Formal Discussion Of: Somatic Mutations and Carcinogenesis'

Formal Discussion of: Somatic Mutations and Carcinogenesis'

HOWARD J. CURTIS Biology Department, Brookhaven National Laboratory, Upton, New York

SUMMARY A great deal of evidence is now available indicating that mutations play a dominant role in the aging process. Because carcinogenesis is one facet of the aging process, mu tations must play a major role here also. However, carcinogenesis certainly does not consist of the simple induction of a mutation in one cell which confers the property of uncontrolled growth on that cell and its progeny. There must be some general condi tions, as well as a specific event, before carcinogenesis can take place. It is postulated that mutations in one form or another are responsible for both conditions.

The mutation theory of carcinogenesis is old and well of the 1st generation (3). We then scored chromosome known to all workers in the field. It is an extremely aberrations in liver cells of mice by inducing cell division attractive theory, and there are many pieces of evidence by partial hepatectomy and examining anaphase figures which argue in its favor. Radiation, which is a very po (14). (Fig. 1). The percentage of cells containing aberra tent mutagenic agent, is also a very effective carcinogen. tions is taken as an index of the mutations present in these Most, if not all, of the chemical carcinogens are also muta cells. gens. The virus theory of carcinogenesis is well estab With the use of this technic a number of different situa lished in certain special cases, and in a very real sense it is tions have been investigated. First it was found that a facet of mutation theory. aberrations increase steadily with age (Chart 1). Next, On the other hand, there are many puzzling phenomena following a dose of X-radiation, there are a large increase which cause one to doubt this theory. For example, if in aberrations and a very slow return to normal (Chart 1). the theory is true, why does it take as long as 40 years It is known that chronic X- or â€˜y-radiationis only about after a person receives a dose of radiation for a tumor to 25 % as effective in shortening the life-span as an acute appear? If a mutation to carcinogenicity is produced, dose of the same total size. It is found that the same is one would expect it to manifest itself very soon thereafter. true for the production of chromosome aberrations (Chart There is also the question of whether mutations, especially 2). It is also known that for neutron irradiation, chronic spontaneous mutations, can be produced in somatic cells. irradiation is just as effective in shortening the life-span A number of years ago it was noted that the life expec as acute irradiation. The same has been found for the tancies of animals exposed to ionizing radiation are short production of chromosome aberrations (8) (Chart 3). ened in proportion to the radiation dose received. It It is well known that some inbred strains of mice are appears that the animals die from the same diseases and quite short-lived and some quite long-lived. It was in about the same proportion as normal animals, but they found that chromosome aberrations increase quite rapidly contract the diseases sooner. In investigating this phe in one short-lived strain and quite slowly in a long-lived nomenon, it seemed that a very likely explanation would strain (4) (Chart 4). be that the radiation induced mutations in the somatic Thus in every situation tried so far there is a direct cells which caused a lack of efficiency and, thus, deteriora correlation between the degree of life shortening and the tion in organs whose cells are not capable of cell division development of somatic mutations. It should be kept in and cancer in organs whose cells regularly undergo mitosis. mind that irradiated animals die, to a very considerable In order to test this idea, it was necessary to develop a extent, from cancer, so that one can equally well say that method for estimating mutations in somatic cells. For this we made an analogy with plants, where, because factors which cause increased somatic mutations also quan somatic cells differentiate to form germ cells, chromosome titatively increase cancer production. aberrations in somatic cells can be related to true muta One of the problems raised by this work is the long de tions scored in the next generation. It was found that lay between cause and effect. Thus a dose of radiation the mutations scored in the 2nd generation are propor given to a young adult mouse may produce serious chro tional to chromosome aberrations scored in somatic cells mosomal damage in almost every cell in the body; yet the mouse appears unaffected and may not show signs of 1 The research reported in this paper was carried out at Brook havenNational Laboratoryunder the auspices ofthe U. S. Atomic aging until many months later. At first glance, this situa Energy Commission. tion seems to contradict the theory, but in the light of 1305

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@ 2@ Â£T@TTIITTT DAYSFROMSTARTOF EXPERIMENT 8 9 0 IF 2 CHART 3.â€”Chromosome aberrations in liver cells of (a) mice AGE - MONTHS subjected to chronic neutron irradiation, (b) mice subjected to CHART 1.â€”Chromosome aberrations in liver cells as a function acute neutron irradiation, and (c) control mice. The dashed line of age in normal mice and in mice that had received a large dose of gives the rate of build-up of aberrations in Group a mice which X-rays. The curves show the steady increase in the number of would be expected if chronic irradiation were as effective in pro chromosome aberrations (mutations) with natural aging and the ducing aberrations as acute is, and it will be seen that the experi dramatic increase and slow return to normal following X-rays mental points fall closely on this line. The data show that there is no chromosomal healing following even small doses of neutrons [from Stevenson and Curtis (14)]. [from Curtis et at. (8)1. IIIIII â€¢STRAIN A/HEJ 100 - 0STRAIN C57BL/6J U) 100 z (I)z YOUNG MICE 0 0 â€”â€”â€”â€”OLDBREEDERS I- 80 - ,frâ€”-â€”.---@ @ 80 w w , 60 - , @ 60 Ui 0 U) 0 40 - -@ â€”. ._oâ€” â€”-0@ 0 U) 0 0 40 oâ€”___o_â€¢@__ 1' I 0 C) I 20 - C) 20 a! <@T@7TI I _0 100 200 300 400 500 600 700 AGE - DAYS 60 90 120 150 180 210 240 270 CHART4.â€”Chromosome aberrations in liver cells of normal mice AGE - DAYS of 2 different inbred strains as a function of age. The median life CHART 2.â€”Chromosome aberrations in liver cells of (a) mice span of each strain is indicated by the arrows. In each case the solid lines represent animals 8 weeks of age at the start of the ex subjected to chronic 7-irradiation, (b) mice given a single dose of periment, and the broken lines, old breeding animals about 1 year X-rays, (c) normal mice. The dashed line shows the rate of old at the start of the experiment [from Crowley and Curtis (4)]. build-up which would be expected for mice of Group a if chronic irradiation were as effective in producing chromosome aberrations (DNA)1 â€”Ã¸'(RNA)1-â€”e@(Protein)1 as acute irradiation is. Since the experimental curve has a much smaller slope, it is concluded that chromosome healing takes place following small doses of radiation [from Curtis and Crowley (7)]. (DNA)2 â€”@(RNA)2 â€”@(Protein)2

modern theory of the role of the nucleus in cell function, (DNA)3 â€”@(RNA)3 â€”.@(Protein)3 it may actually strengthen it. A schematic representation of the control of cellular I I I @ function is given in Chart 5. It is believed that there is I I 1 DNA2 molecule of each species, 10â€”100RNA molecules (DNA)@â€”.@(RNA)@---.'@ (Protein)@ of each, and more than 1000 protein molecules of each. CHART5.â€”Schematic representation of the control of cell func Thus if a mutation occurs which destroys a single DNA tion. The chromosomes contain the different individual DNA molecule, the cell can still function for a long time on molecules, each of which synthesizes the corresponding RNA molecules, which in turn synthesize the corresponding proteins. 2 The abbreviations used are: DNA, deoxyribonucleic acid; A mutation can be considered as the elimination of 1 or more DNA RNA, ribonucleic acid. molecules, which eventually eliminates that molecular line.

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DAYS FROM START OF EXPERIMENT CHART6.â€”Chromosome aberrations in liver cells of both irradiated and control mice, and with and without carbon tetrachloride injections every 45 days (arrows) , which destroyed about half the liver. The standard error of each point is indicated. This induced mitosis caused a relatively slow return to control levels [from Curtis et al. (9)]. stored RNA and proteins. Indeed this seems to be the plant cells, and it now seems even more potent for animal case, and one can cite a number of instances in which cells. Thus there are 2 mechanisms within the cell for cells have been observed to function and even to undergo getting rid of mutations; only one can operate in the fixed a number of mitoses before dying from a lethal mutation postmitotic cells, but both can operate in cells in active (5). This would then seem to adequately explain the turnover. long delay between the induction of chromosomal damage The importance of this result for the present discussion and its manifestation. lies in the fact that it gives a great hope for future cancer Another problem, already mentioned, is that of chro research. It means that chromosome structure is by no mosome healing. One can imagine that as a group of means a fixed entity. It is apparently quite unstable, cells undergoes cell division, the cells containing muta and it achieves what stability it has by a very considerable tions are gradually eliminated and the cell line thereby ability for self-repair. Experiments with labeled thymi kept pure. Indeed, this undoubtedly happens in the dine show that this compound is rather readily incorpo mammal in such tissues as bone marrow. However, rated into the DNA of interphase cell nuclei, and as readily there certainly must be more to it than that to account lost (10). This indicates an exchange reaction, which in for the observed chromosomal stability, especially in such turn indicates that the DNA structure must be quite nondividing tissues as the liver. To investigate this, we labile. With our increasing knowledge of protein struc produced a high percentage of chromosome aberrations in ture and DNA structure, one can easily imagine that the livers of mice by a dose of X-rays and, following this, methods will be found, perhaps in the very near future, gave the mice a dose of carbon tetrachloride to destroy for stabilizing chromosome structure in the mammal. about half the liver every 45 days (9) (Chart 6). It will One is very tempted to regard aging and carcinogenesis be noted that the aberrations were eliminated faster with as 2 separate processes, as if cancer were some unhappy active cell division than without it, but not nearly fast accident. Indeed it does appear that, in general, tissues enough to allow us to say that the 1st cell division elimi which accumulate mutations do not develop cancer, and nates a mutation. On the contrary, it must take an aver vice versa. However, many lines of evidence seem to mdi age of more than 4 divisions to eliminate an aberration. cate that carcinogenesis is merely one form of the aging In the normal liver there is very little cell division, but process. Jones (13) has collected data from a great many the aberrations produced by X-rays are eliminated never different populations and on many diseases. He finds theless. This, combined with the evidence from chronic that, in general, in a population which has a short life -y-ray irradiation, leads to the conclusion that chromo expectancy the causes of death are accelerated as compared somes are capable of a great deal of self-repair. This is a to a long-lived population. process which has long been known to be operative in It is very interesting that the recent studies on the rela

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. 1308 Cancer Research Vol. 25, September 1965 tion between longevity and smoking have shown that may be correct, one can imagine other specific mechanisms cigarette smoking tends to accelerate a great many causes which would be equally plausible. of death, such as cancer of the pancreas and coronary Recent work of Wald et al. (15) has shown that at least thrombosis, which would hardly seem to be related to some, if not all, radiation-induced mouse leukemia is smoking. There seems to be some general aging process caused by either the creation or the activation of a virus, initiated by smoking which affects the organism in such a which then incorporates itself into a particular chromo way that it is more susceptible to all diseases. some of some lymphocytes. This is an important con In a recent study by Johnson et al. (12) it is shown that cept, and it causes one to wonder just what a mutation is. rats kept at a cold teniperature increase their metabolism considerably in order to maintain their body temperature, REFERENCES and their life expectancy is considerably decreased. The 1. Berenblum, I., and Trainin, N. New Evidence on the Mecha interest ing thing is that, with a minor exception, all forms nism of Radiation Leukaemogenesis. In: R. J. C. Harris (ed.), of death including cancer are accelerated. Cellular Basis and Aetiology of Late Somatic Effects of As has already been mentioned, ionizing radiation tends Radiation, pp. 41â€”56.NewYork: Adademic Press, Inc., 1963. to accelerate all causes of death. 2. Berg, B. N., and Simms, H. S. Nutrition and Longevity in the It has been known for many years that if rats or mice Rat. II. Longevity and Onset of Disease with Different Levels of Food Intake. J. Nutr., 71: 255-63, 1960. are restricted in their caloric intake, their life expectancy 3. Caldecott, R. S. Seedling Height, Oxygen Availability Storage will be greatly prolonged. In a recent study, Berg (2) and Temperature : Their Relation to Radiation Induced has shown that all causes of death are delayed in the re Genetic and Seedling Injury in Barley. In: Effects of Ionizing stricted animals. Radiation on Seeds. Vienna: International Atomic Energy Agency, 1961. All these pieces of evidence indicate that when the gen 4. Crowley, C., and Curtis, H. J. The Development of Somatic eral condition of an animal deteriorates (ages), it becomes Mutations in Mice with Age. Proc. NatI. Acad. Sci. U. S., more susceptible to all diseases, and that until this condi 49: 626â€”28,1963. tion is met, the induction of these diseases, including can 5. Curtis, H. J. Biological Mechanisms Underlying the Aging cer, is unlikely. Process. Science, 141: 686â€”94,1963. 6. . The Late Effects of Radiation. Proc. Am. Phil. Soc., On the other hand, one cannot ignore the apparent 107: 5â€”10,1963. specific causes of cancer, such as smoking and lung cancer. 7. Curtis, H. J., and Crowley, C. Chromosome Aberrations in It then seems evident that we must consider both general Liver Cells in Relation to the Somatic Mutation Theory of and specific causes of cancer, and perhaps both factors Aging. Radiation Res., 19: 337-44, 1963. 8. Curtis, H. J., Tilley, J., and Crowley, C. The Cellular Differ must be present for cancer initiation to take place. Beren ences between Acute and Chronic Neutron and Gamma Ray blum and Trainin (1) have ably presented evidence for a Irradiation in Mice. In: Biological Effects of Neutron and 2-factor theory of carcinogenesis, indicating that it is Proton Irradiation, Vol. II, pp. 143â€”55.Vienna: International necessary to have an initiator and a promoter in order to Atomic Energy Agency, 1964. 9. . The Elimination of Chromosome Aberrations by Cell achieve cancer induction. It could well be that they are Division. Radiation Res., 22: 730â€”34,1964. discussing substances which create the general and specific 10. Devik, F., and Halvorson, K. Observations by Biochemical condit ions for carcinogenesis. Analyses and Autoradiography on Labelled DNA in the Nor Returning now to the radiation problem, it is apparent mal and Regenerating Liver of Mice. Nature, 197: 148-50, 1963. 11. Failla, G. The Mutation Theory of Aging. Ann. N. Y. Acad. that radiation may play 2 roles in the carcinogenic story. Sci., 71: 1124â€”32,1958. The production of mutations in the cells of the body may 12. Johnson, H. D., Kintner, L. D., and Kibler, H. H. Effects of very well create the basis for the general conditions neces 48Â°Fand 83Â°Fon Longevity and Pathology of Male Rats. J. sary for carcinogenesis and at the same time produce the Gerontol., 18: 29â€”36,1963. mutation necessary for the production of the specific can 13. Jones, H. B. A Special Consideration of the Aging Process, I)isease and Life Expectancy. Advan. Biol. Med. Phys., 4: cer. The present experiments certainly show that the 281â€”336,1956. general conditions for cancer induction can be delayed 14. Stevenson, K. G., and Curtis, H. J. Chromosomal Aberrations for a large fraction of the life-span of the animal after in Irradiated and Nitrogen Mustard Treated Mice. Radiation they are created by radiation, and the specific mutation Res., 15: 774â€”84,1961. 15. Wald, N., Upton, A. C., Jenkens, V. R., and Borges, W. H. can wait for an equal time until the general conditions Radiation Induced Mouse Leukemia: Consistent Occurrence are met. Failla (1 1) has proposed a more specific mecha of an Extra and a Marker Chromosome. Science, 14$: 810â€”13 nism to account for this 2-step process, and whereas his 1964.

FIG. 1.â€”Photomicrograph of an abnormal dividing liver cell exhibiting 3 bridges and 3 fragments [from Curtis (6)].

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Cancer Res 1965;25:1305-1309.

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