] and the action of morphogenetic fields, selection pressures, and other factors influencing cell turnover and differentiation [4, 38]. Neither initiation nor promotion can be considered to depend entirely on mutational changes. Both involve dynamic interactions among cells, leading to cellular adaptation, selection, and ultimately the outgrowth of increasingly autonomous clones [4, 33, 39].

EFFECTS ON THE AGE-DISTRIBUTION OF NEOPLASIA In comparison with the tumors induced experimentally by potent chemical carcinogens or viruses, the tumors induced by radiation tend to have long latencies. 'Preneoplastic' cells, on the other hand, may be detectable by appropriate transplantation assays long before the appearance of the tumors themselves [40-42]. The latent period between irradiation and the appearance of neoplasia in different species varies as a function of the natural life span of the species [43]. The latent period within a given species varies with age at the time of irradiation and with the type of neoplasm, generally being shorter for leukemia than for solid tumors. The types of leukemia that are induced are also agedependent [18, 44], presumably reflecting corresponding age-differences in the incidence of 'spontaneous' leukemias of different types, e.g. with increasing age at irradiation during adult life, the relative risk of human

730

A. C. UPTON

leukemias (all types combined except the chronic lymphatic type) remains more or less constant, while the absolute risk increases with age at irradiation [18]. Furthermore, most cases of chronic granulocytic leukemia appear within 5-15 yr in a wave-like pattern (Fig. 1). The patterns for other types of radiation-induced leukemia, although not identical, also are wave-like (Fig. 1). The differences among leukemias in age-distribution and latency point to differences in pathogenesis which remain to be explained. The comparatively short latency of most leukemias, as compared with solid cancers, may conceivably reflect the relatively rapid turnover and disaggregated nature of hemopoietic stem cells, which may impair their ability to repair certain types of radiation-induced damage and make them less subject to feed-back regulation, as compared with cells in solid tissues [35]. In contrast to the leukemias, the breast cancers induced by irradiation during childhood have not appeared until more than 30 yr later, when the exposed women have reached the age at which breast cancers characteristically appear in the general population [45]. The data imply, therefore, that carcinogenesis in the female breast can be initiated by irradiation but depends for its expression on the promoting effects of age-related hormonal stimulation. In this context, it is noteworthy that susceptibility to the induction of breast cancer appears to decrease with advancing age at irradiation during adult life [46]. With most other solid cancers, similarly, the induced cases do not appear to be distributed in wave-like pat-

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Pio. 2. Incidence of radiation-induced cancer (excluding leukemia) in relation to attained age in different birth cohorts of heavily-irradiated Nagasaki A-bomb survivors, as compared with the baseline incidence in the general population of Japan. Data for A-bomb survivors [63] denoted by dashed curves, with age of each cohort at time of irradiation as indicated. Data for the general population of Japan [64] denoted by the solid curve.

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terns after irradiation (osteosarcomas excluded). Instead, the annual excess of such cases appears to increase with time as a constant fraction of the underlying baseline incidence (Figs 2 and 3), implying that carcinogenic effects are promoted (or expressed) to a greater degree in the elderly than in the young. This pattern implies a relationship between carcinogenesis and the biology of aging which deserves to be explored further.

DIFFERENCES AMONG ORGANS AND CELLS 5

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FIG. 1. Temporal distribution of radiation-induced leukemias

in A-bomb survivors, in relation to type of leukemia and age at the time of irradiation [62].

Although, in general, the overall incidence of induced cancers increases as a function of time after irradiation, in parallel with the age-specific 'spontaneous' baseline incidence, the excess of a given type of cancer appears to bear no constant relationship to the baseline incidence (Table 3). Chronic lymphatic leukemia, for example, is relatively common in western populations, but its incidence has not been detectably increased by wholebody irradiation [18]. Likewise, reticulum cell sarcoma is relatively common in aging mice of many strains, but its incidence has not been detectably increased by irradiation in such animals [3]. These and other striking differences among organs and cells in susceptibility to

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TABLE 3.

COMPARATIVE SENSITIVITIES OF DIFFERENT TISSUES TO RADIATION CARCINOGENESIS*

Site or type of cancer Breast (female) Lung, bronchus Colon Stomach Leukemia Thyroid gland Urinary tract Pancreas Lymphoma, multiple myeloma Liver, biliary tract Brain, central nervous system Esophagus Pharynx Salivary glands Skin Uterus and cervix Ovary Parathyroid gland Bone Cranial sinuses Larynx Mesothelium Connective tissue, including heart Testis Prostate Other

Spontaneous incidencet (per 106 per yr) male female -690 310 120 805 20 310 105 170 50 60 50 15 10 > 1000 --<5 10 <5 80 <5 20 35 560 120

900 230 340 70 60; 60 130 90 150 50 40 20 5 10 < 1000 440 140 <5 5 <5 15 <5 15 --120

* From [3, 18, 60]. + Values represent rounded averages for all ages and races [61]. $ Excluding chronic lymphatic leukemia.

Estimated radiation-induced excess cases (per 106 per yr per rem)

(> 1)

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20

the carcinogenic effects of radiation, as opposed to other causes, remain to be unexplained. DOSE-INCIDENCE

RELATIONSHIPS

Although some variation in dose-incidence relationships is to be expected in view of the diversity of effects through which radiation can affect the probability of neoplasia, the relationship between incidence and dose varies from one type of neoplasm to another, and not all neoplasms within a given species or strain are increased in frequency by irradiation [3, 47]. For most neoplasms, the relationship between incidence and dose, dose rate, and LET is generally consistent with the pattern shown in Fig. 4. This is the pattern one would expect if the induction of a mutation or chromosome aberration sufficed to initiate carcinogenesis in a suitably susceptible surviving cell [48]. Substantially different patterns, however have been observed with certain neoplasms, which remain unexplained for the most part. The markedly sigmoid curve for osteosarcomas induced by internally deposited radium-226 has been interpreted in terms of a three-stage induction process, involving two initiating steps followed by a promoting step [37]. The anomalously low RBE of neutrons for induction of tumors in the mouse ovary, which persists even at low dose rates, has been interpreted to implicate damage to the cell membrane in radiation-induced killing of oocytes [49]. The paradoxically high effectiveness of exposure at low-dose-rates to fast neutrons [50] or gamma rays [51] for transforming cells in vitro, e.g. Fig. 5, cannot be explained satisfactorily without further data [52]. Comparable protraction and/or fractionation effects in vivo [53-55] likewise remain to be accounted for, although promoting effects mediated by cell killing have been implicated in some instances [47, 55]. In this connection, it is noteworthy that in-vitro transformation itself appears to be a multistep process which, although initiated with single-hit kinetics, may be affected by a diversity of factors, including influences known to promote or inhibit carcinogenesis in vivo [56, 57]. Experiments in which irradiated cultures have been subcultivated at various dilutions after their growth to confluence have been interpreted to imply that transformation is a relatively late-occurring event, the probability of which increases with the number of cell

40

60

80

I00

DOSE, cGy

FIG 5. Frequency of transformation as a function of dose in C3H 10T 1/2 cells exposed to fission neutrons at different dose rates [50].

divisions following irradiation; the data also imply that essentially every irradiated cell is 'initiated' and potentially transformable [7, 56]. In-vivo experiments with irradiated 'clonogens' point to similar conclusions [35]. Efforts to describe the relationship between incidence and dose must take into account the temporal distribution of the induced neoplasms as well as their total number. With radiation, as with many chemical carcinogens, the average induction time under conditions of daily exposure can be represented by the expression: c = dt"

(2)

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