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Glaxo Wellcome Research and Development (T.M.W., P.B., H.R.B., S.A.J., B.H., H.S., S.W., D.M.), Research Triangle Park, North Carolina 27709; and the Department of Pharmacology and Cancer Biology, Duke University Medical School (J.D.N., B.L.W., I.A., D.P.M.), Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Dr. D. P. McDonnell, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical School, Durham, North Carolina 27710. E-mail: McDon016{at}acpub.duke.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using an in vitro approach, we and others have determined the likely mechanism for the cell-selective agonist/antagonist activity of tamoxifen (3, 4, 5, 6). Importantly, it was shown that tamoxifen induces a conformational change within the ER distinct from that induced by estradiol (5, 16). Furthermore, determination of the sequences within ER required for transcriptional activity indicated how these specific ligand-receptor complexes were differentially recognized by the cellular transcriptional machinery. Specifically, it was shown that ER contains two activation domains, AF-1 (activation function-1) and AF-2, that permit its interaction with the transcription apparatus. The relative contributions of these AFs to overall ER efficacy differ from cell to cell (3, 5, 6). Estradiol was determined to function as both an AF-1 and an AF-2 agonist, in that it exhibited maximal activity regardless of which AF was dominant in a given cellular environment. Tamoxifen, on the other hand, functions as an AF-2 antagonist, inhibiting ER activity in cells in which AF-2 is required or is the dominant activator (3, 5, 6). Conversely, tamoxifen functions as an agonist when AF-1 alone is required (5, 6). Subsequently, based on their relative AF-1/AF-2 activities, we were able to define four mechanistically distinct groups of ER modulators: full agonists (i.e. estradiol), two distinct classes of partial agonists represented by tamoxifen and raloxifene, and the pure antagonists, of which ICI182,780 is a representative member (5, 6). These results provide a mechanistic explanation for the observed differences in the biological activities of some ER modulators and indicate that the mechanisms by which ER operates in different tissues were not identical. Interestingly, the agonist activity exhibited by ER modulators, such as estrogen and tamoxifen, in these in vitro systems reflected their activities in the reproductive tracts of whole animals. This correlation did not extend to bone, however, where estradiol, tamoxifen, and raloxifene, which display different degrees of AF-1/AF-2 agonist activities, all effectively protected against bone loss in the ovariectomized (OVX) rat model. Thus, with the exception of the steroidal pure antiestrogens (i.e. ICI182,780), all known classes of ER modulators appear to protect against bone loss in humans and relevant animal models, whereas they display different degrees of estrogenic activity in other tissues (8, 13, 17, 18, 19). It appears, therefore, that there is some unique feature of these compounds that alone is sufficient for bone protection, but insufficient for activity in other cells. This suggests that ER is used in different ways in various target cells, and that dissection of the underlying pathways responsible for this activity may permit the development of tissue selective ER modulators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and cotransfection assays
HepG2 cells were maintained in MEM (Life Technologies, Grand
Island, NY) plus 10% FCS (Life Technologies). Cells were plated in
24-well plates (coated with gelatin) 24 h before transfection. DNA
was introduced into the cells using Lipofectin (Life Technologies).
Briefly, triplicate transfections were performed using 3 µg total
DNA. For standard transfections, 500 ng pCMV-ßGAL (normalization
vector), 1500 ng reporter (variable), and 1000 ng receptor (pRST7-hER)
(21) were used for each triplicate. Incubation of the cells with
Lipofectin proceeded for 3 h, at which time medium was removed,
and cells were washed with PBS and then induced with the appropriate
hormone diluted in phenol red-free medium containing 10%
charcoal-stripped calf serum (Hyclone, Logan, UT). Incubation with
hormone continued for 48 h, after which cells were lysed and
assayed for luciferase and ß-galactosidase activity as previously
described (22).
Uterotropic assay in immature rats
Twenty-one-day-old female Sprague-Dawley rats (30–35 g) were
obtained from Taconic Laboratories (Germantown, NY). Animals were
sorted randomly into treatment groups of five, and average weights were
recorded for each treatment group. Weights were recorded on each
treatment day. GW5638 or tamoxifen was prepared in 100% ethanol as a
10-fold stock solution and stored at -70 C until the day of treatment.
On the treatment day, drug was diluted in 0.5% methyl-cellulose, with
a viscosity of 2% at 25 C (400 centipoises; Sigma). Oral treatment by
gavage was based on a total volume of 10 ml/kg BW. Estradiol (Sigma)
was prepared in sesame oil, mixed in a glass homogenizer (either
dissolved or in suspension), aliquoted, and frozen at -70 C until
administration. Subcutaneous treatment was based on a total of 2 ml/kg
BW. Animals were gavaged (GW5638) or injected (estradiol) for 3 days.
On day 4, animals were killed by CO2 asphyxiation, body
weights were obtained, and uteri were removed, blotted, and weighed.
Data were expressed as uterine weight per BW.
Bone mineral density (BMD) studies
Animal preparation.
Sprague-Dawley rats, 90 days of age,
were anesthetized with isofluorane (4% induction, 2% maintenance),
OVX or sham operated, and randomly assigned to groups (n = 7)
treated from days 1–28 postsurgery by oral gavage with vehicle alone,
estradiol, or GW5638 in 0.5% methyl-cellulose. At death, animals were
killed with CO2, body weights were recorded, and uteri were
removed and weighed. Uterus, vagina, and mammary tissue were fixed in
10% neutral buffered formalin. Samples for histological processing
were taken from the midpoint of each uterine horn. Tissue samples were
embedded in paraffin, stained with hematoxylin and eosin, and evaluated
microscopically by a board-certified veterinary pathologist. Lumbar
vertebrae (L1–L4) and both left and right tibiae were excised. Total
blood cholesterol was measured (Roche Biomedical Laboratories, Palo
Alto, CA).
Dual energy x-ray absorptiometry.
A Hologic QDR-2000 bone
densitometer with a regional high resolution software package was used
for dual energy x-ray absorptiometry. Default scan length, width, line
spacing, and point resolution were set at 2, 0.75, 0.01, and 0.005 in.,
respectively. The densitometer was calibrated daily using a
hydroxyapatite spine platform. Excised tibiae were placed in a 1-cm
deep water bath with tibia and fibula positioned horizontally. For
in vivo scans, rats were anesthetized with isofluorane and
placed in a supine position with the spine parallel to the long axis of
the densitometer table. The scan leg was taped in position parallel to
the long axis of the table, and the tibia was scanned to the junction
with the femur. A region of interest in the tibia was analyzed with
subregional software, focusing on a 2-mm wide zone beginning 3 mm
distal to the growth plate.
Peripheral quantitative computed tomography (QCT).
Computed
tomography scans were performed on a peripheral QCT (XCT-960A, Norland,
White Plains, NY). Four- to 5-mm sections were scanned with a voxel
size of E (0.148 mm) and a step of 0.5 mm. A 3- to 5-mm section distal
to the growth plate was analyzed using contmode, 2/peelmode,
5/cortmode. Measurements of total, trabecular, and cortical BMDs were
obtained. The excised tibiae were placed in a 1-cm deep water bath with
the tibia and fibula positioned horizontally to ensure that the bone
could be scanned vertically. Rats were anesthetized with isofluorane,
and the leg was positioned so that the image of the femur-tibia and
tibia-fibula junctions on scout view could be located and used as
landmarks for computed tomography scans.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently synthesized a series of novel
triphenylethylene-derived ER ligands (20). Preliminary analysis of
these compounds in vivo indicated that the relative
activities of these compounds in bone and uterus were not identical,
reflecting possible mechanistic differences (20). Consequently, we
performed a blinded assay of these compounds on ERwt in HepG2 cells on
the C3 promoter and determined that all but two compounds were
mechanistically indistinguishable from tamoxifen (Norris, J. D., and D.
P. McDonnell, unpublished results). Two compounds, however, GW5638 and
GW7604, demonstrated a sufficiently different profile in this system
from other ER ligands to warrant further investigation. Interestingly,
these compounds are structurally identical to each other, except that
GW7604 is the 4-hydroxylated version of GW5638 (Table 1
). Using an in vitro competitive
radioligand binding assay, we demonstrated that both of these compounds
exhibit high affinity ER interactions. Specifically, GW5638 and GW7604
demonstrated Ki values of 50.4 ± 5.4 and 15.5 ±
1.4 nM, respectively. Under the same conditions
17ß-estradiol was shown to have a Ki value of 6.3 ±
0.4 nM. Although we have not studied the metabolism of
GW5638, it is likely that it is converted to the higher affinity
compound GW7604 in vivo in the same manner as tamoxifen is
converted to the higher affinity metabolite 4-hydroxytamoxifen (23). A
comparison of the agonist activity of these compounds to representative
members of each of the four established groups of ER ligands is shown
in Fig. 1A. In this assay, tamoxifen acts as a partial
agonist of ER when assayed on the C3 promoter, achieving 45% the
efficacy of estradiol. When analyzed in the same manner, raloxifene and
the pure antagonist ICI182,780 do not demonstrate agonist
activity, but inhibit the basal transcriptional activity of
the C3 promoter. Recently, we have determined that the
basal activity of the C3 promoter is ER dependent, although ligand
independent (24). As both raloxifene and ICI182,780 inhibit
ligand-dependent and -independent activation of ER, they appear to be
operating as inverse agonists in this environment. However, both GW5638
and its putative metabolite GW7604 do not demonstrate any agonist or
antagonist activity on this promoter, displaying a fingerprint
previously unrecognized. We concluded that in an environment in which
tamoxifen displays partial agonist activity, the tamoxifen analogs
GW5638 and GW7604 are functionally inactive.
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). In this assay ICI182,780, GW 5638, and GW7604
are functionally inactive. These data indicate that GW5638 (and
GW7604), function in a manner distinct from that of the previously
defined classes of ER mixed agonists and antagonists (5). One possible,
although unlikely, explanation for these initial results is that the
compounds were metabolized (or in some way prevented from binding to
the receptor), thus explaining their inactivity in this assay. This
issue was addressed by assessing the abilities of GW5638 and GW7604 to
inhibit the agonist activity of estradiol and tamoxifen exhibited on
ERwt and ER-TAF1, respectively, and to reverse the inverse agonist
activity of ICI182,780. As shown in Fig. 2A, estradiol
functioned as a full agonist, and tamoxifen functioned as a partial
agonist on ERwt when assayed on the C3 promoter in HepG2 cells.
Importantly, the agonist activity manifested by either tamoxifen or
estradiol was inhibited by both GW7604 and GW5638. Thus, these
compounds were functioning as antagonists on the receptor in a manner
distinct from that of tamoxifen. We performed a similar analysis using
ER-TAF1 in place of ERwt (Fig. 2B). As expected, both GW5638 and GW7604
were capable of inhibiting estradiol- and tamoxifen-induced ER-TAF1
transcriptional activity. Interestingly, raloxifene exhibited partial
agonist activity on ER-TAF1 (Fig. 1B), an activity that was inhibited
by both GW5638 and GW7604 (Norris, J. D., and D. P. McDonnell,
unpublished results). Cumulatively, the data presented to date indicate
that GW5638 and its putative in vivo metabolite, GW7604, are
mechanistically unique ER modulators that do not display agonist
activity in vitro, but can inhibit the agonist activity of
estradiol, tamoxifen, and raloxifene. Although their profiles in some
of these assays resemble that of the pure antagonist class of ligands,
these compounds are distinct from steroidal antagonists such as
ICI182,780 as they do not display inverse agonist activity (Fig. 1A).
To confirm that GW5638 and GW7604 were mechanistically distinct from
ICI182,780, we measured the ability of these compounds to reverse the
inverse agonist activity demonstrated by ICI182,780. The results of
this analysis are shown in Fig. 2C. Specifically, we observed that the
basal activity of the human C3 promoter was suppressed 10-fold upon the
addition of ICI182,780 and that this could be completely reversed by
coaddition of GW7604 and partially reversed by GW5638. One possible
explanation for the mechanistic differences observed is that GW5638 and
GW7604 interact with ER and inhibit its ability to interact with DNA.
This was addressed using a modified ER (ER-VP16) to report on the
nuclear localization and the DNA binding status of ER within a cell
after ligand binding. This modified protein behaves exactly like ER in
all respects except that it activates transcription upon interaction
with an estrogen response element independent of the nature of the
bound ligand (5). Using this reagent we have shown that all classes of
ER ligands, including ICI182,780, GW5638, and GW7604, facilitate
efficient interactions of ER with target DNA (5) (Asplin, I., and D. P.
McDonnell, unpublished results). Thus, we conclude that GW5638 and
GW7604 interact with ER in vivo and demonstrate a
pharmacology distinct from that of other known ER modulators. It is
implied, therefore, that the unique properties of GW5638 and GW7604 are
manifest at some step downstream of DNA binding. Because of the unique
properties of these compounds, a series of whole animal studies to
examine their activities in the skeletal, cardiovascular, and
reproductive systems was initiated.
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indicate that significant bone
loss occurred in the lumbar spines of OVX animals over the course of
the 28-day study compared with that in control sham-operated animals,
whereas BMD was maintained in OVX rats treated with estradiol.
Significantly, GW5638 demonstrated dose-dependent bone protective
activity, being as effective as estradiol at a concentration of 3
µmol/kg (1 mg/kg). This is similar to the dose of tamoxifen required
for bone protection in the same model (13, 19, 28). The bone protective
activity observed was not restricted to the lumbar spine, as analogous
results were obtained when tibial BMD was accessed (Fig. 3B).
Specifically, using the same experimental protocol, it was demonstrated
that GW5638 was effective at maintaining total bone mass, with a very
pronounced effect in the trabecular compartment. This was interesting
in light of the fact that estrogen has previously been shown to
regulate bone turnover in this compartment (27). Together, these data
suggest that GW5638, a novel compound devoid of classical ER agonist
activity when assayed in vitro, functions as an efficient ER
agonist in bone.
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indicated that even at the lowest concentration tested, GW5638 was as
effective as estradiol in decreasing serum cholesterol levels.
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). When
administered orally, as a single agent, GW5638 did not display any
significant activity over the control value. Note in particular that
this compound was inactive in this assay at 10 µmol/kg, 3 times the
amount required for bone protection (Fig. 3). In contrast, tamoxifen
demonstrated uterotropic activity at doses as low as 0.1 µmol/kg. We
extended these studies to show that GW5638, but not tamoxifen, could
completely inhibit the agonist activity of estradiol in these rats,
confirming that this compound is a pure antagonist in this tissue under
the conditions of the assay.
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, indicate that at doses up to 3 times that required
for bone protection, GW5638 displays minimal uterotropic activity.
Importantly, however, we did not notice any significant differences in
the total body weight of OVX rats treated with GW5638 vs.
that of sham-operated animals. Interestingly, we did observe a very
small, dose-independent, increase in uterine wet weight after OVX. This
is similar to what has been reported by others in rats treated with
raloxifene, in which the activity has been attributed to an increase in
water imbibition (12, 14, 19). In addition to measurements of uterine
wet weight, we performed a histological examination of the uteri
harvested from the same animals [Fig. 7, A–F (low
magnification), and Fig. 8, A–D (high magnification)]. In this
analysis we observed the uterine epithelial cells in rats treated with
GW5638 exhibited a dose-related hypertrophy, whereas the stroma
demonstrated a marginal increase in intercellular connective tissue and
ground substance. At the highest doses of GW5638 (3-fold higher than
that required for bone protection), the epithelial hypertrophy observed
was comparable to that of estradiol-treated uteri, whereas the stromal
response and eosinophilic infiltration were less than those observed in
estradiol-treated rats (compare Fig. 8, C and D).
Cumulatively, these data indicate that GW5638 possesses marginal ER
agonist activity in the uterus, whereas in bone it functions as an ER
agonist. Thus, GW5638 is a unique ER modulator that manifests ER
agonist and antagonist activity in a tissue-selective manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of process-selective compounds in other systems has been accomplished by targeting specific isoforms of receptors or enzymes that are expressed in a tissue-restricted manner. This approach is unlikely to succeed for ER, as it appears as though most of the actions of estrogen are manifest through a unique receptor. Although a second ER (ERß) has been identified in rat prostate, it is unclear at this stage if it plays a role in estrogen endocrinology (35). The challenge, therefore, is to develop selective estrogen receptor modulators that possess tissue-specific ER agonist activity(s). This would not appear possible within the confines of classical receptor theory, where ligand functions merely as a switch converting a receptor from an inactive to an active state (7). An extension of this tenet is that the biological activity of a compound reflects only its receptor binding affinity and its associated pharmacokinetic properties (7). However, data presented here and in other studies indicate clearly that classical receptor theory is not sufficient to explain the activity of the known ER ligands (36, 37). Although this divergence was first observed in studies of the nonsteroidal ER ligands, such as tamoxifen, it has been extended to other modulators, implying that the manner by which the ER-ligand complex is recognized or used is not identical in all cells (36, 37, 38). These results and those of others firmly establish that different ligands acting through the same receptor can manifest different biologies in different cells (5, 38, 39, 40). The results of this study show that GW5638, a compound that is mechanistically distinct from known ER modulators, functions as an agonist in bone and in the cardiovascular system, but not in the uterus, further solidifying this concept.
The role of ER in bone and the properties of an ER modulator required for bone protection are unclear. The results of previous studies and those presented here indicate that several classes of ER ligands can protect against bone loss in OVX rats (8, 13, 17, 19, 20). However, the property that these compounds have in common and that is responsible for their bone protective activity remains elusive. We do know that the interaction of the latent receptor with any high affinity ER ligand induces a structural alteration in the receptor that promotes its dissociation from inhibitory heat shock proteins (5, 16). However, as the pure antagonist ICI164,384 effectively activates ER (5), but does not protect against bone loss, it appears as if some process downstream of activation is also required for ER activity in this tissue (Baer, P., T. M. Willson, and D. Morris, unpublished results) (27). In this signal transduction pathway, after estrogen binding, ER undergoes spontaneous dimerization, permitting the receptor to interact with DNA in a high affinity manner. It may be at this stage where divergence occurs between those ER modulators that protect against bone loss and those that do not. Although remaining controversial, it appears as if the pure steroidal antiestrogens, such as ICI164,384 and ICI182,780, interfere with the interaction of ER with DNA (41). The mechanism of this inhibitory activity is complex, involving a combination of changes in nuclear-cytoplasmic shuttling of ER, impaired receptor dimerization, and acceleration of the rate of ER turnover (25, 26, 41). Clearly, however, some ICI182,780-activated ER associates with its target DNA in vivo. In contrast, all other classes of ER ligands, including GW5638 (Asplin. I., and D. P. McDonnell, unpublished results), efficiently deliver ER to DNA and do not induce turnover of the receptor. Thus, the ability to activate ER and deliver the receptor to DNA is an activity that correlates well with bone protection. It is also worth noting that all ER ligands examined to date that protect against bone loss are also effective in suppressing ovariectomy-induced increases in serum cholesterol. The fact that four distinct classes of ER modulators act as agonists in both systems implies that the mechanism by which ER acts in bone and the cardiovascular system is biochemically linked.
Considering the data presented in this study and previously, we propose
the following models to explain the mechanism of action of ER in bone.
I) Although GW5638 does not display ER agonist activity in our
reconstituted in vitro transcription systems under
conditions where tamoxifen, raloxifene, and estrogen are agonists, it
is possible that on targets relevant to bone protection GW5638 is an
agonist. II) Alternatively, the mechanism by which ER acts in bone may
be different from that in classical ER targets, such that the
definition of agonist must be considered only in reference to the
target cell or a specific process. Thus, if the role of estradiol
within a target cell in bone is to deliver ER to DNA and in some way
alter the transcription rate of a target gene(s) by disrupting the
activity of a negative regulator, then any agent that delivers ER to
DNA would be an agonist. It is worth noting that the only class of ER
modulators examined to date that does not appear to protect against
bone loss in OVX rats is the steroidal pure antiestrogens
(i.e. ICI182,780), compounds that do not efficiently promote
ER-DNA interactions (41). III) A third model for which there is a
considerable amount of supporting preclinical data is the proposal that
activated ER can inhibit the actions of a positive regulator of a gene,
an event requiring activated receptor but not DNA binding. One example
of this activity is regulation of the cytokine interleukin-6 (IL-6)
(42). It has been shown recently that in OVX mice, serum IL-6 levels
rise, leading to an increase in the relative number of osteoclasts to
osteoblasts and a subsequent loss of bone mass (42). The link between
this activity and bone was established when it was shown 1) that
administration of neutralizing antibodies to IL-6 was sufficient to
inhibit osteoclast formation in primary bone cell cultures derived from
neonatal murine calvarie (43), and 2) that mice bearing a genetic
deletion of the IL-6 gene did not demonstrate ovariectomy-induced bone
loss (44). Although other events in addition to IL-6 up-regulation are
likely to be involved in osteoporosis, the observation that the IL-6
promoter is down-regulated upon administration of estrogen makes it a
useful system with which to study ER action. Recently, the molecular
basis of ER regulation of the IL-6 promoter has been determined.
Specifically, it has been shown that both nuclear factor-
B and
C/EBPß are required for IL-6 promoter activity (45, 46, 47).
Interestingly, ER, in the presence of estradiol, physically interacts
with both the rel homology domain of nuclear factor-B and
the b-Zip region of C/EBPß, blocking their ability to interact with
DNA and subsequently lowering the transcriptional rate of the IL-6
promoter. Mutations within ER that block DNA binding do not affect the
ability of the receptor to block IL-6 production (46). Although the
activity of tamoxifen, raloxifene, or GW5638 has not been tested in
this system, it is likely that they would manifest activities similar
to that of estradiol because they activate ER without inducing receptor
turnover (46). Given what we know about the mechanism of the bone
protective ER modulators and considering the data on IL-6 regulation,
it is possible that classical ER agonist activity is not required for
bone protection. Regardless of which of these models is correct, the
underlying observation is that the role of ER is not identical in all
targets. Thus, by targeting specific activities of ER, tissue (or
process)-specific agonists can be developed.
The identification of GW5638, a compound mechanistically distinct from tamoxifen, suggests that it may have utility in the treatment of de novo and acquired resistance to tamoxifen observed in ER-positive breast tumors (48, 49). In support of this hypothesis, we have shown that this compound, like the steroidal pure antiestrogen ICI182,780, will inhibit the partial agonist activity of tamoxifen and raloxifene in vitro. Additionally, as this compound has less uterotropic activity than tamoxifen, it may be useful as a breast cancer chemopreventative. From a more general perspective, the work presented here and data from other supporting studies clearly demonstrate that ER transcriptional activity is affected by both the nature of the bound ligand and the environment in which the receptor operates, providing a mechanistic basis for the tissue-selective actions of different ER modulators.
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Acknowledgments |
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Footnotes |
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Received December 31, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-substituted
analogs of the antiprogestin RU486 induce a unique conformation in the
human progesterone receptor resulting in mixed agonist activity. Proc
Natl Acad Sci USA 93:8739–8744This article has been cited by other articles:
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  B. M. Wittmann, A. Sherk, and D. P. McDonnell Definition of Functionally Important Mechanistic Differences among Selective Estrogen Receptor Down-regulators Cancer Res., October 1, 2007; 67(19): 9549 - 9560. [Abstract] [Full Text] [PDF]
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 J. H. Kim, M. H. Lee, B. J. Kim, J. H. Kim, S. J. Han, H. Y. Kim, and M. R Stallcup Role of aspartate 351 in transactivation and active conformation of estrogen receptor {alpha} J. Mol. Endocrinol., December 1, 2005; 35(3): 449 - 464. [Abstract] [Full Text] [PDF]
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 D. P. McDonnell The Molecular Pharmacology of Estrogen Receptor Modulators: Implications for the Treatment of Breast Cancer Clin. Cancer Res., January 15, 2005; 11(2): 871s - 877s. [Abstract] [Full Text] [PDF]
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 W. Shao, E. K. Keeton, D. P. McDonnell, and M. Brown Coactivator AIB1 links estrogen receptor transcriptional activity and stability PNAS, August 10, 2004; 101(32): 11599 - 11604. [Abstract] [Full Text] [PDF]
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 M. A. Iannone, C. A. Simmons, S. H. Kadwell, D. L. Svoboda, D. E. Vanderwall, S.-J. Deng, T. G. Consler, J. Shearin, J. G. Gray, and K. H. Pearce Correlation between in Vitro Peptide Binding Profiles and Cellular Activities for Estrogen Receptor-Modulating Compounds Mol. Endocrinol., May 1, 2004; 18(5): 1064 - 1081. [Abstract] [Full Text] [PDF]
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J. M. Hall and K. S. Korach Stromal Cell-Derived Factor 1, a Novel Target of Estrogen Receptor Action, Mediates the Mitogenic Effects of Estradiol in Ovarian and Breast Cancer Cells Mol. Endocrinol., May 1, 2003; 17(5): 792 - 803. [Abstract] [Full Text] [PDF]
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 P. Webb, P. Nguyen, and P. J. Kushner Differential SERM Effects on Corepressor Binding Dictate ERalpha Activity in Vivo J. Biol. Chem., February 21, 2003; 278(9): 6912 - 6920. [Abstract] [Full Text] [PDF]
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 K. J. Ho and J. K. Liao Nonnuclear Actions of Estrogen Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1952 - 1961. [Abstract] [Full Text] [PDF]
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A. S. Levenson, K. M. Svoboda, K. M. Pease, S. A. Kaiser, B. Chen, L. A. Simons, B. D. Jovanovic, P. A. Dyck, and V. C. Jordan Gene Expression Profiles with Activation of the Estrogen Receptor {alpha}-Selective Estrogen Receptor Modulator Complex in Breast Cancer Cells Expressing Wild-Type Estrogen Receptor Cancer Res., August 1, 2002; 62(15): 4419 - 4426. [Abstract] [Full Text] [PDF]
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R. C. Dardes, R. M. O'Regan, C. Gajdos, S. P. Robinson, D. Bentrem, A. De Los Reyes, and V. C. Jordan Effects of a New Clinically Relevant Antiestrogen (GW5638) Related to Tamoxifen on Breast and Endometrial Cancer Growth in Vivo Clin. Cancer Res., June 1, 2002; 8(6): 1995 - 2001. [Abstract] [Full Text] [PDF]
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 D. P. McDonnell, C. E. Connor, A. Wijayaratne, C.-y. Chang, and J. D. Norris Definition of the Molecular and Cellular Mechanisms Underlying the Tissue-selective Agonist/Antagonist Activities of Selective Estrogen Receptor Modulators Recent Prog. Horm. Res., January 1, 2002; 57(1): 295 - 316. [Abstract] [Full Text] [PDF]
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C. E. Connor, J. D. Norris, G. Broadwater, T. M. Willson, M. M. Gottardis, M. W. Dewhirst, and D. P. McDonnell Circumventing Tamoxifen Resistance in Breast Cancers Using Antiestrogens That Induce Unique Conformational Changes in the Estrogen Receptor Cancer Res., April 1, 2001; 61(7): 2917 - 2922. [Abstract] [Full Text |
0.005). The ranges of BMDs observed
in sham-operated (shaded bar) and OVX (open
bar) animals are indicated. B, The effect of GW5638 on BMD at
the proximal metaphysis of the tibia in OVX rats was measured by QCT.
The significance of the difference in BMD between OVX and treated rats
(indicated by asterisks) was determined using the
Turkey-Kramer test (P 
