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SUSCEPTIBILITY T O TRANSMITTED LEUKEMIA OCCURRING
I N PURE BRED AND HYBRID MICE'
M. D. SCHWEITZER AKD J. FURTH
(From the Department of Pathology, Cornell University Medical College, New York)
Many investigators have called attention to the importance of heredity in
breast cancer, lung tumors, leukemia, and other neoplasms of mice (cf. 1).
Recently, the staff of the Roscoe B. Jackson Laboratory ( 2 ) has demonstrated
the r61e of non-genetic factors and emphasized the need for a reinvestigation
of the part played by heredity in the development of tumors.2 Experiments
have been undertaken in this laboratory to determine the factors responsible
for spontaneous leukemia and for susceptibility to transmissible leukemia.
The studies on susceptibility to transmissible leukemia are described in this
paper.
The older literature concerning leukemia of mammals has been reviewed
by Opie ( 3 ) . It is only within the past decade that the disease has been successfully transmitted in mice. Transmissible strains have been described by
Korteweg (4), Richter and MacDowell ( 5 ) , Mercier and Gosselin ( 6 ) , Krebs,
Rask-Nielsen and Wagner ( 7 ) , Dobrovolskai'a-Zavadskai'a (8), Lawrence and
Gardner ( 9 ) , Lewis (lo), and workers in this laboratory (11).
There are few reports on the heredity of transmissible leukemia. The most
extensive of these is that by MacDowell and Richter ( 1 2 ) . Other workers
have given only fragmentary data, MacDowell and Richter inoculated cells
of their Line 1, originating in the highly leukemic stock C58, into other mice
of the same stock, into mice of the Storrs-Little stock, which had no previous
history of leukemia, and into first and second generation hybrids ( F1and Fp)
and back-cross combinations. These cells failed to grow in Storrs-Little mice
but produced leukemia in almost every mouse of the C58 stock and of the first
hybrid generation. The frequency of the disease among other hybrid combinations, including back-crosses, suggested that a single dominant gene was
responsible for susceptibility. Cells from another C58 mouse (Line A) gave
somewhat different results in hybrid combinations. The percentages of susceptible mice were much smaller and variable. The ratios of susceptible to
resistant mice observed in back-cross generations suggested that from two to
seven genes might be operating.
On the basis of genetic studies of spontaneous leukemias MacDowell and
Richter ( 1 3 ) concluded that a semi-dominant gene determined the incidence
of the disease. Slye ( 14), however, believes that her results are best explained
by assuming a single recessive gene responsible for the occurrence of leukemia
and an additional localization factor.
1This investigation was aided by grants from The Jane Coffin Childs Memorial Fund for
Medical Research, The International Cancer Research Foundation, and The Anna Fuller Fund.
*Earlier observations of Lathrop and Loeb (J. Exper. Med. 28: 475, 1918) suggested the existence of maternal influence on susceptibility to breast tumors.
2 24
SUSCEPTIBILITY TO TRANSMITTED LEUKEMIA I N MICE
225
Following the pioneer investigations of Tyzzer and Little (15) and of Loeb
(19b), there have been numerous studies on hereditary susceptibility of mice
to transplanted tumors. Most of these have been reviewed by Gorer (16).
Bittner’s studies ( 17) on transplantable adenocarcinoma of the mammary gland
parallel the experiments to be reported here, He studied tumors originating
in stocks A (albino) and D (dilute brown) mice and in first generation hybrids
between the two stocks. The tumors originating in the pure stocks could be
transplanted (a) within the strain of origin, ( b ) to the first generation hybrids,
and (c) to mice resulting from matings of these hybrids with the stock of
origin; but these tumors failed to grow in the opposite stock. They grew in a
small percentage of second generation hybrids and in progeny of the mating of
F to the opposite pure stock. The tumors arising in F, hybrids were transmissible to almost all F, animals; the inoculations were successful in only
small percentages of mice of the second filial generation and of both types of
back-cross mating, and failed in each parental stock. Bittner concluded that
the simultaneous presence of seven or eight genes was required for transmission of tumors originating in F, hybrids. The factors influencing the success
of transplantation are numerous and Loeb (19b) doubts that this method is
suitable for analysis of genes.
NOMENCLATURE
In the description of our experiments the following terms are used as indicated:
Stock: Inbred, genetically homogeneous mice. The specific traits of each stock are
given in the text.
Strain: Leukemic cells or fragments of tumor originating in a single stock and maintained by inoculation in related mice, or preserved in the frozen state.
Line: Cells or fragments from a single host, maintained as stated above.
F,: Progeny of mating Ak and Rf, or reciprocally.
F,: Progeny of mating F, with F,.
F,: Progeny of mating F, with F,.
Back-cross: Progeny of mating F, with either parental stock.
MATERIAL
AND METHODS
Two stocks of mice, designated as Ak and Rf respectively, and hybrids between them were studied. Both stocks have been inbred in this laboratory
since 1928. Their origin has been described elsewhere (1 la). Stock Ak is
noteworthy for a high incidence of spontaneous leukemia among members
surviving seven months or longer, the frequency being approximately 70 per
cent in the past eight generations. Additional evidence that the strain is
genetically homogeneous is afforded by the observation that the incidence of
leukemia is the same within a litter whether or not the parents had the disease.
Lymphatic leukemia is the type commonly occurring in stock Ak. In the Rf
stock leukemia is rare; if it occurs, it is usually myeloid or monocytic. The
frequency in this strain has been approximately 1 or 2 per cent.
Inoculations were made with leukemic cell suspensions obtained from the
spleen, lymph nodes, or leukemic tumors, according to procedures previously
described ( l l c ) . Young adult mice, approximately five to eight weeks old,
were used in the transmission experiments.
226
M. D. SCHWEITZER AND J. FURTH
All but two of the transmissible strains studied were of the lymphoid type.
One strain of Ak leukemia (Akh 106) was myeloid and the Rf strain (No.
385) was monocytic (18).
TRANSMISSION
EXPERIMENTS
Table I, a survey of the transmission experiments, shows the results of
inoculation of leukemias occurring in pure stocks and in hybrids into pure
bred mice and into hybrids of known genetic constitution. The table shows
that leukemia occurring in hybrids behaves like that arising in Ak mice.
TABLE
I: Transmissibility of Mouse Leukemia *
Leukemia occurring in
Stock A t
I
1
Stock Rf
I
1
FI Hybrid Fa Hybrid Fa Hybrid
R X Ak
Fi X Rf
I
_
_
-
Stock A k . . . . . . . . . . . .
Stock R f . . . . . . . . . . . .
F1 Hybrids.. . . . . . . . .
FI Hybrids.. . . . . . . .
F1 Back-cross with Ak
.
Excellent
Failed
Excellent
Excellent
-
Failed
Excellent
Failed
Failed
-
Excellent
Excellent
Excellent
Excellent
Excellent
Good
Fair
Variahle
Fair
Good
Fair
Fair
Fair
Fair
Fair
Excellent Fair
Excellent
Poor
Poor
-
-
* Designation of the success of inoculation: excellent = approximately 100 per cent; good = 75
per cent: fair 50 per cent: poor = 25 per cent; failed = 0 per cent.
=i
Transmission of Leukemia Occurring in Stock A k : Transmission of leukemia occurring in mice of stock Ak is regularly successful within the same
stock and just as regularly fails in Rf hosts (Table 11). The malignant
lymphocytes of an Ak mouse were successfully transferred to nearly all F,
hybrid mice tested and in half of the progeny of F, mice mated to Rf.
TABLE
11: Transmission Exfieriments with A k Leukemia *
Mice
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Ak stock
Rf stock
Fi
Fa
FJ
Ak X FI
Rf X FI
Ak X Fa
Rf X Fz
Ak X (Fi/Rf)
Rf X (FiIRf)
Rf X (FJAk)
Number
of mice
injected
145
29
20
43
77
33
53
11
58
7
12
24
Successful
inoculations
Expectation
Number of
experiments
performed
25
4
2
5
10
7
8
2
10
1
1
2
* The transmissible strain used in these experiments (Akf 5 , lymphatic leukemia) is usually
fatal in about ten days.
The expected values are computed on the hypothesis that a single dominant
gene is responsible for susceptibility to transmitted leukemia. Accordingly,
all mice receiving a leukemic gene from one or both parents should be susceptible, all others resistant. Mice in rows 1, 3, 6, 8 and 10 are expected to
be susceptible, those in row 2 resistant. In rows 4, 5 and 12, mendelian
227
SUSCEPTIBILITY TO TRANSMITTED LEUKEMIA IN MICE
segregation should yield three susceptible among every four mice; half of the
mice in rows 7 and 9 should be susceptible and one of four in row 11.
Most of the results shown in Table I1 are consistent with the interpretation
that a single dominant gene determines the fate of injected leukemic cells.
For Fzand F, mice this interpretation should yield a frequency of 75 per cent
susceptibility. Results with F, mice are close to this predicted value (72 per
cent of 43 injected mice). For F3 mice, however, the figures are definitely
lower (50 per cent of 77 injected mice). The back-crosses of F, and F, to the
Rf stock also yielded susceptibility values below the 50 per cent expectation
(38 per cent of 53 injected mice and 36 per cent of 58 injected mice, respectively). The significance of these findings is discussed below.
Transmission of Leukemia Occurring in Stock Rf: Data for attempted
transfers of Rf leukemia comparable to the preceding series are presented in
Table 111. The strain used (Rfb 385) is monocytic leukemia, which is usually fatal in about three weeks. The results obtained are compatible for the
most part with the single gene hypothesis.
TABLE
I I I : Transmission ExPeriments with Rf Leukemia
Mice
Ak stock
Rf stock
Fi
Fa
Fa
Rf X Fi
Rf X Fa
Ak X Fs
Rf X (Fi/Rf)
Number
of mice
injected
23
33
18
41
52
13
16
4
5
Successful
inoculations
Expectation
0
27 or 28 (83%)
18 (100%)
21 ( 51%)
32 ( 62%)
13 (100%)
14 ( 88%)
3 ( 75%)
5 (100%)
0
33 (100%)
18 (100%)
31 75%)
39 ( 75%)
13 (100%)
16 (100%)
2 ( 50%)
5 (100%)
Number of
experiments
performed
4
9
4
5
7
2
2
1
1
Transmission of Leukemia Occurring in F , Hybrids: Table IV shows the
results of transmission experiments obtained with F, leukemias. Transfer
failed in all of 29 Rf animals tested, but was successful in 55 of 56 Ak mice
and in 17 of 18 F, mice that were injected.
TABLE
IV: Transmission Experiments w’th FIHybrid Leukemia
Mice
Number
of mice
injected
Successful
inoculations
Expectation
Number of
experiments
performed
Four strains of lymphoid leukemia were used in these experiments. Since
the results were essentially the same, they are presented in one table. Three
strains (Ha 14, Ha 16, Ha 17) originated in mice resulting from the mating
Ak X Rf; the fourth (Hb 4 ) came from the reciprocal mating.
228
M. D. SCHWEITZER AND J. FURTH
Transmission of F z Leukemias: The results of inoculation with leukemia
originating in the F, hybrids (Strain Hb Sol), shown in Table V, are similar
to those of leukemia originating in F, and Ak mice.
TABLE
V: Transmission Experiments w'th Fe Hybrid Leukemia
Mice
Number
of mice
injected
Ak stock
Rf stock
Fi
FZ
19
20
6
60
65
8
29
Fa
Ak X FI
Rf X FI
Ak X Fz
Rf X Fa
Ak X (FdRf)
5
6
13
Successful
inoculations
17 ( 89%)
0
6 (100%)
28 ( 47%)
30 ( 46%)
4 ( 50%)
10 ( 35%)
1 ( 20%)
2 ( 40%)
10 ( 77%)
Expectation
Number of
experiments
performed
19 (100%)
0
6 (100%)
45 ( 75%)
46 ( 75%)
8 (100%)
15 ( 50%)
5 (100%)
3 ( 50%)
13 (100%)
4
4
1
6
10
1
6
1
1
2
F, X A k Leukemia: A few series of inoculations of leukemia arising in the
mating of F, with Ak (Strain Ha 230) are given in Table VI. The results are
similar to those of the Ak, F, and F, leukemias.
TABLE
VI: Transmission Experiments m'th R X Ak Leukemia
Mice
Number
of mice
injected
Successful
inoculations
Expectat ion
Number of
experiments
performed
Comment: These experiments indicate that all leukemias occurring among
hybrids resemble genetically the leukemias occurring in the pure leukemic
stock. They will be spoken of collectively as Ak-type leukemias. If the results with the hybrid leukemias are considered alone, the number of successful
inoculations falls short of numerical agreement with the predicted values for
a single dominant gene. Conspicuously higher values were obtained only in
an occasional small test, and can be explained by chance occurrence. These
findings are in agreement with the hypothesis of dominance, since all hybrid
combinations have a substantial proportion of susceptible individuals. The
deficiency observed may be interpreted as due to the presence of two dominant genes governing susceptibility, or to some other unknown factors interfering with the results of inoculations. Further experiments are needed to
clarify these observations.
VARIABILITY
According to the hypothesis that susceptibility to transmissible leukemia is
determined by a single dominant gene, all individuals fall into three genotypes,
with both Ak and Rf types of leukemia. With reference to Ak-type leukemia,
the genotypes are AA, Aa, aa, the first two being susceptible, the last resistant.
The different matings will result in different susceptibility frequencies among
SUSCEPTIBILITY TO TRANSMITTED LEUKEMIA IN MICE
229
the progeny. The genetic hypothesis can also be tested by determining the
variability of the success of inoculations. For example, with both Ak and Rf
leukemias it is to be expected that %e of the F, X F, matings will yield
progeny all of which are susceptible, that I/le will yield resistant offspring,
?hoffspring with 75 per cent and % with 50 per cent susceptibility.
Twenty-two combinations were tested in a total of 196 experiments, each
with an average of 6.1 animals. No variation is postulated in 150 experiments, and only twelve of these gave conspicuously divergent results. More
widespread differences were encountered in forty-four experiments in which
variability was expected. For example, the average susceptibility of F, mice
is expected to be 75 per cent and the range 0-50-75 and 100 per cent. The
sixteen experiments performed yielded the following results :
Mice
No.injected
8
Percent 3.
88
No.
+
Total number.
Individual experiments
7
13
4
30
4
3
75
13
5
38
7
0
0
5
4
80
8
6
75
Observed
Predlcted
129
72
56
129
7 8 8 6 9 9 9
8
6
5
4
2
6
4
4
5
100 86 63 50 33 66 44 44 63
7
7
91
75
Inasmuch as the variability predictions are based on the genotypes of the
particular parents, additional experiments were performed with litter mates.
The results of the thirty-one experiments made, while not inconsistent with
the genetical hypothesis, were not conclusive.
TABLE
VII 1 Duration of Life of Individual Mice of Different Genotypes after Inocdation with Cells
of Strain Akf 5
Mice
injected
Ak stock
Fz
Fa
FI X Rf
Fa X Rf
FI X Ak
FI/Rf X Rf
June 15, 1938
Sept. 3, 1938
days
9, 10,10
8,9,9,9,9,10
8,8,10,10,10,11, 19
9,9,9,9,10,10,16,23
9,9,9,9,10,10
days
11,12,12,12
-
-
9,9,9,9,9,9,9,10,10,11, 11, 13,16
9,9,9,11
-
12, 16
9,9,9,9,9,23
DURATION
OF ILLNESS
IN RELATION
TO HEREDITY
Many investigations have shown that when mice are inoculated under
standardized conditions the duration of illness is characteristic for each transmissible neoplasm, although it often decreases in the course of successive passages. Earlier investigators, however, gave little attention to the genetic constitution of the host, and the question arose whether the genotype of the latter
would influence the duration of illness.
The present study has shown that the genotype of the host does not influence the course of disease. This may be illustrated from data with strain
Akf 5. From its origin in February 1937 until June 6, 1938, the average
duration of illness was 13.7 days (twenty observations). Beginning with the
series started on June 15, 1938, and continuing to date, the length of life after
230
M. D. SCHWEITZER AND J. FURTH
inoculation was abruptly shortened to approximately 10 days. The length of
life after inoculation has since been as follows:
Numberof days
Number of mice
8
11
9
40
10
20
11
7
12
5
13
2
15
1
16
3
19
1
23
2
33
1
Ninety per cent of the animals (83 of 93) succumbed between the eighth and
twelfth days following inoculation.
In an experiment performed on June 6, 1938, three Ak animals died after
14, 14 and 15 days, and three F1animals after 14, 14, and 16 days.
The results of two experiments performed June 15 and September 3 respectively are summarized in Table VII.
These data indicate that the genotype of the host exerts no influence on
the duration of leukemia.
DISCUSSION
The Genetic Basis of Susceptibility to Leukemia: The data on transmission of leukemia originating in pure stocks of mice are consistent with the interpretation that dominant genes, single or possibly double, are responsible for
susceptibility to the disease (Tables I1 and 111). These results confirm the
findings of MacDowell and Richter with their Line I. Loeb (19) and other
investigators (cited by Gorer, 16) have suggested that many genes of both
parental stocks are involved in susceptibility of hybrids to transmissible neoplasms and that the genetic differences are not specific for tumors. Previously this problem of specific genetic factors for susceptibility could not be
directly ascertained, inasmuch as the tumors studied originated in only one of
the parental stocks. The leukemias of hybrid origin now available permit a
further analysis of this problem. While the tissue genes of hybrids are derived in equal number from both parent stocks, all leukemias of hybrid origin
thus far investigated conform to the pattern of Ak leukemias (Table VIII).
This is indicative of the presence of specific genes governing susceptibility to
leukemia. The results are incompatible with the theory that genes of mixed
origin determine susceptibility (Table VIII) .
TABLE
VIII: Predicted Ratios for Susceptibility of Mice to Leukemias Originating i n Hybrids Corn.
pared with Ratios Found
Per cent of successful inoculations on basis of the fallowing possibilities
concerning the genetic constitution of leukemic cells
Results
observed
Mice injected
Ak type, single
dominant
Rf type, single
dominant
Miped,
dominant
Mixed,
recessive
Per cent
+
-.___
Ak stock
Rf stock
FI
F2
100
0
100
75
0
100
100
0
0
100
75
Intermediate
0
0
100
100
96
0
97
44-72
It follows from the hypothesis of Loeb that leukemias of hybrids (e.g. F,)
should be more readily transmissible to Fzand other hybrid mice than to mice
of either parental stock. But the leukemias arising in hybrids studied by us
are more easily transmitted to Ak mice than to F, hybrids. The results are
especially conspicuous with Hb 501 strain (Table V). Although this strain
SUSCEPTIBILITY TO TRANSMITTED LEUKEMIA IN MICE
231
of leukemia originated in an F, mouse, it was more successfully transferred to
Ak and F, mice than to F, mice. These ex
ts lend strong support to
the view that specific hereditary differences
n the Ak and Rf strains
which are responsible for susceptibility to transmitted leukemia are segregated
in the hybrids. The hybrid leukemias thus far observed were of the Ak type,
but it is possible that occasionally leukemias of the Rf type may arise among
the hybrids.
Bittner's experiments with transmissible breast tumors arising in hybrids
gave quite different results from those here reported. The tumors originating
in either parental stock gave results comparable to ours. The tumors arising
in F, mice, however, failed to take in the parental stocks, although they were
grafted with some success into F, and other hybrid mice; this agrees with the
postulated values for factors of mixed origin shown in the third and fourth
columns of our Table VIII. These findings support the opinion of Loeb that
the genetic factors for adenocarcinomata as observed by Bittner are not necessarily tumor-specific. Grafting of normal tissues might yield similar results.
In our experiments, however, specific genetic susceptibility for transmitted
leukemia appears necessary for the success of inoculations.
With regard to neoplasms originating in a pure stock, our transmission experiments are comparable to those of previous investigators. The study of
tumors originating in hybrids contributes essential data concerning the mode
of inheritance of susceptibility to transmissible neoplasms.
Are Susceptibility Factors for A k and Rf Leukemia Allelomorphic? If
Ak leukemic factors were dominant over Rf leukemic factors or vice versa, then
every mouse susceptible to a strain from one stock should be resistant to strains
of the other and every mouse in each hybrid combination should succumb to
one. The fact that both leukemic factors are dominant is inconsistent with
this supposition. Furthermore, if susceptibilities for these two types of leukemia were each determined by dominant and allelomorphic genes (single or
multiple), the F, and back-cross percentages in the corresponding columns of
Tables I1 to VI should add up to a maximum of unity (100 per cent), but this
is not the case, the values being consistently above 100 per cent.
Maternal Influences: Although many transfers were made in reciprocal
crosses, no significant differences were observed. I n the experiments with F,
hybrid leukemias four strains were tested and in one the maternal line was Rf,
in three Ak. These experiments are not sufficiently extensive, however, to
disclose small maternal differences.
Genetic Behavior of Leukbmia of Different Histological Types: The Rfb
385 strain studied is monocytic leukemia, Akh 106 is myeloid, all other strains
are lymphoid. The myeloid strain Akh 106 gave results comparable to those
recorded for Akf 5 and other Ak leukemias of lymphoid type. Our data do
not disclose any difference concerning the susceptibility factors for leukemias
of different cell types.
SUMMARY
AND CONCLUSIONS
Spontaneous leukemias originating in the highly leukemic stock Ak, in
stock Rf in which the incidence of leukemia is low, and in first generation and
other hybrids, were inoculated into mice of each of the pure stocks and of
232
M. D. SCHWEITZER AND J. FURTH
various hybrid combinations. The leukemias arising in different hybrids behaved in transmission experiments like the Ak leukemias.
All of these leukemias can be transmitted to almost every member of the
leukemic stock Ak and to F, generation hybrids, but not to members of stock
Rf. All hybrid combinations tested have a substantial proportion of susceptible individuals, indicating dominance of inheritance; but further investigation
is required to determine if one or two dominant genes are responsible for susceptibility.
The duration of illness and the anatomical characteristics of leukemia are
not modified by the genotype of the host.
The susceptibility factors of both Ak and Rf leukemias are not allelomorphic.
Evidence is presented that these genetic factors are specific for susceptibility to transmissible leukemia, and differ from those that, according to Loeb,
determine susceptibility for normal tissue grafts.
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