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J. Biol. Chem., Vol. 275, Issue 21, 15885-15894, May 26, 2000
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From the Departments of
Received for publication, December 8, 1999, and in revised form, March 2, 2000
Clinical studies have shown that estrogen
replacement therapy (ERT) reduces the incidence and severity of
osteoporosis and cardiovascular disease in postmenopausal women.
However, long term estrogen treatment also increases the risk of
endometrial and breast cancer. The selective estrogen receptor (ER)
modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and
agonistic responses when bound to the ER. Their predominantly
antagonistic actions in the mammary gland form the rationale for their
therapeutic utility in estrogen-responsive breast cancer, while their
agonistic estrogen-like effects in bone and the cardiovascular system
make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity
than either estrogen or tamoxifen. To identify additional SERMs, a
method to classify compounds based on differential gene expression
modulation was developed. By analysis of 24 different combinations of
genes and cells, a selected set of assays that permitted discrimination
between estrogen, tamoxifen, raloxifene, and the pure ER antagonist
ICI164384 was generated. This assay panel was employed to measure the
activity of 38 compounds, and the gene expression fingerprints (GEFs)
obtained for each compound were used to classify all compounds into
eight groups. The compound's GEF predicted its uterine-stimulatory
activity. One group of compounds was evaluated for activity in
attenuating bone loss in ovariectomized rats. Most compounds with
similar GEFs had similar in vivo activities, thereby
suggesting that GEF-based screens could be useful in predicting a
compound's in vivo pharmacological profile.
Determining the function of proteins encoded by all human genes in
normal and pathological states is a current challenge faced by the
scientific community now that the cloning and sequencing of a large
percentage of the expressed human genomic sequences has been
accomplished. This effort to determine function will be facilitated by
recent advances in the methodology used to simultaneously monitor the
expression of hundreds to thousands of genes (1, 2) in various
phenotypic states (e.g. normal versus neoplastic, diseased, or activated cells). Such studies will lead to the discovery of specific genes that are regulated under pharmacological or pathological conditions and may suggest strategies for the development of novel therapeutics. A major advance in reducing time and expense in
drug discovery would be achieved if the gene expression changes elicited by drug treatment of cultured cells could be correlated with
in vivo pharmacological or therapeutic activity. This is a
major challenge considering the many factors that influence in
vivo pharmacology (e.g. cell- and tissue-specific
therapeutic and toxicologic effects as well as pharmacokinetics).
To test the feasibility of using in vitro gene expression
profiles to identify and characterize compounds, we chose a
drug-induced biological response that is known to be dependent on
gene expression modulatory events: the estrogen receptor
(ER).1
Clinical studies have shown that estrogen replacement therapy (ERT)
reduces the incidence and severity of osteoporosis and cardiovascular
disease in postmenopausal women (for reviews see Refs. 3 and 4).
However, long term estrogen treatment also increases the risk of
endometrial and breast cancers (5). Therefore, our goal was to find a
new method for identifying selective estrogen receptor modulators
(SERMs) (6) that could be used to reduce the severity of osteoporosis
in postmenopausal women without causing endometrial hyperplasia (and
the concomitantly increased risk of uterine cancer). We sought an
in vitro gene expression profile that would enable us to
find compounds with selective in vivo activities. We did not
require that the genes whose expression was monitored would necessarily
encode proteins that are critical mediators of the desired or measured
in vivo effects of the drug. Rather, the genes only needed
to serve as reporters of the drug's activity. Unlike other approaches
to SERM identification that are based on trans-activation assays using
mutated or chimeric ER and estrogen-responsive promoters driving
reporter genes (7), our studies asked whether compounds could be
distinguished by their different abilities to modulate the mRNA
levels of multiple endogenous genes.
The anti-estrogen tamoxifen (Tam) has "estrogenic" activity in the
bone and the cardiovascular system (for review see Ref. 8) even though
it is an antagonist of estrogen action in the breast. More recent data
has demonstrated that another anti-estrogen, raloxifene (Ral), has ER
agonist effects in reducing the severity of postmenopausal osteoporosis
(9, 10). Ral has less pronounced stimulatory effects on the endometrium
(11) than Tam or estrogen, thereby suggesting its potential advantage
over Tam as ERT for postmenopausal women. Because they have different
in vivo effects, we expected that Tam and Ral would be
distinguishable from each other and from estrogen by differences in the
gene expression changes they elicit. Indeed, studies have shown that
both Tam and Ral have cell- and tissue-specific effects on
transcriptional activation mediated by the ER. For Tam, there is a
large body of evidence supporting its estrogen agonist activity in
regulating endogenous gene expression (12), whereas there is less known for Ral (13). To be useful in distinguishing SERMs, the cells and
genes monitored in response to compound treatment should enable the
generation of a different "gene expression fingerprint" (GEF) for
estrogen, Tam, and Ral. Ideally, the resultant GEF should also be able
to predict the compound's in vivo biological effects.
We describe here the development of a method to classify
compounds based on differential gene expression. Thirty-eight
compounds were tested and grouped into classes based on their GEF. The
endometrial-stimulatory activities and anti-osteoporotic efficacies of
some of the compounds were evaluated to determine the predictability of
the in vitro "fingerprint" for in vivo
activity. The results demonstrate that the majority of compounds
with similar GEFs also have similar in vivo activities.
Cell Culture and Compound Treatment--
The human breast cancer
cell lines MCF7, MDA361, and ZR75-1, the human hepatoma cell line
HepG2 (The Wistar Institute; American Type Culture Collection (ATCC)
HB8065), and the rat pituitary cell line GH3 were obtained from the
ATCC (Manassas, VA). The Fe33 (14) hepatoma ER-transfected FTO-2B cell
line was provided by Dr. Hilgenfeld (Heidelberg, Germany), and the BG-1
human ovarian carcinoma cell line (15) was obtained from Dr. J. Boyd
(University of Pennsylvania).
MDA-231 ER transfectant E-28 human breast cancer cells (16) were
routinely cultured in phenol red-free alpha-modified minimum essential
medium (MEM; Life Technologies, Inc.) supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM
MEM nonessential amino acids, 1.0 mM sodium pyruvate, 50 µg/ml gentamycin (all from Life Technologies, Inc.), 1.0 µg/ml
insulin (Sigma), and 5% dextran-coated charcoal (DCC) (16)-treated
fetal bovine serum (FBS) (Intergen). Cells were plated at approximately
40% confluency (1.5 × 106/plate) in 150-mm culture
dishes. Following an overnight cell attachment, the medium was changed
to include 0.2% ethanol or the test compounds and cultured for an
additional 48 h before harvest.
GH3 rat pituitary cells were routinely cultured in Dullbecco's
modified Eagles' medium (DMEM):F10 (1:1) medium containing 12.5%
horse serum, 2.5% FBS, 25 mM HEPES, 2 mM
L-glutamine, and 50 µg/ml gentamycin sulfate at 37 °C,
5% CO2. Under these conditions, the cells were partially
adherent, and both adherent and nonadherent cells were maintained
during the passaging of the cells. For the measurement of mRNA
expression, cells were seeded (106 per 100-mm dish) in
culture medium without phenol red and containing DCC-treated serum.
After 3 days, the medium was changed to one containing 0.2% ethanol or
the test compounds, and the cells were further incubated for 2 days
before harvest.
BG-1 ovarian carcinoma cells were cultured in DMEM:F12 (1:1) medium
containing 10% FBS, 2 mM L-glutamine, and 50 µg/ml gentamycin sulfate. For measurement of mRNA expression
levels, cells were cultured for 24 h in phenol red-free medium
containing 5% DCC-treated FBS prior to plating in the same medium at a
density of 2 × 106/150-mm plate. The following day,
the medium was changed to include 0.2% ethanol or the test compounds
and cultured for an additional 72 h before harvest.
ZR75-1, MCF7, and MDA361 cell lines were routinely cultured in
alpha-modified MEM supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM MEM nonessential amino
acids, 1.0 mM sodium pyruvate, 50 µg/ml gentamycin, 1.0 µg/ml insulin, and 10% FBS. Cells were plated (ZR75-1: 1.5 × 106/100-mm plate; MCF7: 2 × 106/150-mm
plate; MDA361: 5 × 106/100-mm plate) in phenol red
and insulin-free media containing 5% FBS-DCC for the assays. Following
an overnight cell attachment, the medium was changed to include 0.2%
ethanol or the test compounds and cultured for an additional 24 h
(ZR75-1), 48 h (MDA361), or 72 h (MCF7) before harvest.
The HepG2 human hepatoma cells were transfected with an ER
The ER-transfected human mammary epithelial cells (B5-ER) were cultured
and assayed for gene expression changes according to protocols
previously described (16). Compound treatment was for 72 h.
17 Determination of Relative ER Binding Affinities--
Competition
experiments were performed as described (18). Briefly, cytosol prepared
from rat uterus was incubated for 2 h at room temperature with 50 nM 3H-labeled estradiol either in the presence
or absence of unlabeled estradiol for the standard curve, or with test
compounds at appropriate concentrations. Displacement curves were
established and the relative binding affinity (RBA) was determined as
the percentage of estradiol binding as described (19).
RNA Isolation, Northern, and Slot Blot Analyses--
At the end
of the compound treatment time, cell monolayers were harvested into
Ultraspec (Biotecx Laboratories, Houston, TX) or RNeasy (Qiagen Inc.,
Santa Clarita, CA) RNA isolation reagent (Biotecx Laboratories) and
processed according to the manufacturer's suggested protocol. For the
Northern blots performed for evaluations of the initial gene/cell
combinations, total RNA (20 µg) was analyzed using standard methods.
Slot blots were employed for measuring gene expression changes in the
selected GEF assay panel; total RNA prepared from duplicate cell
treatments (MDA-231 ER, 10 µg; GH3, 1.0 µg; Fe33, 0.5 µg) was
spotted onto a Zetaprobe-GT nylon membrane using a 48-well slot blot
apparatus attached to a vacuum manifold. Hybridization of the membranes
to [32P]dCTP-labeled probes was carried out as described
previously (20). Quantitation of the specific hybridization signals was performed using a PhosphorImager (Fuji Medical Systems, Inc.). Any
variation in sample loading was normalized to the
glyceraldehyde-3-phosphate dehydrogenase mRNA levels determined
following hybridization of membranes that were treated with a boiling
solution of 0.1× SSC/0.5% SDS to remove the original probe. The ratio
of the signal intensities in compound-treated samples relative to
controls provided the value for fold change used in the assessment of
the compound activity for each assay. Changes in mRNA levels
greater than or equal to 1.5-fold were scored as positive
(interexperimental standard deviations were typically 10-15%).
Progesterone Receptor and pS2 Reverse Transcriptase-Polymerase
Chain Reaction Assays--
The measurement of progesterone receptor
(PR) in all cell lines and of pS2 in the B5-ER and MDA-ER cell lines
was performed using reverse transcriptase-polymerase chain reaction
(RT-PCR). All RNA samples were diluted to 20 ng/µl in
diethylpyrocarbonate-treated water. RT-PCR was performed using 100 ng
of total RNA. The reaction mixtures contained 5 units of
rTth DNA Polymerase (Perkin-Elmer; Foster City, CA), 1× EZ
buffer (Perkin-Elmer), 2.5 mM Mn(OAc)2, 300 µM dNTPs (mixture from Amersham Pharmacia Biotech), and
10 pmol of each biotinylated primer in a final volume of 50 µl. PCR primers, synthesized by Synthetic Genetics (San Diego, CA), were PR#1
(5'-GTC AGT GGG CAG ATG CTG TAT TT) and PR2 (5'-AAC TTC AGA CAT CAT TTC
TGG AAA TTC) to give a 426-base pair (bp) PCR product; PS2#1 (5'-CGT
GAA AGA CAG AAT TGT GGT TT) and pS2#2 (5'-TCA GAG CAG TCA ATC TGT GTT
GT) generated a 299-bp product. Amplification consisted of a 30-min RT
step at 60 °C immediately followed by 33 cycles of a two-step PCR
reaction (95 °C for 15 s, 60 °C for 45 s) and a final
7-min extension at 60 °C in a Perkin-Elmer Thermocycler 9600. Following PCR, 1/20 reaction volume was analyzed using
streptavidin-coated 96-well microplates (Xenoprobe, Gaithersburg, MD)
and oligonucleotide probes specific for the PCR target (i.e.
PR: 5' horseradish peroxidase (HRP). The probe was coupled to either
HRP or alkaline phosphatase and addition of either colorimetric
(tetramethylbenzidine for HRP; KPL, Gaithersburg, MD.) or
chemiluminescent (chloro-5-substituted adamantyl-1,2-dioxetane phenyl
phosphate for alkaline phosphatase; Tropix, Bedford, MA) substrates
permit quantitation of 300-500 initial copies of specific RNA template
in a 20- to 100-ng total RNA sample. RNA samples were tested in
triplicate, and the level of mRNA in each sample was extrapolated
from a standard curve generated using purified mRNA transcribed
in vitro. Changes in mRNA levels were scored as positive
if they were greater than or equal to 3-fold. The typical coefficient
of variation for the PR/BG-1 assay was 5-10%.
Uterine Histomorphometric Analysis--
For determination of
uterine stimulatory activity, immature, 19- to 21-day-old female Wistar
rats (Schering AG, Berlin, Germany), weighing 45-50 g, were given
daily subcutaneous injections of compounds or vehicle for 3 days. The
compounds were dissolved in a vehicle consisting of 10% ethanol in
arachis oil or a mixture of benzylbenzoate/castor oil (1:4). On day 4, the animals were weighed and euthanized by carbon dioxide asphyxiation.
The uteri were excised and placed in neutral buffered 3.7%
formaldehyde for a minimum of 24 h. The uteri were then embedded
in paraffin, cut into 4-µm transverse sections, and stained with
hematoxylin and eosin, and the sections were evaluated for luminal
epithelial cell height as described (21, 22). The difference in
epithelial cell height between the estrogen (0.3 µg of E2 per animal)
and vehicle-treated groups was calculated and expressed as 100%. The activity of the compound of interest as a percentage of E2 was calculated as:
Bone Protection Assays--
For determination of efficacy in
preventing bone loss, 3-month-old female rats (Harlan
Sprague-Dawley/Schering AG breeding facilities) were ovariectomized
under ether anesthesia and treated immediately after surgery. Compounds
were applied once daily subcutaneously in benzyl benzoate/castor oil
(1:4) or arachis oil/ethanol (95:5). Control groups (sham-operated and
ovariectomized animals treated with vehicle) and treatment groups
consisted of eight animals each. Animals were sacrificed 4 weeks after
surgery. Left and right tibia were processed for bone mineral density
measurements as described (23). Bone mineral density was measured in
the secondary spongiosa of the proximal tibia 5 mm distal from the joint using peripheral quantitative computed tomography. The activity of the compound of interest as a percentage of E2 was calculated according as:
Selection of Estrogen-responsive Genes and Cells--
Cell lines
were chosen based on prior knowledge of their responsiveness to
estrogen treatment (e.g. growth stimulation/inhibition, gene
expression modulation, and the presence of a receptor protein that
binds estrogen specifically and with high affinity). Included were cell
lines that express endogenous ER (GH3 pituitary adenoma (24); BG-1
ovarian carcinoma (15); MCF7, ZR75-1, and MDA361 breast carcinomas
(25)) and cells stably transfected with ER
To determine which of the genes and cell lines showed measurable
responses to estrogen treatment, cells were grown in estrogen-free culture medium and treated with the natural ligand, E2, or the nonmetabolizable estrogen EE for varying lengths of time from 3 to
72 h. Analysis of the levels of mRNA for the genes of interest gave an estimate of the kinetics of the response to E2 or EE treatment and an indication of the optimal conditions to measure the
responsiveness of each gene. An example is shown for regulation of
CBG mRNA levels in HepG2-ER2 cells (Fig.
1). Maximal stimulatory effects were observed by 48 h of treatment.
Gene/cell combinations (i.e. assays) that responded to E2 or
EE treatment with at least 3-fold changes in mRNA level
relative to vehicle-treated controls are shown in Table
I. The degree of estrogen-responsiveness
in this panel ranged from 3- to 450-fold, but most of the assays
showed less than a 15-fold response to estrogen treatment (for fold
change plus estrogen, see Table I).
Identification of Informative Gene/Cell Combinations for Compound
Discrimination--
Based on the assumption that there would be a
limit to the number of distinguishable mechanisms by which a
ligand-bound ER could modulate gene expression, we performed studies to
determine whether a subset of these gene/cell combinations would be
sufficient to differentiate known pharmacologically distinguishable
compounds. We tested compounds with known ER-binding properties and
activities in animal models for endometrial hyperplasia and bone
protection. Two key compounds were the partial agonists, Tam (the
4-OH-tamoxifen (HT) derivative was used in the initial studies) and
Ral, because they have distinctly different in vivo
activities compared with each other and with estrogen. One full agonist
(2-hydroxy-estradiol (2HE)), three putative partial agonists (RU39411
(RU) (34), centchroman (Cen) (35), ZK119010 (119) (36), and a pure
antagonist, ICI164384 (ICI) (37)) were also included in these initial studies.
Since 1.0 µM or lower concentrations of compounds
elicited maximal responses in most of the assays (data not shown), all
compounds were tested at 1.0 µM. The response of each
gene/cell combination to compound treatment was scored by measuring
steady-state levels of mRNA by Northern, slot blot, or RT-PCR
analysis (Table I). The degree of stimulation of gene expression by
each of these compounds differed as a function of the gene and cell
type. In some cases, the partial agonists HT, RU, and Cen had the same effectiveness as estrogen in stimulating gene expression
(i.e. in TRHR/GH3, TGF Selection of Nonredundant Gene/Cell Combinations--
Additional
studies were performed to determine which of the "redundant" assays
were most amenable to screening strategies (e.g. highest
reproducibility and extent of change relative to control). Based on
these considerations, PR/BG-1 (pattern I), PRL/GH3 (pattern II),
TGF
The response of each compound in the four assays generated a GEF
characteristic of each compound (seen as + and
The concentration dependence of the gene expression response for E2,
EE, HT, Ral, and ICI in each of the four assays is shown in Fig.
2. EC50 values for EE and E2
were 0.01-0.1 nM in the PR/BG-1 (Fig.
2A) and PRL/GH3 (Fig. 2C) assays and
were significantly greater in the TGF Classification of Additional Compounds using Selected
Gene/Cell Assays--
These four gene/cell combinations were
employed to characterize 29 additional compounds (Table
III), including some that are known ER
agonists (e.g. estrone and dihydroequilen (39)) and are
structurally related to either 17
Compounds were tested for their effects on gene expression at multiple
doses ranging from 10
The largest group is the one containing compounds that were active in
all four assays (i.e. group 1). Further resolution within this group is possible by considering the activity of each compound relative to E2 (%E2 in Table IV). The equine estrogens, dihydroequilen and dihyodroequilenin, are distinguishable from E2, EE, E1, and E3 by
their significantly decreased activity in the PR/BG-1 and IGFBP-1/Fe33 assays. The two 11
The remaining compounds were inactive in at least one of the GEF
assays. Group 2 contains compounds that were active in all but the
IGFBP-1/Fe33 liver cell assay, whereas group 3 contains compounds that were devoid of activity in the PR/BG-1
gene/cell combination but gave significant responses in the
IGFBP-1/Fe33 cell assay. The other groups contain compounds
that were inactive in the PR/BG-1 assay but showed differing
activities in the other three assays.
To further elucidate the mechanisms responsible for the different gene
expression modulatory activities of compounds in groups 2-7, most of
the compounds were tested for their abilities to antagonize
estrogen-stimulated gene expression changes in the assays where they
had no activity. None of the compounds in group 2 could antagonize the
stimulatory effect of EE (5 × 10
The two "standard" SERMs in our experiments were Tam and Ral. Tam
and the other compounds in its group (i.e. group 3) have distinctly different GEF from Ral and other members of group 5, all of
which have no activity in the TGF Determination of the Predictive Ability of the GEF
Classification for in Vivo Effects--
Through our analysis of gene
expression patterns, we found that E2, Tam, Ral, and ICI have
distinctly different GEFs (Table IV; groups 1, 4, 5, and 8, respectively). Because E2, Tam, and Ral have "estrogenic" effects
on bone, but differ in their abilities to stimulate growth in the
mammary gland and uterus, these data are consistent with the idea that
compounds with selective in vivo effects could be
distinguished by differential gene expression profiles in
vitro. To determine whether classification by GEF could provide a
guide for selection of weak versus strong stimulators of the
endometrium, we evaluated the uterine stimulatory activity of most of
the compounds found in groups 2-8. We assumed that the compounds
with activity in all four assays (group 1) would be strongly
uterotropic, in agreement with published reports for members of those
groups such as E3 (42) and dihydroequilen (39).
For these studies, we compared the activity of the compounds at doses
ranging from 0.1 to 10 mg/kg/day to a replacement dose of E2 in a 3-day
uterine growth assay and measured changes in the height of the uterine
epithelial cells. The response observed at the maximally effective dose
of each compound is shown in Fig. 3A. All of the compounds in
groups 2 and 3 elicited endometrial stimulatory effects that were equal
to or better than E2 treatment. Most of the compounds in groups 4 and 5 were similar to or less potent than Ral in increasing uterine
epithelial cell height. There was no apparent distinction for this
in vivo parameter between members of these two groups, which
were active in two of the four GEF assays. The two exceptions were Tam
and ZK183955, both of which co-classified with poorly active compounds
by their in vitro GEFs but were more stimulatory than E2 in
the in vivo evaluation. The single members of groups 6 and 7 were very different in their in vivo effects on endometrial
cell height; ZK185427 was highly effective in stimulating the
endometrium and ZK186217 was weakly effective. All but one of the
compounds in the "inactive" class (i.e. group 8) that
includes the pure ICI antagonists were very weak or inhibitory in the
endometrial stimulation assay. The only active compound, ZK183659,
produced a moderate stimulatory response only at the highest dose
tested (i.e. 10 mg/kg/day).
To determine whether compounds that grouped with Ral (group 5) would
have anti-osteoporotic activities, the four compounds in this group
that poorly increased endometrial cell height were tested in
ovariectomized female rats. Fig. 3B compares the activities of compounds in this group at their maximally effective doses to the
effects observed with a replacement dose of E2. Ral is 71% as
effective as E2 in reducing bone loss in this model; the other
compounds were also significantly capable of attenuating bone loss,
albeit not as effectively as Ral. One compound from group 8 was also
tested (RU 58688) and showed no effectiveness in ameliorating bone loss
in this model.
Our search for novel SERMs is an example of the use of gene
expression profiles in a strategy to discover new drug leads. The
results demonstrate that it is possible to group compounds based on
differences and similarities in the pattern of gene expression changes
that they elicit. Furthermore, the feasibility of employing gene
expression profiles to guide selection of compounds for in vivo biological studies is suggested by the separation of
compounds with highly stimulatory effects on the endometrium from those that were poorly effective on the basis of their GEFs. The observation that all of the poorly active members of one group of compounds were
also effective in ameliorating bone loss in an ovariectomized rat
provides additional support that this approach can predict in
vivo pharmacological and therapeutic profiles.
In the example described here, we evaluated 24 gene/cell combinations
comprising 10 different known estrogen-responsive genes in eight cell
lines representing four cell types (i.e. liver, pituitary,
mammary, and ovarian). To determine which of the gene/cell combinations
would be most useful in discriminating between different compounds, a
small group of compounds that had known abilities to bind to the ER and
to elicit various estrogenic responses in vivo was used.
Included in this group were the two SERMs, Tam and Ral, because they
have distinguishable in vivo biological activities in
comparison with estrogen and with each other.
We found five different patterns of gene expression activity in the 24 gene/cell combinations in response to the nine initial compounds
evaluated (Table I), suggesting that many gene/cell combinations report
the same "redundant" information. This may be due to an inherent
limitation in the number of molecularly distinct mechanisms by which
the ER can be activated by ligand binding (for reviews see Refs. 43 and
44). The majority of the gene/cell combinations were responsive to
estrogen and the other full agonists but not to any of the partial
agonists tested. There was at least one gene in each of the 10 cell
lines that was modulated only by estrogen and the full agonists,
whereas genes in only two of the cell lines were regulated by Ral. This may be indicative of a requirement for cell-specific factors for Ral
responsiveness. The contribution of cell-specific factors to regulation
of gene expression by E, HT, and Ral is also suggested by the
observation that the same gene (i.e. pS2) can be
differentially modulated by these compounds in different cell types or
in distinct cell lines from the same cell type (e.g. breast
cancer cell lines ZR75-1 and MDA-ER). In addition, we found that
different genes in the same cells responded differentially to these
compounds, thereby underscoring the importance of gene-specific factors
in determining sensitivity to compound treatment.
From among these 24 gene/cell combinations, a nonredundant panel of
four assays was selected that would enable discrimination between E,
Tam, and Ral as well as identify compounds that had similar and
different activities from them (Table II). Evaluation of 29 additional
compounds using these four assays led to their separation into eight
distinct groups (Table IV). Twelve of the 38 compounds tested were
grouped with E2 and EE, because they had activity in all four assays.
Nineteen compounds were active in one to three assays, and seven were
devoid of activity in any assay. The compounds that were classified
into groups 3-7 were of particular interest, because they included the
standard SERMs, Tam and Ral. None of the members of these groups could
induce PR gene expression in the BG-1 ovarian cells but were
distinguished by differing abilities to modulate gene expression in the
other three assays. Other studies have shown that the transcriptional responses of Tam and Ral can be distinguished by their activities in
trans-activation assays using wild type and mutant ER and the complement C3 promoter driving a reporter gene (45) but did not reveal
the existence of other classes of partial agonists. The experiments
described here demonstrate the use of multiple endogenous gene
expression readouts in different cells to generate a GEF for each
compound that ensures the discrimination between Tam- and Ral-like
compounds. In addition, our data suggest that endogenous gene
expression assays may uncover classes of ER modulators that could not
be identified using other methods.
The in vivo evaluation of compounds for their maximal
uterine stimulatory responses was designed to mirror the approach used in the in vitro analyses. The results for compounds in
groups 3-8 revealed that there was good correlation between their GEFs and endometrial-stimulatory activity. All of the compounds in group 3, which included HT, a major active metabolite of tamoxifen, were
equivalent to or more stimulatory than a replacement dose of E2. The
compounds in this group were structurally diverse, had varying degrees
of affinity for ER An additional point can be made from these data with respect to the
importance of metabolism in in vivo pharmacology. Tam was
placed into group 4 by its in vitro gene expression profile even though its in vivo effectiveness clearly categorized it
with HT and the other potent endometrial stimulators of group 3. These results suggest that Tam was inactive in the IGFBP-1/Fe33
assay either because the 4-hydroxylated metabolite of Tam is essential for the transcriptional activation of the IGFBP-1 promoter
or because HT has 50-fold greater affinity for the ER than Tam. It is
possible that the same explanation could apply to the other exception,
ZK183955. This compound is a tamoxifen derivative that may
undergo similar metabolic conversion in vivo. These data
also emphasize the limitations of in vitro screens that are
not able to measure effects of compound metabolites or have
insufficient sensitivity to score effects of compounds with low
affinities for their biological targets.
To further assess the strength of GEF-based screening for predicting
in vivo biological activity, we determined whether the compounds that were grouped together with Ral were similarly effective in protecting against bone loss in female rats following estrogen deprivation by ovariectomy. All of the compounds in the Ral group demonstrated significant bone-protective activity. This activity, coupled with the weak to poor endometrial stimulatory effects of these
compounds, suggests that they would be potential leading candidates for
optimization as novel SERMs.
These results support the concept that gene expression profiles can be
employed to classify compounds with respect to a reference standard
such as estrogen. However, the number of compounds tested in this study
did not permit statistical evaluation of the correlation between
compound classification and in vivo effects for the members of all compound groups. Additional studies using a larger number of
compounds are required to determine whether the four gene/cell assays
employed in this screen are sufficient to adequately categorize compounds for their in vivo effects.
The integration of high density cDNA array hybridization methods
for the identification of genes and cells to be employed in such
screens will greatly facilitate this process. It should be possible to
use gene expression profiles in the search for new chemical or
biological entities that have similar functions to any compound used as
a reference. In addition to small molecules, mimetics of proteins and
peptides as well as expressed partial or full length cDNAs,
oligonucleotides, or antisense molecules can be identified by screening
compounds that cause changes in gene expression that are similar to
those elicited by such effectors. In situations where large chemical
data bases and corresponding pharmacological and pharmacokinetic data
are available, a reverse approach that evaluates in vivo
characterized compounds in GEF screens similar to those described here
can be carried out. The juxtaposition of known in vivo
efficacy and side effects with the results of GEF analysis can be
employed to derive GEFs that are predictive for pharmacology, toxicity,
and perhaps even pharmacokinetic effects.
We are grateful for gifts of HEGO plasmid
(Dr. P. Chambon), Fe33 rat hepatoma cells (Dr. Hilgenfeld), BG-1
ovarian carcinoma cells (Dr. J. Boyd), and 184B5 cells (Dr. M. Stampfer). We thank J. Simmons, D. Allen, and K. Oliver for technical
assistance. Special thanks are extended to Dr. P. Johnson for valuable
discussions and comments on the manuscript.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Dept. of Cancer
Research, Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804. Tel.: 510-669-4174; Fax: 510-669-4245; E-mail:
deb_zajchowski@berlex.com.
**
Present Address: InforMax Inc., 6010 Executive Blvd., North
Bethesda, MD 20852.
Published, JBC Papers in Press, March 15, 2000, DOI 10.1074/jbc.M909865199
The abbreviations used are:
ER, estrogen
receptor;
SERM, selective ER modulators;
Tam, tamoxifen;
Ral, raloxifene;
GEF, gene expression fingerprint;
MEM, minimal essential
medium;
DCC, dextran-coated charcoal;
PR, progesterone receptor;
HRP, horseradish peroxidase;
E2, 17
Identification of Selective Estrogen Receptor Modulators by Their
Gene Expression Fingerprints*
§,,,
**,,,,,,,, Cancer Research,
¶ Cardiovascular Research, Biologics Discovery Research,
and §§ Animal Pharmacology, Berlex Biosciences,
Richmond, California 94804 and Schering
Research Laboratories, Schering AG 13342 Berlin, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expression vector (i.e. pSV2neo/CMV-ER (16), except that the ER cDNA corresponds to the HEGO sequence (17) or the pRc/RSV (Invitrogen) expression vector containing the HEGO cDNA) and
selected in 1000 µg/ml G418 (Life Technologies, Inc.) prior to
isolation of stable ER-expressing clones, ER1 and ER2. The ER levels in these clones are approximately 200-400 fmol/mg of protein, as measured
by saturation binding analysis using radiolabeled estradiol (data not
shown). Nuclear immunocytochemical staining was observed with
antibodies against the ER and a 65-kDa protein band by immunoblot analyses (data not shown). The HepG2-ER cells were cultured in EMEM
(Life Technologies, Inc.), supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM MEM nonessential amino
acids, 1.0 mM sodium pyruvate, 50 µg/ml gentamycin, and
10% FBS. All cells were plated (HepG2-ER: 4 × 106/100-mm plate; Fe33: 2.5 × 105/150-mm
plate) in phenol red and insulin-free media containing 5% FBS-DCC for
the assays. Following an overnight cell attachment, the medium was
changed to include 0.2% ethanol or the test compounds and cultured for
an additional 72 h.
-Estradiol (E2), 17-ethinyl estradiol (EE), estrone, and
estriol were purchased from Sigma. All other compounds were synthesized in the chemistry department at Schering AG (Berlin). Stock solutions (10 mM) of all of the chemicals were prepared in
Me2SO and serially diluted in absolute ethanol.
The average luminal epithelial cell height in vehicle-treated
rats was 21.1 ± 8.2 µm and 68.6 ± 15.4 µm in E2-treated rats.
![%x=<FR><NU>100 [<UP>Height </UP>(<UP>test compound</UP>)−<UP>height </UP>(<UP>vehicle</UP>)]</NU><DE><UP>height </UP>(<UP>E2</UP>)−<UP>height </UP>(<UP>vehicle</UP>)</DE></FR>](/content/vol275/issue21/fulltext/15885/img001.gif)
(Eq. 1)
The average bone mineral density, BMD, of
ovariectomized animals was 294.7 ± 19.9 mg of
calcium/cm3 and 388.5 ± 29.3 mg of calcium/cm3
for E2-treated animals. Standard deviations were less than 10% for the
compound treatments.
![%x=<FR><NU>100 [BMD (<UP>test compound</UP>)−BMD (<UP>vehicle</UP>)]</NU><DE>BMD (<UP>E2</UP>)−BMD (<UP>vehicle</UP>)</DE></FR>](/content/vol275/issue21/fulltext/15885/img002.gif)
(Eq. 2)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(breast carcinoma
MDA231-E28 (called MDA-ER) (16), human mammary epithelial cell line
184B5-E1 (called B5-ER) (16), human heptoma HepG2-ER1 and ER2, and rat
hepatoma Fe33 (14)). Known estrogen-responsive genes, including the
52-kDa cathepsin D (52kD (26)), growth hormone
(GH) (27), prolactin (PRL) (28), progesterone
receptor (PR) (29), pS2 (16), transforming growth
factor alpha (TGF) (30), insulin-like growth factor
binding protein 1 (IGFBP-1) (31), corticosteroid-binding
globulin (CBG), amphiregulin (32), and thyrotropin-releasing
hormone receptor (TRHR) (33) were also chosen for our studies.
View larger version (61K):
[in a new window]
Fig. 1.
Time course of EE-dependent
CBG gene expression in HepG2-ER2 cells. Total RNA
was isolated from HepG2-ER2 cells at the indicated time points
following treatment with EE or vehicle (VC). Northern blots
were prepared with 20 µg of RNA and analyzed for mRNA levels of
CBG (top gel) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, bottom gel). The
CBG signal intensities (averages of duplicates) were
normalized for glyceraldehyde-3-phosphate dehydrogenase levels and are
represented in the graph as fold change in gene expression relative to
the vehicle control at the corresponding time point.
Activity of selected compounds in modulating gene expression
5-fold; +,
<5-fold; 
, inactive. Activity cut-offs were 2-fold for Northern
analyses, 3-fold for RT-PCR, and 1.5-fold for slot blots.
/B5-ER,
TGF/MDA-ER). But, in most instances, the full agonists
(i.e. E2, EE, 2HE) were significantly more active in
enhancing gene expression than the other compounds. Analysis of 24 different gene/cell combinations with these nine compounds revealed
that most of the assays provided redundant information (seen as the
same pattern of activity across the series of compounds in Table I).
Five major activity patterns across this set of compounds were
discernable among all of these assays when two activity states
(i.e. active and inactive) were considered (patterns I-V on the right side of Table I). Response
pattern I was found in most of the genes and cell
types tested, whereas the other patterns were seen less frequently.
There were only four assays that registered responses to Ral treatment
(i.e. those in patterns II and
V), whereas HT was active in nine assays (i.e.
those in patterns II-V). Interestingly, some compounds
exhibit cell type-dependent abilities to activate the same
gene, as illustrated by the differential response of 52kD
gene expression to treatment with the partial agonists HT, RU, and Cen
in ZR75-1 mammary carcinoma compared with BG-1 ovarian carcinoma
cells. Furthermore, the same compounds can have different effects on
different genes within the same cell (e.g. compare
52kD and TGF- in the B5-ER cells).
/MDA-ER (pattern III), and
IGFBP-1/Fe33 (pattern V) were selected
to use in screens of additional compounds. We did not include the assay
for pattern IV, because the estrogen response was only 4- to
6-fold.
patterns of activity in the row corresponding to each compound in Table II). The GEFs of the full agonists, E2
and EE, are identical to each other but different from those of HT, RU,
and Cen, which all have the same GEF. Importantly, the different GEFs
of E2, Ral, and HT demonstrate that these assays discriminate between partial ER agonists that have distinct in vivo
activities.
Compound classification by selected GEF assays
/MDA-ER
(i.e. 1 and 10 nM, respectively; Fig. 2B) and the IGFBP-1/Fe33 assay (i.e.
0.5 and 50 nM, respectively; Fig. 2D). The large
difference in the potency of E2 and EE in the Fe33 liver cells has
previously been described (38) and is most likely due to metabolism of
E2 in the liver cells. The maximal response obtained with HT and Ral in
both PRL and IGFBP-1 gene modulation was
significantly less than that observed with EE or E2 consistent with
their partial agonist effects on these genes. The ER-mediated nature of
the stimulatory responses of E, HT, and Ral was verified by
demonstrating antagonism of these effects with the ICI compound (data
not shown).
View larger version (20K):
[in a new window]
Fig. 2.
Concentration dependence of gene expression
changes in selected GEF assays. Cells were treated with vehicle,
E2 (open squares), EE (open circles), HT
(diamonds), Ral (solid squares), or ICI
(triangle) at the indicated concentrations. The fold change
in mRNA levels relative to vehicle control is graphically depicted
for each gene/cell combination. A, PR mRNA
levels were measured using RT-PCR analysis following treatment of BG-1
ovarian carcinoma cells for 72 h. B, TGF
mRNA levels were quantitated by slot blot analysis using 10 µg of
total RNA following treatment of MDA231-ER cells for 48 h.
C, PRL mRNA levels were measured by slot blot
analysis of total RNA (1.0 µg) prepared from 48-h compound-treated
GH3 pituitary cells. D, IGFBP-1 mRNA levels
were quantitated in total RNA (0.5 µg) isolated from Fe33 liver
hepatoma cells treated for 48 h with the indicated
compounds.
-estradiol or 17-estradiol. Others are derivatives of Tam (e.g. 3-hydroxy-tamoxifen or
Droloxifene (40)) and Ral (e.g. ZK182254) or of the
pure anti-estrogen, ICI164384 (e.g. ICI182780 (41)). The
chemical structural classes of the remaining compounds are listed in
Table III. The relative ER binding affinities (RBA) of these compounds
(Table IV) ranged from those that were
equivalent or better than E2 (e.g. EE, Ral, and 167466) to
very weak competitors (e.g. ZK155843 and ZK182491) of E2
binding.
Chemical structure classification of compounds employed in these
studies
Compound activities in GEF assays
10 to 106
M and the fold changes in gene expression (at the maximally
effective dose) relative to vehicle-treated control cells were
tabulated (Table IV). Each compound has a characteristic GEF that was
obtained from their activities in modulating gene expression in the
four assays. Compounds were then grouped according to the similarity of
their GEFs. Group 1 includes E2 and EE as well as other compounds with
activity in all four assays, whereas group 8 contains compounds, such
as ICI, that were inactive in all assays. The grouping was independent
of the compound's chemical structure classification. For example,
group 5 includes a benzothiophene (Ral), a triphenylethylene derivative
(ZK183955), two 11-substituted estradiol derivatives (ZK167466 and
180686), and a compound with a 7-substituent similar to ICI (ZK180686).
-substituted estradiol
derivatives ZK166780 and ZK166781 are particularly intriguing because
they maintained full activity in the TGF/MDA-ER assay but
were less effective in the other three assays. These differences are
not simply explained by the ability of the compounds to bind to the ER,
because ZK166780 and ZK166781 have binding affinities that are nearly
equivalent to E2 and E1 (i.e. RBA of 44 and 12 relative to
100 and 14, respectively; Table IV).
10 M)
in the IGFBP-1/Fe33 assay when tested at 100-fold molar
excess, whereas all of the tested compounds in groups 3-8 inhibited
E2-induced responses in the PR/BG-1 assay (data not shown).
These results suggest that compounds in groups 3-7 are classical
partial agonist-antagonists of the ER, whereas the compounds in group 2 are either weak agonists or are metabolized in the Fe33 liver cells.
/MDA-ER assay.
Interestingly, Tam and its more active metabolite HT were separated
from each other by the inactivity of Tam in the Fe33 liver cell assay
and by Tam's weaker response in the TGF/MDA-ER gene/cell
combination (i.e. 44% versus 110% for HT). The
two compounds in groups 6 and 7 had significant activity in only
one assay, in either the PRL/GH3 or the
IGFBP-1/Fe33 gene/cell combination.
View larger version (19K):
[in a new window]
Fig. 3.
Endometrial-stimulatory and bone-protective
activities of GEF-sorted compounds. A, compounds from
groups 2-8 of Table IV were tested at concentrations ranging from 0.03 to 3.0 mg/kg/day in immature female rats (5 rats per treatment group).
Stimulatory effects on uterine epithelial cell height (as percentage of
E2) are provided in comparison to a replacement dose of E2 (0.3 µg
per animal) for the maximal responsive dose of each compound. The
corresponding doses (in milligrams/kg/day) were 0.05 (for RU39411), 0.1 (for HT, ZK167466, 17 -E2, RU58688), 0.3 (for Tam, Ral, ZK183955,
ZK180686, and ZK186217), 3.0 (for Droloxifene, Cen, ZK119010, ZK182254,
ZK184104, ZK185157, ZK183819, and ZK182956), and 10 (for ZK22496,
ZK185704, and ZK183659). B, the compounds from group 5 that
were poorly active in the uterine growth assay were evaluated at doses
ranging from 0.04 to 1.0 mg/kg/day for bone-preserving efficacy in
ovariectomized female rats (8 rats/treatment group). Bone mineral
density (BMD) in the proximal tibia following 4 weeks of
treatment was measured using peripheral quantitative computed
tomography. Compound activity was assessed relative to the effects
observed with a bone-protective dose of 1.2 µg/kg/day E2, which was
set at 100%. The activity at the maximally effective doses (in
milligrams/kg/day: for ZK167466, 0.4; for Ral, ZK180686; and for
ZK184104, 1.0) of each compound are shown. The response of RU58688 (at
0.4 mg/kg/day; from group 8) is provided for comparison.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and were similar only in their ability to score
positively in the same three gene/cell combinations. The compounds in
group 8, which includes ICI, were inactive in all GEF assays and all
but one of them weakly stimulated or inhibited uterine growth in
immature rats. The remaining groups contain compounds, including Ral,
with activity in only one or two assays; most of these compounds had
considerably less endometrial stimulatory activity than members of
group 3. Thus, compound sorting by GEF analysis can be used to predict
compounds that would have a high probability of being very active in
increasing endometrial luminal epithelial cell height (i.e.
those with activity in three or more assays). However, the number of
assays in which a compound is active did not always predict the
strength of the uterine stimulatory response for the poor to moderately
active compounds. Of the two compounds representative of single assay
hits, one of them (i.e. ZK185427, Fig. 3A) was
more effective in the endometrial stimulation assay than the compounds
that were active in two assays.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-estradiol;
EE, 17-ethinyl
estradiol;
HT, 4-OH-tamoxifen;
ICI, ICI164384;
PRL, prolactin;
TGF, transforming growth factor ;
IGFBP-1, insulin-like growth factor
binding protein-1;
TRHR, thyrotropin-releasing hormone receptor;
RU, RU39411;
Cen, centchroman;
bp, base pair(s);
RT-PCR, reverse
transcriptase-polymerase chain reaction.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Lockhart, D. J.,
Dong, H.,
Byrne, M. C.,
Follettie, M. T.,
Gallo, M. V.,
Chee, M. S.,
Mittmann, M.,
Wang, C.,
Kobayashi, M.,
Horton, H.,
and Brown, E. L.
(1996)
Nat. Biotechnol.
14,
1675-1680
2.
Schena, M.,
Shalon, D.,
Davis, R. W.,
and Brown, P. O.
(1995)
Science
270,
467-470
3.
Genazzani, A. R.,
and Gambacciani, M.
(1999)
Maturitas
32,
11-17
4.
Rosano, G. M.,
and Panina, G.
(1999)
Drugs Aging
15,
219-234
5.
Pritchard, K. I.
(1998)
Recent Results Cancer Res.
152,
22-31
6.
Mitlak, B. H.,
and Cohen, F. J.
(1999)
Drugs
57,
653-663
7.
McDonnell, D. P.,
Clevenger, B.,
Dana, S.,
Santiso Mere, D.,
Tzukerman, M. T.,
and Gleeson, M. A.
(1993)
J. Clin. Pharmacol.
33,
1165-1172
8.
Grainger, D. J.,
and Metcalfe, J. C.
(1996)
Nat. Med.
2,
381-385
9.
Black, L. J.,
Sato, M.,
Rowley, E. R.,
Magee, D. E.,
Bekele, A.,
Williams, D. C.,
Cullinan, G. J.,
Bendele, R.,
Kauffman, R. F.,
Bensch, W. R.,
Frolik, C. A.,
Termine, J. D.,
and Bryant, H. U.
(1994)
J. Clin. Invest.
93,
63-69
10.
Bryant, H. U.,
and Dere, W. H.
(1998)
Proc. Soc. Exp. Biol. Med.
217,
45-52
11.
Sato, M.,
Rippy, M. K.,
and Bryant, H. U.
(1996)
FASEB J.
10,
905-912
12.
Cai, W.,
Hu, L.,
and Foulkes, J. G.
(1996)
Curr. Opin. Biotechnol.
7,
608-615
13.
Yang, N. N.,
Bryant, H. U.,
Hardikar, S.,
Sato, M.,
Galvin, R. J.,
Glasebrook, A. L.,
and Termine, J. D.
(1996)
Endocrinology
137,
2075-2084
14.
Kaling, M.,
Weimar-Ehl, T.,
Kleinhans, M.,
and Ryffel, G. U.
(1990)
Mol. Cell. Endocrinol.
69,
167-178
15.
Geisinger, K. R.,
Kute, T. E.,
Pettenati, M. J.,
Welander, C. E.,
Dennard, Y.,
Collins, L. A.,
and Berens, M. E.
(1989)
Cancer
63,
280-288
16.
Zajchowski, D. A.,
and Sager, R.
(1991)
Mol. Endocrinol.
5,
1613-1623
17.
Tora, L.,
Mullick, A.,
Metzger, D.,
Ponglikitmongkol, M.,
Park, I.,
and Chambon, P.
(1989)
EMBO J.
8,
1981-1986
18.
Fuhrmann, U.,
Slater, E. P.,
and Fritzemeier, K. H.
(1995)
Contraception
51,
45-52
19.
Korenmann, S.
(1970)
Methods Enzymol.
36,
49-52
20.
Liu, H. L.,
Golder-Novoselsky, E.,
Seto, M. H.,
Webster, L.,
McClary, J.,
and Zajchowski, D. A.
(1998)
Mol. Endocrinol.
12,
1733-1748
21.
Branham, W. S.,
and Sheehan, D. M.
(1995)
Biol. Reprod.
53,
863-872
22.
Fritzemeier, K.-H.,
and Hegele-Hartung, C.
(1999)
in
Handbook of Experimental Pharmacology; Estrogens and Antiestrogens
(Oettel, M.
, and Schillinger, E., eds), Vol. 135
, pp. 54-64, Springer-Verlag, Heidelberg
23.
Sato, M.
(1995)
Bone
17,
157S-162S
24.
Haug, E.,
Naess, O.,
and Gautvik, K. M.
(1978)
Mol. Cell. Endocrinol.
12,
81-95
25.
Engel, L. W.,
and Young, N. A.
(1978)
Cancer Res.
38,
4327-4339
26.
Westley, B. R.,
and May, F. E.
(1987)
Nucleic Acids Res.
15,
3773-3786
27.
Martinoli, M. G.,
Veilleux, R.,
and Pelletier, G.
(1991)
Acta Endocrinol.
124,
83-90
28.
Rhode, P. R.,
and Gorski, J.
(1991)
Mol. Cell. Endocrinol.
82,
1-9
29.
Read, L. D.,
Snider, C. E.,
Miller, J. S.,
Greene, G. L.,
and Katzenellenbogen, B. S.
(1988)
Mol. Endocrinol.
2,
263-271
30.
Bates, S. E.,
Davidson, N. E.,
Valverius, E. M.,
Freter, C. E.,
Dickson, R. B.,
Tam, J. P.,
Kudlow, J. E.,
Lippman, M. E.,
and Salomon, D. S.
(1988)
Mol. Endocrinol.
2,
543-555
31.
Molnar, P.,
and Murphy, L. J.
(1994)
J. Mol. Endocrinol.
13,
59-67
32.
Martinez-Lacaci, I.,
Saceda, M.,
Plowman, G. D.,
Johnson, G. R.,
Normanno, N.,
Salomon, D. S.,
and Dickson, R. B.
(1995)
Endocrinology
136,
3983-3992
33.
Kimura, N.,
Arai, K.,
Sahara, Y.,
and Suzuki, H.
(1994)
Endocrinology
134,
432-440
34.
Gottardis, M. M.,
Jiang, S. Y.,
Jeng, M. H.,
and Jordan, V. C.
(1989)
Cancer Res.
49,
4090-4093
35.
Hall, T. J.,
Nyugen, H.,
Schaueblin, M.,
and Fournier, B.
(1995)
Biochem. Biophys. Res. Commun.
216,
662-668
36.
Nishino, Y.,
Schneider, M. R.,
Michna, H.,
and von Angerer, E.
(1991)
J. Endocrinol.
130,
409-414
37.
Wakeling, A. E.,
and Bowler, J.
(1987)
J. Endocrinol.
112,
R7-R10
38.
Diel, P.,
Walter, A.,
Fritzemeier, K. H.,
Hegele-Hartung, C.,
and Knauthe, R.
(1995)
J. Steroid Biochem. Mol. Biol.
55,
363-373
39.
Bhavnani, B. R.
(1998)
Proc. Soc. Exp. Biol. Med.
217,
6-16
40.
Eppenberger, U.,
Wosikowski, K.,
and Kung, W.
(1991)
Am. J. Clin. Oncol.
14 Suppl. 2,
S5-S14
41.
Wakeling, A. E.,
Dukes, M.,
and Bowler, J.
(1991)
Cancer Res.
51,
3867-3873
42.
Katzenellenbogen, B. S.
(1984)
J. Steroid Biochem.
20,
1033-1037
43.
Parker, M. G.
(1995)
Vitam. Horm.
51,
267-287
44.
Katzenellenbogen, J. A.,
O'Malley, B. W.,
and Katzenellenbogen, B. S.
(1996)
Mol. Endocrinol.
10,
119-131
45.
McDonnell, D. P.,
Clemm, D. L.,
Hermann, T.,
Goldman, M. E.,
and Pike, J. W.
(1995)
Mol. Endocrinol.
9,
659-669
46.
Pons, M.,
Michel, F.,
Crastes de Paulet, A.,
Gilbert, J.,
Miquel, J. F.,
Precigoux, G.,
Hospital, M.,
Ojasoo, T.,
and Raynaud, J. P.
(1984)
J. Steroid Biochem.
20,
137-145
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