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in human breast cancer cells
Departments of Pharmaco-Biology and
1 Cell Biology, University of Calabria, 87030 Rende (Cs), Italy
(Requests for offprints should be addressed to S Andò; Email: sebastiano.ando{at}unical.it)
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Abstract |
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, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER; (2) they activate the agonist-dependent activation function (AF) 2 of ER and ERß in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17ß-estradiol for binding to ER and ERß; and – as a biological counterpart of the aforementioned abilities – (6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.
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Introduction |
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) are phenolic compounds that occur naturally in grapes and red wine (MacDonald et al. 1998, Teugo et al. 1998). The total amount of PIC and MYR in red-grape wines has been reported to be up to 15 mg/l (MacDonald et al. 1998, Burns et al. 2000, Vuorinen et al. 2000, Cantos et al. 2003), however the biotransformation of the abundant red wine component named resveratrol contributes to increase PIC concentrations at tissue level (Piver et al. 2004). Previous studies have demonstrated that PIC is a natural tyrosine kinase inhibitor (Geahlen & McLaughlin 1989) exerting repressive effects in leukemia, melanomas and colorectal cancer cell lines (Wieder et al. 2001, Wolter et al. 2002, Larrosa et al. 2004), while MYR has been identified as a potent topoisomerase II repressor similar in activity to the epipodophyllotoxin widely used in cancer therapy (Markovits et al. 1989, Azuma et al. 1995, Chang et al. 1995). Several epidemiological investigations have suggested that the consumption of red wine is associated with a variety of health benefits due to the estrogenic activity of certain bioactive components (Stampfer et al. 1988, Frankel et al. 1993, Bertelli et al. 1995, Goldberg et al. 1995, Gronbaek et al. 1995, Pace-Asciak et al. 1995). It has been largely reported that 17ß-estradiol (E2) mainly acts by binding to and transactivating estrogen receptor (ER) and ERß, however different transduction pathways involved in the estrogen signaling have recently received a great deal of attention (Katzenellenbogen et al. 2000, Safe 2001, Maggiolini et al. 2004, Simoncini et al. 2004). A large body of evidence has demonstrated that E2 and E2-like compounds elicit stimulatory effects on breast cancer, the leading cause of tumor deaths among women in Western countries (Hoskins & Weber 1994, Eisen & Weber 1998, Lopez-Otin & Diamandis 1998, Maggiolini et al. 2001). Therefore, much effort has been addressed to ascertain the ability of natural compounds to interact with ER and to trigger breast tumor progression. In this regard, it should be taken into account that wine consumption even in moderate amounts is directly associated with the occurrence of breast carcinoma particularly in post-menopausal women (van den Brandt et al. 1995, Feigelson et al. 2001). Notably, in a multicentre Italian case-control study an increased risk of breast cancer was attributed to wine ingestion compared with other types of alcoholic beverages, explaining up to 12% of breast tumor cases in Italy (Ferraroni et al. 1998). In the present study, we have used as model systems the hormone-sensitive MCF7 and endocrine-independent SKBR3 breast cancer cells in order to provide new insight into the molecular mechanisms by which the wine-derived PIC and MYR behave as estrogen-like compounds. In a concentration range physiologically achievable with a moderate wine consumption, both phytochemicals elicited growth stimulatory effects in MCF7 cells as a biological counterpart of their agonistic activity for ER.
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Materials and methods |
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E2, PIC, MYR and cycloheximide (Cx) were purchased from Sigma, while the ER antagonist ICI 182,780 (ICI) was obtained from Tocris Chemicals (Bristol, UK). Hydroxyflutamide (OHF) and ZK 98299 were a gift from Schering (Berlin, Germany). All compounds were solubilized in dimethylsulfoxide, except E2 which was dissolved in ethanol.
Plasmids
Firefly luciferase reporter plasmids used were XETL (Bunone et al. 1996) and TFF1/pS2-ERE (Berry et al. 1989) for the ERs and GK1 (Webb et al. 1998) for the Gal4 fusion proteins. The reporter plasmid XETL carries firefly luciferase sequences under the control of an estrogen response element (ERE) upstream of the thymidine kinase promoter. TFF1/pS2-ERE (a gift from V. Giguère, McGill University, Quebec City, Canada) contains the ~1050 bp TFF1/pS2 promoter preceding the luciferase reporter of pGL3 (Berry et al. 1989). As an internal transfection control, we co-transfected the plasmid pRL-CMV (Promega) that expresses renilla luciferase enzymatically distinguishable from firefly luciferase by the strong cytomegalovirus enhancer/promoter. Gal4 chimeras Gal-ER, Gal-ER(V), Gal-ER(R), Gal-ER(L), Gal-ER(543/4A), Gal-ER(
F) and Gal-ERß were expressed from plasmids GAL93.ER(G), GAL93.ER(V), GAL93.ER(R),GAL93.ER(L), GAL93.ER(ML543/4AA), GAL93. ER(F) and GAL93.ERß respectively. They were constructed by transferring the coding sequences for the hormone-binding domain (HBD) of ER (amino acids 282–595) from HEG0 (Bunone et al. 1996), pCMVh ERG400 V (Aumais et al. 1997), pCMVhERG521R (Ekena et al. 1996), pCMVhERL525A (Ekena et al. 1996), a PCR-mutagenized intermediate with the point mutations M543A-L544A, a PCR fragment lacking the coding sequences for the F domain, and for the ERß HBD (C-terminal 287 amino acids) from plasmid pCMV5-hERbeta (a gift from J.-Å. Gustafsson, Karolinska Institute, Huddinge, Sweden) respectively, into the mammalian expression vector pSCTEVGal93 (Siepel et al. 1992).
Cell culture
Wild-type ER-positive MCF7 human breast cancer cells were a gift from E. Surmacz (Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, USA), the ER-negative SKBR3 human breast cancer cells and the ER-containing human uterine Ishikawa cells were a gift from D. Picard (Department of cell biology, University of Genève, Switzerland). MCF7 and Ishikawa cells were maintained respectively in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM/F12 both without phenol red supplemented with 10% fetal bovine serum (FBS) (Invitrogen); SKBR3 cells were maintained in RPMI 1640 without phenol red supplemented with 10% FBS (Invitrogen).
Transfections and luciferase assays
A total of 105 MCF7, SKBR3 and Ishikawa cells were transferred into 24-well plates with 500 µl of regular growth medium/well the day before transfection. Medium lacking phenol red as well as serum was used on the day of transfection, which was performed by Fugene6 reagent (Roche) with a mixture containing 0.5 µg reporter plasmid, 5 ng pRL-CMV and 0.1 µg effector plasmid where applicable. After 6 h, treatments were added and cells were incubated for a further 18–20 h. Luciferase activity was then measured with the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s recommendations. Firefly luciferase activity was normalized to the internal transfection control provided by renilla luciferase activity. Luciferase activity of cells receiving vehicle was set as 1-fold induction, upon which the results of treatments were calculated.
RT-PCR
MCF7 cells were grown in 10 cm dishes to about 60% confluence in regular growth medium and then were switched to medium lacking phenol red as well as serum for 24 h. Thereafter, treatments were added for 24 h and mRNA expression was evaluated by semi-quantitative RT-PCR as we have previously described (Maggiolini et al. 1999). The primers for ER, estrogen-inducible trefoil factor TFF1/pS2 (Brown et al. 1984), cathepsin-D (Cavailles et al. 1991), estrogen-responsive RING finger protein (EFP) (Ikeda et al. 2000) as well as for the internal control acidic ribosomal phosphoprotein 36B4 (Laborda 1991) were: 5'AATTCAGATAATCGACGCCAG3' (ER forward) and 5'GTGTTTCAACATTCTCCCT CCTC3' (ER reverse);
5'TTCTATCCTAATACCATCGACG3' (pS2 forward) and
5'TTTGAGTAGTCAAAGTCAGAGC3' (pS2 reverse); 5'AACAACAGGGTGGGCTTC3' (cathepsin-D forward) and 5'ATGCACGAAACAGACTGTGC3' (cathepsin-D reverse);
5'CGTGTGGATTTGTGTGTGGACG3' (EFP forward) and 5'CCCCGAGGTGGAACTGAACC3' (EFP reverse);
5'CTCAACATCTCCCCCTTCTC3' (36B4 forward) and
5'CAAATCCCATATCCTCGTCC3' (36B4 reverse); these primers yielded products of 345, 210, 303, 162 and 400 bp with 20, 15, 20, 20 and 15 PCR cycles respectively.
Western blotting
MCF7 cells were grown in 10 cm dishes to about 60% confluence in regular growth medium and then were switched to medium lacking phenol red as well as serum for 24 h. Thereafter, treatments were added for 24 h before lysis. In the case of Cx, cells were pretreated for 3 h prior to the addition of ligands. Cells were lysed with 300 µl of 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1% SDS, and a mixture of protease inhibitors containing 1 mM aprotinin, 20 mM phenylmethyl-sulfonyl fluoride and 0.2 M sodium orthovanadate. Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham), probed overnight at 4 °C with the antibodies F-10 against ER and ß-actin (both from Santa Cruz Biotechnology, CA, USA), and then revealed using the ECL System (Amersham).
Immunocytochemical staining
MCF7 cells were maintained in medium lacking phenol red as well as serum for 3 days, treated for 1 h and then fixed in fresh paraformaldehyde (PFA; 2% for 30 min). After PFA removal, hydrogen peroxide (3% in methanol for 30 min) was used to inhibit endogenous peroxidase activity. Cells were then incubated with normal horse serum (10% for 30 min) to block the non-specific binding sites. Immunocytochemical staining was performed using as the primary antibody a mouse monoclonal immunoglobulin (Ig) G generated against the human C-terminus of ER (F-10, Santa Cruz Biotechnology; 1:50 overnight at 4 °C). A biotinylated horse-anti-mouse IgG (1:600 for 60 min at room temperature) was applied as the secondary antibody (Vector Laboratories, Burlingame, CA, USA). Subsequently, the amplification of avidin-biotin-horseradish peroxidase complex (ABC complex/HRP; Vector Laboratories; 1:100 for 30 min at room temperature) was carried out and 3,3'-diaminobenzidine tetrachloride dihydrate (Vector Laboratories) was used as a detection system. Cells were rinsed after each step with Tris-buffered saline (0.05 M Tris–HCl plus 0.15 M NaCl, pH 7.6) containing 0.05% Triton-X100 (TBS-T). In control experiments cells were processed replacing the primary antibody with mouse serum (Dako S.p.A., Milan, Italy) or using a primary antibody pre-absorbed (48 h at 4 °C) with an excess of purified ER protein (PanVera Corp., Madison, WI, USA).
Ligand binding assay for ERs
The ability of PIC and MYR to compete with [3H]E2 for binding to ER and ERß was evaluated and compared with that of E2. Two picomoles of purified recombinant human ER and ERß proteins (PanVera Corp., Madison, WI, USA), each in 20 mM HEPES, pH 7.4, 1.5 mM EDTA, 0.5 mM dithiothreitol and 10% (v/v) glycerol, were incubated with 1 nM [2,4,6,7-3H]E2 (72 Ci/mmol; Amersham) in the presence of serial dilutions of unlabeled E2, PIC or MYR for 20–22 h at 4 °C. Bound and free radioligands were separated on Sephadex G-25 PD-10 columns. The amount of receptor-bound [3H]E2 was determined by liquid scintillation counting (OptiPhase, HiSafe 3 and 1409; Wallac, Inc., Gaithersburg, MD, USA). Relative counts per minute were plotted against the concentrations of the ligand, and data were evaluated with the use of a non linear, four-parameter logistic model to estimate the median effective concentration (EC50) value.
Proliferation assays
For quantitative proliferation assays 1 x 104 MCF7 cells were seeded in 24-well plates in regular growth medium. Cells were washed extensively once they had attached and were incubated in medium without serum for 24 h. On the second day, the medium was changed and supplemented with 5% charcoal-stripped (CS)-FBS. Ligands were added at this point, thereafter the 5% CS-FBS medium was renewed every day together with treatments. On day 6, cells were trypsinized and counted in a haemocytometer using the trypan blue exclusion method.
Statistical analysis
Statistical analysis was performed using ANOVA followed by Newman–Keuls testing to determine differences in means. Data are expressed as means ± S.D. P values <0.05 were considered significant.
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We first aimed to evaluate whether a transiently transfected ER reporter gene is able to respond to PIC and MYR. The reporter plasmid XETL carries firefly luciferase sequences under the control of an ERE upstream of the thymidine kinase promoter. As an internal transfection control, we co-transfected a plasmid that expresses renilla luciferase, which is enzymatically distinguishable from firefly luciferase, from the strong cytomegalovirus enhancer/promoter. Using MCF7 cells, which express ER but no ERß as judged by RT-PCR (data not shown), PIC and MYR were able to induce a substantial XETL expression from the concentration of 10 nM (Fig. 2A). The ER antagonist ICI abolished the activation by E2 and both compounds (Fig. 3A), whereas the antiandrogen OHF, the antiprogestin and antiglucocorticoid ZK 98299 had no effect (data not shown), suggesting that PIC and MYR transactivate ER directly. Next, using the natural pS2 ERE-containing promoter (Berry et al. 1989) we confirmed that both compounds were able to induce the ER-mediated transcriptional activity (Fig. 2B), which was no longer noticeable in the presence of ICI (Fig. 3B). For comparison, we performed the same transfection experiments in uterine Ishikawa cancer cells showing that PIC and MYR activated the endogenous ER (Fig. 2C) in a direct manner since ICI inhibited the transcriptional response also in this cellular context (Fig. 3C).
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and ERß in a heterologous system
To provide mechanistic evidence on the ability of PIC and MYR to transactivate ER and to examine the response of ERß, we turned to a heterologous system using SKBR3 cells lacking ERs. Chimeric proteins consisting of the DNA-binding domain (DBD) of the yeast transcription factor Gal4 and the ER or the ERß HBDs containing the ligand-dependent activation function (AF) 2 were both activated by PIC and MYR although with a different potency compared with E2 (Fig. 4A). Hence, the HBD of each ER isoform was sufficient to allow the functional response, while the Gal4-DBD fused to the AF1 domain of ER did not elicit transcriptional effects upon all treatments (Fig. 4B). Next, we examined the response of diverse ER HBD mutants in the context of Gal4 proteins. The two-point mutations L525A and G521R that require a high E2 concentration for a substantial transactivation (Ekena et al. 1996) elicited very low activity upon PIC and MYR (Fig. 4B and C). The AF2 mutant M543/L544A failed to respond to all treatments (Fig. 4C), while the point mutation G400 V and the mutant lacking the F-domain at the C-terminal HBD core exhibited a strong response to E2, PIC and MYR (Fig. 4D). Taken together, our results argue that mutations of ER in the hormone binding pocket are able to alter the transcriptional activity induced by E2 as well as that of the compounds tested.
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Upon ligand binding, ER undergoes conformational changes allowing the formation of receptor dimers and the interaction with specific EREs contained within the regulatory region of target genes (Tsai & O’Malley 1994, Beato et al. 1996, Pratt & Toft 1997). Performing an immunocytochemical assay we aimed to evaluate the ability of short PIC and MYR treatments (1 h) to elicit a nuclear localization of the endogenous ER in MCF7 cells. No detectable ER immunoreactivity was observed in basal experimental conditions obtained by maintaining cells in medium lacking phenol red as well as serum for 3 days (Fig. 5). Only in the nuclear compartment did E2, PIC and MYR produce strong staining signals and these were abrogated in the presence of the ER antagonist ICI 182 780 (Fig. 5). No signals were observed after replacing the anti-ER antibody by irrelevant rabbit IgG or by using the primary antibody pre-absorbed with an excess of receptor protein (data not shown). These findings further supported the functional activity elicited by compounds tested in transfection experiments.
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mRNA and protein levels
Levels of ER in breast cancer cells are autoregulated by E2 through different mechanisms which include a reduced transcription rate of the ER gene together with an increased turnover of the ligand-activated protein (Santagati et al. 1997). Hence, to evaluate whether PIC and MYR are also able to modulate the mRNA of ER in MCF7 cells we performed a semiquantitative RT-PCR standardizing its expression by the housekeeping gene 36B4. Interestingly, both compounds induced a receptor down-regulation (Fig. 6A and B) which was observed even at the protein level depending on new synthesis (Fig. 6C and D) (El Khissiin & Leclercq 1999, Nonclercq et al. 2004).
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As aforementioned, the E2/ER-activated complex regulates the expression of genes that contain ERE sequences within the promoter regions (Truss & Beato 1993). Following the same experimental procedure described above, we documented the ability of PIC and MYR to up-regulate, like E2 the mRNA expression of target genes – such as pS2/TIFF1, cathepsin D and EFP E2, (Fig. 6A and B
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Competition for ER binding
The results obtained strongly suggested that PIC and MYR are ERs for ligands. This issue was examined directly by a competition binding experiment with purified recombinant human ER and ERß proteins. Our data demonstrated that both compounds compete with the radiolabeled E2 tracer for binding to the ERs in a concentration-dependent manner, however they are poor binders particularly for ER in comparison to ERß (Fig. 7). The latter phenomenon is in agreement with a previous report on ER isoform-specific preferences in binding to environmental estrogens (Kuiper et al. 1997).
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Having clearly demonstrated that PIC and MYR are functional activators of ER, we evaluated in a time course study the proliferative response of MCF7 cells to increasing concentrations of both ligands. Cells were lysed on the indicated days, counted in a hemocytometer, and results obtained upon treatments were compared with those of untreated cells. Notably, both compounds were able to induce progressive proliferative effects (Fig. 8A) which were no longer noticeable in presence of ICI (Fig. 8B), suggesting that even the growth stimulation is mediated by an ER-dependent mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingReferences |
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in hormone-dependent MCF7 and Ishikawa breast and uterine cancer cells respectively; (2) to activate the ligand-dependent AF2 domain of ER and ERß in the context of chimeric proteins with the Gal4 DBD in ER-negative SKBR3 breast cancer cells; (3) to elicit responses similar to E2 using different expression vectors for ER mutated in the AF2 domain; (4) to rapidly induce a nuclear immunodetection of ER; (5) to regulate ER mRNA and protein levels as well as the expression of estrogen target genes such as TFF1/pS2, cathepsin D and EFP; (6) to compete with E2 for binding to purified ER and ERß proteins; (7) to stimulate the proliferation of MCF7 cells at concentrations that may be physiologically achievable through a moderate wine intake.
In recent years, the biological properties of natural estrogen-like compounds have attracted increasing interest for the potential beneficial effects on human health (Cotton 1994, Safe 1995, Lopez-Otin & Diamandis 1998). In this regard, a reduced incidence and mortality from a variety of diseases has been associated with moderate wine consumption (Fuhrman et al. 1995, Goldberg et al. 1995, Whitehead et al. 1995, German & Walzem 2000, Gronbaek et al. 2000), which is considered a relevant source of estrogenic compounds like the well-characterized resveratrol (Kopp 1998). In order to ascertain the mechanisms involved in the potential protective role exerted by resveratrol, many studies have been performed demonstrating its ability to regulate lipid metabolism and platelet function as well as to trigger apoptotic events in different cancer cells (Bagchi et al. 2001, Bianchini & Vainio 2003, Pervaiz 2003). However, other investigations evaluating the potential estrogen-like properties of resveratrol have documented an agonistic activity for ER including the ability to stimulate the proliferation of breast tumor cells (Gehm et al. 1997, Basly et al. 2000, Nakagawa et al. 2001, Levenson et al. 2003, Gehm et al. 2004). These findings are reminiscent of those reported for other abundant dietary flavonoids (Hsieh et al. 1998, Maggiolini et al. 2001 and references therein), and may suggest that the biological actions of resveratrol are either mediated by ER or involve ER-independent mechanisms mainly in relationship to the cellular environment and levels of exposure (Gehm et al. 1997, Mgbonyebi et al. 1998, Ashby et al. 1999, Lu & Serrero 1999, Bowers et al. 2000, Damianaki et al. 2000, Bhat et al. 2001). Hence, phytoestrogens and other natural compounds are potentially responsible for diverse pharmacological effects, which depend on bioavailability as well as metabolic clearance rates influencing consequently ER agonism/antagonism and even anti-cancer activity. In this respect, it is worth noting that resveratrol can undergo hydroxylation to yield PIC via different cytochrome P450 enzymes like CYP1B1 which has been found overexpressed in a wide variety of human tumors (Potter et al. 2002, Piver et al. 2004). Taking into account that PIC also derives directly from wine intake, we performed a panel of in vitro assays designed to better define its estrogen-like properties together with those of MYR, an additional phytochemical occurring abundantly in red wine. At a low concentration range physiologically achievable through wine consumption, both compounds stimulated the growth of hormone-sensitive breast tumor cells as ER activators. Moreover, the results obtained in transfection experiments with Gal4 chimeras suggested that PIC and MYR are AF2 agonists alongside other selective estrogen receptor modulators (SERMs) grouped as class I estrogens, according to the molecular classification previously proposed (Jordan et al. 2001). The ability of ligands to modulate intracellular levels of ER is important for ER pharmacology. The human ER is a substrate for ubiquitination in the presence of E2 and the exposure to different ligands has been shown to lower ER levels by a proteosome-mediated proteolysis (Nawaz et al. 1999). The down-regulation of ER following treatments with PIC and MYR in MCF7 cells further supported their agonistic activity observed also through a ligand-dependent receptor degradation process. Moreover, despite a low binding affinity for ER both compounds up-regulated the expression profiles of diverse estrogen target genes providing a reliable assessment of the transcriptional efficacy exhibited in the context of natural DNA response elements.
A large body of evidence has suggested that ER-mediated estrogen signaling plays a major role in the etiology and progression of breast carcinoma (Ciocca & Fanelli 1997, Lopez-Otin & Diamandis 1998). Consequently, ER antagonists have been extensively utilized as adjuvant endocrine treatment to prevent such malignancy and/or its recurrence (Early Breast Cancer Trialists Collaborative Group 1998, Cosman & Lindsay 1999). However, about half of ER-positive tumors fail to respond to antiestrogen therapy from the beginning and most of the remaining ones eventually acquire pharmacological resistance. In this context, it should be noted that E2 and diverse phytoestrogens irrespective of ER expression trigger rapid genomic and non-genomic events (Brownson et al. 2002, Maggiolini et al. 2004) which, at least in part, contribute to the failure of antiestrogen treatment as well as to breast tumor progression. On the basis of the aforementioned observations and the present findings, the estrogenic properties of phytochemicals studied might be considered as a risk factor in postmenopausal women who habitually consume red wine (van den Brandt et al. 1995, Ferraroni et al. 1998, Feigelson et al. 2001, Poschl & Seitz 2004). However, taking into account that PIC and MYR exert inhibitory effects in different tumor cells (Markovits et al. 1989, Azuma et al. 1995, Chang et al. 1995, Wieder et al. 2001, Wolter et al. 2002, Larrosa et al. 2004), their concentrations as well as the cellular context may direct various biological responses as has been previously reported for resveratrol and other phytoestrogens (Gehm et al. 1997, Hsieh et al. 1998, Basly et al. 2000, Maggiolini et al. 2001, Nakagawa et al. 2001, Bianchini & Vainio 2003, Pervaiz 2003). Herein, we have provided mechanistic insight into the estrogenic potential of PIC and MYR that may be involved in the beneficial as well as the adverse effects associated with wine consumption. In this vein, further investigations are needed to elucidate the level of absorption and exposure to these phytochemicals, and the dose-dependent properties as well as the activity exerted in vivo in estrogen target tissues such as breast, uterus and bone.
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References |
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Received 26 April 2005
Accepted 13 June 2005
Made available online as an Accepted Preprint 8 July 2005
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