From the Département de Biochimie, Université de Montréal, Montréal, Québec, Canada H3C 3J7
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ABSTRACT |
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Anti-estrogens like hydroxytamoxifen (OHT) have mixed agonist/antagonist activities, leading to tissue-specific stimulation of cellular proliferation. Partial agonist activity of OHT can be observed in vitro in endometrial carcinoma cells like Ishikawa. Here, we have compared several anti-estrogens (including extensively characterized OHT and pure anti-estrogens such as ICI164,384 and RU58,668, which are devoid of uterotrophic activity) for their capacity to stimulate promoters containing estrogen response elements (EREs) or AP1-binding sites (12-O-tetradecanoylphorbol-13-acetate response elements, TREs), the two types of DNA motifs known to mediate transcriptional stimulation by estrogen receptors. Assays were performed in Ishikawa cells either by transient transfection or by using cell lines with stably propagated reporter vectors. In transient transfection experiments, none of the anti-estrogens displayed agonist activity on the promoters tested. In contrast, significant transcriptional stimulation was observed with low concentrations of OHT and RU39,411 in Ishikawa cells stably propagating reporter constructs containing a minimal ERE3-TATA promoter. In addition, micromolar concentrations of OHT, but not of RU39,411, stimulated stably propagated AP1-responsive reporter constructs. No transcriptional stimulation of ERE- or TRE-containing promoters was observed with the pure anti-estrogens ICI164,384 and RU58,668. These results indicate that the presence of estrogen response elements in promoters is sufficient to mediate cell-specific agonism of anti-estrogens at the transcriptional level, and that stimulation of AP1 activity may be restricted to a subset of anti-estrogens possessing agonist activity on EREs. In addition, our results suggest that transient transfections do not fully recapitulate in vivo conditions required to observe agonist activity of anti-estrogens.
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INTRODUCTION |
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The estrogen 17-estradiol
(E2)1 regulates gene
transcription by binding to the estrogen receptor (ER), which interacts
with specific target DNA sequences known as estrogen response elements (EREs). When bound to DNA the ER stimulates transcription via two
transcription activation domains, AF-1 and AF-2 (1-3). AF-1 is located
in the poorly conserved N-terminal A/B domain of the ER (2, 4), whereas
AF-2 is found in the C-terminus of region E, the hormone-binding region
(5-8). Binding of E2 to the ER is thought to induce a conformational
change in the hormone binding domain, stimulating its transactivation
properties.
Different types of synthetic compounds have been developed that are capable of antagonizing ER action in reproductive tissues and, in particular, of blocking estradiol stimulation of cellular growth in breast and uterine tissues. These anti-estrogens act by competing with E2 for binding to the ER and block ER-mediated activation of transcription when co-administered with hormone (9). However, tamoxifen, one of the most widely used anti-estrogens in breast cancer treatment (10), can induce uterine cell growth in vivo in animal models (11) and in humans (12, 13). Hydroxytamoxifen also induces cellular proliferation (14, 15) and transcription of endogenous estrogen target genes such as the progesterone receptor (PR) gene in human endometria and cultured human endometrial carcinoma cells (15, 16). Other anti-estrogens, like ICI164,384 (17) or the more recently developed RU58,668 (18), were reported not to stimulate uterine cell growth and may therefore prove more appropriate for breast cancer therapy (17).
To better understand the mechanisms of tissue-specific estrogenic activity of anti-estrogens, we compared transcriptional activation of ERE-containing reporter constructs by full or partial anti-estrogens in the estrogen-dependent breast carcinoma cell line MCF7 and in the endometrial carcinoma cell line Ishikawa. We also assessed whether anti-estrogens may regulate transcription of target genes through AP1-binding sites (TPA response elements, TREs) rather than, or as well as, through EREs. Indeed, estrogenic stimulation of promoters containing TRE sites has been documented (19-22), a possible mechanism being direct interaction between ER and AP1 components (22). Assays used for investigating the contribution of EREs or TREs in transcriptional stimulation by anti-estrogens with partial agonist activity included, in addition to transient transfection assays in MCF7 or Ishikawa cells, direct hormonal stimulation of reporter vectors stably propagated as episomes in these cell lines. The latter assay was selected because of increasing evidence for mechanistic links between transcriptional stimulation and reorganization of chromatin structure. Comparison of the two types of assays and implications for the mechanism of cell-specific agonism by tamoxifen are discussed.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Materials--
17-Estradiol (E2) was purchased
from Sigma; RU58,668 (RUf) and RU39,411 (RUp) were generous gifts from
Dr. D. Philibert, Hoechst-Marion-Roussel, Romainville, France.
ICI164,384 was kindly provided by Dr. T. Willson, Glaxo-Wellcome
Research Institute, Research Triangle Park, NC. OHT and TAM were
purchased from Sigma. Cell culture media, fetal bovine serum, G418, and
hygromycin B were purchased from Life Technologies, Inc.
Plasmid Recombinants-- Expression vectors pSG5, pSG5-HEG0, and reporter recombinants Vit-tk-CAT (23) and STR-CAT (equivalent to construct 84-CAT in Ref. 24) were kindly provided by Dr. P. Chambon (Illkirch, France). ERE3-tk-CAT was constructed by insertion of three copies of double-stranded oligonucleotides containing the 15-bp Xenopus vitellogenin A2 ERE sequence (25) flanked by HindIII and XbaI sites between the HindIII and XbaI sites of pBLCAT8+. ERE3-TATA-CAT was constructed in several steps from GRE5-CAT (26). First, the BglII site upstream of the CAT gene in GRE5-CAT was deleted by filling-in with Klenow, creating GRE5-CAT[-BglII]. A fragment containing the ERE3-TATA promoter was then excised from the vector ERE3-pAL10 (27) by digestion with Asp-718, end-filling with Klenow fragment, and digestion with BamHI; this fragment was inserted into GRE5-CAT[-BglII] which had been digested with SacI, treated with Klenow fragment, and then digested with BamHI to remove the GRE5-TATA promoter. By taking advantage of the unique XhoI and BglII sites upstream and downstream from the three EREs, respectively, these motifs were removed and replaced by multimerized oligonucleotides containing a consensus TPA response element (TRE, Fig. 1A), creating TRE2-TATA-CAT and TRE6-TATA-CAT. ERE3-TATA-CAT/EBV, TRE2-TATA-CAT/EBV, and TRE6-TATA-CAT/EBV were obtained by removal of XbaI fragments containing the whole minimal promoter-CAT gene transcriptional unit from the parental vectors and insertion into GRE5-CAT/EBV also digested by XbaI (28).
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Cell Culture and Transfections--
MCF7 and Ishikawa cells were
grown in -minimum Eagle's medium supplemented with 10 and 5% fetal
bovine serum (FBS), respectively, and switched to phenol red-free (29)
DMEM supplemented with 5% charcoal-treated FBS 72 h before
plating for transient transfections. Cells were divided into 10-cm
plates (1.5 million cells/plate) and transfected using the
calcium-phosphate coprecipitation method (30) with 15 µg of DNA (1 µg of expression vector where applicable, 2 µg of CAT reporter
vector, 2 µg of internal standard vector RSV-LacZ, and Bluescribe
M13+ to 15 µg). After 20 h, medium was changed twice to remove
precipitate, and hormones were added for a further 24 h (as
indicated in figure legends). Cells were harvested by scraping in 1 ml
of phosphate-buffered saline 1×, followed by centrifugation at 2,500 rpm for 10 min. Extracts were prepared by three cycles of
freeze-thawing in 0.25 M Tris-HCl, pH 8.0, and standardized
for
-galactosidase activity. CAT activity was determined by
incubation of protein samples for 1 h with 0.25 µCi of
[3H]chloramphenicol and N-butyryl-CoA (0.2 mg/ml), followed by extraction with xylene, and liquid scintillation
counting (31). To test for regulation of progesterone receptor
expression, cells maintained in phenol red-free medium supplemented
with charcoal-treated serum were further incubated for 5 days in the
absence or presence of estradiol or of anti-estrogens. Medium was
changed every 2nd day, and hormones were added every day. On day 5, cells were plated (1.8 million cells in 10-cm dishes) and transfected
in duplicate with 2 µg of GRE5-TATA-CAT plasmid (26), 1 µg of
internal control RSV-LacZ, and Bluescribe M13+ as carrier DNA (total 15 µg). Progesterone was added 1 day later, after removal of
calcium-phosphate precipitates by two consecutive washes. Cells were
harvested 24 h later, and CAT activity was assayed as described
above. All CAT assays were reproduced a minimum of three times.
Generation and Hormonal Treatment of Stably-transfected Cell
Lines Derived from Ishikawa and MCF7 Cells--
Ishikawa or MCF7 cells
were transfected with 15 µg of ERE3-TATA-CAT/EBV, TRE2-TATA-CAT/EBV,
or TRE6-TATA-CAT/EBV (10-cm plates, 1.5 million cells). Forty eight
hours after transfection, cells were passaged into 15-cm plates using
medium containing 150 µg/ml hygromycin B and maintained in this
medium for about 2 weeks until disappearance of all cells in control
non-transfected plates (28). Surviving cells in each 15-cm plate were
then pooled, propagated, and tested for estrogen or TPA induction of
CAT activity. Different pools of cells carrying the same reporter
plasmid were found to behave similarly. For generation of stable cell
lines containing non-episomal TRE-based reporter vectors, Ishikawa
cells were cotransfected with 15 µg of TRE6-TATA-CAT vectors and 1.5 µg of neomycin resistance gene expression vector Rc/RSV (Invitrogen).
48 h after transfection, cells were trypsinized and replated into
selection medium (-minimum Eagle's medium containing 5% FBS and 1 mg/ml G418). Two weeks later, individual clones were selected,
expanded, and tested for stimulation of CAT activity by incubation with
TPA (100 ng/ml) for 24 h. Established cell lines were subsequently
maintained in medium containing half the concentration of antibiotic
used for selection. For hormonal treatment, cells were preincubated for
72 h in medium without phenol red, supplemented with
charcoal-treated serum. Incubation with estrogen or anti-estrogens was
then carried out for 24 h except when indicated otherwise.
Reverse-transcription-PCR Amplification of Actin and CAT
mRNAs--
Ishikawa-ERE3/EBV cells maintained in phenol red-free
DMEM, 5% charcoal-treated FBS were incubated with E2 (25 nM), OHT (100 nM), or ethanol for 8 h
before harvesting and isolation of total RNA by CsCl gradient
centrifugation. 3 µg of total RNA were precipitated, resuspended in 6 µl of 1× DNase I digestion buffer (Promega) containing 0.5 units of
DNase I, incubated for 15 min at 37 °C and for 10 min at 75 °C,
and then transferred on ice. DNase I-treated RNAs (2 µl) were
reversed-transcribed using Superscript II RNase H reverse transcriptase
(Life Technologies, Inc.) and 0.5 µM random hexamer in a
20-µl final volume of RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM dithiothreitol supplemented with 0.5 mM
dNTPs) at 37 °C for 1 h, followed by 75 °C for 10 min.
Aliquots of resulting cDNAs (2 µl) were amplified by PCR using
Taq DNA polymerase (Amersham Pharmacia Biotech) in a 50-µl final volume of 1× Taq buffer supplemented with 0.2 mM dNTPs and 0.5 µM forward and reverse CAT
or -actin primers as follows: CAT forward,
5'-CCGCCTGATGAATGCTCATCCG-3', and CAT reverse,
5'-GCATTCTGCCGACATGGAAGCC-3';
-actin forward,
5'-GCTGTGCTATCCCTGTACGC-3', and
-actin reverse, 5'-GCCATGGTGATGACCGGC-3'.
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RESULTS |
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Differential Effects of Anti-estrogens on the Electrophoretic Migration of Estrogen Receptor-DNA Complexes-- Partial anti-estrogens such as hydroxytamoxifen are thought to induce a specific conformation of the estrogen receptor, which differs from both those of unliganded and estrogen-liganded ER. As a result, interaction between ER and estrogen or OHT differentially affects electrophoretic mobilities of ER·ERE complexes in gel shift assays. Full anti-estrogens like ICI164,384 induce yet another conformation, resulting in a migration that is closer to that of unliganded ER (32).
We used a gel shift assay to examine ER conformational changes induced by two other anti-estrogens, RU39,411 (RUp) and RU58,668 (RUf), which are both 11-
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Differential Induction of Progesterone Receptor Gene Expression by
Anti-estrogens in Ishikawa Cells--
Hydroxytamoxifen was previously
reported to induce expression of the estrogen target gene human
progesterone receptor in Ishikawa cells (15, 16). We decided to
investigate whether RUp, which similarly affects ER migration in gel
shift assays, can also induce expression of PR in Ishikawa and MCF7
cells. Cells were pretreated with estrogen or anti-estrogens prior to
transient transfection with GRE5-TATA-CAT/EBV (Fig. 1), followed by
addition of progesterone for 24 h (see "Experimental
Procedures"). No stimulation of CAT activity by progesterone could be
observed in extracts of cells in the absence of treatment with estrogen
or anti-estrogens (Fig. 3A, lane
2). In cells treated with estrogen or with the ER agonist moxestrol, the 11-methoxy derivative of ethynyl estradiol (36, 37),
CAT activity was induced over 20-fold in the presence of progesterone
(Fig. 3A, lanes 4 and 12). In cells pretreated
with OHT, progesterone-induced CAT expression levels were 12% of those obtained with estrogen pretreatment (Fig. 3A, lane 6). The
anti-estrogen RUp induced PR expression to comparable levels (Fig.
3A, lane 10), whereas no stimulation could be detected
following incubation with RUf (Fig. 3A, lane 8). Contrary to
what was observed with Ishikawa cells, none of the anti-estrogens
assayed detectably stimulated PR transcriptional activity in MCF7 cells
using this assay (Fig. 3B, compare lanes 5-10 to
lanes 1 and 2). These observations confirm that
partial agonist activity of anti-estrogens on expression levels of
endogenous estrogen target genes can be observed in Ishikawa cells (15,
16).
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Lack of Agonist Activity of Anti-estrogens in Transient Transfection Assays of ERE-containing Reporter Vectors-- In order to analyze the mechanisms of transcriptional regulation by anti-estrogens with partial agonist activity in Ishikawa cells, we examined whether synthetic estrogen-responsive promoters can be stimulated by these anti-estrogens in transient transfection assays (Fig. 4). Three estrogen-sensitive reporter recombinants, ERE3-TATA-CAT/EBV, ERE3-tk-CAT, or Vit-tk-CAT (Fig. 1), were transiently transfected into Ishikawa cells in the absence of cotransfected estrogen receptor expression vector (Fig. 4A). Anti-estrogens did not detectably stimulate CAT expression from ERE3-TATA-CAT/EBV (Fig. 4A, lanes 3-6), ERE3-tk-CAT (Fig. 4A, lanes 10-13), or Vit-tk-CAT (Fig. 4A, lanes 17-20) under conditions where estradiol stimulated these reporter constructs 35-, 10-, and 25-fold, respectively (Fig. 4A, lanes 2, 9, and 16). Similar results were obtained when CAT assays were repeated with 10 times more extract than in Fig. 4A to confirm that none of the anti-estrogens tested stimulated CAT activity higher than vehicle (data not shown).
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Detection of Cell-specific Agonist Activity of Anti-estrogens Using a Stably Propagated ERE3-TATA-CAT Episomal Reporter Vector-- The failure to detect significant transcriptional activation by OHT or other anti-estrogens in transient transfections raised the possibility that in vivo conditions required for agonist activity of OHT are not fully reconstituted in this assay. Therefore, we established Ishikawa and MCF7 cell lines stably propagating the ERE3-TATA-CAT/EBV episomal plasmids, selecting pools of transfected cells by addition of hygromycin B in cell culture medium (see "Experimental Procedures"; note that for brevity cell lines stably propagating ERE3-TATA-CAT/EBV episomal plasmids will be indicated by the suffix -ERE3/EBV). Stimulation with estrogen (25 nM, 24 h) of Ishikawa-ERE3/EBV cells led to a marked stimulation of CAT expression levels (~17-fold, Fig. 5A, lane 2). No stimulation was observed with the pure anti-estrogens RUf or ICI (100 nM, Fig. 5A, lanes 5 and 6). In contrast, OHT and RUp stimulated CAT activity to ~22 and 30% of the levels obtained with estrogen, respectively (Fig. 5A, lane 3 and 4). These results were obtained with different pools of Ishikawa-ERE3/EBV cells generated by two independent rounds of selection. On the other hand, neither the partial anti-estrogens OHT and RUp nor the full anti-estrogens ICI or RUf stimulated CAT expression more than background in MCF7-ERE3/EBV cells; note, however, that the fold stimulation by estrogen was lower in stably transfected MCF7 cells than in Ishikawa-derived cell lines (Fig. 5B, lanes 1-7).
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Minimal Promoters Containing AP1-binding Sites Are Not Activated by Estrogen or Anti-estrogens in Transient Transfection Assays of Ishikawa Cells-- Previous reports have described induction of AP1 activity by estrogen treatment in several cell lines (19-22) and by anti-estrogens in transient transfection assays of Ishikawa cells (22). To test whether TRE motifs are sufficient to mediate stimulation by estrogen and anti-estrogens in Ishikawa cells, we transiently transfected reporter vectors containing a minimal promoter composed of six TRE motifs upstream of a TATA box (Fig. 1) or a reporter vector containing the AP1-responsive rat stromelysin promoter, STR-CAT (Fig. 1, see also Ref. 24). Stimulation of expression from the STR-CAT reporter vector could be observed in the presence of estrogen (3-fold, compare lane 2 to lane 1 in Fig. 6A) but not in the presence of anti-estrogens. TPA-stimulated CAT expression levels 5-10-fold, and no additional increase was obtained in the presence of estrogen or anti-estrogens (Fig. 6A, lanes 5-8). No stimulation of the minimal TRE6-TATA-CAT reporter constructs could be observed after incubation of the transiently transfected cells with either estrogen or anti-estrogens (Fig. 6B). When the same promoter was incorporated into an episomal vector, a lower basal activity was observed (10-fold lower, data not shown) and a weak stimulation by estrogen (2.5-fold) could be detected in the absence but not in the presence of TPA (Fig. 6C, compare lanes 2 and 6 to lanes 1 and 5). Anti-estrogens had no effect on CAT expression levels directed from this reporter construct (Fig. 6C, lanes 3 and 4, and 7 and 8).
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Micromolar Concentrations of OHT, but Not of Other Anti-estrogens, Can Stimulate Transcription from Minimal Promoters Containing TRE Motifs in Stably Transfected Ishikawa Cells-- To investigate further the potential role of anti-estrogens in stimulation of the AP1 signaling pathway, cell lines were derived from Ishikawa cells by stable transfection of TRE6-TATA-CAT vectors (clonal selection by integration into the cellular genome) or of the episomal vectors TRE6-TATA-CAT/EBV or TRE2-TATA-CAT/EBV.
Two clones obtained by selection for integration of the TRE6-TATA-CAT reporter vector responded to TPA stimulation (100 ng/ml) by an increase in CAT expression (3-7-fold; Fig. 7A, compare lane 5 to lane 1 for Ishikawa-TRE6 #29). Ishikawa-TRE6 clone 29 was further used to investigate whether estrogen and/or anti-estrogens can stimulate CAT activity under the same tissue culture conditions as used for the Ishikawa-ERE3/EBV cell lines (i.e. phenol red-free DMEM supplemented with 5% charcoal-treated FBS). Treatment with estradiol or anti-estrogens did not significantly modulate CAT expression in these cells, either in the presence or in the absence of TPA (Fig. 7A, lanes 2-4 and 6-8). Similar results were obtained with the other clone (data not shown). Note that estrogen did stimulate expression from an ERE3-hsp68-LacZ reporter vector transiently transfected in Ishikawa-TRE6 cells, demonstrating that lack of induction of the AP1 pathway in these cell lines was not due to loss of ER function (data not shown).
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DISCUSSION |
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Mechanisms underlying the partial agonist activity of anti-estrogens are still poorly understood (9). In this report, we have investigated whether the partial agonist activity of anti-estrogens in Ishikawa cells can be mediated at the level of regulation of gene expression by typical estrogen response elements and/or by TPA response elements, which can mediate estrogen stimulation in some promoters (19-22). Although transcriptional activation of ERE-containing promoters by OHT has been documented using transient transfection assays in a number of cell lines, including HeLa cells and chicken embryo fibroblasts (3), agonist activity of OHT and RU39,411 was not observed in transiently transfected Ishikawa cells using minimal promoters containing EREs (ERE3-TATA) or more complex promoters (ERE3-tk, Vit-tk). Failure to detect transcriptional activation of ERE-containing promoters by OHT in transiently transfected Ishikawa cells is in agreement with previous observations (22). Contrary to results obtained with transient transfections, we observed significant levels of transcriptional activation by OHT and RUp in Ishikawa, but not in MCF7 cells, when the ERE3-TATA-CAT/EBV reporter vector was stably propagated as an episome. These results correlate well with the observed agonist effect of these two anti-estrogens on expression levels of the endogenous progesterone receptor (note that the human progesterone receptor upstream sequences contain a half-palindromic TGACC motif but no consensus EREs, Ref. 38). In addition, OHT and RUp, although structurally unrelated, induced similar shifts in mobility of ER·ERE complexes in gel retardation assays. The migration of these complexes was found to be distinct from those formed in the presence of the full antagonists RUf and ICI.
Differences observed in transcriptional activity in the presence of anti-estrogens in transient transfection assays and using "reporter cell lines" may reflect the different status of the reporter vectors, which are present at a lower copy number when maintained as episomes (generally less than 50 copies per cell, Ref. 39) and are incorporated into chromatin to a higher degree (40) compared with transiently transfected reporter plasmids. These results suggest that stimulation of ERE-mediated transactivation by anti-estrogens requires cofactor(s) limiting in amounts or availability in transient transfection assays in Ishikawa cells, whereas estrogen-liganded ER may recruit other, non-limiting cofactors (41, 42). Of interest is the fact that capacity to remodel chromatin structure via histone acetyltransferase or deacetylase activities has been attributed to an increasing number of nuclear receptor co-activators and co-repressors (42-49), demonstrating that incorporation of target promoters into chromatin is an integral part of the mechanism of transcriptional activation by nuclear receptors. It is not clear at present whether ER can interact with these cofactors in vivo when bound by anti-estrogens with partial agonist activity. It is possible that cofactors specific to OHT-bound ER, such as the newly described co-activator L7/SPA (50) may mediate the agonist activity of this anti-estrogen. Future functional characterization of cofactors interacting with ER in the presence of OHT and RUp should provide insights into the molecular mechanisms of action of anti-estrogens with partial agonist activity.
Low concentrations of OHT or RUp, which were sufficient for activation of ERE3-TATA promoters in stably propagated vectors, did not yield detectable transcriptional stimulation of promoters containing TRE sites inserted upstream of a TATA box either in transient transfection assays or using stably propagated vectors. Others have previously documented transcriptional activation of the TRE-containing collagenase promoter by anti-estrogens in transiently transfected Ishikawa cells (22). Discrepancy between these and our results could be due to the promoter context of TRE elements. Along the same line, our results indicate that promoter context influences stimulation of AP1-responsive promoters by estrogen in transient transfection assays. Alternatively, differences in cell lines or transfection methods could be the source of this discrepancy. Side-by-side comparison of cell lines carrying episomal reporter vectors whose promoters differed only by the response elements present in the minimal synthetic promoters confirmed that transcriptional stimulation by anti-estrogens like RUp or OHT could be mediated by estrogen response elements but not TPA response elements at low concentrations of anti-estrogens. Stimulation of AP1-responsive promoters could only be observed in serum-free medium using 5 µM OHT or TAM but not estrogen or other anti-estrogens. Whether this effect is mediated by estrogen receptors or is initiated at the cellular membrane remains to be investigated.
In conclusion, our results suggest that transcriptional stimulation by anti-estrogens can be mediated by consensus EREs in Ishikawa cells, this observation being consistent with the absence of TRE sites in the promoters of genes whose expression can be induced by hydroxytamoxifen in the uterus (38, 51). In addition, TREs are also capable of mediating transcriptional stimulation by anti-estrogens, although in a manner that is restricted by both the nature and the concentration of the anti-estrogen. Finally, while transient transfection has proven to be a powerful tool for analyzing intracellular signaling pathways, our study emphasizes that this assay only partially recapitulates the conditions required for initiation of transcription in vivo.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. D. Philibert (Roussel-Uclaf, Romainville, France) and to Dr. T. Willson (Glaxo-Wellcome, Research Triangle Park, NC) for the gift of reagents and to Dr. P. Chambon (Illkirch, France) for providing reporter plasmids and ER expression vectors. We also thank Dr. J. White (McGill University, Montreal, Canada) for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Grant MT-13147 from the Medical Research Council of Canada and grants from the Cancer Research Society Inc. and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.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.
The first two authors contributed equally to this work.
§ Supported by a Chercheur-Boursier award from the Fonds de Recherche en Santé du Québec. To whom correspondence should be addressed: Dépt. de Biochimie, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Qué, Canada H3C 3J7. Tel.: 514-343-6111 (ext. 5173); Fax: 514-343-2210; E-mail: maders{at}bch.umontreal.ca.
1 The abbreviations used are: E2, estradiol; EBV, Epstein-Barr virus; ER, estrogen receptor; ERE, estrogen response element; PR, progesterone receptor; FBS, fetal bovine serum; TAM, tamoxifen; OHT, 4-hydroxytamoxifen; ICI, ICI164,384; RUp, RU39,411; RUf, RU58,668; CAT, chloramphenicol acetyltransferase; tk, thymidine kinase; STR, rat stromelysin promoter; Vit, Xenopus vitellogenin A2 promoter; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; RT, reverse transcriptase; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; GRE, glucocorticoid response element(s).
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REFERENCES |
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