[CANCER RESEARCH 34, 3253-3257,
December 1974]
Estrogen Receptor Content and Hormone-responsive Growth
of Mouse Mammary Tumors
MelsSluyserand RoberthaVan Nie
Departments ofBiochemistry
[M. S.] and Biology [R. V. N.J, The Netherlands Cancer Institute, Amsterdam, The Netherlands
SUMMARY
Mammary tumors were induced with estrone and proges
terone in ovariectomized GR mice. Nearly all of the induced
tumors were hormone dependent and remained so for the next
few serial transplantations,
after which they became first
hormone responsive and finally hormone independent. These
tumors were grafted into castrated mice that were treated with
estrone and progesterone and into castrated mice that did not
receive hormones. The tumors elicited in the hormone-treated
castrates by hormone-dependent or hormone-responsive grafts
had high estrogen receptor levels, but those obtained after
grafting with hormone-independent
tumors had low estrogen
receptor levels. The tumors obtained in hormone-untreated
castrates after grafting with hormone-responsive or hormone
independent tumors had low estrogen receptor contents. All
tumors
derived
from castrated
hosts not given hormones
were
found to be hormone independent and to have low estrogen
receptor contents.
These results indicate that in this model system only the
cells of a tumor graft that are devoid of an estrogen receptor
are able to multiply in the absence of estrone and proges
Correspondingly,
cessation of this treatment again led to
regression.
Hormone-dependent
mammary tumors can be induced in
GR mice or in their F1 hybrids by treating ovariectomized
mice continuously with estrone and progesterone (R. Van Nie,
unpublished data). After transplantation, nearly all of these
tumors retain their hormone-dependent
behavior during the
1st transplant generation. During subsequent transplantations,
a decrease in degree of hormone responsiveness generally
occurs, which eventually leads to complete hormone inde
pendence. Since it is possible to compare the behavior of 1
particular tumor in different mice under varying hormonal
conditions, this model system has certain advantages above the
system that uses mice that are alternatingly pregnant and
nonpregnant. We have used this system to examine the
relationship between hormone-responsive growth and estrogen
receptor content in more detail.
MATERIALS AND METHODS
Animals. Castrated male or female mice of the inbred GR/A
terone.The possible
bearingof thisfindingon thelossof strain were used in all experiments (8).
hormone responsiveness of mouse mammary
successive transplant generations is discussed.
tumors during
INTRODUCTION
The growth rate of certain types of mammary tumors in
inbred strains of mice is influenced by endocrine factors.
Gardner (3) reported that some mammary tumors of C57 X
CBA F1 hybrids grew during pregnancy but regressed after
parturition. Foulds (2) revealed 2 main types of behavior, i.e.,
“unresponsive―
tumors which grew independently of preg
nancy and the postpartum period, and “responsive―
tumors
which grew during pregnancy but regressed after parturition.
The content of estrogen receptors in pregnancy-dependent and
-independent tumors of GR/A mice was assayed by Terenius
(10), who reported that the pregnancy-dependent
tumors
contained such receptors, while the independent tumors had
low levels or an absence of receptors.
Van Nie and Thung (1 1) showed that responsive tumors
which had regressed after parturition could be made to recur
by combined treatment with estrone and progesterone.
Received April 29, 1974; accepted July 5, 1974.
DECEMBER
Induction of Tumors. Three-month-old female GR/A mice
were ovariectomized and, on the same day, treatment with
estrone and progesterone (both from N. V. Organon Co., Oss,
The Netherlands.) was started. Estrone was dissolved in
ethanol (2 mg/ml) and the solution was added to the drinking
water to give a final concentration of 0.5 jig/ml. Progesterone
was administered in pellets introduced s.c. in the neck region
of the mouse. The dose was 3 pellets (2.7 mg progesterone per
pellet) per animal per week. After 3 to 4 months, mammary
tumors were found usually measuring about 10 mm in
diameter. Tumor lines were established by transplantation
from 13 different tumors.
Transplantation
of Tumors. For serial transplantation,
tumor tissue was minced with scissors and suspended in 0.14
M NaCI. Portions
of this suspension
were grafted
s.c. in the
right flank of 020 X GR F3 hybrid mice (0.5 ml/mouse). The
mice had been orchiectomized or ovariectomized about 1
week previously. For testing of hormone dependence, tumor
tissue was inoculated into 2 or more castrated female or male
mice that received no hormone treatment. Two or more
castrated animals, treated with estrone and progesterone as
above, served as controls. The animals were checked regularly
for 3 months. If within this period no tumor appeared in the
animals
that
did
not
receive
hormones,
but
tumors
1974
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1974 American Association for Cancer Research.
grew
3253
M. Sluyser and R. Van Nie
progressively in 1 or both of the treated animals, the tumor
tested was considered hormone dependent. However, if the
tumor grew in the nontreated animals as well, and the time of
tumor appearance was equal to that in the treated group, the
tumor was considered hormone independent. Tumors that
were transplantable into nontreated animals but appeared
more than 1 week earlier in the hormone-treated animals were
called hormone responsive. Hormone-dependent tumors were
transplanted every 4 to 6 weeks; hormone-independent tumors
were transplanted every 2 to 4 weeks. The tumors used for
transplantation were derived from mice treated with estrone
and progesterone, except in 1 experiment mentioned later.
Estrogen Receptor Assay. Estrogen receptor levels were
assayed
in the cytosol
according
modified
using 500-mg
to the method
by Korsten
samples of tumor
of Korenman
and Persijn
tissue
and Dukes (5), as
(6). In brief,
this method
consists of measuring estradiol-3H binding in the presence and
absence of nafoxidine using appropriate controls. The differ
ence between the values obtained (which is taken as a measure
of estrogen receptor content) was calculated per mg tumor
tissue. Tumor tissues to be used for assay were stored at —70°
Materials. Estradiol-l7@3-6,7-3H with specific radioactivity of
56 Ci/mmole was obtained from the Radiochemical Centre,
Amersham, England. Radioactivity was counted in the Packard
Tri-Carb Model 544 liquid scintillation spectrometer. The
radioactivity was corrected for quenching.
Statistical Analysis. Statistical analysis of the data obtained
was carried out with the Wilcoxon test (7, 12).
RESULTS
Estrogen Receptor Levels. Thirteen mammary tumor lines
(designated A, B, C, . . . M) were established by transplanta
tion. Each time, the grafts were derived from hormone-treated
animals. Chart I shows a typical result obtained when the
estrogen receptor contents of several transplant generations of
one such tumor line were assayed. Tumor tissue of the 2nd
transplant generation of this line (tumor line J) was inoculated
into 3 mice treated with estrone and progesterone and into 3
mice not treated with these hormones. Tumors appeared soon
in the hormone-treated animals and reached weights of about
2.5 g 19 days after grafting
(transplant
generation
3). When
theanimals
werekilledandtheestrogen
receptor
levels
ofthe
tumors were assayed, these were found to be high. In contrast,
tumors appeared late in the hormone-untreated animals, and
tumor weights of 2.5 g were not reached until about 35 days
after grafting. The estrogen receptor content of these tumors
was found to be low. Tumors of transplant generations 5
through 8 appeared soon after inoculation and reached weights
of about 2 g in 15 days, whether or not estrone and
progresterone were administered. The estrogen receptor levels
of these tumors were found to be low irrespective of whether
hormones
were given. Transplant
generation
4 yielded
values
with respect to growth and estrogen receptor content, which
were intermediate between those obtained with transplant
generation 3 and transplant generations 5 through 8.
Chart 2 summarizes the results obtained with all 13
mammary tumor lines studied. It shows that, after hormone
3254
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ESTRADIOL- 3H BINDING (cpm/ mg tumor tissue)
Chart 1. Estradiol-3 H binding of mouse mammary tumors obtained
in estrone + progesterone-treated castrates (.) and in untreated
castrates (o). The tumors were transfer generations 3 to 8 of tumor line
J. The grafts used to induce transfer generation 3 were hormone
responsive; those used to induce transfer generations 4 to 8 were
hormone independent.
dependent tumors were grafted in hormone-treated
mice,
tumors with high estrogen receptor levels were obtained (31
mice studied; average estradiol-3H binding, 148 cpm/mg tumor
tissue). No tumors were obtained in mice untreated with
hormones. After hormone-responsive tumors were grafted in
hormone-treated animals, tumors with high estrogen receptor
levels were also obtained (47 mice studied ; average estradiol
@Hbinding, 147 cpm/mg tumor tissue). Statistical analysis by
the Wilcoxon test revealed a high probability (p 0.47) that
this content did not differ significantly from that of the
tumors obtained after grafting with the hormone-dependent
tumors. In contrast, when hormone-responsive tumors were
grafted in animals not given hormones, tumors with low
amounts of estrogen receptor were obtained (47 mice studied;
average estradiol-3H binding, 33 cpm/mg tumor tissue). The
statistical probability that this content equaled that of the
tumors obtained after grafting hormone-responsive tumors in
hormone-treated mice is very low (p < 0.01). Finally, the
hormone-independent
transplant generations elicited tumors
with low estrogen receptor levels, whether or not the rodents
in which they were grafted received hormone treatment ; the
average estradiol-3H binding values of tumors found in 20
hormone-treated
and 26 untreated mice were 37 and 36
cpm/mg tumor tissue, respectively (p
0.26). Tumors
obtained in mice not given estrone and progesterone after
grafting with hormone-responsive tumors had low estrogen
receptor levels, equal to those obtained after grafting with
hormone-independent tumors (p 0.54).
As shown in Table 1, a tumor from line I, carried for 9
generations in hormone-treated
hosts, grew faster and had
more estrogen receptors when grafted in estrogen + proges
CANCER RESEARCH VOL. 34
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1974 American Association for Cancer Research.
Estrogen Receptors
terone-treated mice than when grafted in untreated hosts. In
contrast, a tumor from the same line I, but carried in
hormone-untreated hosts, showed no difference in growth rate
or receptor content whether transplanted in hormone-treated
or untreated hosts.
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were grafted
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(p
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Progesterone
treatment
alone
in mice treated
with hormones,
it took
3 to S
weeks to obtain tumors weighing about 1 to S g. In contrast,
when hormone-independent
tumors were grafted in the
@OCO
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animals
also had no effect on tumor growth or estrogen receptor levels
(p = 0.23). When line I tumors carried in hormone-untreated
hosts were grafted in mice, combined estrone and progesterone
treatment of the hosts did not affect the estrogen receptor
levels or growth of the tumors (p
0.15), nor did estrone
administration alone have any effect (p 0.82).
Growth Rate of Tumors. Chart 3 shows the age at which the
tumors were harvested and the weights of these tumors. It can
be seen that, when hormone-dependent or -responsive tumors
D 2.
E 3.
0
Tumors
Table 1 also shows the results of giving estrone alone or
progesterone alone. Line I tumors carried in hormone-treated
hosts were transplanted into mice that were given either
estrone or progesterone. Treatment with estrone alone did not
significantly affect tumor growth, and the receptor content of
tumors grown in estrone-treated
animals did not differ
significantly from the receptor content of tumors grown in
untreated
4-
B 4.
C 4.
in MouseMammary
oo••
o....
animals,
•
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0
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tumors
weighing
1 to S g were obtained
after only 2
to 3 weeks. This indicates that independent mammary tumors
had a relatively higher growth rate than did hormone-depend
ent or -responsive tumors.
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DISCUSSION
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The understanding
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5,
6.
7,
8.
00
(c)
is important
cancer. For
•I
0
•
o. oo
100
200
300
ESTRADIOL-3H BINDING (cpm/mg tumor tissue)
Chart 2. Estradiol-3 H binding of mouse mammary tumors obtained
in estrone + progesterone-treated castrates (.) and in untreated
castrates (o). The tumors of several transplant generations (1 to 12) of
13 different tumor lines (A, B, C, . . . M) were assayed. The grafts used
to induce the tumors were hormone dependent (a), hormone responsive
(b), and hormone independent (c).
of hormonal influence on tumor growth
in the elucidation
of the pathogenesis of breast
this study the hormone-responsive
mammary
tumors in GR mice may be a useful model. The present
experiments were designed to test the relationship between
estrogen receptor levels in tumors and the response of these
tumors to hormonal influence. In particular, these studies were
undertaken to investigate the progressive loss of hormone
responsiveness of mammary tumors during successive trans
plantations.
The data presented here can be summarized as follows.
When a hormone-responsive mammary tumor was inoculated
into a castrated mouse that received treatment with estrone
and progesterone, a tumor with a high estrogen receptor level
Table 1
Estrogen receptor content of mammary tumors obtained in castrated mice and hormone-treated castrated mice
after inoculation ofgrafts obtained from a hormone-treated castrated donor or a castrated donor
tissue)'@p@'Hormone-treated
Graft donorSeriesHormone
castrated mouse1None
treatment
of host•
Estrone + Proges
terone
Estrone
Progesterone20
0.23Castrated
Estradiol-3H binding (cprn/mgturnor
115
161
95
95
77
81
72
85
28
76
47
47 35 55 65 32
0.16
10614 840 10114 11240 7027 2566 4050 38<0.012
mouse2None
Estrone+Proges
51
23
15
6
22
terone
Estrone19
06
00
117
00 12120.15
0.82
a Each value r Tpresents 1 mouse.
b Statistical
pr@'b )ility
calculated
with the Wilcoxon
test, compared
with the castrated hosts without
hormone
treatment
of the same series.
DECEMBER
1974
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3255
M. Sluyser
and R. Van Nie
population. However, the subsequent change to hormone
independence is accompanied by a large increase in the
percentage of autonomous cells in the tumor.
The assumption that hormone-responsive tumors are mixed
populations of hormone-dependent
and autonomous cells is
4also consistent with our observation that tumor grafts derived
from hormone-untreated castrates were invariably found to be
Cl)
hormone-independent
when tested by transplantation. This
4
implies that all the tumors designated by open circles in Charts
0
I and 2 probably were autonomous, since all had grown in the
3.
•
I-.
absence
of hormones; this again is consistent with the absence
•
A
0
of an estrogen receptor in these tumors.
0
Mi
The mammary tumors in GR mice described in this paper,
•.•@
an,
which
were originally obtained by treating castrates continu
2.
.• OR
A
•
••
@••03 AA
c@ o
ously
with
estrone and progesterone, also required both of
•
A
0
0
A
00
•
OAO
these hormones in order to remain hormone-responsive during
•• A
A
•
• 0
•A
successive transplantations.
Treatment of the hosts with
A
A
0
estrone
alone
or
progesterone
alone did not appear to be
•
•
sufficient stimulus.
The data in this paper confirm the findings of Terenius (10)
with pregnancy-dependent
tumors, indicating a relationship
between the growth and estrogen receptor content of
I
I
I
I
mammary tumors in GR mice. On the other hand, Shyamala
10
20
30
40
(9) has reported that certain spontaneous tumors in GRS/A
DAYS AFTER TRANSPLANTATION
mice, which are independent of estradiol, contain cytoplasmic
Chart 3. Tumor weights at the indicated number of days after
receptors but no intranuclear localization of the hormone.
grafting. Tumors obtained in estrone + progesterone-treated castrate
However,
the properties of mouse mammary tumors depend to
mice after grafting with hormone-dependent tumors (is), hormone
a large extent on the way these tumors are induced. Therefore,
responsive tumors (o), and independent tumors (.).
the spontaneous hormone-independent
tumors studied by
appeared within a few weeks. In contrast, when a hormone
Shyamala may have different characteristics from those of the
responsive mammary tumor was inoculated into a hormone
estrone + progesterone-induced and -dependent tumors studied
untreated castrated mouse, a tumor appeared after a much by us. The hormones of the pituitary gland (e.g., prolactin) are
longer period of time and this tumor did not contain an not necessary for the growth of the mammary tumors
estrogen receptor. This result indicates that, in the absence of described in the present paper. Thus, in hypophysectomized
mice treated with estrone + progesterone, the grafted tumors
hormones, only the autonomous (i.e., hormone-independent)
tumor cells were able to proliferate, whereas in the presence of grew progressively. Furthermore, the presence of a pituitary
hormones both the autonomous tumor cells and the tumor graft under the . kidney capsule of the hosts treated with
estrone + progesterone did not have significant influence on
cells that require hormones for growth were able to multiply.
the growth of the transplanted tumors (unpublished results).
In other words, our data are consistent with the assumption
that hormone-responsive mammary tumors in GR mice are The observation of very thin uteri in the female mice killed 3
months after ovariectomy demonstrated only a very low
mixed populations
of hormone-dependent
and hormone
secretion of endogenous adrenal estrogens.
independent cells.
When hormone-dependent
or -responsive tumors were
At present it is not clear whether the level of cytoplasmic
estrogen receptor can be taken as a measure of the number of grafted in hormone-treated castrates, tumors took longer to
cells in the tumor that contains the receptor. In order to appear than when hormone-independent
grafts were used
substantiate this conclusion, other methods such as radioau
(Chart 3). This suggests that autonomous tumor cells in this
tography should be utilized. However, if this assumption is model system multiply at a faster rate than do hormone
correct, our data indicate that in hormone-responsive tumors dependent tumor cells. The more rapid proliferation of
the autonomous cells probably constitute only a minor autonomous tumor cells may perhaps contribute to the loss of
fraction of the total tumor mass. Evidence for this conclusion
hormone responsiveness of these mammary tumors during
is that, when tumors growing in hormone-treated hosts are successive transplant generations.
compared, the receptor content ofhormone-responsive tumors
These results may also be of interest with respect to
is not detectably
lower than the receptor content of hormone-responsive human mammary carcinomas. It has been
hormone-dependent
tumors. In contrast, the difference in shown that the presence of estrogen receptor in human breast
receptor content between the group of hormone-responsive
cancer can be correlated with the hormone responsiveness of
tumors and that of independent tumors is very marked (Chart the tumor (1 , 4). However, in these cases ovariectomy of the
2). This suggests that the initial change from hormone
patient may have only a temporary beneficial effect, due to
dependency to hormone responsiveness is accompanied by the fact that by this treatment only the tumor cells that
only a slight increase of autonomous cells in the tumor contain an estrogen receptor are destroyed.
5-
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3256
CANCER RESEARCH VOL. 34
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Estrogen Receptors in Mouse Mwnmary Tumors
The authors gratefully acknowledge the excellent technical assistance
5. Korenman, S. G., and Dukes, B. A. Specific Estrogen Binding by
the Cytoplasm of Human Breast Carcinoma. J. Clin. Endocrinol.
Metab., 30: 639—645, 1970.
of Saskia Uhlenbroek and of J. Dc Moes. We also thank the N. V.
6. Korsten, C. B., and Persijn, J. P. A Simple Assay for Specific
ACKNOWLEDGMENTS
Organon Company for their gift of estrone and progesterone.
Estrogen Binding Capacity in Human Mammary Tumors. Z. Kim.
Chem. Klin. Biochem., 10: 502—508,1972.
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DECEMBER
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1974
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1974 American Association for Cancer Research.
3257
Estrogen Receptor Content and Hormone-responsive Growth of
Mouse Mammary Tumors
Mels Sluyser and Robertha Van Nie
Cancer Res 1974;34:3253-3257.
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