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Endocrinology |
Robert H. Lurie Comprehensive Cancer Center [J. M. S., H. L., V. C. J.] and Department of Surgery [D. J. B.], Northwestern University Medical School, Chicago, Illinois 60611, and Signal Pharmaceuticals, San Diego, California 92121 [J. W. Z.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) and silences
activating function 2 (AF2) in the ligand binding domain, but
activating function 1 (AF1) at the other end of the ER remains
constitutive and is considered to be ligand independent. Amino acid
D351 in the ligand binding domain appears to be critical for
interactions with the antiestrogenic side chain of antiestrogens. We
have devised an assay to evaluate the biological activity of 351 mutant
ERs and antiestrogens at the transforming growth factor
(TGF) gene in situ (J. I. MacGregor
Schafer et al., Cancer Res., 59:
4308–4313, 1999). The substitution of glycine for aspartate at
position 351 results in the conversion of the 4-OHT:ER complex from
estrogen-like to completely antiestrogenic. In cells stably expressing
D351G ER, the ER retains responsiveness to estradiol (E2)
and also retains antiestrogenic responsiveness to both raloxifene and
ICI 182,780. The relative binding affinity of E2 for D351G
ER (0.77 ± 0.17 x 10-9
M) is comparable with wild-type ER (0.42 ± 0.08 x 10-9 M). In addition,
the D351G ER retains the ability to bind SRC-1 in the presence of
E2, thus D351G ER AF2 activity has not been compromised. We
also used a cell line stably expressing an ER with a triple mutation in
helix 12 (D538A, E542A, and D545A) that ablated AF2 activity, which
resulted in decreased effects of E2, suggesting that both
AF1 and AF2 activity are required for maximal estrogen activity in
MDA-MB-231 cells. Interestingly, the triple mutation also completely
reduced the estrogen-like actions of 4-OHT. We propose that a specific
mutation at amino acid 351 can allosterically silence AF1 in the
4-OHT:ER complex by either preventing the binding of coactivators or
encouraging the binding of a corepressor molecule. We suggest that the
4-OHT-specific site responsible for estrogen-like actions can be
referred to as AF2b. This binding site would consist of at least four
carboxylic acids at amino acids 351 and 538, 542 and 545 in helix 12 to
permit coactivator docking for gene activation. The AF2b site is
distinct from AF2 for E2 action. Further studies will
provide insight into the estrogen-like actions of tamoxifen in select
tissues and breast tumors and identify a significant mechanism of drug
resistance to tamoxifen. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The active metabolite of tamoxifen, 4-OHT (7, 8, 9) , is used in the laboratory as an antiestrogen to study the molecular actions of tamoxifen. The metabolite has a high binding affinity for the ER and competes with E2 for the LBD (7 , 9 , 10) . Two transcriptional domains, AF1 and AF2 (11) , have been identified in the ER to bind coactivators and initiate gene transcription. The binding of E2 to the ER causes activation of AF1 and AF2, but 4-OHT silences AF2 activity in the LBD (12, 13, 14) . It is hypothesized that AF1 activity is constitutive and ligand independent.
The recent resolution of the crystal structure of the ER LBD with Ral (15) and 4-OHT (16) has provided an invaluable insight into the activation of AF2 in ER by E2 and silencing by antiestrogens. An estrogen binds within the hydrophobic pocket of the LBD and is sealed inside by helix 12. This conformational change in the LBD activates AF2. In contrast, 4-OHT and Ral prevent helix 12 from sealing the binding pocket. The inappropriate repositioning of helix 12 prevents the binding of the coactivator GRIP1 and silences AF2 (16) .
Antiestrogens require an alkylaminoethoxy side chain to block estrogen
action (17)
. Early hypotheses proposed that the
antiestrogenic side chain must specifically interact with the LBD to
prevent sealing of the hydrophobic pocket (18
, 19)
. The
crystal structure of the Ral:ER complex demonstrates that the
antiestrogenic side chain interacts with aspartate at position 351 in
the ER (15)
, thereby suggesting the importance of this
site for antiestrogen action. The crystal structure of the 4-OHT:ER
complex shows the side chain having a loose interaction with amino acid
351 (Ref. 16
; Fig. 1
). By coincidence, a D351Y ER, isolated from a tamoxifen-stimulated
tumor line, had already been identified (20)
and found to
change the pharmacology of Ral from a complete antiestrogen to a
partial estrogen (21
, 22)
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gene in situ
(25)
. Surprisingly, the 4-OHT:ER complex is as effective
as the E2:ER complex at inducing mRNAs for TGF
(26)
. In contrast, Ral is a complete antiestrogen with the
wild-type receptor (22)
. We propose that the difference in
actions of 4-OHT and Ral results from distinct differences in the
crystallographic structure of the 4-OHT:ER complex and the Ral:ER
complex. The side chain of Ral interacts with D351, but the side chain
of 4-OHT is in a different position so that the interaction with D351
is tenuous (Fig. 1)
. Because we have demonstrated previously that a
natural substitution at D351 can enhance estrogenicity of an
antiestrogen receptor complex (21
, 22)
, we believed that
this amino acid position in the ER was significant for further study.
We have substituted aspartate with glycine at amino acid 351 in the ER
to determine the impact on the agonist activity of partial
antiestrogens by removing the charge and the side chain at position
351. Our hypothesis was that the receptor complex could lose the
estrogenic property that partial antiestrogens contain. We were
surprised to discover that a glycine substitution silences both AF1 and
AF2 when the efficacy of the 4-OHT:ER D351G is evaluated at the
TGF gene in situ. Antiestrogenic activity of
D351G is retained.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis.
The glycine mutation (pSG5D351GER) at amino acid 351 was introduced
using QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,
CA) using pSG5HEGO (a wild-type ER expression vector generously
provided by Professor Pierre Chambon, University of Strasbourg,
France) as the template. The primers constructed are as
follows: 5' primer (5'-GGGCTTACTGACCAACCTGGC
AGGCAGGGAGCTGGTTCAC-3'; the underlined nucleotide was
changed to make the D351G mutation) and a 3' primer
(5'-CCCGAATGACTGGTTG GACCGTCCGTCCCTCGACCAAGTG-3').
pSG5ER/G3m (D538A/E542A/D545A) expression vector was generated as
described above (28)
. The pair of primers used was:
5'-GCCCCTCTATGCCCTGCTGCTGGCGATGCTGGCCGCCCACCGCC-3'/5'-GGCGGTGGGCGGCCAGCATCGCCAGCAGCAGGGCATAGAGG-GGC-3'.
The point mutations are underlined. pGEX-
SRC-1 is an expression
vector for a GST fusion protein containing amino acids 759-1061 of
SRC-1 (29)
. The SRC-1 fragment with BamHI and
EcoRI sites was generated by PCR and inserted into pGEX-2TK
(Pharmacia). All sequences were confirmed by sequencing analysis (ABI
automated sequence and USB sequencing kit).
Stable Transfections.
MDA-MB-231 cells were stably transfected by electroporation. Cells
(5 x 106) were mixed with 10 µg
of pSG5D351GER or pSG5ER/G3m (an ER with an inactivating triple
mutation in AF2) mutant ER expression vector and 1 µg of
constitutive neomycin resistance expression vector (pBK-CMV) and were
electroporated (950 µF; 330V) using a Bio-Rad Gene Pulser II.
Briefly, after electroporation, cells were cultured in estrogen-free
media for 48 h to recover. Then, neomycin-resistant cells were
selected by using media containing 500 µg/ml G418. Colonies were
isolated and propagated in G418-containing media. Neomycin-resistant
clones were screened, identified, and characterized for ER expression
levels using both Northern and Western blot analysis.
Western Blot Analysis.
Cells were seeded into T-75 cm2 tissue culture
flasks and treated for 24 h with hormone or drug. At the
conclusion of the experiment, cells were trypsinized and pelleted. The
pellet was resuspended in protein extraction buffer [0.5% NP40, 2%
glycerol, 1 mM DTT, 1 mM EDTA, 150
mM NaCl, 50 mM Tris (pH 7.4), 1 mM
EGTA, 3 mM phenylmethylsulfonyl fluoride, 25 µg/ml
leupeptin, 9 µg/ml aprotinin, 25 µg/ml trypsin inhibitor, and 25
µg/ml t-chymotrypsin]. Samples were incubated on ice with
intermittent vortexing for 30 min and then pelleted. Supernatant was
then collected and stored at -80°C. Protein concentration was
measured using the Bio-Rad Protein Assay kit, and equal amounts of
protein were run in a standard Western blot protocol. The ER primary
antibody used was AER311 (Neomarkers, Fremont, CA), and ß-actin
antibody AC-15 (Sigma Chemical Co., St. Louis, MO) was used to
standardize loading. The appropriate secondary antibody conjugated with
horseradish peroxidase kit was used to visualize bands using an ECL
visualization kit (Amersham Corp., Arlington Heights, IL). The membrane
was wrapped in plastic wrap and exposed to Kodak X-OMAT film for
10 s to 1 h.
Northern Blot Analysis.
Analysis of TGF mRNA expression was assessed by Northern blots.
Briefly, total RNA was isolated from S30, JM-6, and G3m cells using the
TRIZOL reagent (Life Technologies, Inc.) after treatment with a range
of concentrations of E2, 4-OHT, Ral, and ICI
182,780 for 24 h. A human TGF cDNA probe derived by
EcoRI digestion of a TGF-containing plasmid was a
generous gift from Dr. R. Derynck, (Genentech, San Francisco, CA).
Bands were quantitated densitometrically using ImageQuaNT analysis
(Sunnyvale, CA). Comparisons in TGF induction relative to ß-actin
induction were analyzed by ANOVA. The two tailed t test was
used to analyze differences between treatments using StatMost 2.5
(Datamost Corp., Salt Lake, UT).
In Vitro Protein-Protein Interaction.
A GST-pull down assay was performed as described previously (30
, 31)
using 35S-labeled wild-type ER and
35S-labeled D351G ER, which were made from
pSG5HEGO and pSG5D351GER, respectively, using an in vitro
transcription-coupled translation system (Promega Corp., Madison, WI).
Ligand Binding Assay.
Ligand binding assays in stable transfectants were performed as
described previously (32)
. For saturation binding assays,
the stable transfectants were incubated with increasing concentrations
of [3H]E2 (Amersham
Corp., Arlington Heights, IL) for 2 h at room temperature. To
determine nonspecific binding, each concentration of
[3H]E2 was competed
with 400-fold excess of unlabeled DES. The specific binding was
obtained by subtracting the nonspecific binding from the total binding.
For competition binding assays, the stable transfectants were incubated
with 10-9 M
[3H]E2 with increasing
concentrations of different ligands including 4-OHT, Ral, or ICI
182,780 for 2 h at room temperature. The data were analyzed by
GraphPad Prism (GraphPad Prism Software, Inc., San Diego, CA).
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RESULTS |
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. In addition, to determine the effects of E2 and
antiestrogens on D351G ER expression, we treated S30 (Fig. 3A
) and JM-6 cells (Fig. 3B)
for 24 h and found that 4-OHT treatment has no effect on D351G ER expression.
Both E2 and ICI 182,780 reduced ER expression
(Fig. 3)
.
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. We found that the ligand affinity of wild-type ER compared with D351G
ER was not statistically different for E2
(Kd 0.42 ± 0.08 versus
0.77 ± 0.17 nM). Ral was the only
antiestrogen that had a significantly lower binding affinity for D351G
ER compared with the wild type (EC50
1.04 ± 0.22 versus 13.24 ± 1.45 nM; P < 0.05). From these
studies, it is also important to point out that the concentrations of
4-OHT used for the pharmacology studies saturated the binding sites
(Fig. 4)
.
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mRNA expression beginning at
10-10 M in JM-6 cells
(Fig. 5)
. These data are comparable with results published previously
(21)
with D351 ER in S30 cells.
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mRNA
induction, we first compared single concentrations
(10-7 M) of 4-OHT, Ral, and ICI
182,780 with E2 (10-9
M). In S30 cells, 4-OHT induced TGF transcription to the
same extent as E2 (Fig. 6A, B)
. In contrast, although E2 was
capable of inducing TGF in JM-6 cells, 4-OHT was not estrogenic, as
evidenced by a diminished ability to induce TGF gene transcription
(Fig. 6
C, D). In both S30 and JM-6 cells, neither Ral nor
ICI 182,780 induced TGF mRNA transcription. These data alone,
however, do not prove that 4-OHT, Ral, and ICI 182,780 are, in fact,
binding to the ER. This could be an explanation for the lack of agonist
activity. To address this question, we used combinations of
E2 (10-9
M) and antiestrogens (10-7
M) and determined whether the antiestrogen
blocked TGF mRNA induction (Fig. 7)
. We found that 4-OHT and ICI 182,780 continued to be antiestrogens
because they inhibit E2 induction of TGF. The
sum of the data from Figs. 3
and 4
and Table 1
demonstrate that
differences in ligand binding affinity or ER protein stability cannot
explain the difference in the intrinsic activity of 4-OHT between
wild-type and D351G ER. The substitution of glycine at position 351
silences transcriptional activity of the 4-OHT:ER complex.
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. Both 4-OHT and ICI 182,780 were effective
antiestrogens at 10-8 M;
however, a concentration of 10-7
M Ral was required to block
E2 action (Fig. 8D)
. This was
consistent with a reduced EC50 for Ral (Table 1)
.
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SRC-1 pull-down
experiments using 35S-labeled D351 ER and D351G
ER after treatment with vehicle, E2, 4-OHT, or
ICI 182,780 (Fig. 9
). After vehicle and E2 treatments, an
increase in binding to SRC-1 occurred compared with antiestrogen
treatments. Similar binding patterns were observed for D351 and D351G
ERs. A possible explanation for the SRC-1 binding in the vehicle lane
is that rabbit reticulocyte lysate was used that may contain
E2. This is supported by the fact that all
antiestrogens used prevented binding of SRC-1. These data support the
view that the AF2 activity is still functioning in the D351G ER, and
loss of 4-OHT agonist activity is not a result of the loss of AF2
function in the D351G ER as a whole.
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mRNA to compare S30 and JM-6 cells with G3m cells (Fig. 10)
. We treated these cells with vehicle, E2, or
4-OHT and measured TGF transcription and found that 4-OHT was unable
to activate TGF transcription through an ER with an inactivated AF2,
suggesting that an intact helix 12 is also necessary for the agonist
activity of 4-OHT. In addition, when treated with
E2, the TGF induction in G3m cells was
reduced, demonstrating that E2 requires both AF1
and AF2 for full activity; thus, the induction by
E2 was through the AF1 alone.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene
target in situ.
We have focused our attention on amino acid 351 because of the finding
that a natural mutation D351Y, isolated from a tamoxifen-stimulated
breast tumor (20)
, could convert Ral from an
antiestrogenic complex to an estrogenic complex at a TGF
gene target in the context of the MDA-MB-231 cells (21
, 22)
. Naturally, we wondered whether the reverse would be true,
i.e., by removing the charge from amino acid 351, could the
4-OHT:ER complex convert from being estrogen-like to antiestrogenic? If
so, we could dissect out differences between the promiscuous 4-OHT:ER
complex and the E2:ER complex.
In an early publication (25)
, we proposed an assay
that could distinguish between tamoxifen-like, Ral-like, and ICI
182,780-like compounds. The assay methodology compared and contrasted
the activation of the TGF gene in MDA-MB-231 cells
stably transfected with cDNAs from the wild-type and D351Y ER.
Unfortunately, it was not possible, at that time, to distinguish the
tamoxifen-like compounds from estrogens because both groups of
compounds fully activated the TGF gene target
(26)
with either wild-type or D351Y ER. With the
validation of the D351G assay system, we can now select estrogens,
tamoxifen-like, Ral-like, and ICI 182,780-like compounds by comparing
the wild type transfectant with the D351Y and D351G transfectants.
Although we believe that the application of our observation will help
to identify new SERMs for clinical applications, we suggest that our
finding provides an important new tool to identify novel coactivators
and corepressors of ER function. Identification of a molecular
mechanism to explain the promiscuous estrogenic actions of the 4-OHT:ER
complex could potentially provide one of perhaps many reasons for the
target site-specific actions of SERMs. Additionally, if a molecular
mechanism for the estrogen-like action of tamoxifen at the ER could be
established, then perhaps this knowledge could be used to counter the
development of tamoxifen-stimulated breast cancer growth as a form of
drug resistance.
Previous studies (12 , 13) suggest that the agonist actions of 4-OHT may be a function of the target tissue as well as the promoter structure of the target gene. For convenience, several groups have used yeast cells to conduct transient transfection experiments (10 , 33) , but sometimes variable and inconsistent results are reported. An expression system that uses a yeast copper metallothionine promoter and ubiquitin fusion technology to express ER in yeast was unable to show that antiestrogens could block E2 action at cotransfected ß-galactosidase reporter (34) . However, the antiestrogen action alone did not produce a full estrogen-like response; therefore, the explanation for the negative findings is unclear. In contrast, Wrenn and Katzenellenbogen (35) noted that 4-OHT was a full agonist using a ß-galactosidase reporter gene; however, the ER used for these pharmacology studies (34 , 35) was the G400V ER, which is known to be unstable (33) . G400V ER has a reduced affinity for E2 compared with wild-type ER and an enhanced estrogenicity with 4-OHT in stable transfectants of MDA-MB-231 cells (36) . Paradoxically, 4-OHT is an antiestrogen with no estrogen-like properties when G400V is transiently transfected into Chinese hamster ovary cells using a catechol acetyl transferase reporter gene (35) .
To establish assays of potential physiological relevance, the C3 promoter system has been ligated to a luciferase reporter gene. Importantly, the complex promoter has enhanced estrogen-like properties in the context of human hepatocellular carcinoma (HepG2) cells cotransfected with wild-type ER. The promoter system is physiologically relevant because E2 stimulated expression of the complement component in luminal epithelial cells of the rat uterus, but expression is not prevented by the coadministration of a number of antiestrogens including tamoxifen (37) . In fact, the triphenylethylene type antiestrogens stimulates C3 mRNA (37) , although the pure antiestrogen ICI 164,384 could block both E2 and 4-OHT-stimulated C3 (38) .
We initially used transient transfection of ER cDNAs into ER negative
breast cancer cell lines and used a simple ERE luciferase reporter
system to study ligand activity. Results were variable, and 4-OHT was
not a full agonist, even when MDA-MB-231 cells were used (data not
shown). It is clear that the agonist actions of a SERM:ER complex can
only become apparent in the correct cellular context if a complex gene
promoter such as C3 is used (13)
. We have taken another
approach by studying ER actions at a TGF gene target
in vitro in the context of an MDA-MB-231 breast cancer cell
(23)
. We reasoned that these cells would be replete with
relevant transcription factors to regulate the TGF gene,
a known growth factor involved in cell proliferation. As a result, we
stably transfected MDA-MB-231 breast cancer cells with cDNAs from D351G
to compare and contrast with our established cell lines
(23)
.
The wild-type and D351G ER have similar affinities for
E2, and there is a concentration-related
inhibition of [3H]E2 with
antiestrogens. However, the fact that the stable transfectant, JM-6,
with D351G ER had no agonist actions with 4-OHT compared with wild-type
ER mandated that the stability of the ER was determined. Earlier
studies with G400V ER demonstrated that the mutant receptor was turned
over rapidly compared with the wild-type ER (33)
;
therefore, it was possible that rapid receptor destruction was
responsible for the loss of agonist activity for 4-OHT. This approach
was particularly important because the pure antiestrogen ICI 182,780 is
known to facilitate the rapid destruction of ER
(39)
. As a result, ICI 182,780 probably exerts its pure
antiestrogenic activity by producing both an inappropriate shape for
the receptor complex and rapid destruction of complexes. However, we
show the D351G is not destroyed (Fig. 3)
by 4-OHT; therefore, this is
unlikely to be the explanation for a decrease in the estrogen-like
properties of the complex. A D351V mutation has been analyzed
previously in the context of yeast cells (35)
. The D351V
mutant was isolated as one of a series of mutations generated randomly
by formic acid treatment (35)
. Interestingly, whereas
4-OHT was a full agonist with wild-type ER, the D351V ER did not
possess estrogen-like actions. Although the ligand binding affinity or
the antiestrogenic activity was not determined in yeast, D351V ER was
antiestrogenic with 4-OHT in Chinese hamster ovary cells.
We have noted previously that the antiestrogens EM-652
(25)
and Ral (22)
silence both AF1 and AF2 in
the wild-type ER in the context of the MDA-MB-231 cells. It is,
therefore, not inconsistent that we now find that the mutation D351G in
the LBD can silence AF1 and AF2 activity with 4-OHT. The allosteric
interaction of AF2 with AF1 was further investigated using an ER with
an inactivating triple mutation in AF2 (G3m) stably transfected into
MDA-MB-231 cells. We show that this mutation, which changes three amino
acids that are carboxylic acids to uncharged alanines, also resulted in
the ablation of 4-OHT agonist activity. This led us to deduce that both
aspartate at 351 and an intact helix 12 are required for the partial
agonist activity of 4-OHT (Fig. 10)
. This result with helix 12
mutations is consistent with previous results using HepG2 cells and a
C3 promoter (13)
. In addition, the agonist activity of
E2 in the G3m cells is reduced compared with
D351G, suggesting that classical AF2 activity maybe affected in JM-6
cells when E2 is a binding ligand (Fig. 10)
.
We thought that it is important to compare and contrast the D351 and
D351G ERs to be confident that the silencing of AF1 with 4-OHT in D351G
is really the result of the single amino acid change in the LBD. We
have shown that the binding characteristics for
E2, 4-OHT, and ICI 182,780 are similar, but
we took the comparison one step further. We show, using GST-pull down
experiments, that D351G ER is still capable of binding the coactivator
SRC-1 when bound to E2 but not when ligated with
4-OHT (Fig. 9)
. This provides evidence that AF2 is still functional in
JM-6 cells for E2 action but is silenced for
4-OHT. For the future, the important goal is to discover the precise
relationship of the AF1 and AF2 sites in the ER to identify site
interactions and coactivator clusters. Unfortunately, the
crystallographic structure is unhelpful, because only the LBD minus the
F region has been crystallized (15
, 16)
. At present, it is
also not possible to perform pull-down assays with the whole ER. A
method is required to investigate the interaction of coactivators and
corepressors with the whole receptor to isolate novel proteins.
Nevertheless, on the basis of our experimental evidence, we believe it
is appropriate to propose a model of ER pharmacology as a framework for
further study.
Recently, Norris et al. (27) have identified peptides that can distinguish between E2:ER complexes and 4-OHT:ER complexes. Different peptides can block E2 and 4-OHT agonist activities, thereby, demonstrating that agonism occurs by different mechanisms at different surfaces. In other words, coactivator binding could be ER complex specific. The repositioning of helix 12 to seal E2 into the LBD enables coactivators such as GRIP1 to bind in the AF2 domain (16) . The blocking of this site by helix 12 with either 4-OHT (16) or Ral (15) as the binding ligand raises the issue of a second site that could bind a coactivator molecule. It is interesting to speculate that coactivator binding for 4-OHT:ER complexes could involve a point attachment in the region 300–351 with AF1. The region 282–351 has been noted previously by Norris et al. (40) to be a potential transactivating site on ER that they have termed AF2a.
The antiestrogens 4-OHT and Ral have a side chain that interacts with
aspartate 351 (Fig. 1)
. However, the interaction of the tertiary
nitrogen of 4-OHT with the carboxylic acid is tenuous at best; thus, we
propose that unlike Ral, the charge on the aspartate 351 is not
neutralized. Recently, we have resolved the space-filling structure of
the ER dimer with either DES or 4-OHT as the binding ligand (Fig. 11)
. We have identified aspartate 351 as a surface amino acid that can
potentially interact with other coactivator molecules. Fig. 11
illustrates the profound difference on the external surface of the
DES:ER or 4-OHT:ER complex. We suggest that the difference in
the positions of the side chain of 4-OHT and Ral results in the
creation of a potential docking site for coactivators for the 4-OHT:ER
complex. Although it is unlikely that this is the only docking site,
which may also include the AF1 site and helix 12, we suggest that the
loss of charge at aspartate 351 by the substitution of glycine is
critical for the loss of estrogenicity in the 4-OHT complex. We are
currently addressing the structure-function relationships of this novel
target on the surface of the ER.
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FOOTNOTES |
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1 Supported in part by Department of Defense
Breast Cancer Training Grants DAMD 17-94-J-4466, DAMD 17-96-1-6169, and
Breast Cancer Program Development Grants P20 CA65764 and RO1-CA56143.
We are deeply grateful for support from the Lynn Sage Breast Cancer
Research Foundation of Northwestern Memorial Hospital and the Avon
Breast Cancer Research Fund. 
2 To whom requests for reprints should be
addressed, at Northwestern University Medical School, Robert H. Lurie
Comprehensive Cancer Center, 303 East Chicago Avenue, 8258 Olson
Pavilion, Chicago, IL 60611. Fax: (312) 908-1372.
3 The abbreviations used are: ER, estrogen
receptor ; 4-OHT, 4-hydroxytamoxifen; Ral, raloxifene; TGF,
transforming growth factor; SERM, selective estrogen receptor
modulator; AF1, activating function 1; AF2, activating function 2; LBD,
ligand binding domain; EC50, effective 50% inhibitory
concentration; GST, glutathione S-transferase; DES,
diethylstilbestrol.
Received 11/ 3/99. Accepted 7/25/00.
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REFERENCES |
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mRNA in MDA-MB-231 breast cancer cells stably expressing the oestrogen receptor. Br. J. Cancer, 77: 1812-1819, 1998.[Medline]
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 A. Ring and M. Dowsett Mechanisms of tamoxifen resistance Endocr. Relat. Cancer, December 1, 2004; 11(4): 643 - 658. [Abstract] [Full Text] [PDF]
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 M. H. Herynk and S. A. W. Fuqua Estrogen Receptor Mutations in Human Disease Endocr. Rev., December 1, 2004; 25(6): 869 - 898. [Abstract] [Full Text] [PDF]
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 C. Osipo, H. Liu, K. Meeke, and V. C. Jordan The Consequences of Exhaustive Antiestrogen Therapy in Breast Cancer: Estrogen-Induced Tumor Cell Death Experimental Biology and Medicine, September 1, 2004; 229(8): 722 - 731. [Abstract] [Full Text] [PDF]
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 S. T. Pearce, H. Liu, I. Radhakrishnan, M. Abdelrahim, S. Safe, and V. C. Jordan Interaction of the Aryl Hydrocarbon Receptor Ligand 6-Methyl-1,3,8-trichlorodibenzofuran with Estrogen Receptor {alpha} Cancer Res., April 15, 2004; 64(8): 2889 - 2897. [Abstract] [Full Text] [PDF]
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H. Liu, E.-S. Lee, C. Gajdos, S. T. Pearce, B. Chen, C. Osipo, J. Loweth, K. McKian, A. De Los Reyes, L. Wing, et al. Apoptotic Action of 17{beta}-Estradiol in Raloxifene-Resistant MCF-7 Cells In Vitro and In Vivo J Natl Cancer Inst, November 5, 2003; 95(21): 1586 - 1597. [Abstract] [Full Text] [PDF]
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D. Bentrem, J. E. Fox, S. T. Pearce, H. Liu, S. Pappas, D. Kupfer, J. W. Zapf, and V. C. Jordan Distinct Molecular Conformations of the Estrogen Receptor {alpha} Complex Exploited by Environmental Estrogens Cancer Res., November 1, 2003; 63(21): 7490 - 7496. [Abstract] [Full Text] [PDF]
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 E. V. Jensen and V. C. Jordan The Estrogen Receptor: A Model for Molecular Medicine Clin. Cancer Res., June 1, 2003; 9(6): 1980 - 1989. [Abstract] [Full Text] [PDF]
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 S. K. Mishra, A. Mazumdar, R. K. Vadlamudi, F. Li, R.-A. Wang, W. Yu, V. C. Jordan, R. J. Santen, and R. Kumar MICoA, a Novel Metastasis-associated Protein 1 (MTA1) Interacting Protein Coactivator, Regulates Estrogen Receptor-{alpha} Tr |
