CHROMOSOMAL AND EXTRACHROMOSOMAL INFLUENCE IN RELATION TO THE INCIDENCE OF MAMMARY TUMORS IN MICE

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1 CHROMOSOMAL AND EXTRACHROMOSOMAL INFLUENCE IN RELATION TO THE INCIDENCE OF MAMMARY TUMORS IN MICE WILLIAM S. MURRAY New York State Institute for the Study of Malignant Disease, B. T. Simpson, Director AND C. C. LITTLE Roscoe B. Jackson Memorid Laboratory In 3 the authors published a report of their study of the genetics of mammary tumor incidence in mice (). Two inbred strains, one of which (dilute brown) gives an incidence of 0. per cent spontaneous mammary tumors in virgin females and the other of which (C Black) had given no mammary tumors in either breeding or virgin females, were used as the parents of an outcross. The cross was made reciprocally and the following numbers of virgin females were raised: No. of animals 3 3 How animals were obtained Dilute brown female X C Black male C Black female X dilute brown male dbf, female X dbf, male BdF, female X BdF, male Abbreviation for cross dbf, BdF, dbf, BdF, These animals were observed until the appearance of mammary tumor or until they died of other causes. After due allowance was made for the age of each population, the following conclusions were drawn: () The dbf, animals showed a significantly higher incidence of mammary tumors than did those of the BdF, generation; 3. per cent as against.0 per cent. () This difference persisted in the F, generations, as follows: dbf, 3. per cent, BdF,. per cent. (3) Since the chromosomal constitutions of these females were theoretically the same, the observed difference in the incidence of mammary tumor must have been due to extrachromosomal influence. () Since this difference persisted in the two F, generations, the chromosomal constitutions of which were theoretically the same, its chief basis is obviously transmitted in some manner by influence outside the chromosomes. () The occurrence of some mammary tumors in both F, and F, generations indicated that the possibility of some chromosomal influence in the genesis of mammary tumors was not excluded. () The difference in tumor incidence in the reciprocal crosses of both F, and F, generations lasted throughout all age periods and was strikingly constant. () The relative importance of what appears to be extrachromosomal influence in the incidence of mammary tumors in the mice studied is approximately six times that of the possible chromosomal influence. In the abbreviations used the first letter indicates the female and the second letter designates the male. Small d indicates the dilute brown high-tumor strain and large B the C Black nontumor strain. No dominance or recessiveness is indicated by the use of large or small letters. 3

2 INCIDENCE OF MAMMARY TUMORS IN MICE 3 It was evident, from this study, that those animals whose mothers or grandmothers were of the high-cancer strain were much more disposed to mammary tumors than those whose mothers and grandmothers were of the non-cancer strain. Since the chromosomal constitutions in the reciprocal F, generations were theoretically identical, consisting of one-half from the dilute browns and one-half from the blacks, and since mammary cancer appeared in both F, generations, it was reasoned that this disease is inherited, to some extent, through the chromosomes. Inasmuch as the mammary tumor incidence in the F, generations was practically identical with that observed in corresponding F, generations (although the chromosomal content of the animals varied because of the reassortment of genes in the F, generations), it was impossible to determine whether the tumors which were occurring in the animals of the F, generations had any relationship to the concentration of chromatin from one stock or the other. It was therefore decided to raise further generations in which the chromosomal constitutions should be manipulated in such a manner as to concentrate the chromatin of the high-tumor stock in animals which received their extrachromosomal influence from the non-cancer stock, and to concentrate the chromatin of the non-cancer stock in animals which received their extrachromosomal influence from the high-tumor stock. It should then be possible to determine the chromosomal influence in the inheritance of these tumors. GENETIC THEORY UPON WHICH THE EXPERIMENT IS BASED Theoretically, if we represent the F, generation as being a two-factor cross, the genetic formula of the high-cancer strain may be indicated as AABB and that of the low cancer strain as aabb. All of the F, animals will have the formula AaBb, since they derive one-half of their chromatin from the cancerous and one-half from the non-cancerous parent.' This situation will exist no matter how many genes are involved. In the FL generations, however, we have a variety of genotypes: AB Ab ab ab AB Ab ab ab AABB AABb AaBB AaHb ~ ~ _ ~ AABb AAbb AaBb Aabb - ~ ~ - AaBB AaBb aabb aabb ~ AaBb Aabb aabb aabb Totaling the number of times each letter occurs, we get: A; B; a; b. From this Punnett's square, it will be noticed that in the aggregate one-half of the chromatin of this generation is inherited from the high-cancer stock (ha; B) and one-half from the low-cancer stock (a; b). The chromosomal constitutions of the animals, however, are not identical, as they were in In this and following illustrations dominance and crossing over are not considered. The distinguishable somatic characters in the actual crosses segregated remarkably close to expectation.

3 3 WILLIAM S. MURRAY AND C. C. LITTLE - TABLE I: Derivation and Formdae of Crosses Chromosomal Extrachromosomal Cross Female Male complex influence Number d ba drfi Rlk RdFi RdFi d ba RdFi t3f X Blk X dnfi X dba X RdFi X dba X RdFi X Rlk x Blk cccc cccc cccc cccc cccc cccc cccc cccc E 3 E e 3 e e 0 E C 0 E the F, generations, since we have (due to segregation and reassortment of the genes) individuals with a variety of the possible combinations of these letters. Inasmuch as we are trying to measure a physiological condition rather than a morphological one, as is usual in genetic studies, and since we cannot recognize these different genotypes, it is not possible in the F, generations to determine whether or not cancer of the breast is more prevalent in animals of any one of the possible types. That is, it is not possible to tell whether the concentration of the chromatin from the cancer strain has any effect upon the incidence of the breast tumors which appear. It is a well established principle in breeding that back-crossing to a homozygous parent tends to concentrate the chromatin of the homozygous strain in the offspring. Thus, if F, females from the black female X dilute brown male cross, which have the formula AaBb, are crossed to a homozygous black male which has the formula aabb, we have: A3 Ab all ab ab Aanb I Aabb aallb I aabb Totaling the number of times each of these letters appears, we find that three-fourths of the chromatin came from the non-cancer stock (a; b) and that one-fourth (A; B) came from the high-cancer strain. If now we use CCCC to designate the chromosomal complex of the cancer stock and cccc to indicate that of the non-cancer stock (regardless of how many factors are involved), the F, and F, generations may be represented as having the following formulae: High cancer female X non-cancer male CCcc E3 dbf, Non-cancer female X high-cancer male CCcc e BdF, dbf, female X dbf, male CCcc E dbf, BdF, female X BdF, male CCcc e BdF, Table I summarizes the crosses made to date and gives the formula assigned to each. From this table it is evident that we now have, for comparative purposes, four generations of the formula CCcc and two each of the formula CCCc and Cccc. The last four crosses (A, B, C, and D) present a variety of chronio- Capital E in the formula represents the extrachromosomal influence derived from a highcancer mother and small e indicates extrachromosomal influence from a non-cancer mother.

4 INCIDENCE OF MAMMARY TUMORS IN MICE 3 *Stock Number Observed tumors Formula Per cent of cancer brown TABLE : Incidence of Mammary Cancer C Dilute BdFi BdFz A drfl drf, ~ R _ ~ ~ ~ 3 3 CCCCE CCccE CCccE CCcce.0 I D CCcce CCCce CCCcE Cccce CcccE soma concentrations in animals with different extrachromosomal influence (E or e). Tabulating these crosses according to the percentage of mammary cancer incidence, Table I is obtained. From this, it will be seen that the hybrid generations group themselves, according to the percentage of mammary tumors which they develop, into two very distinct classes: those which have in the vicinity of 3. per cent cancerous animals and those which have per cent or less. By observing the table further, it will be seen that all of the crosses which go to make up the high-cancer group, regardless of their chromosomal constitution, have capital E appended to their formula. That is, they all derived their extrachromosomal influence from mothers or grandmothers of the highcancer stock. All of the classes with per cent or less of cancer, on the other hand, have the formula containing small e, which indicates their derivation from females of the non-cancer stock. In respect to the measurement of the chromosomal influence, critical crosses are the A and B, C and D. It will be noticed that there was just as much cancer in D, which derived three-fourths of its chromatin from the noncancer strain as there was in B, which derived three-fourths of its chromatin from the high-tumor strain. In a similar way, A may be compared with B and C with 33. Grouping the stocks in this manner, the cancer percentage is radically different, although the chromosomal constitutions are the same, in each of the two groups. If the chromosomal constitutions had any effect, A might be expected to be like B and C like D. These findings corroborate conclusion 3 of our first publication: that the extrachromosomal influence is much more potent in the transmission of the disposition to have mammary cancer than is the chromosomal influence. They also tend to eliminate the chromosomal factor as one which plays a part in the transmission of the tendency to have mammary tumors. As has been pointed out previously (), a simple percentage used to express the cancer incidence may not mean very much. The element of time, as expressed by the age to which the animals lived, must be taken into consideration. It is this time element, as manifested by the limits of the incidence curves of the various inbred strains of animals, which is one of the better indicators of transmitted influences upon the physiological condition of the animals. Thus in the breeding colony of the dilute brown mice, the curve of mammary tumor incidence is known to begin at the fourth month and to end

5 0 WILLIAM S. MURRAY AND C. C. LITTLE TABLE : Number of Mice Alive at the Beginning of Each Age Period Age in Dilute A, 3 C D months brown drf, dbf~ RdF, IidF, Cross Cross Cross Cross - * Total i 3 abruptly at the twenty-second month. The mean age for the appearance of tumor is 0. months, with a standard deviation from this mean of. months. From this, we may say that of the breeding females in this population, per cent of those set aside as breeders will develop mammary cancer, the great majority of them between the 3th and the 3th days. This stock might thus be said to have a critical period of five months during which the females are particularly susceptible to this disease, while before this period and after it the danger is not so great. When these data are rearranged, however, it becomes evident that this is not the case. If, for instance, the percentage of those animals living at the beginning of each age group which develop mammary cancer during or following that age period is calculated, it is found that the chance of these females

6 INCIDENCE OF MAMMARY TUMORS IN MICE TABLE IV: Number of Mice with Mammary Cancer Age in a Dilute A,B., C D months brown dbfl dbf BdFl BdF, Cross Cross Cross Cross Total I having tumors increases steadily as they grow older. A female which lives to the beginning of the seventh month has chances in 00 of becoming cancerous before she dies, whereas a female which lives to the beginning of the thirteenth month has chances in 00 of developing this same type of tumor. It therefore becomes imperative, if we are to compare the out-cross stocks accurately, that the age to which the animals attain be taken ihto consideration in any computations which are made. The various strains of mice die off at different rates and are made up of samples of different size (Tables I and IV), so that it is difficult to see from the crude survival or death rates how they compare as to mammary cancer death rates. Some of the differences in numbers of mammary cancer deaths are due to the differences in the numbers of mice exposed to the risk of mammary cancer, i.e. age distribution of the population (Table ). If, therefore, we multiply the cancer death rates of the individual strains (Tabe.V) by a standard population (Table VI) which is composed of all the animals involved, we get a number of deaths which would theoretically occur in this population if the animals in it died as do those in any one of our experimental strains (Table VII). By adding these theoretical cancer death rates and di-

7 I WILLIAM S. MURRAY AND C. C. LITTLE TABLE V: Derivation of the Mamnzary Cancer Rate for the Various Age Periods for Virgin Dilute Brown Females ~ -. No. alive at No. having Age in beginning mammary cancer Cancer ratc months of period during period for period, ,0., , 3, TAIKE VI: Derivation of Standard Mammary Cancer Rate for Virgin Dilute Brown Females ~ - -- Age in months Theoretical number Standard ' Mammary cancer of mammary population death rate, dbr. cancer deaths , , , ??.!& = 0. dead of mammary cancer per 0,000 population viding by the total standard population, we get one figure for each strain, which represents the standard cancer death rate per 0,000 population. These figures are directly comparable from one strain to another.

8 Age in months INCIDENCE OF MAMMARY TUMORS IN MICE 3 TABLE VII: Standardizing Population Composed of Dilute Brown Virgin Females, dbf,, dbf,, BdF,, BdF,, A, B, C, and D Crosses Standard POPUlation Theoretical number of mammary cancer deaths Virgin dilute drfl dbf BdF HdF A B C ID brown Total Standard rate per 0, When these standard cancer rates for the various crosses are tabulated, as in Table VIII, it becomes apparent that the virgin dilute brown stock had more than twice as much cancer of the breast as did the females of the dbf, and dbf, crosses; four times as much as the females of the B and D crosses; one hundred times as much as the A and C crosses. Transcribing these ratios to percentages, using the standard rate of the honiozygous cancer strain

9 WILLIAM S. MURRAY AND C. C. LITTLE - TABLE VIII: Standard Cancer Rates dbfr BdFi Stock dba dbf, BdFn Formula Standard cancer rate per 0,000 Percentage of cancer (dba as unity) CCCCE CCccE CCccE CCcce.3.. CCcce.. - A l c XCce.. CCCcE Cccce CcccE 3..0 as unity, it becomes evident that in the first and second generations of the cross derived from the high-cancer females (dbf, and dbf), the mammary cancer incidence is approximately per cent of that of the parent stock. In the reciprocal cross derived from females of the non-cancer strain (BdF, and dbf,), the cancer incidence is reduced to per cent of that shown by the high-tumor strain. Thus we have four classes in which the chromatin of the two parent stocks is equally distributed. These arrange themselves in two groups which show significantly different rates of tumor occurrence. Comparing in the same way the two back-cross generations in which the chromatin of the high-cancer stock was concentrated (A and B), it is found that the A cross had only per cent of the cancer rate of the high tumor stock and that B, which had the same chromosomal make up as A, had per cent as much cancer of the breast as the high-tumor strain. In the same manner, the two generations which received per cent of their chromatin from the non-cancer strain (C and D> may be compared. Here it is found that D had per cent as much cancer as did the high-tumor stock and that in the C cross cancer of the breast was practically non-existent. Comparing A and C, it is found that they have almost identical cancer rates in spite of the fact that their chromosomal complexes vary greatly. The same thing is found to be true of the B and D crosses, From this, it is evident that concentration of the chromatin of the highcancer strain in these hybrids has little if any effect upon their cancer rates. EFFECTS OF FURTHER CONCENTRATION OF HIGH-CANCER CHROMATIN AND OF NON-CANCER CHROMATIN UPON THE INCIDENCE OF MAMMARY TUMORS As has been demonstrated by Wright (3) and others, each successive backcross between a heterozygous and a homozygous animal reduces the number of remaining heterozygous genes by one-half. Since the dbf, and BdF, generations described in the earlier part of this paper may be said to be composed of one-half high-cancer chromatin and one-half non-cancer chromatin, repeated back-crosses of dbf, females and their female progeny to homozygous noncancerous males gradually concentrate the chromatin of the non-cancerous animals in this stock, which originally derived its extrachromosomal influence from the dilute brown high-cancer stock. In a similar manner, by repeatedly back-crossing BdF, females and their female progeny to dilute brown highcancer males, the chromatin of the high-cancer stock is gradually concentrated

10 INCIDENCE OF MAMMARY TUMORS IN MICE TABLE IX: Mammary Cancer Death Rates Age in Dilute months brown dbfl dbf BdFI BdFs A B C D lo , OO.0.0.0, , , , , , , , , ,03.00, in animals which received their extrachromosomal influence from the black non-cancer mother. The system of matings used to concentrate the chromatin was as follows: % Black chromatin I Female Male Female Male dbfl X Blk st BC X Blk nd BC X Blk 3rd BC X Blk th BC x Blk th BC X Blk th BC x Blk th BC X Blk th BC x Blk BdFl X dbr st BC X dbr nd BC X dbr 3rd BC X dbr th BC X dbr th BC X dbr th BC X dbr th BC X dbr th BC X dbr Dilute brown chromatin

11 WILLIAM S. MURRAY AND C. C. LITTLE TABLE X: Crosses Made Name of cross Female Male Formula Should be like: S cccce X cccc cccce Original non-cancer strain and U cross T cccce X dil. br. CCccE dbf, dbf and V cross U crcce X Black cccce Original non-cancer strain and S cross V cccce X CCCC CCccE dbfi, dbf and 'r cross W x CCCCe x cccc CCCCe X dil. br. CCcce CCCCe BdFI, BdF and Y cross Original high-cancer strain and Z cross Y CCCCe X Black CCcce BdF, BdFl and W Z CCCCe X CCCC CCCCe Original high-cancer strain and X cross TABLE XI: Computation of Mammary Cancer Rate in Population Composed of Four Early Crosses Carrying Extrachromosomal Factor Age in Number alive at beginning of period Total Mammary months tumors cancer dbfi dbfz " B " " D " Total rate 0 3 ' , ,00.0.o ,0

12 INCIDENCE OF MAMMARY TUMORS IN MICE TABLE XII: Computation of Tumors to be Expected in First Out-crosses Not Carrying the Extrachromosomal Factor, Had They Been Like Crosses Carrying This Factor Number alive at beginning of age Cancer rate: Theoretical number Age in period : BdFI, dbfi, dbf,, if BdFl etc., is months BdFa, A, C B, D like dbfl etc , , , , , Theoretical number - ' = 0 Number observed ' ' After eight generations of these matings, animals were obtained which may be said to have the formulae CCCC and cccc. The first of these should be like the dilute brown high-cancer strain and the second should be like the Black non-cancer stock, since they have the same chromosomal complexes as these stocks. Inasmuch as the strain cccc was derived from a dilute brown high-cancer female, a large E may be attached to its formula (cccce); and since the strain CCCC was derived from a Black non-cancer female, it may have a small e attached to its formula (CCCCe). With these back-cross derivatives it is possible to make a number of crosses (Table X), which may be compared with one another and with those reported previously. Eflect of Eight Successive Back-cross Generations upon the ExtrachromosomaE Influence: In previous experiments those crosses which derived their extrachromosomal influence from high-cancer mothers (dbf,, dbf, B and

13 WILLIAM S. MURRAY AND C. C. LITTLE TABLE XIII: Computation of Tumors to be Expected in Eighth Generation Bwk-crosses Not Carrying the Extrachvomosoinal Factor, Had They Been Like Early Out-crosses Carrying This Factor Alive at beginning Mammaiy cancer Age in months of period: S, T, U, V rate: dbf,, dbf, B and D Theoretical number for S, T, U, V , S , ,0. - = Theoretical number of tumors S, T, U, V crosses would have had, had they been like other crosses with capital E in their formulae (dbfi, dbf, B and D) = Observed number of tumors D), when combined, give a population of 3 animals living to the beginning of the fifth month. Four hundred fifty-four of these developed tumors of the breast. If the monthly rates of cancer appearance for this group are computed (Table XI), and are then multiplied by the number of mice alive at the beginning of the various age periods in those crosses which derived their extrachromosomal influence from the non-mammary cancer mothers, it is possible to obtain a theoretical number of tumors which would have been observed in this latter group had they developed tumors at the same rate as did the animals with large E appended to their formulae. When this is done (Table XII) it is found that, had these two groups been the same, mammary tumors would have been found in the BdF,, BdF,, A, and C crosses. Actually, mammary tumors were observed. This indicates that animals which derive their extrachromosomal influence from cancerous mothers have ten times (/) as much mammary cancer as do animals which have the same chromosomal complexes but which receive their extrachromosomal influence from non-cancerous mothers. This is in animals which are not more than two generations removed from the inbred stocks.

14 NCIDENCE OF MAMMARY TUMORS IN MICE TABLE XIV: Computation of Tumors to be Expected in Eighth Generation Back-crosses Carrying Extrachrontosomal Factor, Had They Been Like Early Out-crosses Carrying This Factor Alive at beginning Cancer rate: Age in of period: W, dbf, dbf, Theoretical number months x. Y. z B and D for W-. X. Y. Z OO , ,0.03.0, , = Theoretical number of tumors W, X, Y, Z would have had, had they been like other crosses with large E in their formulae = Observed number For comparison with these, we now have four crosses (S, T, U, V) which are eight generations removed from the inbred stocks but which originally received their extrachromosomal influence from high-cancer mothers, large E ; and four crosses (W, X, Y, Z) which are eight generations removed from the inbred stocks which derived their extrachromosomal influence from noncancerous mothers. When those carrying large E are grouped and the theoretical rate of mammary cancer is determined (Table XIII) on the basis of their likeness to those crosses carrying large E in the first experiment, it is found that mammary tumors should have appeared. Actually were observed. The incidence of cancer, therefore, was only 3 per cent of that of the former group. When this same procedure is applied to the group W, X, Y, Z (Table XIV), it is found that, had this group been like dbf,, dbf,, B, and D, it would have had mammary tumors. Four were actually observed. The percentage of tumors was the same as for the S, T, U, V group although the extrachromosoma formulae were different. Summarizing these findings: when due allowance is made for the ages to which the mice attain, the crosses which are eight generations removed from

15 0 WILLIAM S. MURRAY AND C. C. LITTLE TARLE XV: Computation of Tumors to be Expected in Eighth Generation Buck-crosses of Formula CCcc, Had They Been Like Early Out-crosses of the Same Formula - Alive at beginning Age in of period: T, Tumor rate of Theoretical number months v, w, y drf, drf of tumors , 3.OO , , , , Rate of dbf, dbfz -_ =- Rate of BdFI, BdFz = number of mammary tumors which should have been obtained had T, V, W, Y been like earlier crosses with same formula the inbred stocks show almost identical rates of mammary cancer, whether their extrachromosomal influence was derived from the high-cancer or the non-cancer stock. We therefore conclude that, whatever the extrachromosomal influence which makes mice derived from high-cancer females ten times as tumorous in the first out-cross generations as are those derived from non-cancerous mothers, eight generations of back-crossing eliminates it completely. We may therefore drop the large E and small e from the formulae of these last eight crosses and consider only their chromosomai formulae. When the S to Z crosses are segregated according to their chromosomal formulae, they arrange themselves in three groups : () The first of these (S and U) have the formula cccc, They should, therefore, be similar to the original non-cancer strain, since they have the same chromosomal formula. Inasmuch as no mammary tumors were found in either of these strains (neither the original strain nor the eighth generation

16 INCIDENCE OF MAMMARY TUMORS IN MICE TABLE XVI: Distribution of Observed Data for Eighth Generation Back-crosses Age in month: Number alive at the beginning of the period * S T U v W X Y Z Total a () ()3 3 () () (0 ( ()3 () 3 () () * Tumors in parentheses. derivatives of similar chromosomal complex), no further comparison is necessary. () The second group (T, V, W, Y), which is comprised of all crosses having the formula CCcc, may be compared with those generations of the original out-cross which had the same formula but which were not subject to the extrachromosomal influence (BdF, and BdF,). When this is done (Table XV), it is found that mammary tumors should have been observed in the population available for study. Seven mammary tumors were observed, or per cent of the number which might have been expected had the tendency to mammary cancer been transmitted through the chromosomes. (3) The third group (X and Z), which includes all crosses having the formula CCCC is comparable to the virgin females of the high-cancer strain. When the animals in this population are subjected to the same mathematical treatment, using the rate of dbf, and dbf, and converting according to the proportion of tumors at the bottom of Table XVII, it is found that mammary tumors should have appeared. Actually 3 were observed. Since it has been demonstrated that the extrachromosomal influence carried by the virgin dilute browns is ten times as great as any chromosomal factor in the first generation out-crosses, it seems that one-tenth of or is the number of tumors with which the number observed, 3, is comparable. That is, there were one-fifth as many tumors observed as might have been expected

17 WILLIAM S. MURRAY AND C. C. LITTLE TABLE XVII: Computation of Tumors to be Expected in Eighth Generation Back-crosses of Formula CCCC, Had They Been Like First Hybrid Generations of Formula CCCC No. alive at Theoretical number Age in beginning of Cancer rate: of tumors if like months period: X, Z dbfi, dbfz dbfi, dbfv 0.00.a00,003, $ O ' X 00 = = the number of tumors which should have been found had X, Z been like the high-tumor stock. Observed number = 3 had the tendency to develop mammary tumors been transmitted through the chromatin. It therefore seems reasonable to conclude: () Some extrachromosomal influence, which is ten times as powerful as any possible chromosomal factor, is instrumental in determining whether or not mammary cancer appears in the first out-cross generations. () This extrachromosomal influence becomes non-effective after eight generations of back-crossing. (3) Concentration of the chromatin of the high-mammary-cancer strain does not return the cancer incidence to that obtained in the first hybrid generation or that in the original cancer strain. () The tendency to have mammary cancer is not mendelian in nature. LITERATURE CITED. MURRAY, W. S., AND LITTLE, C. C.: Genetics 0: -, 3.. MURRAY, W. S.: Am. J. Cancer 0: 3-3, WRIGHT, S.: Genetics : -,.