A Fusion Protein of the Estrogen Receptor (ER) and Nuclear Receptor Corepressor (NCoR) Strongly Inhibits Estrogen-Dependent Responses in Breast Cancer Cells
Pei-Yu Chien,
Masafumi Ito,
Youngkyu Park,
Tetsuya Tagami,
Barry D. Gehm and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT
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Nuclear receptor corepressor (NCoR) mediates
repression (silencing) of basal gene transcription by nuclear receptors
for thyroid hormone and retinoic acid. The goal of this study was to
create novel estrogen receptor (ER) mutants by fusing transferable
repressor domains from the N-terminal region of NCoR to a functional ER
fragment. Three chimeric NCoR-ER proteins were created and shown to
lack transcriptional activity. These fusion proteins silenced basal
transcription of the ERE2-tk-Luc reporter gene and inhibited the
activity of cotransfected wild-type ER (wtER), indicating that they
possess dominant negative activity. One of the fusion proteins
(CDE-RD1), containing the ER DNA-binding and ligand-binding domains
linked to the NCoR repressor domain (RD1), was selected for detailed
examination. Its hormone affinity, intracellular localization, and
level of expression in transfected cells were similar to wtER, and it
bound to the estrogen response element (ERE) DNA in gel shift assays.
Glutathione-S-transferase pull-down assays showed that
CDE-RD1 retains the ability to bind to steroid receptor coactivator-1.
Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion
protein eliminated silencing and dominant negative activity. Thus, the
RD1 repressor domain prevents transcriptional activation despite the
apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators.
Transcriptional silencing was incompletely reversed by trichostatin A,
suggesting a histone deacetylase-independent mechanism for repression.
CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231
breast cancer cells and repressed the growth of T47D cells when
delivered to the cells by a retroviral vector. These ER-NCoR fusion
proteins provide a novel means for inhibiting ER-mediated cellular
responses, and analogous strategies could be used to create dominant
negative mutants of other transcription factors.
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INTRODUCTION
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The estrogen receptor (ER) belongs to the superfamily of nuclear
receptors, which regulate the transcription of specific target genes.
The ER binds to the estrogen response element (ERE), a palindromic DNA
sequence in the promoters of estrogen-regulated genes (1, 2), and
activates transcription in response to its ligand, estradiol
(E2). Several functional domains have been identified in
the ER (3), including transactivation domain 1 (TAF-1, regions
A/B), a DNA-binding domain (region C), a hinge region (region D), and
transactivation domain 2 (TAF-2)/hormone-binding domain (region E). In
most cases, both TAF-1 and TAF-2 are required for full
hormone-stimulated activity. It has been shown recently that there are
two different isoforms of ER, ER
(4, 5) and ERß (6, 7), that are
encoded by different genes. These two isoforms are highly similar in
the DNA-binding domain and, to a lesser degree, in the ligand-binding
domain (6, 7).
Hormone-induced gene transcription can be enhanced by coactivators,
including steroid receptor coactivator-1 (SRC-1) (8, 9), SRC-2 [also
called TIF2 (transcriptional intermediary factor) and GRIP-1
(glucocorticoid receptor interacting protein 1 ) (10, 11)], SRC-3
[also called p/CIP (p300/CBP cointegrater- associated protein),
RAC3 (receptor-associated coactivator 3), ACTR (activator of thyroid
receptor), and AIB1) (amplified in breast cancer 1) (12, 13, 14, 15),
and CREB binding protein (CBP)/p300 (16, 17), among others. Recently
determined crystal structures of nuclear receptors reveal that helices
3, 5, and 12 of the ligand binding domain form a hydrophobic groove
that serves as the interaction surface for an hydrophobic
-helical
segment (LXXLL) in coactivators (18, 19). A translocation of helix 12
is induced by ligand binding and plays a critical role in the
recruitment of coactivators (18). Therefore, recruitment of
coactivators to TAF-2 of nuclear receptors is responsible for
ligand-dependent gene activation. Coactivators also interact with the
N-terminal (TAF-1) region of nuclear receptors (20, 21), and this
region contributes to ligand-independent gene activation (22). SRC-1
increases ER transcriptional activity by enhancing the interaction
between the TAF-1 and TAF-2 domains of ER (23). In addition,
coexpression of CBP/p300 and SRC-1 synergistically stimulates ER
transcriptional activity (24). The conformational changes induced by
ligand binding to ER recruit a coactivator complex containing SRC-1,
CBP/p300, and p300/CBP-associated factor (P/CAF) (25). These
coactivators have recently been found to have intrinsic histone
acetyltransferase (HAT) activity (12, 26, 27, 28). HAT acetylates histones,
a process that is proposed to alter the chromatin structure and
increase the accessibility of DNA to transcription factors and the
basal transcription machinery (29, 30), thereby enhancing the rate of
gene transcription (31). HAT also acetylates components of the basal
transcription complex (32), in addition to histones.
On the other hand, in the absence of ligand, several nuclear receptors
suppress basal gene transcription by recruiting corepressors (CoRs).
Nuclear receptor corepressor (NCoR) (33) and silencing mediator for
retinoic acid and thyroid hormone receptors (SMRT) (34, 35) have been
intensively studied in the context of the unliganded thyroid hormone
receptor (TR) and retinoic acid receptor (RAR). The amino acid
sequences of NCoR and SMRT show approximately 40% identity (35). NCoR
contains three transferable repressor domains in its N-terminal region
(36), of which the first (RD1) appears to be the most potent (33). SMRT
is a shorter protein and has two repressor domains in its N-terminal
region, which corresponds to the third repressor domain of NCoR (37).
Unliganded TR and RAR recruit a multicomponent CoR complex which, in
addition to NCoR or SMRT, also contains mSin3, a mammalian homolog of a
yeast transcription CoR (38, 39), and histone deacetylase (HDAC)
(36, 37). HDAC deacetylates histone H3 (40, 41), stabilizing the
structure of chromatin, and inducing repression of basal gene
transcription. Based on these recent findings, it is believed that
chromatin remodeling is a key event in the regulation of gene
transcription by nuclear receptors and other transcription factors
(reviewed in Refs. 42, 43, 44, 45, 46).
In contrast to TR and RAR, suppression of ER-mediated basal
transcription by CoRs has not been observed. However, there is evidence
that ER interacts with CoRs in vitro. In
glutathione-S-transferase (GST) fusion pull-down assays,
SMRT binds to ER in a hormone-independent manner (47). NCoR
coimmunoprecipitates with ER weakly without ligand and more strongly in
the presence of tamoxifen (48), an ER-mixed antagonist/agonist (49, 50).
Dominant negative mutants of ER, which block the function of the
wild-type ER (wtER), have been created by chemical or site-directed
mutagenesis (51, 52, 53, 54). These mutants retain DNA binding and dimerization
properties, but they are deficient in transcriptional activation,
allowing them to function as antagonists at ERE binding sites. As the
RD1 of NCoR retains its repressor activity when fused to DNA binding
proteins such as Gal4 (33), we hypothesized that dominant negative
variants of ER could be produced by fusing the NCoR repressor domains
to the ER.
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RESULTS
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A schematic diagram of the proteins used in this study is shown in
Fig. 1
. The wild-type ER contains six
functional domains (A to F). The CDE fragment used in this study is a
truncated form that contains the DNA binding domain, hinge region, and
TAF-2/ligand binding domain. NCoR contains repressor domains 1, 2, and
3 (RD1, RD2, and RD3) in its N-terminal region (36) and two C-terminal
interaction domains (ID1 and ID2), which interact with the ligand
binding domain of nuclear receptors like TR and RAR (33, 55). RD1 was
fused to the C terminus of CDE to create CDE-RD1 and to the N terminus
to create RD1-CDE. The whole repressor region of NCoR, containing all
three of the repressor domains, was fused to the N terminus of CDE to
create RD13-CDE.

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Figure 1. Schematic Diagram of the wtER, NCoR, and Fusion
Proteins
The functional domains of wtER (AF) and NCoR (RD1, RD2, RD3, ID1, and
ID2) are displayed. CDE, a truncated form of ER, contains the
DNA-binding domain (C), hinge region (D), and TAF-2/ligand binding
domain (E). Repressor domain 1 (RD1) of NCoR, the most potent repressor
domain, was fused to the C terminus of CDE to create CDE-RD1 or to the
N terminus to create RD1-CDE. The whole repression region (RD13) was
fused to the N terminus of CDE to create RD13-CDE. The amino acid
residues corresponding to functional domains are indicated.
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Effect of Repressor Domain(s) on the Transcriptional Activity of
Fusion Proteins
The transcriptional effects of various ER constructs were examined
using the estrogen-responsive reporter, ERE2-tk109-luc, in ER-negative
TSA-201 cells (Fig. 2
). In the absence of
transfected ER, E2 did not stimulate the reporter activity
in these cells. Cotransfection with an expression vector encoding wtER
conferred 10-fold estrogen responsiveness. Control transfections with
empty expression vectors, or with plasmids containing unrelated
proteins, did not alter estrogen responsiveness, either in the absence
or presence of the wtER (data not shown). The truncated ER variant,
CDE, also conferred estrogen responsiveness, but the
E2-stimulated reporter activity was about half that
obtained with wtER. The activity of CDE presumably represents
transcription stimulated by the TAF-2 domain in the absence of TAF-1.
In contrast, the fusion protein CDE-RD1 showed no estrogen-induced
stimulation of reporter gene expression. RD1-CDE and RD13-CDE were
also deficient in E2 responsiveness (data not shown). These
data suggest that the fusion proteins are not expressed, are defective
in E2 binding, or that the repressor domain(s) inhibit the
activity of TAF-2, resulting in the loss of E2-induced
transcriptional activity. CDE-RD1 was chosen for more detailed
characterization (see Discussion).

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Figure 2. Fusion Protein CDE-RD1 Is Transcriptionally
Inactive
TSA-201 cells were transfected with 500 ng/well ERE2-tk109-luc reporter
plasmid and 50 ng/well of control vector (pCMX-CAT), pCMX-ER (wtER),
pCMX-CDE, or pCMX-CDE-RD1. After transfection, cells were treated and
assayed as described in Materials and Methods. Results
of all luciferase assays are shown as the mean ± SD
of quadruplicate transfections.
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Expression of CDE-RD1
The extreme N-terminal domain of NCoR has been shown to
target NCoR for proteasomal degradation (56). Therefore, the N-terminal
repressor domain might target the fusion protein for degradation,
resulting in poor expression. To examine this possibility, the presence
of CDE-RD1 in transfected cells was confirmed using immunocytochemistry
and Western blotting. Immunostaining using the anti-ER antibody H222
showed that wtER was expressed in the nuclei of TSA-201 cells
transfected with pCMX-ER (Fig. 3A
). Both
CDE and CDE-RD1 were also expressed in the nuclei of the transfected
cells (Fig. 3
, B and C). In contrast, no staining was detected in cells
transfected with empty vector (Fig. 3D
) or when control rat IgG was
used in place of H222 (data not shown).

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Figure 3. CDE-RD1 Is Expressed in Transfected Cells
AD, TSA-201 cells were transfected and subjected to
immunostaining as described in Materials and Methods.
Photomicrographs are shown at 400x magnification. Staining is visible
mainly in nuclei, as indicated by arrowheads. E, Western
blot. Nuclear extracts were prepared, blotted, and probed as described
in Materials and Methods. Lanes 14 represent cells
transfected with no ER, wtER, CDE, and CDE-RD1, respectively. The scale
at left indicates the position and molecular mass (kDa)
of marker proteins.
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Nuclear extracts of transfected TSA-201 cells were also analyzed by
Western blot (Fig. 3E
). Antibody H222 revealed similar levels of wtER
and CDE-RD1 (lanes 2 and 4) and a greater level of expression of CDE
(lane 3). The mobilities of the wtER, CDE, and CDE-RD1 bands are
consistent with their predicted molecular masses of 66, 45, and 82 kDa,
respectively. Two faint bands of smaller molecular mass were detected
in lanes 2 and 4. These bands may result from proteolytic degradation
of wtER (lane 2) and CDE-RD1 (lane 4). No ER expression could be
detected when the cells were transfected with empty vector (lane 1).
The immunostaining and Western blotting demonstrate that the expression
and localization of CDE-RD1 in transfected TSA-201 cells are similar to
that of wtER. Thus, lack of CDE-RD1 activity is not simply due to
degradation.
Hormone Binding to CDE-RD1
Although CDE-RD1 contains the ER ligand-binding domain, it is
possible that the attachment of RD1 could interfere with estrogen
binding, accounting for the loss of transcriptional activity. We
therefore examined the affinity of the fusion protein for estrogen.
CDE-RD1 was synthesized by in vitro
transcription/translation, and Scatchard analysis of
125I-estradiol binding yielded a Kd of 0.38
nM (data not shown), which is comparable to that of the
wtER (0.5 nM) (57). Thus, the absence of transcriptional
activity by CDE-RD1 is not due to loss of hormone binding.
DNA binding of CDE-RD1
The inactivity of CDE-RD1 could also be caused by loss of
DNA binding. The ability of CDE-RD1 to bind to the ERE
in vitro was examined using electrophoretic mobility shift
assays. Nuclear extracts from TSA-201 cells transfected with expression
vectors for wtER, CDE, CDE-RD1, or no ER (empty vector) were incubated
with a radiolabeled ERE probe (Fig. 4
).
The binding of wtER to the ERE probe is shown in lane 2. The CDE
fragment also binds to the ERE and exhibits more rapid migration,
consistent with the lower molecular mass of CDE (lane 3). Conversely,
the larger CDE-RD1 protein produced a more slowly migrating band (lane
4), which was also less intense. Preincubation with anti-ER antibody
(AER308) supershifted the wtER, CDE, and CDE-RD1 bands, confirming the
identity of the proteins (data not shown). The bands were also
eliminated by a 100-fold excess of nonradioactive probe, indicating
specific binding to the ERE (data not shown). These and other
experiments confirm that CDE-RD1 binds to DNA, although the amount of
binding was consistently reduced, suggesting weaker affinity, or
reduced stability, of the protein-DNA complex.

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Figure 4. CDE-RD1 Binds to the ERE
Nuclear extracts from TSA-201 cells transfected with empty vector (no
ER), wtER, CDE, or CDE-RD1 were incubated with 32P-labeled
ERE and analyzed by PAGE as described in Materials and
Methods.
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Coactivator Binding to CDE-RD1
Because the ligand binding domain of ER is fully intact in
CDE-RD1, the fusion protein might still be able to bind SRC-1. We
examined this possibility using GST pull-down assays. GST was fused to
the central region [amino acid (a.a.) 661 to a.a. 855] of SRC-1,
which contains three LXXLL (where L is leucine and X is any a.a.)
motifs (also called NR boxes) that are important for binding to nuclear
receptors, including ER (11, 21, 58, 59, 60). 35S-labeled wtER
or CDE-RD1 was incubated with GST, or with the GST-SRC-1 fusion
protein, in the presence or absence of E2 (Fig. 5
). Unmodified GST bound very little wtER
or CDE-RD1. In contrast, GST-SRC-1 bound wtER in an
E2-dependent manner, reflecting hormone-dependent
association of the receptor and coactivator. Similar results were
obtained with CDE-RD1, indicating that the presence of the repressor
domain does not preclude binding to SRC-1. Thus, CDE-RD1 is
transcriptionally inactive despite its ability to bind DNA, ligand, and
coactivators.

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Figure 5. CDE-RD1 Interacts with GST-SRC-1 in
Vitro
35S-labeled wtER (upper panel) or CDE-RD1
(lower panel) were applied to glutathione-agarose beads
pretreated with bacterially expressed GST or GST-SRC-1 in the presence
or absence of 10 nM E2. Bound proteins were
eluted, resolved by 10% SDS-PAGE, and visualized by autoradiography.
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Inhibition of wtER Transcriptional Activity by ER-NCoR
Fusions
All three fusion proteins (CDE-RD1, RD1-CDE, and RD13-CDE) were
examined for their abilities to inhibit the activity of wtER.
ER-deficient TSA-201 cells were transfected with an estrogen-responsive
reporter plasmid, a constant amount of wtER expression vector, and
varying amounts of the fusion protein vectors, such that the ratio of
chimera/wt ER ranged from 0.1 to 10. After transfection, the cells were
treated with or without E2. As shown in Fig. 6A
, each of the fusion proteins inhibited
wtER activity in a dose-dependent manner. In cells transfected with a
10-fold excess of CDE-RD1 or RD1-CDE vectors, E2-induced
transcription was almost completely (
98%) inhibited. RD13-CDE
produced substantial inhibition, but it was not as potent as the other
two fusion proteins. When the reporter gene was replaced with tk109-luc
(the same reporter without an ERE), E2 did not increase
transcription, and the activity of the reporter was not affected by the
addition of the fusion proteins (data not shown). These data indicate
that fusion proteins of RD1 and CDE have a strong dominant negative
effect with respect to the wtER and that the dominant negative effect
is dependent on the presence of the ERE.

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Figure 6. Repressor Fusion Proteins Block wtER Activity, and
This Dominant Negative Activity Requires DNA Binding
TSA-201 cells were transfected with 500 ng/well ERE2-tk109-luc, 5
ng/well pCMX-ER, and 0.5 to 50 ng/well of fusion protein expression
vectors in panel A or 5 or 50 ng/well of DNA binding mutant in panel B,
resulting in the indicated range of fusion/wt vector ratios. Empty
vector was added to provide equal amounts of total DNA in each well. C,
The dominant negative effect of CDE-RD1 is specific for ER. TSA-201
cells were transfected with 100 ng/well GRE-tk109-luc, 5 ng/well
RSV-GR, and 50 ng/well empty vector (pCMX), wtER, or CDE-RD1. Six hours
after transfection, cells were treated with 100 nM
dexmethasone or control vehicle and assayed for luciferase activity
48 h later.
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To further investigate whether the dominant negative activity of
CDE-RD1 requires DNA binding, a DNA binding-deficient version of
CDE-RD1 was created by introducing three point mutations (E203G, G204S,
and A207V) into the C region of CDE-RD1. These three amino acids in
nuclear receptors have been shown to play a key role in DNA binding by
the ER (61, 62). The introduction of the DNA-binding domain mutations
into CDE-RD1 eliminated ERE binding in electrophoretic mobility shift
assays, but did not affect the expression level of the fusion protein
in Western blot (data not shown). In transient transfection assays,
when wtER was cotransfected with CDE-RD1 at a ratio of 1:1, the wtER
activity was strongly inhibited, whereas no repression was observed
when wtER was cotransfected with DNA-binding mutant (Fig. 6B
). These
results confirm that full dominant negative activity by the fusion
protein is dependent on DNA binding. Notably, the DNA binding-deficient
mutant produced some inhibition when transfected at 10-fold excess over
wtER. This inhibition may be mediated by protein-protein interactions.
Mutating the DNA-binding domain also abolished the basal silencing
effect of the fusion protein (data not shown).
The effect of CDE-RD1 was also tested with respect to glucocorticoid
receptor (GR)-mediated transcription to examine whether its inhibitory
activity was specific for the ER, or also occurred with other steroid
receptors. In TSA-201 cells transfected with RSV-GR (a GR expression
vector) and a glucocorticoid-responsive reporter (GRE-tk109-luc),
treatment with dexamethasone induced a 3.8-fold increase in luciferase
activity (Fig. 6C
). Cotransfection of wtER expression vector with GR at
a 10:1 ratio reduced (37%), but did not eliminate, the response to
dexamethasone. This inhibition may be due to competition for
transcriptional cofactors (squelching). In contrast, cotransfection
with CDE-RD1 expression vector did not inhibit dexamethasone-induced
activity, and dexamethasone induced a 4.2-fold increase of reporter
activity. Thus, CDE-RD1 does not suppress GR-mediated transcription,
suggesting that its dominant negative effect is specific for
ER.
Comparison of CDE-RD1 with Other Dominant Negative ER Mutants
Several dominant negative ER mutants have previously been created
by chemical and site-directed mutagenesis (52, 53, 54). We compared the
dominant negative activity of CDE-RD1 to two of these mutants: L540Q, a
point mutation in the TAF-2 domain, and ER1536, a C-terminal
truncation of the TAF-2 domain. TSA-201 cells were cotransfected with
the ERE2-tk109-luc reporter plasmid, wtER, and expression vectors for
L540Q, ER1536, or CDE-RD1 (at ratios to the wtER ranging from 1 to
10) (Fig. 7
). At a ratio of 1:1, CDE-RD1
exhibited about twice the inhibitory activity of either L540Q or
ER1536. At a ratio of 10:1, CDE-RD1 completely blocked wtER activity,
whereas the inhibition by L540Q and ER1536 was partial.

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Figure 7. Comparison of CDE-RD1 with Other Dominant Negative
ER Mutants
TSA-201 cells were transfected with 500 ng/well ERE2-tk109-luc, 5
ng/well of pCMX-ER, and 550 ng/well of expression vectors for L540Q,
ER1536, or CDE-RD1 producing mutant/wt ratios ranging from 1 to 10,
as indicated. Empty vector was added to make the total DNA in each well
equal. Cells were treated and assayed as described in Materials
and Methods.
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Inhibition of ER-Dependent Responses in Breast Cancer Cells
Since dominant negative ER mutants might be useful for inhibiting
estrogen action in breast cancer cells, we examined the dominant
negative effect of CDE-RD1 in two breast cancer cell lines. ER-negative
MDA-MB-231 cells were cotransfected with wtER and CDE-RD1. At a ratio
of 1:1, E2 induction of wtER activity (assessed by the
reporter ERE2-tk109-luc) was inhibited by 50%, and a 10:1 ratio
produced 85% inhibition (Fig. 8A
). In
ER-positive T47D breast cancer cells, CDE-RD1 inhibited endogenous ER
activity in a dose-dependent manner (Fig. 8B
). Using 100 ng of CDE-RD1
expression vector, estrogen-stimulated reporter activity was decreased
by 70%. Inhibition was almost complete (>90%) using 500 ng of the
CDE-RD1 expression vector. These data show that CDE-RD1 exhibits a
dominant negative effect with respect to endogenous receptor in breast
cancer cells.

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Figure 8. CDE-RD1 Inhibits wtER Transcriptional Activity in
Breast Cancer Cells
A, MDA-MB-231 (ER-negative) breast cancer cells were transfected with 1
µg/well ERE2-tk109-luc, 5 ng/well of wtER, and 5 ng/well or 50
ng/well of CDE-RD1 expression vectors resulting in 1:1 or 10:1
CDE-RD1/wt ratios. After transfection, cells were treated and assayed
as described in Materials and Methods. B, T47D
(ER-positive) breast cancer cells were transfected with the
ERE2-tk109-luc (2 µg/well) and the CDE-RD1 expression vector (0500
ng/well). Forty-eight hours after transfection, cells were treated with
1 nM E2 or control vehicle and assayed for
luciferase activity.
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To study the effect of CDE-RD1 on estrogen-stimulated growth of breast
cancer cells, a retroviral delivery system was used to achieve more
quantitative introduction of the mutant into the cells. T47D cells were
transduced with medium collected from packaging cells transfected with
empty (control) or CDE-RD1-containing retroviral vectors and assayed
for cell growth. CDE-RD1 mRNA was detected by RT-PCR in cells
transduced with CDE-RD1 retrovirus but not with empty retrovirus (data
not shown), confirming delivery of the CDE-RD1 gene. As shown in Fig. 9
, at assay day 7, the cells transduced
with control retrovirus grew well in response to E2
(>400% increase over day 1). In contrast, the cells transduced with
CDE-RD1 retrovirus proliferated significantly slower in the presence of
E2 (90% increase over day 1) and did not grow at all in
the absence of E2.

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Figure 9. CDE-RD1 Suppresses the Growth of Breast Cancer
Cells
T47D cells were transduced with control or CDE-RD1-containing
retrovirus as described in Materials and Methods. The
cells were treated with 1 nM E2 or control
vehicle, and the cell density was determined at the indicated
intervals. Each point represents the mean ± SD of
five replicate wells.
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Silencing Effect of CDE-RD1
Unliganded TR and RAR repress or silence basal gene transcription
via interaction with the NCoR complex (33, 36, 55, 63). Since
transcriptional silencing is mediated by the repressor domains of NCoR,
we tested whether CDE-RD1 might also repress basal transcription of
ER-responsive genes. The activity of the ERE2-tk109-luc reporter gene
in TSA-201 cells transfected with control vector (100 ng) and treated
with control vehicle (ethanol) established the level of basal
expression (indicated by dashed line in Fig. 10A
). Transfection with ER (100 ng)
produced higher reporter activity, even in the absence of
E2. This may represent transcriptional activity by the
unliganded receptor (64), or it may be due to traces of estrogen, even
though the cells were cultured in estrogen-depleted medium for 4 days.
In contrast, transfection with CDE-RD1 decreased reporter activity
below the basal level. This suppression was dose-dependent, with 100 ng
of expression vector producing a 70% decrease in activity (Fig. 10A
).
When CDE-RD1 was cotransfected with ER, the unliganded ER activity was
also inhibited by CDE-RD1 (Fig. 10B
). This basal inhibitory effect was
not detected when tk109-luc was used instead of ERE2-tk109-luc (data
not shown), indicating that it requires the presence of the ERE. Thus,
incorporation of RD1 into ER produces a protein that silences basal
reporter gene transcription and unliganded wtER activity.

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Figure 10. CDE-RD1 Silences Basal and Unliganded wtER Gene
Transcription
A, TSA-201 cells were transfected with 500 ng/well ERE2-tk109-luc and
100 ng of control or wtER expression vectors, or 1, 10, or 100 ng of
pCMX-CDE-RD1. Basal activity is indicated by a dashed
line. B, TSA-201 cells were transfected with 500 ng/well
ERE2-tk109-luc and 5 ng of control or wtER expression vectors, or 5 ng
of wtER expression vector plus 50 ng of pCMX-CDE-RD1. Six hours after
transfection, fresh estrogen-depleted medium was added and cells were
assayed as described in Materials and Methods.
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Mechanism of Silencing by CDE-RD1
As previously described, NCoR recruits HDAC (36), which inhibits
the transcription of many promoters. We used the HDAC inhibitor,
trichostatin A (65, 66, 67, 68, 69), to examine the role of HDAC in the action of
CDE-RD1. TSA-201 cells were transfected with the ERE2-tk109-luc
reporter plasmid and control vector, wtER, or CDE-RD1, as shown in Fig. 11
. In the absence of ER (control), the
reporter activity was unaffected by E2, and the fold
induction by trichostatin A was similar in the unliganded and
E2-treated groups (23- and 27-fold, respectively). This
induction suggests that the reporter construct is sensitive to
trichostatin A, independent of the ER. In cells transfected with wtER,
both ligand-independent and E2-stimulated activity were
strongly increased by trichostatin A, but the fold increase was less
for the E2-treated group (14-fold vs. 27-fold).
In cells transfected with CDE-RD1, trichostatin A increased basal
transcription, but the fold increase was smaller than that in control
cells, and E2 produced no further induction (15-fold and
11-fold for the unliganded and E2-treated groups,
respectively). The inability of trichostatin A to completely reverse
silencing by fusion protein RD1 suggests that the inhibition by RD1 may
involve an HDAC1-independent mechanism.

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Figure 11. Effect of the HDAC1 Inhibitor, Trichostatin A, on
Transcriptional Silencing
TSA-201 cells were transfected with 500 ng/well ERE2-tk109-luc and 5
ng/well of control, or wtER, or 55 ng CDE-RD1 expression vectors. Cells
were treated with or without 1 nM E2 and with
or without 100 nM trichostatin A. Results are shown as the
mean ± SD of quadruplicate transfections.
Inset, Same data plotted on expanded scale to facilitate
comparison of different ER regimens in the absence of trichostatin A.
|
|
 |
DISCUSSION
|
---|
Several dominant negative ER mutants have been created previously
by chemical or site-directed mutagenesis (51, 52, 53, 54). These mutants
inhibit ER stimulation of target genes, presumably by competing for
binding to the ERE, or by forming inactive dimers with the wtER.
Recently, studies of dominant negative mutants of the TR revealed an
unanticipated role for CoRs in their inhibitory activity (70, 71). For
example, mutations in the TR that specifically disrupt binding to NCoR
or SMRT almost completely eliminate dominant negative activity of
naturally occurring mutants that cause resistance to thyroid hormone
(70, 72). These features suggest that the recruitment of CoRs is an
important component of inhibition by these mutants. Because the wtER
interacts relatively weakly with CoRs (47, 48), we reasoned that the
dominant negative activity of ER mutants might be enhanced if they were
fused to CoRs.
Three different fusion proteins between ER and NCoR were created in an
effort to identify a configuration that would retain ER properties such
as dimerization and DNA binding, but allow the repressor domains to
contribute to transcriptional inhibition. The smaller proteins, CDE-RD1
and RD1-CDE, contained one of the repressor domains (RD1) of NCoR fused
to either the carboxy terminus or the amino terminus of a truncated
form of the ER (CDE). In another mutant, all three repressor domains
(RD13) were fused to the amino terminus. The CDE region of the ER was
used, rather than the full-length ER, for several reasons. Elimination
of the TAF-1 domain (A/B region) was anticipated to reduce the
constitutive transcriptional activity conferred by this region (3, 73).
As shown in Fig. 2
, CDE retained about half of the transcriptional
activity of the wtER. These experiments also suggest that this
truncated form of the receptor retains essential functions of the ER,
such as estrogen binding and the ability to bind and activate target
genes. We also eliminated the F domain from these constructs. Although
the function of this domain is not fully understood, it may play a role
in functional differences mediated by agonists and antagonists (74, 75). Deletion of the A/B and F domains permitted creation of fusion
proteins with molecular masses similar to that of wtER. The predicted
molecular mass for the fusion protein with RD1 is 82 kDa, which is
close to that of wtER (66 kDa). In contrast, the predicted size of the
RD13-CDE fusion protein is much greater (233 kDa). In electrophoretic
mobility shift assays, CDE-RD1 (Fig. 4
) and RD1-CDE (data not shown)
retained dimerization and ERE binding, whereas little or no DNA binding
was seen with the larger RD13-CDE fusion protein (data not shown).
For this reason, we concentrated the studies on CDE-RD1, which also
retained E2 binding, nuclear targeting, and a near-normal
level of protein expression (Fig. 3
). It is possible that the larger
fusion protein (RD13-CDE) blocks ERE binding or is unstable under
conditions of the gel shift assay.
The addition of the RD1 domain suppressed the activity of the truncated
ER, in both the absence and presence of E2. Current models
hold that the binding of ligand induces conformational changes in
nuclear receptors that release the repressor complex (for receptors
like TR or RAR) and allow the recruitment of a coactivator (CoA)
complex that stimulates gene transcription. In the ER, helices 3, 5,
and 12 (18, 76, 77) and several residues in the ligand-binding domain,
such as Leu417 and Glu420 (78), are important for the interaction with
the coactivator, SRC-1. These regions of the ER are intact in CDE,
which retains ligand-dependent transcriptional activation (Fig. 2
).
Therefore, it is intriguing that the repressor domain of NCoR is
sufficient to suppress the transcriptional activity of the TAF-2
domain. One possible explanation is that the repressor domain, which
cannot be released from the fusion protein, precludes the recruitment
of SRC-1. However, the GST pull-down assay demonstrated that CDE-RD1
retains interactions with SRC-1 (Fig. 5
). Since GST pull-down assay is
a simplified system and can only reflect in vitro behavior,
we cannot rule out the possibility that the repressor domain might
still preclude the recruitment of SRC-1 or other components of the CoA
complex in vivo. Based on reporter gene assays, even if
SRC-1 is recruited to CDE-RD1 through a ligand-induced conformation
change, the TAF-2 domain remains inactive, indicating that the
repressor domain might suppress the TAF-2 activity by inhibiting CoA
function. Thus, the repressor complex may inhibit transcription so
strongly that it must be removed before the HAT activity of the CoA
complex can become effective.
Consistent with the irreversible inactivation of the ER by the
repressor domain, fusion proteins such as CDE-RD1 are also effective as
dominant negative inhibitors of wtER. CDE-RD1 inhibits wtER activity
under both liganded (Fig. 6A
) and unliganded conditions (Fig. 10B
).
CDE-RD1 did not inhibit GR-mediated transcription (Fig. 6C
), indicating
that its dominant negative effect is specific for the ER. When compared
with other dominant negative ER mutants, such as L540Q (a point
mutation) and ER1536 (a carboxy-terminal truncation), CDE-RD1
appeared to exert more potent inhibitory activity (Fig. 7
). It is
notable, however, that the ER dominant negative mutants are reasonably
effective in the absence of the RD1 fusion protein. It is possible that
these mutants interact with cellular CoRs more effectively than the
unliganded wtER, but this remains to be investigated. These previously
reported dominant negative mutants of ER result from mutation or
deletion in helix 12 of the TAF-2 domain, causing loss of the ability
to recruit coactivators. Our data indicate that CDE-RD1 can interact
with SRC-1 and so represents a novel class of dominant negative ER
mutants.
Given the strong dominant negative activity of CDE-RD1, we decided to
further explore its effectiveness using breast cancer cell lines. As
shown in Fig. 8
, CDE-RD1 inhibited both exogenous and endogenous ER
activity in this model. Furthermore, using retroviral delivery of
CDE-RD1, we were able to demonstrate significant growth suppression of
these breast cancer cells (Fig. 9
). Combined with another recent study
using adenovirus to deliver dominant negative ER mutants into breast
cancer cells (79), these data suggest a potential utility for targeting
ER in the treatment of breast cancer.
In addition to its ability to block E2-mediated
transcription, CDE-RD1 also suppressed basal gene transcription in a
manner reminiscent of that seen with unliganded TR and RAR (80, 81, 82)
(Fig. 10A
). Transcriptional silencing by unliganded TR and RAR involves
the recruitment of CoRs such as NCoR and SMRT, which in turn recruit a
complex containing Sin3 and HDAC (36). HDAC deacetylates
histone, a process that is proposed to stabilize chromatin structure
and thereby suppress gene transcription (reviewed in Refs. 42, 43, 44, 45, 46).
Regions within NCoR that have been shown to mediate the interaction
with Sin3 include an amino-terminal SIN interaction domain (SID 1, a.a.
254312) and a second domain (SID 2, a.a. 18291940) (36, 63). The
RD1 (a.a. 1312) fragment studied here includes SID1. Therefore,
fusing RD1 with ER might be expected to recruit SIN3 and suppress gene
transcription by a mechanism that involves the recruitment of HDAC.
Based on this idea, we used trichostatin A, an inhibitor of HDAC1, to
assess its effect on transcriptional suppression by CDE-RD1 (Fig. 11
).
Trichostatin A increased the activity of the reporter gene, even under
control conditions, suggesting that the promoter is repressed under
basal conditions by histone deacetylation. However, the effect of
trichostatin A on ERE2-tk109-luc activity was much greater in the
presence of E2-stimulated ER, indicating that inhibition of
histone deacetylation enhances transcriptional activation by the ER
(83, 84). Unexpectedly, trichostatin A did not reverse the inhibitory
effect of the repressor domain in CDE-RD1. Similar findings have been
reported for suppression by the retinoblastoma protein (Rb). Rb is a
tumor suppressor protein that inhibits gene transcription, in part
through interactions with E2F, a family of cell cycle transcription
factors (85). Rb has been shown to suppress gene transcription through
both HDAC-dependent (69, 86, 87) and HDAC-independent (87, 88)
mechanisms. The amino-terminal region of NCoR (a.a. 11017) has been
shown to physically interact with basal transcription factors in
vivo and in vitro, and NCoR can inhibit the recruitment
of the TAFII-32 subunit of TFIID by TFIIB (89). RD1 (a.a.
1312) used in our study is included within this amino-terminal
region. Therefore, it is possible that CDE-RD1 might silence gene
transcription through interactions with the basal transcriptional
machinery. CDE-RD1 might also recruit an unidentified HDAC that is
resistant to inhibition by trichostatin A.
In summary, we have demonstrated that fusing a repressor domain from
NCoR to a truncated form of ER can irreversibly silence ER function and
inhibit wtER activity when cotransfected into cells. This strategy
offers a new approach for the study of estrogen action and, with
modification, might be useful for gene therapy for breast, and other
estrogen-responsive, neoplasms. In addition, fusion with CoRs should be
an effective way of generating dominant negative mutants for other
transcription factors or CoAs.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The reporter plasmid ERE2-tk109-luc has been described
previously (90). GRE-tk109-luc was created by deleting the DR4 region
from GRE-tk-DR4-luc, and rous sarcoma virus (RSV)-GR expression vector
was used as previously described (91, 92). The pCMX-NCoR expression
vector was provided by M. G. Rosenfeld (University of California,
San Diego, CA) (33). pSG5-HEGO (wtER expression vector) was provided by
Pierre Chambon (Université Louis Pasteur, Strasbourg, France)
(93). The pCMX-NCoR-RD plasmid, containing the entire repressor domain
region of NCoR, including RD1, RD2, and RD3 (a.a. 11551), was created
by deleting a BstXISalI fragment from pCMX-NCoR
and introducing an artificial EcoRI site. Fusion proteins of
ER and NCoR were created as follows. To create pCMX-RD13-CDE, the CDE
domains of ER (a.a. 181553) were PCR-amplified from pSG5-HEGO by PCR
using the primers,
5'-GATCGGTACCGCGGGCATGGAATTCGAGACTCGCTACTGTGCAGT-3' and
5'-CTAGCTAGGCGGCCGCTAGCGCTAGTGGGCGCATGTAGGC-3'. The PCR product was
digested with EcoRI and NheI and inserted between
the corresponding sites in pCMX-NCoR-RD. To create pCMX-RD1-CDE,
repressor domain 1 (RD1) of NCoR (a.a. 1312) was PCR amplified from
pCMX-NCoR using the primers
5'-GATCGATCGCGGCCGCGCGGGCATGTCAAGTTCAGG-TTATCC-3' and
5'-CTAGGGATCCGCTAGCGAATTCATCATAACGTTGGCAGATTT-3'. Subsequently, the
repressor domain region of pCMX-RD13-CDE was removed by restriction
digestion and replaced with RD1.
pCMX-CDE-RD1 was constructed by ligating the
KpnINotI fragment of CDE and the
NheINotI fragment of RD1 into the pCMX
expression vector. A DNA binding-deficient version of pCMX-CDE-RD1 was
created by introducing three point mutations (E203G, G204S, and A207V)
into the C region of CDE-RD1. pCMX-CDE was created by deleting the RD1
fragment of pCMX-CDE-RD1. pCMX-ER was constructed by cloning an
EcoRI fragment of pSG5-HEGO, containing the entire cDNA for
wtER, into pCMX. The dominant negative ER mutant L540Q was created by
changing codon 537 of the ER gene from TAT (tyrosine) to TAG (stop).
The codon for a.a. 540 was replaced by a stop codon to create the
dominant negative ER mutant, ER1536. pCMX-CAT was constructed by
ligating the NotI fragment of pOP13CAT
(Stratagene, La Jolla, CA), containing the cDNA for
chloramphenicol acetyltransferase (CAT), into the NotI site
of pCMX. The CAT gene product is unrelated to ER or NCoR and was used
as a negative control. All constructs were verified by DNA
sequencing.
Cell Culture
Sera and media were purchased from Life Technologies, Inc. (Gaithersburg, MD) and Sigma (St. Louis, MO).
TSA-201 cells, derived from human embryonic kidney 293 cells (94) were
grown in DMEM supplemented with 10% FBS. MDA-MB-231 (ER-negative)
cells and T47D (ER-positive) cells, provided by V. Craig Jordan
(Northwestern University Medical School, Chicago, IL), are human breast
carcinoma cell lines. MDA-MB-231 cells were cultured in Eagles MEM
supplemented with 5% calf serum, nonessential amino acids, and 10
mM HEPES. T47D cells were cultured in RPMI 1640
supplemented with nonessential amino acids and 10% FBS. Penicillin
(100 U/ml) and streptomycin (100 µg/ml) were included in all media.
Four days before transfection, cells were harvested using phenol
red-free trypsin-EDTA and cultured in estrogen-depleted media (prepared
without phenol red and supplemented with sera extracted three times
with dextran-coated charcoal).
Transfections and Luciferase Assays
Cells were transferred to 12-well plates in estrogen-depleted
medium 1 day before being transfected with ERE2-tk109-luc and
expression vectors. Within each experiment, the total amount of DNA
transfected into each group of cells was kept constant by the addition
of the pCMX empty vector. TSA-201 cells were transfected with calcium
phosphate and MDA-MB-231 cells with liposomes as previously described
(90, 95). T47D cells were transfected by electroporation, using a
Bio-Rad Laboratories, Inc. (Hercules, CA) Gene Pulser (300
V, 0.4-cm gap cuvette) with a capacitance extender (960 µfarads) and
pulse controller (infinite resistance), and grown in estrogen-depleted
medium for 24 h before treatment. E2 was added to
treatment media as an ethanol stock solution, and ethanol was added to
control wells to produce the same final solvent concentrations
(typically 0.1%). Cells were treated for 40 (TSA-201) or 48 h
(MDA-MB-231, T47D) and then assayed for luciferase activity as
previously described (95).
Electrophoretic Mobility Shift Assays
Labeled probe was prepared by annealing two oligonucleotides
(5'-CAAGTCAGGTCACAGTGACCTGATCAA-3' and 5'-TTGATCAGGTCACTGTGACCTGA-3')
containing the Xenopus vitellogenin A2 ERE and
filling the overhanging ends in the presence of
[
-32P]dTTP (6000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL). Nuclear extracts were prepared
as previously described (96) from TSA-201 cells. The cells were
estrogen-depleted for 4 days, transfected with pCMX (no ER), pCMX-ER,
pCMX-CDE, or pCMX-CDE-RD1 (20 µg DNA/15-cm plate) and incubated in
estrogen-depleted medium for 2 days. Immediately before harvest, cells
were treated for 2 h with 20 nM E2 to
increase nuclear localization of the ER. Protein concentrations of the
extracts were determined with a protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts (11 µg) of nuclear protein
were used in each reaction. Extracts were preincubated with a binding
buffer containing 20 mM HEPES, 40 mM KCl, 1
mM MgCl2, and 11 µg sperm DNA at 4 C for 30
min and then incubated with labeled ERE probe at 4 C for an additional
30 min, in a total volume of 20 µl. The monoclonal anti-ER antibody
AER308 (NeoMarkers, Fremont, CA; Ref. 97) or a 100-fold excess of
nonradioactive ERE probe was added in some of the experiments. The
samples were loaded onto prerun 4% polyacrylamide gels, and
radioactivity was visualized by autoradiography.
Western Blots and Immunocytochemistry
Western blots were performed on the same nuclear extracts
described above. The extracts were fractionated in 415% SDS-PAGE
ready gel (Bio-Rad Laboratories, Inc.) and transferred
onto hydrophobic polyvinylidene difluoride membranes
(Amersham Pharmacia Biotech). Immunodetection was
performed using rat monoclonal ER antibody H222 and a LumiGLO kit
(Kirkegaard & Perry Laboratories, Gaithersburg, MD). The
antibody and protocols for immunoblotting and immunocytochemistry were
kindly provided by Geoffrey L. Greene (University of Chicago, Chicago,
IL). This antibody reacts with an epitope in the E domain of the ER
(98). For immunocytochemical staining, TSA-201 cells were grown in
estrogen-depleted media in 10-cm plates and transfected with 10 µg of
pCMX, pCMX-ER, pCMX-CDE, or pCMX-CDE-RD1. Twenty-four hours after the
transfection, cells were replated in Lab-Tek chambered glass slides
(Nalge Nunc International, Naperville, IL) and treated
with 20 nM E2 for another 24 h. The cells
were washed with PBS and fixed with 4% paraformaldehyde. After being
permeablized with methanol and acetone, cells were incubated with H222
or normal rat IgG in 10% goat serum for 60 min and stained using a
Histostain-Plus kit (Zymed Laboratories, Inc. South San
Francisco, CA) according to the manufacturers directions.
Estrogen Binding Assays
CDE-RD1 and wtER were synthesized in vitro from the
corresponding plasmids using a TNT Coupled Reticulocyte Lysate System
(Promega Corp., Madison, WI). Translation products were
assayed for 125I-estradiol binding by the charcoal
absorption method (99), as previously described (90).
GST Pull-Down Assays
ER and CDE-RD1 were translated in vitro as described
above in the presence of [35S]methionine. The central
region of SRC-1 (a.a. 661855) was fused to GST in the expression
vector pGEX2TK (Pharmacia Biotech, Piscataway, NJ) to
create a GST-SRC-1 fusion protein. GST and GST-SRC-1 were transformed
into Escherichia coli BL21(DES)/pLys, grown to an
OD600 of 0.8 and incubated with 0.1 mM
isopropyl-ß-D-thiogalactopyranoside overnight at
30 C. The bacteria were resuspended in NET buffer (150 mM
NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.4) with
proteinase inhibitors and sonicated for 15 sec. After centrifugation,
the supernatants were incubated with glutathione-agarose
(Sigma) for 30 min at 4 C. The supernatant was removed and
the agarose was washed five times with NET buffer plus proteinase
inhibitors. Expression and purification of fusion proteins were
confirmed by Coomassie blue staining. 35S-labeled wtER or
CDE-RD1 was incubated for 30 min at 4 C with agarose-bound GST or
GST-SRC-1 in the presence of 10 nM E2 or
control vehicle. After washing five times with NET buffer plus
proteinase inhibitors, bound proteins were recovered, resolved by 10%
SDS-PAGE, and visualized by autoradiography.
Construction of Retroviral Vector, Transient Production of
Retrovirus, and Transduction of T47D Cells
CDE-RD1 was inserted into the multiple cloning site of the
retroviral vector pLXSN (CLONTECH Laboratories, Inc. Palo
Alto, CA), and empty vector was used as a control. pLAPSN
(CLONTECH Laboratories, Inc.), a vector with the same
backbone but containing the alkaline phosphatase gene, was used to
optimize transfection and transduction conditions. After optimization,
alkaline phosphatase expression (assayed with Western Blue Stabilized
Substrate for Alkaline Phosphatase; Promega Corp.) was
detected in up to 50% of T47D cells. The Phoenix amphotropic packaging
cell line was purchased from American Type Culture Collection (Manassas, VA) under the authorization of G. P.
Nolan (Stanford University, Stanford, CA). Phoenix cells were grown in
estrogen-depleted medium for 4 days and plated in 10-cm tissue culture
plates 24 h before transfection. CDE-RD1-containing retroviral
vector or control vector was transfected into Phoenix cells by the
calcium-phosphate/chloroquine method following the protocol provided by
G. P. Nolan. Packaging cells were provided with fresh medium
24 h after transfection and incubated at 32 C overnight. Culture
supernatants from packaging cells containing retroviral particles were
collected and centrifuged at 1500 rpm for 5 min to remove cell debris.
T47D cells were estrogen depleted for 4 days and plated at a density of
5 x 105 cells per 10-cm plate 1 day before the
transduction. Transduction was then performed by incubating T47D cells
overnight in viral medium containing 4 mg/ml polybrene
(Sigma). T47D cells were transduced twice to increase
transduction efficiency. pLAPSN was used in parallel to monitor the
transfection and transduction. A portion of the viral medium was used
to transduce NIH3T3 cells (a gift from Richard Longnecker, Northwestern
University, Chicago, IL), and the viral titer was determined as
described in the protocol provided by CLONTECH Laboratories, Inc. The maximum titer used for the transduction was
105 colony-forming units (cfu)/ml.
Growth Assay
T47D cells transduced with control or CDE-RD1 retrovirus were
seeded into four 96-well plates at a density of 3,000 cells per well on
day 0 and placed in a 37 C incubator. The following day (day 1), cell
density in one of the plates was measured by the tetrazolium reduction
assay (Promega Corp., Madison, WI) as described previously (90).
E2 (1 nM) or control vehicle was then
added to the other cells. At 48-h intervals, a plate was taken for
measurement of cell density and fresh media were applied to the
remaining plates.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. G. Rosenfeld for the pCMX-NCoR expression
vectors, Pierre Chambon for the pSG5-HEGO plasmids, Peter Kopp for
GRE-tk109-luc reporter, Ron Evans for the RSV-GR expression vector,
Craig Jordan for breast cancer cell lines, Richard Longnecker for the
NIH3T3 cell line, and Geoffrey L. Greene for the antibody H222 and
protocols used for immunoblotting and immunocytochemistry. We are also
grateful to Wade Johnson for advice with gel shift assays; Eun-Jig Lee
for help with immunocytochemical staining; Joanne McAndrews and Rachel
Duan for helpful discussions; and Jeff Weiss for the comments about the
paper.
 |
FOOTNOTES
|
---|
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry Building 15709, 303 East Chicago Avenue, Chicago, Illinois 60611.
This work was supported in part by NIH Grant DK-42144 and by Department
of Defense Grant USAMRDC B4337379.
Received for publication October 16, 1998.
Revision received August 11, 1999.
Accepted for publication September 9, 1999.
 |
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