[CANCER RESEARCH
45, 6436-6441,
December 1985]
Inhibition by 2-Bromo-a-Ergocriptine
Estrogen-dependent
Rats1
and Tamoxifen of the Growth of an
Transplantable Pituitary Tumor (MtT/F84) in F344
Akihiro Ito,2 Kengo Kawashima, Nariaki Fujimoto, Hiromitsu Watanabe, and Masashi Naito
Department ot Cancer Research, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan
MATERIALS AND METHODS
ABSTRACT
A new transplantable pituitary tumor, designated MtT/F84,
was induced in estrogenized female F344 rats and has been
serially passaged in 17/3-estradiol-treated females. It grew well
in rats treated with estrone, 17/3-estradiol, or estriol but not in
intact females or in rats given progesterone or testosterone.
The growth of MtT/F84 in rats grafted with up to 1.6 x 106
tumor cells and given 17/3-estradiol was inhibited by orally ad
ministered high dose bromocriptine (37.5 mg/kg in food) or by
intraperitoneal injection of tamoxifen citrate but was not inhibited
by low dose bromocriptine (3.75 mg/kg in food).
The tumors grown in intact females contain high amounts of
estrogen receptor, and they were greatly reduced in the tumors
grown either in 170-estradiol or 17/3-estradiol-plus-tamoxifen
loaded rats. However, administration of bromocriptine resulted
in estrogen receptor levels significantly higher than those of
tumors grown in 17/8-estradiol. The existence of dopamine re
ceptor was also confirmed.
Growth inhibition of MIT/F84 either by high dose bromocriptine
or by tamoxifen may be a direct action and may be an estrogen
and dopamine receptor-mediated phenomenon.
INTRODUCTION
2-Bromo-a-ergocriptine (bromocriptine) has a suppressive ef
fect on human prolactinomas (1,9,16,
21 ), as well as experi
mental pituitary (30) and mammary tumors (30, 33); furthermore,
it acts on normal pituitary glands to inhibit the release of prolactin
(10). The mechanism and mode of action, however, are still
incompletely explored. We have reported previously that bro
mocriptine acts cytocidally on human prolactinomas, leaving
necrosis and shrinkage of the tumor cells (8).
However, direct action of bromocriptine on pituitary tumor
cells is still debated. Lamberts and MacLeod (15) and Mac Leod
and Lehymeyer (20) demonstrated growth-inhibitory effects by
ergocriptine on transplantable rat pituitary tumors, but bromo
criptine was not inhibitory.
In this report, we have established a new estrogen-dependent
mammosomatotropic tumor (MIT/F84) transplanted in F344 rats,
and the inhibitory effects of bromocriptine and tamoxifen were
examined on the grafted tumors. Estrogen and dopamine recep
tor contents were examined in the individually treated tumor
cells.
Received 9/26/84; revised 8/20/85; accepted 8/22/85.
' Supported in part by Grants-in-Aid for Cancer Research from the Ministry of
Pellet was made by melting the hormone with cholesterol powder until
fused. Each pellet was weighed and cut to an appropriate size and
placed s.c. in the back. It was replaced with a new one every 12 weeks.
Chemicals. Bromocriptine (Sandoz Ltd., Basel, Switzerland) was
mixed with powdered diet (Ninon Clea Co. Ltd., Tokyo, Japan), and
chow biscuits were prepared under pressure. Final drug concentrations
were 37.5 mg/kg diet (HD3 bromocriptine) and 3.75 mg/kg diet (LD
bromocriptine). They were given orally ad libitum. Consumption of chow
pellet was always the same, either of regular food or of combined food
with HD or LD bromocriptine. The average daily oral dose was 0.75 mg/
rat in a group of rats given HD bromocriptine and 0.075 mg/rat in LD
bromocriptine. Tamoxifen citrate (Stuart Pharmaceuticals, Wilmington,
DE) was suspended in sesame oil and injected i.p. at 0.1 mg/rat/day 3
times/week.
Induction and Transplantation of MtT/F84. The primary pituitary
tumor was induced in a female F344 rat by continuous administration of
2.5-5.0 mg 17/3-estradtol for 10 months. The enlarged pituitary tumor
was asceptically minced into small pieces by curved scissors and grafted
s.c. in the back of the neck in 4 syngeneic female rats, two of which
received 17|8-estradiol simultaneously. It grew only in 170-estradioltreated females with a latency of 27 weeks (Table 1). Thereafter, it has
been mainly propagated in female rats given 2.5-5.0 mg of 17/3-estradiol.
It did not grow in intact females up to the 11th passage except on the
eighth one.
For transplantation, tumor tissue was minced in MEM and gently
shaken for 2 h [in 300 units collagenase/ml (Worthington Biochemical
Co., NJ) containing MEM solution] at 37°Cin a water bath by a modifi
cation of the-method of Gould et al. (12) and Watanabe ef a/. (32). After
washing out of enzyme and debris with fresh MEM, the tumor cell
suspension was treated with 4 ¿¡g
deoxyribonuclease I/ml solution (Sigma
Chemical Co., MO). This procedure removes most of the debris and
RBC. Monodispersed tumor cells were scored with trypan blue exclusion
test. Appropriate numbers of viable tumor cell suspension in 0.03 ml
were mixed with an equal amount of 50% brain homogenate, which was
obtained separately from syngeneic rats and homogenized in MEM. A
mixture of 0.06 ml tumor cell and brain homogenate was inoculated into
1 to 5 different sites per rat at subcutaneous fat pads. They were: the
back of the neck; both sides of the lower lateral back; and both inguinal
regions. "Tumor take" was evaluated by palpating the grafted sites, and
'The
Education, Science, and Culture of Japan.
2 To whom requests for reprints should be addressed.
CANCER
Animals. Female F344 rats (Charles River Japan Co. Ltd., Kanagawa,
Japan) 4 to 6 weeks of age were used throughout the experiment. The
grafted tumors were palpated once every week under light ether anes
thesia. The rats were killed when the grafted tumors became over 4 cm
in average diameter. Tumor size is expressed as a mean of two opposing
diameters.
Steroid Hormones. On transplantation of pituitary tumor cells, rats
were, 1 day in advance, implanted with a pellet containing one of the
following steroid hormones: estrone, 17/3-estradiol, estriol, ethynylestradiol 3-methyl ester, progesterone, or testosterone (Sigma Chemical Nos.
E-9750, E-9000, E-1253, E-5001, P-0130, and T-1750, respectively).
abbreviations
used are; HD, high dose; LD, low dose; MEM, minimal
essential medium; ER, estrogen receptor; DAR, dopamine receptor.
RESEARCH
VOL.
45
DECEMBER
1985
6436
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EFFECT OF BROMOCRIPTINE
ON PITUITARY TUMOR
Table 1
Genealogyof transplantableMIT/F84 in female F344 rats treated with 17ß-estradiol
or 17ß-estradiol
plus bromocriptine
with:Tumorgeneration1234567891011No.
No. of tumors after treatment
tumorcellsof
inoculated
siteGross03.6
per
ofgrafted
(wk.)!l272856511914778170-Estradiol2
sites4(4)"8(8)12(12)25
bromocriptineNT*NT2
(100)e4(100)4(100)4(50)10(100)11(44)14
10»1.0x10«1.6X
x
10«5.4
10s1.3x
x
10s2.0
X1042.0x10*3.3
1043.7
x
10*1.6X
x
104No.
(50f2(25)7(70)3(12)0(0)9(36)20(80)NTNTNon-treated0(0)"0(0)
(25)30(6)75(15)75(15)65(13)75(15)75
(56)15(75)22(88)40(80)33(73)17|8-Estradiol+
(15)70(14)Tumorlatency
" Tumor latency was determined at a time when mean tumor diameter was 1 cm across. Number of tumor take was evaluated at this time.
6 HD bromocriptine was started when tumor cells were inoculated.
c Pieces of tumor tissue were grafted s.c.
" Numbers in parentheses, number of rats examined.
e Numbers in parentheses, percentage.
' NT, not tested.
the minimum average size was recorded as 0.2 cm.
Serum 170-Estradiol and Prolactin Levels. Blood samples were
rone was counted. The result was expressed as the maximum number
of [3H]spiperone binding sites per mg membrane protein.
collected either from the jugular vein or from the abdominal artery under
ether anesthesia, and sera were stored at -20°C until assay. Serum
17/3-estradiol levels were measured by radioimmunoassay with reagents
obtained from the Commissariat a L'Energie Atomique (Gif-sur Yvette,
France). Serum prolactin levels were measured as described previously
with reagents of the NIADDK rat prolactin kit (13).
Measurement of ER in Cytosol. The ER assay was adapted from the
work of Ginsburg ef a/. (11). The experiments were carried out at 04°C.The tumor tissues were weighed and homogenized in Tris-EDTAdithiothreitol buffer (10 rriM Tris:10 mw EDTA:1 HIMdithiothreitol, pH 7.6).
The homogenate was centrifuged at 105,000 x g for 60 min. The
concentration of cytosol protein was measured with the Lowry method
(19).
Aliquots of the cytosol were incubated in [3H]estradiol ([2,4,6,7-3H]
estradici: specific activity, 101 Ci/mmol; New England Nuclear, Boston,
MA) by the range of 0.03-3 nw with and without 2000-fold non-radio
active estradiol (6 MM 170-estradiol; Sigma Chemical No. E-8875). After
incubation at 30°Cfor 30 min, unbounded estradiol was absorbed with
the dextran-charcoal
method [an equal volume of dextran-coated
char
coal suspension (activated charcoal, 3.0 g/liter:dextran, 0.25 g/liter in
Tris-EDTA-dithiothreitol
buffer) added to each of the incubated cytosol
for 10 min], and they were centrifuged at 1000 x g for 10 min. (Activated
charcoal was from the Sigma Chemical Co.; Dextran T-70 was from
Pharmacia Fine Chemicals, Inc.) The supernatant including bounded [3H]
estradiol was counted. Results were expressed as fmol of [3H]estradiol
specific binding sites per mg protein with Scatchard analysis (28).
Measurement of DAR in Cell Membrane. The MtT/F84 tumor was
asceptically excised after the rat was withdrawn of whole blood, and
viable tumor cells were dissected out by scissors (2). The tumor tissue
was placed into ice-cold buffer A (15 mw Tris: 120 rtiM NaCI:5 mw KCI:1
mW MgCI?:2 mw CaClî:0.1%ascorbic acid:12.5 /JM nialamide, pH 7.4),
which was used throughout the receptor study. Tumor tissue was
homogenized in a combination of buffer A and 0.32 M sucrose (6
volumes/wet weight of tissue) and centrifuged at 800 x g for 3 min, and
its supernatant was recentrifuged at 145,000 x g for 60 min; then the
pellet was resuspended in ice-cold buffer A as a cell membrane fraction.
Aliquots of the membrane fraction were incubated in [3H]spiperone
([1 -phenyl-4-3H]spiperidol; specific activity, 39-51 Ci/mmol; New England
Nuclear) in the range of 0.1-1.0
nw with and without 2 UM fluphenazine.
After incubation, unbounded fluphenazine was separated by rapid filtra
tion of What CTF/c filter. The supernatant including bounded [3H]spipe-
RESULTS
Genealogy and Characteristics of MIT/F84. Since primary
pituitary tumor was induced in a 17/S-estradiol-treated rat, it was
always propagated in 17/3-estradiol-treated female rats, and we
examined the transplantability in intact female rats. A newly
established MtT/F84 grew well in 17/3-estradiol-treated females
but rarely in intact females (Table 1). After the third generation,
tumor latencies were stable, and incidence of tumor take was
dependent on the number of tumor cells grafted. Approximate
tumor latency was 5 weeks after inoculation of 106 tumor cells,
7 weeks after 105 cells, and 9 weeks after 104 cells in 17/3estradiol-treated female rats.
The somatotropic activity of MtT/F84 was indicated by the
increase in body weights of animals at time of tumor take.
Average body weights in rats with 17/3-estradiol plus MtT/F84
were significantly increased compared to body weights in rats
with 17^-estradiol alone on 50 days by the appearance of grafted
MIT/F84 (Table 2). The rats with palpable tumors inevitably
showed hyperplastic mammary glands with multiple milk cysts,
and serum prolactin levels increased significantly.
Effect of Various 17/9-Estradiol Doses and Sex Steroid
Hormones on the Tumor Growth. On the ninth passage of MtT/
F84, monodispersed tumor of 3.3 x 104 cells per site were
grafted in 5 sites per rat in animals previously given 0, 0.1, 0.5,
2.5, or 12.5 mg of 17/3-estradiol (Table 3). The incidence of tumor
take was always less than 50% in all groups up to 25 days after
tumor grafting. At 32 days and afterward, the tumor incidence
and the mean tumor diameters increased in correspondence with
the dose of 17/3-estradiol administered. By 39 days in rats given
12.5 mg 17/3-estradiol, incidence of tumor take was 96%, and
the average tumor diameter was 1.5 cm. In the same experiment,
the effect of various sex steroid hormones on tumor growth was
examined. The steroids examined were estrone, 170-estradiol,
estriol, ethynylestradiol 3-methyl ester, progesterone, and tes
tosterone. At up to 25 days, none of the group of rats with
various steroid hormones had more than 48% for tumor takes.
CANCER RESEARCH VOL. 45 DECEMBER
1985
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EFFECT OF BROMOCRIPTINE
ON PITUITARY TUMOR
Table 2
Changein body weight and serum prolactin levels in rats with or without MIT/F84
levelMIT/F84
Av. body wt. and serum prolactin
days after
tumor
graftWt.
of rats
examined10
grafted"17/3-Estradiol
Treatment3
graftWt.230days after tumor
(^g/ml)12.0
±2.9'
(g)122±7.4c(0)ti
±9.3"(100)
134±1.99(0)
148±2.5ft(0)
+
+
17/3-Estradiol+ HD bromocriptine
121 ±1.8(0)
57
17|3-Estradiol
115 ±2.6(0)
152 ±2.4(0)50
Control
+No.
518
a 170-Estradioland HD bromocriptine were started at 39 days of age.
6 Monodispersedtumor cells (1.95 x 104)were inoculated s.c. at 5 sites per rat at 39 days of age.
c Mean ±SE.
d Numbers in parentheses, percentage of tumor take.
8 Versusg and h, significantlydifferent by P < 0.001.
' Versus/ and /, significantlydifferent by P < 0.01.
169 ±3.2(0)Prolactin
0.1 ±0.03Õ
1.4 ±0.31'
0.1 ±0.03
Tables
Effect of various doses of 170-esfrad/o/and sex steroids on the incidence of tumor take and averagetumor size in MtT/F84
sizeHosttreatment170-EstradiolEthynylestradiol3-methyl
Incidenceof tumor take and tumor
days after tumor
graftNo.
days after tumor
graftNo.
days after tumor
graftNo.
Ofgraftedsites25
17/3-estradiol
daysafter
level at 35
ofsera55,Ntf55ND55NDNDSerum
graft(ng/ml)0.2
tumor
oftumors01(4)"18(72)20
oftumors0013
oftumors0012(48)"7(38)6(24)2(8)3(12)12(48)00Size
(cm)0.3
(cm)0.7
(cm)0.0
(5)c25(5)25(5)25(5)25(5)25(5)25(5)25(5)25(5)25(5)25
0.011.2
±
±0.171.4
0.070.3
±
(52f15(60)18(72)7(28)8(32)18(72)00Size
50.6±0.1
0.090.6
±
±0.201.5±0.110.5
0.060.1
±
(80)24
(96)1
0.030.0
±
±0.100.2
0.070.3
±
(44)10(40)20
1
±0.130.5
0.030.0
±
esterEstroneEstriolProgesteroneTestosteroneSteroiddose(mg)a0.00.10.52.512.52.52.52.52.52.5No
0.091.0
±
±0.141.3
0.020.3
±
±0.1639 (80)00Size
±0.15No.
±0.0832
0.02d2.6
±
0.257.6
±
±1.4125.4
4.883.3
±
0.203.3
±
±0.28
3Each steroid hormone was made as a cholesterol pellet under heating indicated herein.
6 Monodispersedtumor cells (3.3 x 104)were inoculated s.c. at 5 sites per rat.
c Numbers in parentheses, number of rats examined.
" Mean ±SE.
8 Numbers in parentheses, percentage.
' ND, not determined.
However, after 32 days, percentage of tumor take and tumor
growth rate were highest in estriol- and 17/8-estradiol-treated
rats, followed by those receiving ethynylestradiol 3-methyl ester
and estrone. Tumors did not grow in rats with progesterone or
testosterone or in control rats up to 39 days.
Effect of Tumor Cell Dosages on Response to HD Bromo
criptine. Two different numbers of tumor cells, 5.4 x 10s and
5.4 x 10", were inoculated into four groups of rats, in which
groups 1 and 2 received 17/3-estradiol alone and groups 3 and
4 received 17/3-estradiol plus HD bromocriptine (Table 4). Inci
dences of tumor take and average tumor size were recorded at
35, 42, and 49 days after grafting. Both parameters were much
smaller in groups 3 and 4 in comparison to groups 1 and 2 on
42 and 49 days. Furthermore, the differences between groups 2
and 4 were much greater than those between groups 1 and 3;
i.e., inoculation of a smaller number of tumor cells resulted in
greater retardation of tumor take.
Dose Effect of Bromocriptine on the Growth of MtT/F84.
Thirty-two rats were divided into 4 groups with 8 rats in each
group (Chart 1). Groups 1 to 3 received 2.5 mg of 17/3-estradiol.
grafting. On 42 and 49 days, incidences of tumor take were 50
and 63% in groups 1 and 3; in group 2, it was only 13.0%. The
average tumor size in group 2 was smallest among groups 1
through 3.
Comparative Effects of HD Bromocriptine and Tamoxifen
on MtT/F84 Growth. The effect of tamoxifen on MtT/F84 cells
was compared with HD bromocriptine by giving the chemicals in
female rats which received 170-estradiol and 1.3 x 103 tumor
cells. The incidences of tumor take and average tumor sizes
were recorded at 53, 73, and 93 days after grafting (Chart 2).
Both parameters were reduced in rats with HD bromocriptine as
compared to rats with 17/3-estradiol alone. Neither rats which
received tamoxifen and 17/î-estradiolnor control rats showed
tumor takes during the period of observation.
Presence of ER and DAR in the Tumor. Since MtT/F84
growth was much dependent on the dose and type of estrogen
(Table 3), we measured ER on tumor cytosol in rats given 17/3estradiol alone, 17/3-estradiol plus HD or LD bromocriptine, and
17/3-estradiol plus tamoxifen. The results indicated average max
Group 4 was the control. Rats in groups 2 and 3 were orally
given HD and LD bromocriptine, respectively, throughout the
experimental period. The incidence of tumor take and average
tumor size were recorded at 35, 42, and 49 days after tumor
CANCER
RESEARCH
imum number of estrogen binding sites (Table 5). The ER level
in 17/3-estradiol-treated tumors was significantly higher than that
in tumors with 17/3-estradiol plus tamoxifen and lower than those
with HD or LD bromocriptine. Among those studied, the highest
cytosolic ER level was noted in the tumors grown in normal
VOL. 45 DECEMBER
1985
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EFFECT OF BROMOCRIPTINE
ON PITUITARY TUMOR
Table 4
Effect of HD brotnocriptine on different cell doses of MIT/F84
sizeGroup
Incidenceof tumor take and tumor
graftNo.
days after tumor
of
grafted tumor
cells
site5.4
per
x 10*
5.4 x 104
10s5.4
5.4 x
days after tumor
graftNo.
graftNo.
days after tumor
of
grafted
sites10
of
oftumors10(100)"
(cm)1.9±0.20C tumors10(100)"(cm)2.5
Treatment1
17/3-Estradiol
+ 0.11
(2f
2
17/3-Estradiol
7(47)
0.4 ±0.16
15(3)
10(67f
1.1 ±0.22
3
17/3-Estradiol+ HD
15(3)15(3)35 7(70)0(0)Size0.6 ±0.1542 10(100)3(20fSize
±0.160.1
1.3
bromocriptine
x 10*No.
4
17/3-Estradid + HD
±0.0549
bromocriptineNo.
8 Numbers in parentheses, number of rats examined.
" Numbers in parentheses, percentage.
c Mean ±SE.
" ND, not determined, because the animals' death of tumor cachexia.
" Versusg, significantly different by P < 0.01.
' Versush, significantly different by P < 0.01.
E
L)
E
U
0)
(U
N
N
•H
of
tumorsND0 (cm)2.2
14(93)'
±0.32
ND5
(33)"Size 0.2 ±0.11
<H
m
en
r_
o
C-
o
E
E
.,1-
-1
--t80
80
60
60
u
x
ta
m
4-1
40
0) o
c §
0)
TJ
jj
•
rH
,4.
U
o
40
f_
O
e
20
20
u 0
35
40
45
50
50
Days
60
70
HO
90
Days
Chart 1. Dose effect of bromocriptine for M1T/F84 in 170-estradiol-treated
female rats. Tumor cells of 1.6 x 10* were grafted s.c. into backs. Points, means
of 8 grafted sites; bars, SE. • •,17/3-estradiol(group 1); •
•,17/Îestradiol + HD bromocriptine (group 2);• •,
17/3-estradiol+ LD bromocriptine
(group 3); •
•,
control (group 4). Incidenceof tumor take in group 2 at 42
days was significantly smaller than that in group 3 (*, P < 0.05).
females.
To evaluate for the inhibitory action of bromocriptine for MtT/
F84 growth, the presence of DAR was looked for in tumor cell
membrane fraction in a rat with 17/î-estradiol.The number of
binding sites and the apparent K¿,determined from Scatchard
plots, were 220 fmol/mg membrane protein and 0.67 nw, with a
dissociation constant of 1.7 x 10~2/min.
CANCER
RESEARCH
Chart 2. Comparative effects of H-bromocriptine and tamoxifen on MtT/F84
growth. Tumor cells of 1.3 x 103were grafted s.c. into backs. Points, averages of
25 grafted sites; bars, SE. • •,17/3-estradiol;•
•,170-estradiol + HD
bromocriptine; • •,170-estradiol + tamoxifen; •
•,control. Inci
dence of tumor take in rats with 17/3-estradiolon 53 days was significantly higher
than those of the other 3 groups. (*, P < 0.05; ", P < 0.01).
DISCUSSION
The present experiment was designed to investigate the direct
effect of bromocriptine on transplanted pituitary tumor cells.
There have been numerous reports concerning the inhibitory or
suppressive effect of bromocriptine on human prolactinomas (9,
16,17, 24). In experiments in vitro with isolated pituitary tumor
cells derived from estrogen-induced pituitary tumor 7315a, pro-
VOL. 45 DECEMBER
1985
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EFFECT OF BROMOCRIPTINE
Table 5
Cytosolic estrogen receptor levels in MIT/F84 treated with bromocriptine or
tamoxifen
Monodispersed turner cells (2.6-4.2 x 104) were grafted s.c.
Treatment17/3-Estradiol
17/3-Estradiol + LD
bromocriptine
17/3-EstradioH- HD
bromocriptine
17/3-Estradiol +
tamoxiten
Non-treatedNo.
of tumors
tested38(6)"
3(3)3(3)
binding sites
(fmcrf/mg
protein)62
±6.6e-"
±11.6*107
93
±11.4s
5(5)
37 ±6.4"
±0.14
0.74
0.070.78
±
±0.11
1.20 ±0.15
157 ±10.4K,(HM)0.99
4(2)Maximum
0.57 ±0.05
" Tumors were examined 20 to 60 days after tumor inoculation, and ranges of
the tumor size examined were from 0.5 to 2.0 cm in diameter.
6 Numbers in parentheses, number of rats examined.
c Mean ±SE.
" Versus e, significantly different by P < 0.05.
lactJn release was inhibited in a dose-dependent manner by the
addition of bromocriptine to the medium (4, 26).
Bromocriptine was found to be a dopamine agonist (7), work
ing as a prolactin inhibitor through the hypothalamo-pituitary
axis. Thus, it works de novo as a tumor inhibitor on spontaneous
(6) and estrogen-induced pituitary tumors in rodents (18, 27).
ON PITUITARY TUMOR
were different. Tamoxifen also markedly inhibited MtT/F84
growth. The cytosol of MtT/F84 cells, which was grown in intact
female rats, contained the highest level of ER. However, it
decreased significantly in the tumor grown in 17/3-estradiol
loaded rats. This decrease was moderately resumed by the
administration of bromocriptine. This observation is similar to the
finding that bromocriptine has been shown to resume ER level
in the anterior pituitary gland, which was previously disconnected
from hypothalamus and had a reduced ER level (3). Since ta
moxifen is known to be an antiestrogen (5), tumor inhibition by
this chemical was confirmed, and it reduced ER level to a level
much lower than that of 17/3-estradiol alone. To understand the
regulatory mechanisms of 17/3-estradiol on MtT/F84 growth, the
next step is to investigate nuclear ER among the tumors grown
in the variously conditioned rats. Though the precise mechanism
of tumor inhibition by bromocriptine is not yet clear, it may be
closely related to the existence of ER and DAR in these tumor
cells. The inhibition of tumor growth observed in bromocriptinetreated rats may be accompanied by the inhibition of prolactin
gene transcription from DMA to messenger RNA (22, 23). Since
tumor cells contain both ER and DAR, the action of bromocriptine
may be through both of these two receptors intertwiningly. The
tumor-inhibitory effect of bromocriptine was found to be depen
However, its effect on extrafossal pituitary tumors was not clear.
Lamberts and MacLeod (15) described refractoriness to dopaminergic inhibition of prolactin release in transplanted pituitary
tumors 7315a and MIT/W15. The apparent contradiction be
tween their and our results could be due to the fact that the
tumors examined by them were known to grow autonomously
in syngeneic rats. In the present experiments, we have inde
pendently established and utilized estrogen-dependent pituitary
tumor strain MIT/F84 through serial passages in 17/3-estradioltreated females. MtT/F84 was always passaged in rats given
17/3-estradiol or 17/3-estradiol plus bromocriptine and in intact
females by inoculating from 103 to 106 tumor cells. MtT/F84
grew well in 17/3-estradiol treated rats, but it grew very slowly
with small frequency in intact females. Tumor growth was also
17/3-estradiol dose dependent. It grew well in rats with 17/3estradiol and estriol and less well in those with ethynylestradiol
3-methyl ester and estrone. Mean serum 17/3-estradiol levels in
rats with those estrogens were much higher than those of intact
females. It is interesting to explore why such a high amount of
17/3-estradiol is necessary to keep the growth of MtT/F84 tumor
cells. Morel ef al. (25) have demonstrated that pharmacological
doses of estradiol inhibited growth of MtT/F4, originally induced
in a chronically estrogenized F344 rat, and that it has a mammosomatoadrenotropic function (31) somewhat similar to MIT/F84,
and they correlated the tumor growth inhibition with the exist
ence of estrogen receptor. The growth of an MtT/F4 cell line had
been shown previously to be dependent on the existence of
estrogen receptor (29). A similar conflicting phenomenon was
found on the occurrence of androgen-responsive MT-W9C from
the conversion of methylchoranthrene-induced,
prolactin-dependent MT9 (14), in which a change of genetic constituents
might have been a causative factor.
Oral administration of HD bromocriptine to 17/3-estradioltreated rats effectively reduced the incidence of tumor take and
average tumor size compared to those in rats treated with 17/3estradiol alone. The effect by HD bromocriptine was compared
with that of tamoxifen, though dose and routes of administration
CANCER
RESEARCH
dent on the number of tumor cells, as well as on the dose of
bromocriptine. These facts also suggest that the action of bro
mocriptine is direct on the tumor cells through DAR.
ACKNOWLEDGMENTS
We thank Drs. Bunzo Sato and Masashi Koga for assaying dopamine receptor
content in tumors, Dr. Fluckkjer at Sandoz, Basel, Switzerland for supplying
bromocriptine, Kyoko Tabuchi and Youko Sakai for their excellent technical assist
ance and maintenanceof a M1T/F84cell line, and Dr. Kelly Clifton for reviewing the
manuscript.
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Inhibition by 2-Bromo-α-Ergocriptine and Tamoxifen of the
Growth of an Estrogen-dependent Transplantable Pituitary
Tumor (MtT/F84) in F344 Rats
Akihiro Ito, Kengo Kawashima, Nariaki Fujimoto, et al.
Cancer Res 1985;45:6436-6441.
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