chromosomal and extrachromosomal influence ......chromosomal influence from the non-cancer stock,...

CHROMOSOMAL AND EXTRACHROMOSOMAL INFLUENCE IN RELATION TO T H E 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 1935 the authors published a report of their study of the genetics of mammary tumor incidence in mice (1). Two inbred strains, one of which (dilute brown) gives an incidence of 50.84 per cent spontaneous mammary tumors in virgin females and the other of which (C57 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 How animals were obtained

113 379 664 68 7

Dilute brown female X C57 Black male C57 Black female X dilute brown male dBF, female X dBF, male BdF, female X BdF, male

Abbreviation for cross 1

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:

(1) The dBF, animals showed a significantly higher incidence of mammary tumors than did those of the BdF, generation; 39.82 per cent as against 6.06 per cent.

( 2 ) This difference persisted in the F, generations, as follows: dBF, 35.54 per cent, BdF, 5.96 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.

(4) 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.

(5) 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.

(6) The difference in tumor incidence in the reciprocal crosses of both F, and F, generations lasted throughout all age periods and was strikingly constant.

(7) 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.

1 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 C57 Black non- tumor strain. No dominance or recessiveness is indicated by the use of large or small letters.

536

INCIDENCE O F MAMMARY TUMORS I N MICE 537

AB

Ab

aB

a b

I t 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 re- lationship 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. I t should then be possible to determine the chromosomal influence in the inheritance of these tumors.

AABB AABb AaBB AaHb

AABb AAbb AaBb Aabb

AaBB AaBb aaBB aaBb

AaBb Aabb aaBb aabb

~ ~ _ _ _ ~

- _ _ _ _ ~ ~ -

_ _ _ _ _ _ _ ~ _ _ _

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 a b

Totaling the number of times each letter occurs, we get: 16A; 16B; 16a; 16b.

From this Punnett's square, it will be noticed that in t h e aggregate one-half of the chromatin of this generation is inherited from the high-cancer stock (1hA; 16B) and one-half from the low-cancer stock (16a; 16b). The chromosomal constitutions of the animals, however, are not identical, as they were in

2 In this and following illustrations dominance and crossing over are not considered. The dis- tinguishable somatic characters in the actual crosses segregated remarkably close t o expectation.

538 WILLIAM S. MURRAY AND C. C. LITTLE

TABLE I : Derivation and Formdae of Crosses -

Chromosomal Extrachromosomal Cross Female Male complex influence Number

d ba

Rlk d R F i

RdFi R d F i d ba R d F i t113F1

X Blk X d n F i X dba X RdFi X dba X RdFi X Rlk x Blk

c c c c c c c c c c c c c c c c c c c c c c c c cccc cccc

E 113 E 664 e 3 19 e 687 e 250 E 252 C 250 E 244

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 recog- nize 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.

I t 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:

A13 Ab all a b

a b Aanb I Aabb 1 aallb I aabb 1 Totaling the number of times each of these letters appears, we find that

three-fourths of the chromatin came from the non-cancer stock (6a; 6b) and that one-fourth (2A; 2B) 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 E 3 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 pur- poses, 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-

cancer mother and small e indicates extrachromosomal influence from a non-cancer mother. Capital E in the formula represents the extrachromosomal influence derived from a high-

INCIDENCE OF MAMMARY TUMORS I N MICE

TABLE 11: Incidence of Mammary Cancer

539

BdFi

379 23

CCcce 6.06

Dilute *Stock 1 brown 1 dRFl 1 dRF, R C I D BdFz A

~ _ _ _ ~ ~ ~ _ _ 687 250 252 250 244 41 6 90 1 83

CCcce CCCce CCCcE Cccce CcccE 5.96 2.40 35.71 0.41 34.00

Number Observed tumors Formula Per cent of cancer

297 113 664 151 45 236

CCCCE CCccE CCccE 50.84 39.82 35.54

soma1 concentrations in animals with (E or e).

different extrachromosomal influence

Tabulating these crosses according to the percentage of mammary cancer incidence, Table I1 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 3.5 per cent cancerous animals and those which have 5 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 consti- tution, have capital E appended to their formula. That is, they all derived their extrachromosomal influence from mothers or grandmothers of the high- cancer stock. All of the classes with 5 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 non- cancer 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 ( 2 ) , a simple percentage used to express the cancer incidence may not mean very much. The element of time, as ex- pressed by the age to which the animals lived, must be taken into consideration. I t 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 in- dicators 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 40 WILLIAM S . MURRAY AND C. C. LITTLE

TABLE 111: Number of Mice Alive at the Beginning of Each Age Period

“ A ” , 6 1 3 ” “ C ” “ D ” Age in Dilute months brown dRF, d B F ~ RdF, IidF, Cross Cross Cross Cross * Total

- 0 1 2 3 4 5 6 7 8 9

10 11 1 2 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

297 297 297 297 297 297 297 297 297 296 293 290 277 245 214 175 151 121 101 70 51 44 32 23 13 4

113 113 113 113 113 113 107 102 98 95 94 93 92 91 88 82 76 70 62 57 50 45 42 36 33 30 22 18 14 13 8 6 2

664 664 664 664 664 664 664 664 663 661 656 594 532 464 413 381 362 34 1 317 296 268 234 207 182 163 141 112 98 80 67 50 35 18

7

379 379 3 79 379 379 379 378 378 375 374 365 357 343 322 291 263 234 216 206 203 193 183 167 154 142 125 114 102 87 74 62 55 39 28 24 19 15 4 1 1

68 7 687 687 687 687 687 687 68 7 685 684 679 666 635 60 1 5 63 516 488 473 458 434 418 395 365 335 312 287 271 240 203 169 136 95 54 26 9 4 1

250 250 250 250 250 250 250 250 250 250 250 250 250 249 249 248 248 244 241 238 235 232 220 2 13 2 04 169 153 141 121 98 77 47 26 5

252 252 252 25 2 252 252 252 252 25 1 25 1 250 249 246 242 233 227 217 201 188 176 163 148 135 123 110 99 74 62 49 34 23 16 10 6 4 4 1

250 250 250 250 250 250 250 250 250 250 250 250 248 241 245 245 241 2 39 236 226 222 217 209 201 184 162 148 133 110 82 61 35 25 13

7 4 4 2 i

244 244 244 244 244 244 244 244 244 244 244 243 240 237 234 232 224 216 210 201 188 170 159 142 126 114 96 81 63 44 24 12 4 3 2

3136 3 136 3136 3136 3136 3.136 3129 3124 3113 3105 3081 2992 2863 2698 2530 2369 2241 2121 2019 1901 1788 1668 1536 1409 1287 1131 990 875 727 581 44 1 30 1 178 88 66 31 21 6 2 1

abruptly at the twenty-second month. The mean age for the appearance of tumor is 10.6 months, with a standard deviation from this mean of 2.65 months. From this, we may say that of the 2 2 5 2 breeding females in this population, 59 per cent of those set aside as breeders will develop mammary cancer, the great majority of them between the 235th and the 394th 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

INCIDENCE O F MAMMARY TUMORS I N MICE 541

TABLE IV: Number of Mice with Mammary Cancer

Age in a Dilute “ A ” ‘,B., “ C ” “ D ” months brown dBFl dBF2 BdFl BdF, Cross Cross Cross Cross Total

5 1 6 7 1 1 8 1 1 9 2 1

10 1 10 11 4 13 1 3 12 12 1 15 1 3 13 19 1 17 1 3 14 23 5 16 2 15 12 5 9 16 18 4 15 4 17 6 5 17 2 2 18 ’ 14 13 1 19 15 3 13 1 20 5 2 14 1 3 21 8 2 11 3 22 4 2 15 4 1 23 2 1 8 2 24 5 2 7 1 3 1 25 1 4 10 1 2 1 26 1 5 1 4 1 27 6 2 1 1 28 6 1 29 2 4 1 2 1 30 3 1 4 31 2 3 1 32 3 1 33 34 35 36 37 1

1 1 1 2 6 4 7

14 9 6 3 4 6 4 I 4 5 7 3 1 1

1

1

2 2 4

1 13 1 23 2 36 3 50 1 51 5 38 5 60 3 44 4 38 6 41 9 38 7 37 6 37 7 24 6 30 5 31 2 17 1 12 6 14 2 12 1 9

7 4

1

having tumors increases steadily as they grow older. A female which lives to the beginning of the seventh month has 65 chances in 100 of becoming cancerous before she dies, whereas a female which lives to the beginning of the thirteenth month has 76 chances in 100 of developing this same type of tumor. It therefore becomes imperative, if we are to compare the out-cross stocks ac- curately, 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 I11 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 111). If, therefore, we multiply the cancer death rates of the individual strains (Tab1e.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 V I I ) . By adding these theoretical cancer death rates and di-


TABLE V: Derivation of the Mamnzary Cancer Rate for the Various Age Periods for Virgin Dilute Brown Females

____I__ ___ - ___ -____ - __ ____ .___________

No. alive a t No. having Age in beginning mammary cancer Cancer ratc months of period during period for period __

8 297 1 ,0034 9 296 2 .0068

10 293 0 11 290 4 .0139 12 277 12 .0433 13 245 19 .0776 14 214 23 .1075 15 175 12 ,0686 16 151 18 .1192 17 , 121 6 .0496 18 101 14 .1386 19 70 15 .2143 20 5 1 5 .0980 2 1 44 8 ,1818 22 32 4 ,1250 23 23 2 .0870 24 13 5 .3846 25 4 1 .2500

-~ -

TAIKE VI: Derivation of Standard Mammary Cancer Rate for Virgin Dilute Brown Females _ _ _ _ ~ _ - - ______- - - - - -__

Age in months

8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Theoretical number Standard ' Mammary cancer of mammary

population death rate, dbr. cancer deaths

3113 3105 3081 2992 2863 2698 2530 2369 2241 2121 2019 1901 1788 1668 1536 1409 1287 1131

.0034

.0068

,0139 .0433 .0776 ,1075 .0686 .1192 .0496 .1386 .2143 .0980 .1818 .1250 .0870 ,3846 .2500

10.58 21.11

41.59 123.97 209.36 271.98 162.51 267.13 105.20 279.83 407.38 175.22 303.24 192.00 122.58 494.98 282.75

347 1.4 1

??..!& = 501.44 dead of mammary cancer per 10,000 population 69229

viding by the total standard population, we get one figure for each strain, which represents the standard cancer death rate per 10,000 population. These fig- ures are directly comparable from one strain to another.

INCIDENCE OF MAMMARY TUMORS I N MICE 543

Age in months

TABLE VII: Standardizing Population Composed of Dilute Brown Virgin Females, dBF, , dBF,, BdF,, BdF,, A , B , C , and D Crosses

0 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Stand- ard

POPU- lation

3136 3136 3136 3136 3 136 3136 3129 3124 3113 3105 3081 2992 2863 2698 2530 2369 2241 2121 2019 1901 1788 1668 1536 1409 1287 1131 990 875 727 581 44 1 301 178 88 66 31 21

6 2 1

Total 69229 Standard rate

per 10,000

_____ Theoretical number of mammary cancer deaths

Virgin dilute dRFl dBF2 BdF1 HdF2 “ A ” “ B ” “ C ” ‘ID” brown

26.60

30.62 10.58 21.11

41.59 32.66

123.97 31.21 209.36 29.68 271.98 143.70 162.51 144.51 267.13 117.88 105.20 151.44 279.83 407.38 99.99 175.22 71.52 303.24 74.06 192.00 73.11 122.58 39.17 494.98 77.99 282.75 150.76

45.05

89.36

100.32

4.69 4.67 4.66

46.83 65.52 80.74 98.75 97.91 55.91 92.78

105.84 82.78 83.45 93.33 78.40

11 1.36 62.00 55.2 1 80.19 44.15 53.55 54.53 34.69 26.46 25.80 29.67

8.38 13.46 8.30 13.46 8.36 13.49

9.11

18.38 19.73 8.91

4.44 9.31 9.30 12.87

27.36

18.32 16.90 6.91

9.52 12.36 6.31 9.05 7.92 6.67 8.61 14.65 6.44

17.15 3.68 6.21 8.36 7.84 6.86 5.93 7.10 12.97 5.48 4.56

1.50

12.42 12.32 12.63 11.97 12.27 23.19 23.76 66.91 33.99 43.52 10.88 72.97 51.17

144.54 49.97 95.02 29.48 64.41 38.36 32.32 56.84 43.81 85.65 67.55 68.72 45.47 7.37 57.91 45.79 69.46 58.56 61.26 79.96 49.65 40.10 20.59 14.09 10.76 14.83 69.21

26.44 18.39

18.81

3471.41 1.529.63 1573.87 188.23 169.46 38.47 1008.56 7.37 857.39

501.44 220.95 227.34 27.19 24.48 5.56 145.68 1.06 123.85

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


TABLE VIII: Standard Cancer Rates

dBFr BdFi --- CCccE CCcce

227.34 27.19

45 5.4

-

Stock 1 dba 1 dBF, BdFn

CCcce

24.48

4.8

Formula Standard

cancer rate per 10,000

Percentage of cancer (dba as unity)

X C c e

5.56

1 .1

CCCCE

501.44

100

CCCcE Cccce

145.68 1.06

27.8 0.2

CCccE

220.95

44

A 1 l c -1-1-

CcccE

123.85

25.0

as unity, it becomes evident that in the first and second generations of the cross derived from the high-cancer females (dBF, and dBF2), the mammary cancer incidence is approximately 45 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 5 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 1 per cent of the cancer rate of the high tumor stock and that B, which had the same chromosomal make up as A, had 28 per cent as much cancer of the breast as the high-tumor strain.

In the same manner, the two generations which received 75 per cent of their chromatin from the non-cancer strain (C and D> may be compared. Here it is found that D had 2 5 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 high- cancer 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 back- cross 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 non- cancerous 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. I n a similar manner, by repeatedly back-crossing BdF, females and their female progeny to dilute brown high- cancer males, the chromatin of the high-cancer stock is gradually concentrated

INCIDENCE OF MAMMARY TUMORS IN MICE 545

TABLE IX: Mammary Cancer Death Rates

Age in Dilute months brown dBFl dBF9 BdFI BdFs “A“ “ B ” “ C ” “ D ”

0 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

.0034

.0068

.0139

.0433

.0776

.lo75

.0686

.1192

.0496

.1386

.2143

.0980

.1818

.1250

.0870

.3846

.2500

.0088

.0098

.0106

.0109

.0110

.0568

.0610

.0526

.0714

.0526

.0400

.0444 ,0476 .0278 .0606 .1333 .0455

.1538

.3333

.0015

.0015

.OO 15

.0152

.02 19

.0282 ,0366 .0387 .0236 ,0414 .0499 .0410 .0439 .0522 .0470 .0725 .0440 .0429 .0709 .0446 .0612 .0750 .0597 .0600 .0857 .1667

.0028

.0029

.003 1

.0093

.0049 ,0052 .0164

.0130

.0074

.0080

.0087

.0196

.0115

.0135 .0161 .0182 .0256

.2500

.0045

.0047

.0050

.0036

.0082

.0042

.0022

.0072

.0110

.0096

.0070 ,0148 .0042

.0118 .0294

.0045

.0049 ,0059 .0065 .0071

.0102

.0040

.0040

.0040

.0081 ,0248 .0172 .0308 .0645 .0448 .0319 ,0170 .0245 .0405 .0296 .0325 ,0455 .0707 .0405 .0161 .0204

.0625

.0041

.0041 ‘ .0083

,0126 .0043 .0216 .0223 .0139 .0190 .0299 .0479 .0412

.0048 .0377 .0493 .0476 ,0439 .0208 ,0123 .0952 .0455 .0417

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

50 75 87.5 93.7 96.9 98.4 99.2 99.6 99.8

Female Male Female Male I1 dBFl X Blk 1st BC X Blk 2nd BC X Blk 3rd BC X Blk 4th BC x Blk 5th BC X Blk 6th BC x Blk 7th BC X Blk 8th BC x Blk

BdFl X dbr 1st BC X dbr 2nd BC X dbr

’ 3rd BC X dbr 4th BC X dbr 5th BC X dbr 6th BC X dbr 7th BC X dbr 8th BC X dbr

Dilute brown chromatin

50 75 87.5 93.7 96.9 98.4 99.2 99.6 99 8


Name of cross

S

T

U

V

W

x

Y

Z

TABLE X: Crosses Made

Female Male

ccccE X cccc

ccccE X dil. br.

crccE X Black

ccccE X CCCC

CCCCe x cccc

CCCCe X dil . br.

CCCCe X Black

CCCCe X CCCC

Formula

ccccE

CCccE

ccccE

CCccE

CCcce

CCCCe

CCcce

CCCCe

Should be like:

Original non-cancer strain and U cross

dBF1, dBF2 and V cross

Original non-cancer strain and S cross

dBFI, dBF2 and 'r cross

BdFI, BdF2 and Y cross

Original high-cancer strain and Z cross

BdF1, BdFl and W

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 months

5 6 7 8 9

10 11 12 13 14 15 16' 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Number alive at beginning of period

dBFi dBFz " B " " D " Total

113 107 102 98 95 94 93 92 91 88 82 76 70 62 57 50 45 42 36 33 30 22 18 14 13 8 6 2

664 664 664 663 661 656 594 532 464 413 381 362 34 1 317 296 268 234 207 182 163 141 112 98 80 67 50 35 18

7

252 252 252 251 251 250 249 246 242 233 22 7 217 201 188 176 163 148 135 123 110 99 74 62 49 34 23 16 10 6 4 4 1

244 244 244 2 44 244 244 243 240 23 7 234 232 224 216 210 201 188 170 159 142 126 114 96 81 63 44 24 1 2 4 3 2

1273 1267 1262 1256 1251 1244 1179 1110 1034 968 922 879 828 777 730 669 597 533 483 432 384 304 259 206 158 105 69 34 16 6 4 1

Total Mammary tumors cancer 1 rate

1

2 1 2

13 15 20 27 26 26 38 34 23 25 29 26 27 20 20 26 11 8

13 8 4 6 3

,0007

.0015

.0008

.0016 ,0104 .0127 .o 180 .0261 .0268 .0282 .0432 .0410 .0411 .0342 .0433 .0452 .0506 .0414 .0463 .0677 .0361 .0309 .0631 .0506 .0381 .0869 ,0882

INCIDENCE OF MAMMARY TUMORS IN MICE 547

TABLE XII: Computation of Tumors to be Expected in First Out-crosses Not Carrying the Extra- chromosomal 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.

5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

1566 1566 1565 1560 1558 1544 1523 1475 , 1418 1348 1272 1209 1171 1140 1100 1067 1021 958 899 825 735 680 61 1 509 413 321 222 134

.0007

.0015

.0008 , .0016

.0104

.0127 ,0180 .0261 .0268 0282 .0432 .0410 .0411 .0342 .0433 .0452 ,0506 .0414 .0463 .0677 .0361 .0309 .063 1 ,0506 .0381 .0869 .0882

1.0962

2.3415 1.2480 2.4928

16.05 76 19.3421

' 26.5500 27.0098 36.1264 36.8704 52.2888 48.0010 46.8540 3 7.6200 46.2011 46.1492 47.9748 37.2186 38.1975

' 49.7595 24.5480 18.8799 32.11 79 20.89 78 12.230 1 19.2918 11.8180

759.

Theoretical number - '159 Number observed 71

- = 10

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 Extrachromo- somaE Influence: In previous experiments those crosses which derived their extrachromosomal influence from high-cancer mothers (dBF,, dBF2, B and

548 WILLIAM S. MURRAY A N D C. C. LITTLE

TABLE XIII: Computation of Tumors t o be Expected in Eighth Generation Bwk-crosses N o t Carrying the Extrachvomosoinal Factor, Had They Been Like Early Out-crosses

Carrying This Factor

Alive a t beginning Mammaiy cancer Age in of period: S , rate: dBF,, dBF2, Theoretical number months T, U, V B and D for S , T, U, V

5 372 .0007 .2604 6 369 7 367 .0015 5.505 8 364 .0008 .2912 9 363 .0016 5808

10 362 .0104 3.7648 11 361 .0127 4.5857 12 361 .0180 6.4980 13 359 .0261 9.3699 14 357 .0268 9.5676 15 354 .0282 9.9828 16 349 .0432 15.0768 17 347 .0410 14.2270 18 343 ,0411 14.0973 19 33 7 .0342 11 S938 20 33 1 .0433 14.3323 21 318 .0452 14.3736 22 299 .0506 15.1294

11.4264 23 276 .0414 24 249 .0463 11.5387 25 222 .0677 15.0294 26 180 .0361 6.4980 27 114 .0309 3.5226 28 44 .063 1 2.7764 29 13 ,0506 .6578 30 2 .0381 ,0762

195.7974

196 - = Theoretical number of tumors S , T, U, V crosses would have had, had they been like

6 = Observed number of tumors other crosses with capital E in their formulae (dBFI, dBF2, B and D)

D), when combined, give a population of 1273 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 com- puted (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, 759 mammary tumors would have been found in the BdF,, BdF,, A, and C crosses. Actually, 7 1 mammary tumors were observed. This indicates that animals which derive their extrachromosomal influence from cancerous mothers have ten times (759/71) 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.

1NCIDENCE OF MAMMARY TUMORS I N MICE 549

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, dBF1, dBF2, Theoretical number months x. Y . z B and D for W-. X. Y . Z

5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

316 311 311 308 308 3 05 303 300 298 298 295 288 281 2 74 2 66 228 198 178 132 91 68 29 13 5

.0007

.0015

.0008

.OO 16

.0104

.0127 01 80 ,0261 .0268 .0282 .0432 .0410 .0411 .0342 .0433 .0452 .0506 ,0414 .0463 .0677 ,0361 .0309 .0631

.1212

,4665 .2464 .4928 .4270

3.8481 5.4000 7.7778 7.9864 8.3190

12.4416 11.5210 10.2614 9.0972 9.8724 8.9496 9.0068 5.4648 4.2133 4.6036 1.0469 .4017 .3155

122.3810

122 = Theoretical number of tumors W, X, Y , Z would have had, had they been like other

4 = Observed number crosses with large E in their formulae

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 non- cancerous 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 196 mammary tumors should have appeared. Actually 6 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 122 mammary tumors. Four were actually observed. The percentage of tumors was the same as for the S, T, U, V group although the extrachromo- soma1 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

5 50 WILLIAM S. MURRAY AND C. C. LITTLE

TARLE XV: Computation of Tumors t o 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 dRF1, dRF2 of tumors

5 360 .0012 .4320 6 356 .0013 ,4628 7 355 .OO 13 .46 15 8 352 .0013 .4576 9 352 .0053 1.8656

10 350 .0106 3.7100 11 350 ,0189 6.6150 12 350 .0256 8.9600 13 347 .0324 11.2428 14 347 .0359 12.4566 15 344 ,0389 13.3816

13.8096 16 336 .0411 17 333 .0438 14.5854 18 328 ,0448 14.6944 19 323 .0425 13.7275 20 307 .0471 14.4597 21 288 .0573 16.5024 22 271 .0522 14.1462 23 237 .0520 11.3240 24 207 .0561 11.6127 25 177 .0885 14.5645 26 122 .0672 8.1984 27 76 .05 1 7 3.9292 28 30 ,0638 1.9140 29 5 30 2

213.6095

Rate of dBF1, dBFz 9 214 Rate of BdFI, BdFz 1 24

- _ - = -

24 = 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 :

(1) 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

Age in month:

4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

INCIDENCE OF MAMMARY TUMORS IN MICE

TABLE XVI: Distribution of Observed Data for Eighth Generation Back-crosses

5 5 1

Number alive at the beginning of the period * S T U v W X Y Z Total

96 111 95 106 95 105 94 105 92 105 91 105 91 104

a 90 104 90 104 89 103 88 103 88 (1)102 87 101 87 100 87 99 85 97 85 95 81 (1)93 73 89 64 85 56 (1)77 51 65 43 50 23 (1)29 8 10 3 5

2

84 83 82 81 81 81 81 81 81 81 80 79 78 77 76 76 75 73 68 62 56 51 38 25 9 5

88 88 87 87 86 86 86 86 86 86 86 85 83 83 81 79 76 71 69 65

(1160 55

(1149 37 17

103 68 103 68 101 67 100 67 98 66 98 66 97 66 97 66 97 64 95 64 95 64 94 64 92 (1)63 90 59 88 (1)56 88 51 83 (1)35

32 31 72

54 17 48 7 38 2

(1)17 2 10 1 3 1

75 .

63 63 63 63 63 63 63 63 63 63 63 63 60 60 60 59 53 49 41 33 22 19 6

82 82 82 81 81 81 79 77 76 76 76 74 73 72 70 68 57 42 34 28 14 9 4 2 1

695 688 680 678 672 671 667 664 66 1 65 7 655 649 63 7 628 61 7 603 559 516 477 408 340 2 90 209 127 49 13 2

* Tumors in parentheses.

derivatives of similar chromosomal complex), no further comparison is neces- sary.

( 2 ) 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 24 mammary tumors should have been observed in the population available for study. Seven mammary tumors were observed, or 28 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 144 mammary tumors should have appeared. Actually 3 were observed.

Since it has been demonstrated that the extrachromosomal influence car- ried 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 144 or 14 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


TABLE XVII: Computation of Tumors t o 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

5 150 .00 12 .1a00 6 149 ,0013 ,1937 7 148 .0013 .1924 8 147 $0013 .1911 9 147 .0053 .7791

10 145 .0106 1.5370 11 143 .O 189 2.7027 12 140 .0256 3.5840 13 140 .0324 4.5360 14 140 .0359 5.0260 15 138 .0389 5.3682 16 136 .0411 5.5676 17 131 .0438 5.7378 18 126 .0448 5.6448 19 119 .0425 5.0575 20 92 .0471 4.3332 21 74 .0573 4.2402 22 65 .0522 3.3930 23 45 .0520 2.3400

' 24 21 .0561 1.1781 25 11 .0885 .9735 26 6 .0672 .4032 27 3 .05 17 .1551 28 1 .0638 .0638

63.3800

63.38 __ X 100 = 144 44 144 = the number of tumors which should have been found had X, Z been like the high-tumor

Observed number = 3 stock.

had the tendency to develop mammary tumors been transmitted through the chromatin.

It therefore seems reasonable to conclude: (1) 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.

( 2 ) 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.

(4) The tendency to have mammary cancer is not mendelian in nature.

LITERATURE CITED

1. MURRAY, W. S., AND LITTLE, C. C.: Genetics 20: 466-496, 1935. 2. MURRAY, W. S.: Am. J. Cancer 20: 573-593, 1934. 3. WRIGHT, S.: Genetics 6: 111-178, 1921.

chromosomal and extrachromosomal influence ......chromosomal influence from the non-cancer stock,...

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