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and ß1
Molecular Oncology Group, Royal Victoria Hospital (A.T., G.B.T., V.G.), the Departments of Biochemistry, Medicine, and Oncology, McGill University (V.G.), Montreal, Québec, Canada H3A 1A1; and the Laboratory of Molecular Endocrinology, CHUL Research Center (C.L., F.L.), Québec City, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Québec, Canada H3A 1A1. E-mail: vgiguere{at}dir.molonc.mcgill.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. Using RT-PCR, we
show that ER and ERß are expressed in mouse mammary glands,
suggesting that both receptors should be considered putative targets
for antiestrogen action in the breast. In cotransfection assays using a
synthetic estrogen-responsive promoter, EM-652 shows no agonistic
activity on ER and ERß transcriptional function and blocks the
estradiol (E2)-mediated activation of both ER and ERß.
EM-652 is also very effective in abrogating E2-stimulated
ER and ERß trans-activation of the pS2 promoter in
HeLa cells. EM-652 does not alter binding of ER and ERß to DNA.
The Ras-mediated induction of ER and ERß transcriptional activity
in the presence of E2 is also completely abolished by
EM-652. In addition, EM-652 blocks the E2-dependent
activation of ER and ERß by the steroid hormone receptor
coactivator-1 as well as the in vitro interaction
between SRC-1 and the ligand-binding domains of both ERs. These results
demonstrate that the novel antiestrogen EM-800 fully impedes AF-1 and
AF-2 activities of ER and ERß and can, therefore, be considered a
potent and pure antagonist of both ER subtypes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-substituted derivatives of
estradiol (E2), such as ICI 164,384 and ICI 182,780, have
been shown to be completely devoid of estrogenic activities and able to
block ligand-independent activation of the ERs, and these types of
antagonists are, therefore, considered to be pure or specific
antiestrogens (6). ICI 182,780 has been evaluated in clinical
trials for the treatment of tamoxifen-resistant breast cancers (7, 8).
Over the past decade, all of the studies on elucidation of the
molecular events underlying the mode of ER action as well as the
antiestrogen-designed therapy have focused on the ER identified and
cloned several years ago (9, 10, 11). Recently, a second ER, designated
ERß, has been described and shown to share common structural and
functional characteristics with ER (5, 12, 13). Based on amino acid
sequence comparison, ERß shares with ER the same modular
structure, composed of six domains (A–F) (14). Domain C, which
contains the two zinc fingers responsible for DNA binding, is the most
conserved, followed by domain E, which is responsible for ligand
binding, homodimerization, and nuclear localization. Domain E also
contains a ligand-dependent activation function (AF-2) involved in
trans-activation by the ERs. A second activation function,
AF-1, resides in the A/B domain and acts in a ligand-independent manner
(15, 16, 17). Both ERs recognize a specific estrogen response element (ERE)
composed of two AGGTCA motif half-sites configured as a palindrome
spaced by three nucleotides (5). ER has also been shown to interact
with a number of coregulators via the AF-2 domain, and these
protein-protein interactions promote transcriptional regulation of
target genes (18, 19, 20, 21). Both OHT and ICI 182,780 prevent the
interactions between steroid hormone receptor coactivators and ER
and ERß (5, 18, 22).
In an effort to develop new and more effective antiestrogens, we have
recently generated a novel nonsteroidal antiestrogen compound with a
high oral bioavailability, designated EM-800 (Fig. 1
). EM-800 was demonstrated to be a
potent estrogen antagonist under different in vitro and
in vivo estrogen-sensitive biological criteria, including
its effects on the proliferation of various human breast cancer cell
lines (23) and histopathological studies of reproductive tissues (24).
Moreover, EM-800 exerts no stimulatory effects on alkaline phosphatase
activity on estrogen-sensitive parameters in human Ishikawa cells
(24a). However, its mechanisms of action at the molecular level remain
to be elucidated. We have, therefore, undertaken the study of the
effect of EM-652, the active metabolite of EM-800, on the
transcriptional functions of both estrogen receptors. The results
presented here demonstrate that EM-652 is a very potent estrogen
antagonist in fully abolishing the E2 responsiveness of
ER and -ß, and that activation of both ERs by H-Ras or the steroid
hormone receptor coactivator-1 (SRC-1) is completely abrogated by
EM-652. EM-800, therefore, represents a novel class of pure estrogen
antagonists that can abolish both ER and ERß transcriptional
activities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
C57 mice were shaved, and mammary glands were dissected from
nonlactating, 17.5-day postcoitum pregnant and lactating females; liver
was isolated from the nonlactating females, and total RNA was extracted
using Trizol reagent (Life Technologies, Grand Island, NY). Five
hundred nanograms of total RNA were used as a template to synthesize
first strand complementary DNA (cDNA) in the presence of specific mouse
ER and ERß primers (see below), using Superscript reverse
transcriptase (Life Technologies) according to the manufacturer’s
protocol. Five microliters of the cDNA were used in a PCR reaction (50
µl total volume) performed with Taq polymerase (Boehringer
Mannheim, Indianapolis, IN) under the following conditions: step 1, 94
C for 5 min; and step 2, 94 C for 45 sec, 58 C for 45 sec, and 72 C for
60 sec, repeated for 30 cycles. Parallel reactions conducted with 25,
35, and 40 cycles confirmed the PCR reactions were within the linear
range. One microliter of the reaction was electrophoresed on a 1.2%
agarose gel, blotted onto a Hybond N+ membrane (Amersham,
Arlington Heights, IL), and hybridized overnight to specific mouse
ER and ERß probes contained within the PCR product. After a high
stringency wash, the membrane was exposed to film for 60 min at room
temperature. The following primers were used in the above analysis: for
mouse ER: first strand cDNA synthesis,
5'-GTCAGCTGTCAAGGACAAGGCAG-3'; PCR reaction,
5'-GTCTAATTCTGACAATCGACGCC-3' and 5'-GGGCTTGGCCAAAGGTTGGCAGC-3'; and
for mouse ERß: first strand cDNA synthesis,
5'-GAATAATCTAGTTATGTAAGCC-3'; PCR reaction,
5'-GAAGAGGAAGCTTGGCGGGAGCG-3' and 5'-TCAGGCAATGCACCTGCTCGCTG-3'. The
expected sizes of the products are 431 and 355 bp for ER and ERß,
respectively.
Plasmids
The expression vectors carrying the cytomegalovirus promoter,
pCMX-mER and pCMX-mERß, and reporter plasmids
vitA2EREBLuc, vitA2ERETKLuc, and pS2Luc
(vitA2, vitellogenin A2; TK, thymidine kinase)
were constructed as previously described (5). H-Ras and
H-RasV12 expression plasmids were gifts from Dr. Morag
Park, and the full-length SRC-1 cDNA (19) was inserted into pCMX for
expression studies.
Cell culture, DNA transfection, and luciferase assay
For transfections, COS-1 and HeLa cells were seeded in six-well
plates in phenol red-free DMEM (Life Technologies) supplemented with
10% charcoal dextran-treated FBS, 100 µg/ml penicillin, and 100
µg/ml streptomycin. At 50–60% confluence, cells were transfected
with 1–2 µg reporter plasmid, 0.5–1 µg receptor expression
vector, 1 µg CMX-ß-galactosidase or Rous sarcoma virus
(RSV)-ß-galactosidase, and 6–7.5 µg pBluescript II KS (Stratagene,
La Jolla, CA) as carrier DNA, using the calcium phosphate-DNA
precipitation method (25). After 8–16 h, cells were washed, and
typically 10 nM E2 or 100 nM
antiestrogens, unless otherwise stated, was added to the growth medium
for 16 h. For luciferase assay, cells were lysed in potassium
phosphate buffer containing 1% Triton X-100, and luciferin was added
for light emission measurement using a luminometer (LKB, Uppsala,
Sweden). Values are expressed as arbitrary light units normalized to
the ß-galactosidase activity of each sample. Typically, transfections
were performed in duplicate for each sample, and results are compiled
as the mean ± SEM of at least three separate
experiments.
Antiestrogen competition studies
The murine ER and ERß proteins were in vitro
transcribed-translated using rabbit reticulocyte lysate (Promega,
Madison, WI) with pCMX-mER and pCMX-mERß templates, respectively;
diluted 12-fold in TEG buffer [10 mM Tris (pH 7.5), 1.5
mM EDTA, and 10% glycerol]; and kept on ice until use.
One hundred microliters of this dilution were used in each competition
reaction at 4 C overnight containing 1 nM
[2,4,6,7-3H]E2 and antiestrogen
concentrations varying from 10-13–10-6
M. Unbound steroids were removed with dextran-coated
charcoal, and counts per min were determined by liquid scintillation
counting. The results were plotted as the percentage remaining bound,
where 100% represents the counts in the absence of antiestrogen.
Electromobility shift assay
mERß and mER were produced using rabbit reticulocyte
lysates. Typically, preincubation was conducted with 5 µl programmed
lysate and 5 nM E2 or 500 nM
antagonists on ice for 30 min in 5 mM Tris, pH 8.0,
containing 40 mM KCl, 6% glycerol, 1 mM
dithiothreitol, 0.05% Nonidet P-40, 2 µg poly(dI-dC), 0.1 µg
denatured salmon sperm DNA, and 10 µg BSA. Then, 0.1 ng
-32P end-labeled probe was added and allowed to
bind for 30 min at room temperature. The entire reaction (20 µl) was
loaded onto a 4% polyacrylamide gel and electrophoresed at 150 V at
room temperature. Gels were dried and exposed overnight at -85
C. The vitA2ERE with the inverted repeat
(underlined),
5'-TCGACAAAGTCAGGTCACAGTGACCTGATCAAG-3' (5)
was used as the probe.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß are expressed in mouse mammary glands, the expression of both ER and -ß
was detected under all physiological conditions studied. ERß
transcript levels were slightly increased in pregnant and lactating
mammary glands, whereas the level of ER transcript remained
unchanged. Total RNA from liver was also tested. As expected, ER
transcripts are present in the liver, whereas ERß transcript could
not be detected under the conditions used.
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and ERß (26, 27).
These effects were also described more recently for ERß (5). We thus
used a reporter plasmid containing one copy of the vitA2ERE
linked to the minimal viral TK promoter (vitA2ERETKLuc) to
study the effects of EM-652 together with a panel of previously
characterized antiestrogenic compounds (Fig. 1) on mouse ER- and
ERß-mediated trans-activation. When used in the absence of
E2, none of the antagonists studied, including the mixed
agonist-antagonist OHT, showed significant agonist activity on
either ER (Fig. 3A) or ERß (Fig. 3B) with the vitA2ERETKLuc reporter. As previously reported
(5, 16), the agonistic effect of OHT was more apparent when a basal
promoter carrying only an ERE with a TATA box
(vitA2EREBLuc) was used in cotransfection studies. As shown
in Fig. 3C, OHT induced a 2.5-fold activation of ER activity using a
vitA2EREBLuc reporter. Under the same conditions, EM-652
had no effect on basal ER activity (Fig. 3C). None of the
antagonists, including OHT, showed agonist activity on basal ERß
transcriptional activity (Fig. 3D).
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, A–D). A similar observation was obtained in
HeLa cells (data not shown), suggesting that EM-652 acts in a cell-type
independent fashion. It is of interest to note that EM-652 reduced,
even below basal levels, the E2 induction of both ER and
ERß in COS-1 cells.
To further evaluate the potencies of the antagonists, we compared their
dose-dependent inhibitions of E2-induced ER and ERß
activity using vitA2ERETKLuc in COS-1 cells (Fig. 4). Compared with ICI182,780, EM-652 was
extremely effective, achieving a complete blockade of the
E2-induced effect of ER (Fig. 4A) and ERß (Fig. 4B) at
doses of 10-8 M and above. Comparison of the
apparent IC50 showed that under the conditions used, EM-652
was more potent in repressing ER activity (IC50 = 2
nM) than was ICI182,780 (IC50 = 20
nM). Both antiestrogens were more effective at inhibiting
ERß function, with IC50 of 0.4 and 8 nM for
EM-652 and ICI182,780, respectively. In addition, lower concentrations
of EM-652 in the 10-10–10-11 M
range contributed to a 25–30% reduction in the E2
responses of both ERs, and even when added at 10-13
M, EM-652 still showed a 20–25% repression (data not
shown).
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and ERß and ERß using
a binding competition assay. Increasing amounts of EM-652 contributed
to reduce the binding of E2 on ER (Fig. 5A) and ERß (Fig. 5B) with apparent
IC50 of 5 x 10-7 and 1 x
10-7 M, respectively. Similar IC50
values of E2 displacement from both ERs were observed with
ICI182,780.
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and ERß to
vitA2ERE and -ß to the ERE was not dependent on the presence
of E2 under the conditions used (compare lanes 2 and 3 and
lanes 7 and 8 in Fig. 6). When EM-652,
EM-800, or ICI 182,780 was used alone, a shifted band of similar signal
intensity was observed compared with unliganded and
E2-bound ERs (Fig. 6). However, both ERE-bound ER and
ERß complexes migrated more slowly with the antagonists compared with
E2, suggesting a change in the conformational state of ER
in the presence of antiestrogens.
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(3, 4, 29)
and serine 60 in mouse ERß (5) through activation of the Ras-MAPK
pathway has been shown to further maximize the E2 responses
of both ERs. To investigate whether EM-652 could efficiently block this
effect, we used the wild-type H-Ras and its dominant active form
H-RasV12 in our transfection experiments, as indicated in
Fig. 7. As observed previously (3, 5),
the addition of H-Ras increased the activity of ER in the presence
of E2, with an even stronger response when
H-RasV12 was used (Fig. 7A). These inductions by both Ras
forms were completely abolished with the addition of EM-652 to the
medium, in the same way as with ICI 182,780, suggesting that EM-652 is
effective in blocking the AF-1 activity of ER. The same experiment
was conducted on ERß, where H-Ras and H-RasV12 augmented
the E2 response in a similar manner (Fig. 7B). Again,
EM-652 and ICI 182,780 abolished the Ras effect on ERß in the
presence of E2. Interestingly, we observed a
ligand-independent effect of Ras on ERß basal activity, where a 2- to
3-fold induction occurred with H-RasV12 (Fig. 7B). On the
other hand, no effect of Ras was seen on basal levels of ER. The Ras
induction of unliganded ERß was blocked by EM-652 and ICI 182,780
(data not shown). We were also interested to test whether EM-652 was
efficient in blocking ER responsiveness on a natural promoter. The pS2
promoter has been extensively studied with respect to its
ER-mediated regulation (30). We previously showed that ERß can
also modulate trans-activation of a reporter gene driven by
the pS2 promoter in HeLa cells, and that the E2 response
was potentiated by H-Ras (5). Figure 7, C and D, demonstrates that the
effects of Ras on liganded ER and ERß activities were completely
abrogated by EM-652. Dose-response analyses were also performed to
further evaluate the potency of EM-652 to inhibit the effect of Ras on
ER activities in the presence of E2. EM-652 was slightly
more effective than ICI 182,780 in blocking H-RasV12
inductions of ER and ERß, especially at lower concentrations (Fig. 7, E and F).
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and
ERß (19, 31). More recently, we have demonstrated that
SRC-1 also stimulates ERß activity through a direct interaction with
its ligand-binding domain (LBD), where the AF-2 domain resides (5). We
took advantage of this effect of SRC-1 to study whether EM-652 could
block the E2-activated AF-2 function of ER and ERß. We
first generated glutathione-S-transferase (GST) fusion
proteins with the E and F domains of mERß (GST-mERßEF) and domains
D–F of mER (GST-mERDEF) for use in GST pull-down experiments
(Fig. 8A). GST-mERßEF and GST-mERDEF
were expressed in Escherichia coli, purified with
GST-Sepharose, and incubated with [35S]methionine-labeled
SRC-1. As shown in Fig. 8A, the LBD of mER interacted weakly with
SRC-1 in the absence of E2 (lane 3), whereas addition of
E2 caused an increase in the interaction between the two
proteins (lane 4). Both EM-652 (lane 5) and ICI 182,780 (lane 6)
efficiently blocked the ligand-dependent SRC-1 interaction, with a
stronger effect for EM-652. A similar inhibition of the
E2-dependent interaction between SRC-1 and the LBD of ERß
was also observed, whereas ICI182,780 was less efficient (see Fig. 8A, lanes 7–10). We also demonstrated that the stimulatory effect of SRC-1
on the E2 responses of both ERs in COS-1 cells was
completely abolished with the addition of EM-652 in the medium, and the
novel antagonist was as efficient as ICI 182,780 in that respect (Fig. 8, B and C). Furthermore, as observed with Ras (see above), SRC-1
enhanced the basal activity of ERß, but not that of ER, in the
absence of ligand. This ligand-independent effect of SRC-1 on ERß was
blocked by EM-652. Similar results were obtained using HeLa cells
transfected with a pS2Luc reporter construct (Fig. 8, D and E).
Dose-response analyses were also performed to further evaluate the
potency of EM-652 to inhibit the potentiating effect of SRC-1 on ER
activities in the presence of E2. EM-652 appeared very
effective in blocking SRC-1 potentiation of ligand-dependent ER and
ERß transcriptional activities, with apparent IC50 values
of 10-10 and 10-9 M, respectively
(Fig. 8, F and G). ICI 182,780 was also a potent inhibitor of the SRC-1
induction of ER and ERß activities, with an IC50 of
10-8 M for both receptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß transcriptional activities.
This pure antiestrogenic profile is of primary importance in
endocrine-based breast cancer therapy, as exemplified by the use of
tamoxifen, which acts as a mixed agonist-antagonist on ER function.
Besides a relatively good clinical record in inducing remission of
ER-positive metastatic breast cancer and in postsurgical adjuvant
therapy, a resistance to tamoxifen, probably due to its intrinsic
agonist properties, does occur, and severe tumor progression ensues in
most patients (32). As a result, the search for pure antiestrogens led
to the discovery of compounds such as ICI 164,384 and ICI 182,780 (6, 33), and now EM-800 (23, 34). Of the two ICI compounds, ICI 182,780 is
regarded as a promising candidate for primary breast cancer treatment
and has been evaluated in phase I and phase II clinical trials (7, 33, 35). Preliminary studies have shown that EM-800 also behaves as a very
potent antiestrogen on different in vivo and in
vitro parameters, including having a strong inhibitory effect on
the proliferation of various human breast cancer cell lines (23).
For these reasons, we were interested in comparing EM-652 with ICI
182,780 on the basis of their potency to block ER-regulated gene
expression at the molecular level. In addition, we tested the effects
of EM-652 and ICI 182,780 on the transcriptional activity of ER and
that of the newly described ERß (5, 12, 13) to fully impede the
estrogen action to target tissues such as the mammary gland, where both
receptors are expressed. We identified ER and ERß messenger RNAs
in mouse mammary glands. The expression of ER in mammary glands had
been largely documented previously (36, 37), but no report had yet
described the expression of ERß in that tissue. Dose-response curves
showed that EM-652 was very potent in blocking the E2
response of ER and ERß activities. This was paralleled by a strong
competition by EM-652 on the binding of E2 to both ERs.
Competitive inhibition of estrogen binding is believed to mediate ER
function impairment by antiestrogens, as depicted for ICI 182,780,
which showed a stronger binding affinity than ICI 164,384 and tamoxifen
on uterine ERs (38, 39). However, despite a clear inhibition of
estrogen binding to target tissues in vivo (40), tamoxifen
only provoked a 60% inhibition of antiuterotropic determination in
immature rats (41), whereas ICI 182,780 produced almost complete
inhibition (38). Our results on the competitive binding of E2in vitro and the abolition of the E2
response of ER activity in vivo support the observation that
EM-652 behaves as a very potent antagonist.
The potency of EM-652 to inhibit ER function was even more
dramatic when the E2 response was maximized through
activation of AF1 and AF2 domains of ERs by Ras and SRC-1,
respectively. Phosphorylation of Ser118 triggered by the
Ras-MAPK pathway has been described for ER and shown to further
increase its E2-stimulated transcriptional activity (3).
Ras also activates liganded ERß, presumably through phosphorylation
of Ser60 (5). Here we show that EM-652 strongly inhibited
the E2-induced ER and ERß activities triggered by
either Ras or its dominant active form RasV12. We observed
a similar pattern with SRC-1. SRC-1 has been described as a general
coactivator for steroid receptors and has been shown to up-regulate
ER-stimulated transcription (19, 42). More recently, we demonstrated
that SRC-1 interacts with ERß and stimulates its transcriptional
activity (5). This interaction occurred with the LBD of both ERs (5, 42). Again, EM-652 was very potent in fully abolishing the
E2 response of ER and ERß enhanced by SRC-1. These
effects were not cell or promoter specific, as demonstrated with the
pS2 promoter in HeLa cells. Hence, EM-652 can be regarded as a pure
antagonist that acts on both activation domains of the ERs.
Interestingly, both Ras and RasV12 induced the activation
of transcription of ERß in the absence of E2. Such
ligand-independent activation of Ras was not observed with ER (Ref.
3 and the present work), although it was reported with EGF treatment
(4). A similar pattern of activation of ERß, but not ER, was
observed with SRC-1. Our previous work (5) has shown that the
SRC-1-induced ligand-independent activation of ERß was not blocked by
OHT, which is an AF-2-specific antagonist (16), suggesting that SRC-1
might interact with other regions of the receptor. A possible target
region for such an interaction might be contained within the
amino-terminal region of ERß, as ICI 182,780 and EM-652 inhibit the
ligand-independent effect of Ras and SRC-1. In contrast to the
DNA-binding domain and LBD, the amino-terminal region is poorly
conserved between ER and ERß. By using amino- and
carboxyl-terminal truncated mutants of ER in transfection
experiments, McInerney et al. (42) observed a
ligand-dependent activation by SRC-1 and hypothesized that SRC-1
might act as an adapter to promote both AF-1 and AF-2 activities of
ER. Our own experiments showed that activation of ER by Ras or
SRC-1 occurred in the presence, but not the absence, of ligand, whereas
both ligand-dependent and -independent effects were observed for ERß.
Certainly, these observations need to be further investigated.
The present work describes the premises of the molecular mode of action
of the novel antiestrogen EM-800 and shows that it is a very potent and
pure estrogen antagonist that fully impedes ER-regulated gene
expression by targeting both ER and ERß. These properties identify
EM-800 as a potential therapeutic agent that would completely deprive
mammary tumors of estrogenic stimulation, thus providing an effective
endocrine therapy for breast cancer.
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Acknowledgments |
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and human
SRC-1 cDNAs, and Morag Park for providing us with H-Ras expression
plasmids. We also greatly acknowledge A. Matthyssen for her expert
technical assistance. |
Footnotes |
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Received July 8, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expressed in the mouse embryo. Mol. Cell
Biol 10:2335–2340
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B. Saville, M. Wormke, F. Wang, T. Nguyen, E. Enmark, G. Kuiper, J.-A. Gustafsson, and S. Safe Ligand-, Cell-, and Estrogen Receptor Subtype (alpha /beta )-dependent Activation at GC-rich (Sp1) Promoter Elements J. Biol. Chem., February 25, 2000; 275(8): 5379 - 5387. [Abstract] [Full Text] [PDF]
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 M. Schapira, B. M. Raaka, H. H. Samuels, and R. Abagyan Rational discovery of novel nuclear hormone receptor antagonists PNAS, February 1, 2000; 97(3): 1008 - 1013. [Abstract] [Full Text] [PDF]
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G. Pelletier, V. Luu-The, A. Charbonneau, and F. Labrie Cellular Localization of Estrogen Receptor Beta Messenger Ribonucleic Acid in Cynomolgus Monkey Reproductive Organs Biol Reprod, November 1, 1999; 61(5): 1249 - 1255. [Abstract] [Full Text]
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 N. Zeps, J. M. Bentel, J. M. Papadimitriou, and H. J.S. Dawkins Murine Progesterone Receptor Expression in Proliferating Mammary Epithelial Cells During Normal Pubertal Development and Adult Estrous Cycle: Association with ER{alpha} and ER{beta} Status J. Histochem. Cytochem., October 1, 1999; 47(10): 1323 - 1330. [Abstract] [Full Text]
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 S. Couillard, M. Gutman, F. Labrie, B. Candas, and C. Labrie Effect of Combined Treatment with the Pure Antiestrogen EM-800 and Radiotherapy on the Growth of Human ZR-75-1 Breast Cancer Xenografts in Nude Mice Cancer Res., October 1, 1999; 59(19): 4857 - 4863. [Abstract] [Full Text] [PDF]
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M. Gutman, S. Couillard, F. Labrie, B. Candas, and C. Labrie Effects of the Antiestrogen EM-800 (SCH 57050) and Cyclophosphamide Alone and in Combination on Growth of Human ZR-75-1 Breast Cancer Xenografts in Nude Mice Cancer Res., October 1, 1999; 59(20): 5176 - 5180. [Abstract] [Full Text] [PDF]
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W. P. Bocchinfuso, W. P. Hively, J. F. Couse, H. E. Varmus, and K. S. Korach A Mouse Mammary Tumor Virus-Wnt-1 Transgene Induces Mammary Gland Hyperplasia and Tumorigenesis in Mice Lacking Estrogen Receptor-{{alpha}} Cancer Res., April 1, 1999; 59(8): 1869 - 1876. [Abstract] [Full Text] [PDF]
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G. B. Tremblay, A. Tremblay, F. Labrie, and V. Giguere Dominant Activity of Activation Function 1 (AF-1) and Differential Stoichiometric Requirements for AF-1 and -2 in the Estrogen Receptor alpha -beta Heterodimeric Complex Mol. Cell. Biol., March 1, 1999; 19(3): 1919 - 1927. [Abstract] [Full Text] [PDF]
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D. Chen, P. E. Pace, R. C. Coombes, and S. Ali Phosphorylation of Human Estrogen Receptor alpha by Protein Kinase A Regulates Dimerization Mol. Cell. Biol., February 1, 1999; 19(2): 1002 - 1015. [Abstract] [Full Text] [PDF]
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 M. A. Shupnik, L. K. Pitt, A. Y. Soh, A. Anderson, M. B. Lopes, and E. R. Laws Jr. Selective Expression of Estrogen Receptor {alpha} and {beta} Isoforms in Human Pituitary Tumors J. Clin. Endocrinol. Metab., November 1, 1998; 83(11): 3965 - 3972. [Abstract] [Full Text]
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T. Ördög, J. R. Goldsmith, M.-D. Chen, M. A. Connaughton, J. Hotchkiss, and E. Knobil On the Mechanism of the Positive Feedback Action of Estradiol on Luteinizing Hormone Secretion in the Rhesus Monkey J. Clin. Endocrinol. Metab., November 1, 1998; 83(11): 4047 - 4053. [Abstract] [Full Text]
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