MAMMARY TUMORS IN MICE IN RELATION TO NURSING1

Transcription

1 MAMMARY TUMORS IN MICE IN RELATION TO NURSING1 JOHN J. BITTNER (From the Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, in cooperation with the United States Public Health Service) In this report we shall consider in more detail the effects of nursing on mammary gland tumor incidence in mice. Some of the data have been published (Bittner, 1936~). Three stocks of mice inbred brother-to-sister have been used in the work. As each strain has been continued by such matings for more than twenty generations, they were considered to be homozygous. CHART 1. OCCURRENCE OF TUMORS IN ONE SUB-LINE OF THE A STOCK Litters which were fostered by low-tumor line females are designated. The A stock (Bittner, 193 1) is descended from a line of albino mice started by Dr. L. C. Strong (1936~). It has a high incidence of mammary tumors and a few primary bronchial carcinomas in breeding females (Bittner, 1935a and b, 19363). The ratios will be given later, as additional observations have been made since the last report. A line of Strong's CBA stock (Strong, 19363) was obtained in The breast tumor incidence was 13.5 per cent in a total of 125 breeding females at an average age of twenty-one months. 1 Read before The American Association for Cancer Research, Chicago, March 24,

2 MAMMARY TUMORS IN MICE IN RELATION TO NURSING 531 Representatives of the C57 Black strain were given to the author by Dr. C. C. Little. The incidence of breast tumors in breeding females of this line is about 1.0 per cent (Cloudman, Bittner and Little, in press). Young born to A stock females were removed as soon as they were recorded (approximately 8:30 each morning) and fostered by females of the CBA or C57 Black stock. These fostered females of the A stock were mated and permitted to nurse their progeny. Thus, only one generation of A stock mice was fostered by low-tumor line females. They are designated as mem- a I5 GEH OF CA CA. ONON-CA. CHART 2. DESCENDANTS FROM THREE A STOCK FEMALES (LITTER MATES) WHICII WERE FOSTERED BY A CBA STOCK FEMALE, SHOWING THE INCIDENCE OF TUMORS bers of the first generation in this report. Mice of subsequent generations were nursed by their A stock mothers.' The fostered and control or normal mice of the A stock were maintained under identical conditions and received the same diet. The pedigree'of the sub-line of the A stock from which one of the litters fostered by a CBA female was descended is given in Chart 1. The diagnosis for each animal is given, and the age at death of the non-tumor mice. The These A stock females were themselves fostered in the case of the first generation and were descended from such animals in subsequent generations.

3 532 JOHN J. BITTNER mother, No , was a member of the 15th successive generation having breast cancer. One of her daughters had 30 descendants in the following three generations, of which 23 or 76.6 per cent developed mammary gland tumors. A sister of No died non-tumorous at 14 months. This mouse, No , had 12 unfostered progeny of which 10 or 85 per cent had TABLE I: Proportion of the A Stock Breedzng Females Fostered by CBA Stock Females to Develop Tumors A Stock Mice Fostered by CBA Females No. Breast Tumor Lung Tumor Non-Tumor 1st Generation 2nd Generation 3rd Generation 4th Generation TOTAL % 35.0% 6.9% 15.4y0 23.1% 44.4% 32.5% 34.5% 23.1% 33.0% 22.2% 32.5% 58.6% 61.5 % 44.0% - TABLE 11: Troportion of A Stock Mice Descended from Mothers Fostered by CBA Femnles to Develop Tumors (Progeny grouped according to diagnosis of mother) A Stock Mice Fostered by CBA Females Progeny of fostered mice according to diagnosis of mothers Breast-tumor mothers Lung-tumor mothers Non-tumor mothers No' Breast Tumors 3 6@.0% 10.5% 0.0% Lung Tumors % 43.1% 0.0% Non- Tumor % 47.4% 100.0% TABLE 111: Comparison of Tumor Incidence and Average Age for Control und Fostered Groufis of A Stock Breeding Females Total number Breast tumors Lung tumors Non-tumorous A Stock Average age Appearance of breast tumors Appearance of lung tumors Death of non-tumorous mice TOTAL Normal or Fostered by Fostered by Controls CBA Females C57 Females % 3.7% 13.1% 11.5 mo mo mo mo % 33.0% 44.0% 11.8 mo mo mo mo % 46.3 % 48.8% 10.0 mo mo mo nio. breast tumors. Chart 2 gives some of the other descendants of tumor female No They are the offspring of three representative females which were fostered by CBA mice. To date 91 breeding females of the CBA fostered series of the A stock have completed their life span. The data for these are presented in Table I

4 MAMMARY TUMORS IN MICE IN RELATION TO NURSING 533 according to generations. The incidence of mammary tumors for the entire group was 23.1 per cent, considerably less than the proportion which died nontumorous (44 per cent). In Table I1 the animals are divided according to the diagnosis of the nine first-generation fostered females. Twenty of the progeny of mothers which had breast cancer were mated; the breast cancer incidence among these was 60 per cent. Fifty-seven offspring of lung tumor mothers were observed and they gave 10.5 per cent mammary tumors. The lung tumor incidence in these two groups was 10 per cent and 42.1 per cent respectively. The 5 offspring descended from non-tumorous fostered mothers all died non-cancerous. l5 r "r;'- NORMAL AGE IN MONTHS AGE IN MONTHS W m I 0 AGE IN MONTHS CHART 3. PROPORTI~N OF CONTROLS AND FOSTERED MICE OF THE A STOCK DYING FROM MAMMARY CANCER (SOLID LINE) OR OF OTHER CAUSES (BROKEN LINE) ACCORDING TO MONTHLY AGE PERIODS In the first group of A stock mice fostered by C57 Black females there were 46 mice. Five are living at the beginning of the twenty-first month. Two or 4.9 per cent of the 41 developed breast tumors, 46.3 per cent had lung tumors, and 48.8 per cent died non-tumorous, as is shown in Table 111. In this table is recorded, also, the proportion of the control or normal females of the A stock to give rise to breast tumors (83.2 per cent) or lung tumors (3.7 per cent), or to die non-tumorous (13.1 per cent). The figures are based on the percentage of the total living four months or longer which ultimately developed growths. The total number observed was 788. Graphic representations of the breast tumor data are presented in Charts 3 and 4. Chart 3 gives the percentage of animals dying from mammary tumors or other causes according to monthly age periods. The number of mice is given for the fostered groups and percentages for the control series. The

5 534 JOHN J. BITTNER curves in Chart 4 show the proportion living to the first of each age period or longer to develop breast tumors. Table I11 gives, also, the average age of the observation of mammary or lung tumors and of death of the non-tumorous mice. All classes had breast tumors at approximately the same age. Lung tumors were recorded at later :; ;so. W u 50 k "A" STOCK. \ NOe~4L AGE IN MONTHS!I 7 0 I1 I3 IS AGE IN MONTHS ages, but the differences noted for the control and fostered classes are probably not significant. Non-tumorous mice of the fostered groups lived from three to four months longer, on an average, than did the non-cancerous control mice. The average age of all the mice fostered by C57 Black mice was nearly five months greater than the controls; for the CBA fostered mice it was three months greater. That the mice of the fostered groups lived longer is again demonstrated in

6 Table IV and Chart 5. The figures and curves express the percentage of the total number living to the beginning of each monthly age period or longer. In the control series 10.5 per cent of the total number of animals lived to be sixteen months old or older, and 8.5 per cent of the breast tumors were observed in mice reaching that age. Of the 83 individuals which reached that age, 67.5 per cent gave rise to breast tumors. Among the animals which were fostered by CBA females 44.9 per cent of the total lived to the above mentioned age. Only 2.4 per cent, however, died of mammary cancer. Sixtythree per cent of the C57 Black fostered series survived to that age and no breast tumors were observed among this group. TABLE IV: Percentage of the Controls and Fostered Groufis of the A Stock Living to the Beginning of Each Monthly Age Period or Longer - Age in Months Controls % Fostered by CBA Females % Fostered by C57 Black Females 46* 100.0% * * 5 mice still living. Summary: Breeding females of the inbred A stock showed a significant decrease in the incidence of mammary gland tumors after being nursed by low-tumor stock females. In general, the progeny of the fostered females developed the same types of tumors observed in their mothers. The mice lived longer than the controls, and a greater proportion showed primary lung tumors or died non-tumorous. The incidence of mammary gland tumors is no higher in mice which have nursed their young than in those which have lost their progeny, thus causing stagnation of milk. This observation was made by Murray (1934) on breeding females of the dilute brown strain. Fekete and Green (1936) found that complete blockage of the nipple had little effect in the development of tumors in stocks which do not inherit the cancer susceptibility. In a preliminary report made by the Staff of the Jackson Memorial Labora-

7 536 JOHN J. BITTNER tory (1933) and later papers by Murray and Little (1935a, b, 1936) and by Bittner (1936d), the theory of an extrachromosomal influence in the transmission of mammary tumor susceptibility was advanced. Articles confirming this work have been published by Korteweg (1934, 1935, 1936), Cloudman and Little (1936), and Bittner (1936~). In every cross between high and low mammary gland tumor stocks the incidence was greater among the progeny which had high-tumor stock mothers. That leukemia follows the same type of inheritance has been stated by MacDowell (1935) and Mac- Dowel1 and Richter (1935). There are three possible methods by which the maternal parent may transmit the extrachromosomal influence to her progeny. (1) Cytoplasmic: As the cytoplasmic content in the sperm germ-cells is practically negligible, any effect inherited through this material must be derived from the ovum. Korteweg (1936) believes that this theory gives the most plausible explanation. (2) During intra-uterine life: This phase is being investigated by Little and Fekete. (3) By means of the mother's milk: MacDowell and Richter (1935) have eliminated this influence in the transmission of leukemia. In this experiment the young born to high-breast-tumor females of the inbred A stock were removed as soon as recorded and fostered by females of low-tumor lines-the CBA and C57 Black stocks. Only one generation of A stock mice was fostered. In succeeding generations the progeny were nursed by their A stock mothers. In the series fostered by females from the CBA stock four generations of mice have been observed. Only breeding females are considered. Ninety-one mice were observed in the CBA fostered series, 46 in the C57 Black fostered group (5 still living); the control group consisted of 788 breeding mice. The CBA and C57 Black fostered groups averaged 14.8 and 16.5 months respectively at the time of death or observation of tumors. The average age of all the control mice was 11.7 months. Thus the mice of the two fostered groups lived longer than the control series; they had the same genetic constitution, and were subjected to the same irritation factors incident to the bearing and nursing of young. Apparently they diverged from the control mice only in that the first generation females obtained their milk while nursing from low-tumor stock females instead of from their A stock mothers. Only nine A stock females were fostered by females of the CBA stock. Their progeny for three generations have been observed. The total number was 91 mice, of which 23.1 per cent developed breast tumors. The incidence of breast tumors among the control A stock mice was 83.2 per cent. One-third of the fostered first generation mice developed mammary gland tumors. The incidence among their 20 progeny was 60 per cent. The progeny of lung tumor mothers had a breast tumor ratio of 10.5 per cent. None of the 5 progeny descended from non-tumorous mothers developed breast cancer. One line descended from a CBA fostered female is of interest. Female No had 15 successive generations of mammary cancer in her direct

8 MAMMARY TUMORS IN MICE IN RELATION TO NURSING 537 ancestry. She was killed when 18 months old and found to have a primary lung carcinoma. Three generations of her progeny have been observed, a total of 32 mice. One had a mammary tumor, 16 developed lung tumors, and 15 died non-tumorous. The progeny lived to an average age of 16.3 months. The mother of fostered female No was cancerous female No An unfostered daughter of No , a sister of No , was also mated. This animal had 32 descendants in the next three generations, 30 of which lived to the average cancer age and 82.1 per cent developed breast cancer. A sister of No (No ) died non-cancerous at 14 months. Ten of her 12 unfostered descendants had breast tumors. These data are presented to show that in the same sub-line from which the above mentioned fostered litter originated, the breast tumor incidence among the control mice approached that for the entire unfostered stock. Also, the proportion of breast cancer observed in the progeny of non-tumorous unfostered females was as high as for breast cancer mothers. This confirms the statement made by Strong (1935) that chance alone or disease may explain the observation of non-cancerous mice in a high-cancer inbred strain. In the one line of fostered mice descended from female No , however, three generations of progeny have been observed in which less than 5 per cent developed breast cancer. Another group of A stock mice has been fostered by C57 Black females. Forty-one have died and 5 are living in the oldest lot. This number has been greatly increased in later work. The animals which have succumbed lived to an average age of 16.5 months. Two or 4.9 per cent had mammary tumors, average age 10 months; 46.3 per cent had lung tumors and 48.8 per cent died non-tumorous. Three C57 Black stock females fostered by A stock females have developed carcinoma of the breast. In addition, one mouse. of the next generation had a tumor of similar type. These four tumors occurred in a small group of fostered mice that numbered about 25, the majority of which are still alive. In the control C57 Black stock only 3 mammary tumors have been recorded in several hundred breeding and virgin females. That some tumors developed in the fostered females of the A stock and their progeny may be due to the manner in which the experiments were carried out. The nipples of the A stock' females were not blocked. Their progeny were recorded only once a day. Thus some of the young may have obtained sufficient milk from the A stock mothers before they were removed to initiate the development of breast cancer. The proportion of fostered mice with mammary tumors may also have some relation to the incidence in the virgin females of the high-cancer line and/or the incidence in the stocks used as foster mothers. Less than 1 per cent of the C57 Black females normally develop breast tumors as compared with 13.5 per cent in the CBA breeding females. The proportion with mammary tumors among the C57 Black fostered A stock mice was considerably less than in the CBA fostered group. Following the fostering by mice from a nearly negative breast-tumor stock, as the C57 Black, the incidence may approximate that for virgin females of the high-cancer stock. This is 4.5 per cent (11 of 244) for the A stock virgin

9 538 JOHN J. BITTNER females (Bittner, and unpub.). In the breeding females of the A stock fostered by C57 Black females the incidence was 4.9 per cent. This work may also indicate that the cancer susceptibility constitution which an animal inherits is not as significant in the development of cancer as the milk which it receives while nursing. In the A stock, however, the production of young, causing secretion of' the ovarian hormones and the stimulation of the mammary glands, is required for the expression of breast cancer in most animals. These results obtained by the foster-nursing of young from a high-breasttumor line by females of low-tumor stock may offer an explanation for the extrachromosomal influence in the transmission of mammary tumors. Lung tumor susceptibility, which may be transmitted through either parent (Bittner, in press), is not affected. Further work has been planned in an attempt to procure evidence as to the nature of the " breast-cancer-producing " influence or influences. CONCLUSIONS These experiments would tend to indicate that some influence is transmitted through the mother's milk which is of prime importance in determining the incidence of breast tumors. The incidence of breast tumors may be significantly decreased by fostering females of a high-breast-tumor stock to lowtumor stock mothers. This work may offer an explanation for the extrachromosomal influence in the etiology of breast tumors. LITERATURE CITED BITTNER, J. J.: Am. J. Cancer 15: 2202, BITTNER, J. J.: Am. J. Cancer 25: 113, l935n. BITTNER, J. J.: Am. J. Cancer 25: 791, BITTNER, J. J.: Proc. Soc. Exper. Biol. & Med. 34: 42, 1936~. BITTNER, J. J.: Am. J. Cancer 27: 519, BITTNER, J. J.: Science 84: 162, 1936c. BITTNER, J. J.: J. Heredity 27: 391, 1936d. CLOUDMAN, A. M., AND LITTLE, C. C.: J. Genetics 32: 487, FEKETE, E., AND GREEN, C. V. : Am. J. Cancer 27 : 513, KORTEWEG, R.: Nederl. tijdschr. v. geneesk. 78: 240, KORTEWEG, R.: Nederl. tijdschr. v. geneesk. 79: 1482, KORTEWEG, R.: Genetica 18: 350, MACDOWELL, E. C. : Science 81 : 418, MACDOWELL, E. C., AND RICHTER, M. N.: Arch. Path. 20: 709, MURRAY, W. S.: Am. J. Cancer 20: 573, MURRAY, W. S., AND LITTLE, C. C.: Science 82: 228, 1935~. MURRAY, W. S., AND LITTLE, C. C.: Genetics 20: 466, MURRAY, W. S., AND LITTLE, C. C. : Am. J. Cancer 2 7 : 516, STAFF, JACKSON MEMORIALABORATORY : Science 78 : 465, STRONG, L. C.: Am. J. Cancer 25: 599, STRONG, L. C.: J. Heredity 27: 21, 1936~. STRONG, L. C.: Brit. J. Exper. Path. 17: 60,