Abstract
Cytochrome P4502C9 (CYP2C9) is an important drug-metabolizing enzyme responsible for the metabolism of approximately 16% of all clinically relevant drugs. It was shown previously that the activity of CYP2C9 in vivo is inhibited by oral contraceptives. The mechanisms of this effect have not been elucidated. We hypothesize that this may occur because of the sex steroid-dependent activation of estrogen receptor α (ERα) with further transactivation of the CYP2C9 gene. Here, we show that the CYP2C9 promoter indeed contains a functionally relevant estrogen responsive element (ERE) half-site at position −149/−145. Its ERα binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ERα into Huh7 hepatoma cells and treated with various ERα ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17β-estradiol (EE), and 17α-ethinylestradiol (ETE). The luciferase activity driven by the wild-type CYP2C9 promoter construct was up-regulated by 4-OHT and R and significantly or marginally suppressed by ETE and EE, respectively. An identical effect was observed in primary hepatocytes treated with these compounds. Mutations introduced into the ERE half-site abolished the observed effects in the Huh7 cells. Electrophoretic mobility-shift assay revealed sequence-specific binding of a nuclear protein to the oligonucleotide containing the ERE half-site, which was identified as ERα by antibody supershift analysis. In addition, the association of ERα with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ERα and its ligands play an important role in the regulation of CYP2C9 expression.
Introduction
CYP2C9 is one of the key enzymes in drug metabolism and is estimated to metabolize approximately 16% of all clinically prescribed drugs. These include phenytoin, tolbutamide, torsemide, losartan, several anti-inflammatory drugs (nonsteroidal anti-inflammatory drugs), and the important anticoagulant warfarin, which is known to have a narrow therapeutic window (Goldstein, 2001; Urquhart et al., 2007; Flockhart et al. 2008). CYP2C9 is the second most abundant hepatic cytochrome enzyme and is polymorphically expressed (Miners and Birkett, 1998). The most important genetic variants in whites include CYP2C9*2 and CYP2C9*3, which occur at a frequency of approximately 7 and 11%, respectively. Carriers of these variants show slower metabolism toward CYP2C9 substrates and a considerably higher risk for adverse drug reactions (Kirchheiner and Brockmöller, 2005).
It is noteworthy that high interindividual variance in CYP2C9 activity within wild-type carriers has been observed, which cannot be exclusively explained by polymorphic expression of the enzyme (Scordo et al., 2001; Sandberg et al., 2004). Drug-dependent CYP2C9 induction may explain some, but not all, of the variations in individual responses seen in patients treated with CYP2C9 substrates, such as warfarin (Peyvandi et al., 2004). Such a drug-dependent induction was shown to be mediated by gene regulatory factors, which are involved in CYP2C9 gene expression. Important transcription factors that are able to trigger substrate-specific CYP2C9 expression include the pregnane X receptor (PXR), the constitutive androstane receptor (CAR), and the glucocorticoid receptor. Chen et al. (2004) showed that PXR mediates the induction of CYP2C9 by rifampicin, hyperforin, and phenobarbital. Furthermore, CYP2C9 induction by dexamethasone and phenobarbital probably is mediated via glucocorticoid receptor and CAR-responsive elements within the CYP2C9 promoter. Hepatocyte nuclear factor-4α (HNF-4α) has also been shown to be a major regulator of CYP2C9 expression (Chen et al., 2005).
Accumulating clinical evidence suggests that CYP2C9 might be regulated by sex hormones. In vivo data of Sandberg et al. (2004) showed that oral contraceptives (OC) are able to inhibit CYP2C9 activity to a significant extent as measured with losartan as a probe drug. In addition to CYP3A4 and members of the CYP1 family, CYP2C9 is (to a lesser extent) involved in the metabolism of 17β-estradiol (EE), suggesting that CYP2C9 might have an impact on the localized metabolic control of estrogen homeostasis (Cheng et al., 2001). Therefore, taking into account all of these data, it cannot be excluded that the parallel intake of estradiol derivatives, especially together with CYP2C9 substrates that have a narrow therapeutic window, e.g., warfarin, will increase the risk for adverse events (Zingone et al., 2009).
Estrogenic and antiestrogenic effects are mediated through two related estrogen receptors, ERα and ERβ, both ligand-dependent transcription factors (van de Stolpe et al., 2004). The ERs exert their transcriptional control via the interaction of receptor dimers with specific DNA sequences such as classic estrogen responsive elements (EREs) (Couse et al., 1997) or ERE half-sites (van de Stolpe et al., 2004). Antiestrogens can act as pure estrogen antagonists [7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780)] or as partial agonists, also known as agonists/antagonists; examples are selective estrogen receptor modulators [SERMs, e.g., tamoxifen, raloxifene (R)], which exert tissue-selective estrogen-like effects.
In this article, we demonstrate for the first time that the estradiol derivatives EE and 17α-ethinylestradiol (ETE) and partial estrogen agonists, the potent tamoxifen metabolite 4-hydroxytamoxifen (4-OHT) and R, are able to modulate CYP2C9 expression via ERα-mediated interaction with the newly identified ERE half-site within the CYP2C9 promoter.
Materials and Methods
Plasmid Constructs.
A 0.5-kb-long fragment of CYP2C9 5′-flanking region was subcloned into the MluI/XhoI site of pGL3basic vector (Promega, Madison, WI) upstream of the luciferase gene (2C9wt_0.5; Table 1 and Fig. 1B). The construct containing destructive mutations within the putative ERE half-site at position −149/−145 was generated from the parental 2C9wt_0.5 construct using the GeneTailor kit (Invitrogen, Carlsbad, CA) (construct 2C9mut_0.5; Table 1 and Fig. 1B).
The expression vector encoding human ERα (pSG5-hERα) was a kind gift of Dr. P. Chambon (Institute for Genetics and Cellular and Molecular Biology, Strasbourg, France). The ERα insert was recloned into the EcoRI site of pcDNA3.1 empty vector (Promega).
Electrophoretic Mobility-Shift Assay.
Nuclear extracts (NEs) were prepared from Huh-7 cells as described previously with slight modifications (Dignam et al., 1983; Nakabayashi et al., 1991). To compare the binding intensity of ERα to the CYP2C9 promoter in dependence of the influence of different ERα agonists, cells were treated with 10−6 M 4-OHT, R, EE, ETE, or vehicle (0.2% ethanol) 24 h before harvesting.
Four different 50-bp-long double-stranded oligonucleotides comprising four different putative ERE half-sites in the CYP2C9 promoter were generated by annealing sense and antisense oligonucleotides (Table 1). Double-stranded oligonucleotides were 32P-labeled (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) using the T4 DNA polynucleotide kinase system (Invitrogen) in a final reaction volume of 25 μl.
The labeling reaction was carried out at 37°C for 15 min. The reaction was stopped with 5 μl of 0.2 M Na2EDTA. Binding reactions were carried out in 4% glycerol, 8 mM HEPES, pH 7.9 (Sigma-Aldrich, St. Louis, MO), 0.6 mM MgCl2, 50 mM NaCl, 2 μg of polydeoxyinosine:deoxycytosine, 12.8 fmol of 32P-labeled double-stranded probe (approximately 20,000 cpm), and 4 μg of Huh-7 cell NEs in a total volume of 25 μl.
Mixtures with NE were preincubated at 37°C for 15 min followed by the addition of the respective labeled double-stranded oligonucleotide. The complete mixture was incubated again at 37°C for 15 min. For competition experiments, 100- and 200-fold excess of the respective unlabeled double-stranded oligonucleotide was added to the probe before the addition of 32P-labeled double-stranded oligonucleotides. For supershift experiments 2 μg of ERα antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reaction before the incubation with labeled oligonucleotides and samples were incubated on ice for 45 min. Five microliters of loading buffer was added, and protein-bound as well as unbound DNA was resolved on a 4% nondenaturing polyacrylamide gel. Dried gels were subjected to autoradiography using a phosphoimager (Fujifilm BAS-1800; Fujifilm, Tokyo, Japan).
Transient Transfections.
Huh-7 cells were grown at 37°C in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). One day before transient transfections 2 × 105 Huh-7 cells were plated into 12-well plates. Two micrograms of the pGL3 expression vector 2C9wt_0.5, 2C9mut_0.5 or pGl3basic empty vector was cotransfected with 0.5 μg of human pcDNA3.1-ERα or 0.5 μg of pcDNA3.1 empty vector (negative control). Transient transfections were carried out in plain medium using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Twenty four hours after transfection medium was replaced with complete medium, and cells were treated as indicated in each figure with 10−10 to 10−6 M 4-OHT, R, EE, and ETE (all chemicals were purchased from Sigma-Aldrich). Cells were harvested and analyzed for luciferase activity 24 h later. In each transfection mixture 6 ng of the plasmid harboring Renilla luciferase gene (pRL-TK Renilla; Promega) was included as an internal control for the transfection efficiency. Luciferase activity is expressed as fold induction/repression in comparison with the vehicle-treated CYP2C9 promoter constructs, which was set to 1. All experiments were performed in triplicate and repeated three times.
ChIP Analysis.
The assay was carried out using a ChIP assay kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. Huh7 cells were grown to 80% confluence in two 75-cm2 flasks for each treatment. Proteins were cross-linked in 1% formaldehyde, washed with 1× phosphate-buffered saline, fixed in glycine solution, and scraped into 2 ml of phosphate-buffered saline supplemented with phenylmethylsulfonylfluoride. Cells were lysed, homogenized, and enzymatically sheared followed by a subsequent immunoprecipitation using ERα antibody (Santa Cruz Biotechnology, Inc.) or control IgG (negative control). The resulting immunoprecipitates and an input sample were subjected to PCR using a primer pair (Table 1) specific for the CYP2C9 promoter region. The primer set amplified a 226-bp fragment that included the investigated ERE half-site at position −149/−145.
Hormone Treatment of Human Hepatocytes.
Human hepatocytes were obtained from Invitrogen. Cells were seeded into 24-well plates at 105 cells/well using Williams' medium E without phenol red supplemented with fetal bovine serum, plating cocktail solution, and dexamethasone. After 5 h cells were treated with 4-OHT, R, EE, and ETE at concentrations of 10−10 to 10−6 M using ethanol as a vehicle. Cells were maintained under these conditions for 24 h until harvesting.
Real-Time PCR.
Total RNA was isolated from human hepatocytes with a RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. cDNA was prepared from the extracted RNA by performing reverse transcriptase reactions with SuperScript II RT (Invitrogen). mRNA levels were determined by a quantitative PCR on a 7500 Fast Real-Time PCR system using specific TaqMan gene expression assays for CYP2C9 and human TATA-box binding protein (TBP) (CYP2C9: HS00426397; TBP: 4333769F; Applied Biosystems, Foster City, CA). TBP was measured as an internal control. The measurements were performed in quadruplicates.
Statistical Analysis.
Differences in luciferase gene reporter activities and mRNA levels in human hepatocytes were statistically evaluated by the one-sample t test using Prism version 5.0 (GraphPad Software Inc., San Diego, CA). A p value threshold of <0.05 was considered statistically significant in all analyses.
Results
ERα Binds Specifically to the ERE Half-Site at Position −149/−145.
Analysis of the CYP2C9 promoter revealed four different putative ERE half-sites at positions −104/−100 (site I), −149/−145 (site II), −1557/−1553 (site III), and −1829/−1825 (site IV) (Fig. 1A). To investigate whether any of these sites interact with nuclear transcription factors and to reveal the identity of these factors we chose the electrophoretic mobility-shift assay (EMSA) as a primary method of analysis. Four different double-stranded 32P-labeled oligonucleotides carrying the putative wild-type or mutant ERE half-sites were incubated with NE from Huh7 cells. Two protein-DNA complexes were formed only with the oligonucleotides specific for binding site II (2C9_wt_site II; Fig. 2A, lane 2) but not with the oligonucleotides 2C9_wt_site I, 2C9_wt_site III, and 2C9_wt_site IV (results not shown). The specificity of the protein-oligonucleotide binding was confirmed by a successful competition with unlabeled wild-type oligonucleotide (Fig. 2A, lanes 3 and 4) and an oligonucleotide carrying a representative ERE consensus sequence (Fig. 2A, lanes 5 and 6). Moreover, incubation with ERα antibody resulted in a significant shift and/or the competition of the specific band (Fig. 2A, lane 1). These results suggest binding of ERα to ERE half-site II at position −149/−145.
4-Hydroxy-Tamoxifen, Raloxifene, 17β-Estradiol, and 17α-Ethinylestradiol Augment the Binding Activity of ERα to the CYP2C9 Promoter.
To investigate whether ER ligands modify the binding affinity to ERE half-site II, Huh7 cells were treated with 10−6 M 4-OHT, R, EE, ETE, or vehicle (0.2% ethanol) 24 h before harvesting. Lane 1 in Fig. 2B shows the ER-specific complex of the 2C9_wt_site II oligonucleotide formed with the nuclear protein from the vehicle-treated Huh7 cells. The binding was strongly augmented when NE from Huh7 cells treated with 4-OHT (Fig. 2B, lane 2), R (Fig. 2B, lane 3), EE (Fig. 2B, lane 4), or ETE (Fig. 2B, lane 5) was used. It is noteworthy that the effect of 4-OHT and R on binding affinity seemed to be stronger compared with the impact of EE and ETE.
Estradiol Derivatives Inhibit and SERMs Activate the CYP2C9 Promoter.
To investigate the functional significance of ERα for the transcriptional activation of the CYP2C9 promoter, a reporter plasmid carrying 0.5 kb of the CYP2C9 promoter was generated (Fig. 1B) and transiently cotransfected into the Huh-7 cells with pcDNA3.1-ERα of pcDNA3.1 empty vector (negative control). Cells were treated with increasing amounts of 4-OHT, R, EE, and ETE (Fig. 3). As demonstrated by the data in Fig. 3, C and D, CYP2C9 promoter activity tended to be up-regulated upon treatment with 4-OHT and R in a dose-dependent manner. Statistically significant changes were observed only with R. EE and ETE were able to suppress CYP2C9 promoter activity in a ligand concentration-dependent manner (Fig. 3, A and B). The effect of ETE was much more pronounced (p < 0.05) compared with the effect of the endogenous ligand EE (statistically not significant). Maximum dose effects were achieved by treatment with 10−6 M 4-OHT and R and with 10−7 M EE and ETE.
The Transcriptional Activity of ERα Is Regulated Via ERE Half-Site II (−149/−145).
The identified ERE half-site II at position −149/−145 was shown to be a target of ERα binding in EMSA. To further investigate whether this particular ERE half-site is responsible for the observed modulatory effects of estradiol derivatives and SERMs on CYP2C9 promoter, a construct with mutations in ERE half-site II was cotransfected with ERα into Huh-7 cells. Cells were treated with 4-OHT, R, EE, and ETE at the concentrations that produced the strongest effects in the experiments shown in Fig. 3. The results of these experiments demonstrate a clear tendency toward a reversal of the observed suppressive effects with estradiol derivatives and the SERM-dependent induction of the CYP2C9 promoter (Fig. 4). These data are well in line with EMSA experiments, which show a clear and specific binding of ERα to the oligonucleotide carrying the wild-type form of ERE half-site II. Altogether, these findings support the hypothesis that ERE half-site II is important for ERα-dependent regulation of CYP2C9 promoter.
ERα Is Associated with the CYP2C9 Promoter in Huh7 Cells.
To investigate whether ERα may indeed associate with the hypothetical ERα binding site at position −149/−145, as suggested by EMSA data, we used the chromatin immunoprecipitation approach. Genomic DNA from Huh7 cells was isolated, cross-linked, enzymatically digested, and immunoprecipitated with ERα antibody. Resulting complexes were amplified by PCR using a primer set encompassing the putative ERα half-site II (Table 1). As shown in Fig. 5 the primer pair was able to amplify a PCR product of 226-bp length when cells were pretreated with 10−7 M ETE (Fig. 5, lane 7). In contrast to that, no specific immune complexes were generated with proteins from cells treated with vehicle or 4-OHT (Fig. 5, lane 10). These data explicitly point to the ERα association with CYP2C9 promoter upon ERα stimulation by ETE.
CYP2C9 Expression Is Inhibited by Estradiol Derivatives and Activated by SERMs in Human Hepatocytes.
The in vitro data presented above suggest a possible transcriptional regulation of CYP2C9 via the ERα-mediated mechanism. To investigate whether this indeed reflects the situation in liver cells, we determined the CYP2C9 expression in human hepatocytes treated with the ERα ligands EE, ETE, 4-OHT, and R in a dose-dependent manner. Quantitative PCR analysis revealed that CYP2C9 mRNA levels were significantly suppressed by ETE and EE at ligand concentrations of 10−7 and 10−6 M, respectively (Fig. 6). In line with the gene reporter results, the effect of ETE on CYP2C9 gene expression was much stronger compared with the effect of EE. In contrast to that, a weak, but significant, induction of CYP2C9 gene expression was observed with 10−6 M 4-OHT. In addition, no significant changes in CYP2C9 mRNA amounts were observed upon the treatment of cells with R.
Discussion
In the present study we demonstrate that ERα, alone or after activation by its ligands 4-OHT, R, EE, and ETE, is able to modulate CYP2C9 promoter activity via a newly detected ERα binding half-site at position −149/−145. This is the first study that reveals the mechanism by which both estradiol derivatives and SERMs are able to modify CYP2C9 activity. The specificity of ERα binding to the CYP2C9 promoter was demonstrated by both EMSA and ChIP analysis, whereas the ligand-dependent regulation of the CYP2C9 promoter was supported by the gene reporter data and real-time PCR results from human hepatocytes.
The transcriptional regulation of CYP2C9 has become a focus of a number of studies. Kawashima et al. (2006) identified and proved experimentally the functional relevance of two direct repeat 1 elements within the first 200 bp of both the CYP2C9 and CYP2C19 promoters. These sites have been shown to bind HNF-4α and be involved in the transactivation of the CYP2C9 promoter. Martínez-Jiménez et al. (2006) demonstrated in experiments with Hela cells that two coregulators of HNF-4α, peroxisome proliferator-activated receptor γ coactivator-1α and steroid receptor coactivator 1, are necessary for significant HNF-4α-dependent up-regulation of CYP2C9 promoter activity. Chen et al. (2005) showed that nuclear receptors such as CAR and PXR, in cross-talk with HNF-4α, play a critical role in transcription factor-mediated CYP2C9 promoter induction by different drugs, e.g., rifampicin. Moreover, Bort et al. (2004) detected several putative binding sites for HNF-3γ, which probably are involved in the transactivation of the CYP2C9 promoter. Our group has reported a functionally relevant double GATA binding site within the CYP2C9 promoter, which is located in a direct neighborhood to the ERE half-site (Mwinyi et al., 2010c), and is similar to two adjacent functionally relevant GATA binding sites detected in the CYP2C19 promoter (Mwinyi et al., 2010b). It remains to be further investigated whether and how ERα interacts with other transcription factors on CYP2C9 promoter regulation, especially with those proteins that bind in the vicinity of the functionally relevant ERE half-site.
Sandberg et al. (2004) observed a significant inhibitory effect of female sex steroids on CYP2C9 enzyme activity in vivo when analyzing the pharmacokinetic data of the CYP2C9 substrate losartan of individuals treated with OC. These in vivo findings are in line with the in vitro observation of Laine et al. (2003) who tested the effect of eight different female sex steroids on CYP2C9 activity using human liver microsomes and evaluated the efficacy of losartan metabolism. Laine et al. (2003) demonstrated that ETE, as well as some of the tested progestin hormones including gestodene and 3-ketodesogestrel, are potent inhibitors of CYP2C9, whereas 17β-estradiol did not show any relevant effect on CYP2C9 activity. These findings match well with our data, showing that the CYP2C9 promoter is significantly down-regulated by ETE, whereas the effect of EE is comparatively weak. It has to be noted that the data reported by Laine et al. (2003), as well as the inhibition observed in our study, were obtained using steroid concentrations that were higher than the OC plasma levels normally observed in vivo (10−9 to 10−10 M). However, because it is quite difficult to estimate the effective intracellular concentrations of steroids, it is common for such in vitro studies to use higher doses of hormones for achieving the maximal effects (van de Stolpe et al., 2004; Higashi et al., 2007). This, and an inhibitory tendency that was observed at the 10−10 M concentration of ETE (although not supported statistically) (Fig. 3A) will make, in our opinion, the plasma levels of OC sufficient for the transcriptional regulation of CYP2C9. In addition, our data suggest that among the two estradiol derivatives tested ETE is probably the most clinically relevant.
It seems that ERα-dependent transcriptional regulation is rather well conserved in the CYP2C subfamily because we were able to report a similar regulatory mechanism for CYP2C19 (Mwinyi et al., 2010a). The functionally relevant ERE half-site detected in the CYP2C19 promoter is apparently the conserved analog of the ERE half-site described here, as confirmed by the alignment of corresponding fragments (not shown). However, along with sequence-specific similarities there are also slight differences between CYP2C9 and CYP2C19 in terms of their ERα-mediated regulation. Unlike CYP2C9, CYP2C19 promoter activity was strongly and significantly suppressed by both ETE and EE. In addition, SERMs were able to slightly transactivate the CYP2C9 promoter, whereas no such effects could be detected in the CYP2C19 study. The observed weak transactivating effect of SERMs on the CYP2C9 promoter is, however, inconsistent with the results of an in vivo study, which showed that tamoxifen significantly inhibits the metabolism of the CYP2C9 substrate losartan (Boruban et al., 2006). Thus, it cannot be excluded that tamoxifen unfolds its overall stronger inhibitory effect via other, not yet investigated mechanisms such as direct inhibition of the CYP2C9 enzyme or prevention of the ERα binding to the CYP2C9 promoter. The latter mechanism seems to be a more likely scenario because it was also supported by the results of ChIP experiments that failed to demonstrate ERα binding upon 4-OHT treatment.
In summary, our data suggest the possibility that the clinically observed inhibitory effect of OC on CYP2C9 activity (Sandberg et al., 2004) is mediated by an ERα-dependent regulation of CYP2C9 gene transcription.
Authorship Contributions
Participated in research design: Mwinyi, Cavaco, Mkrtchian, and Ingelman-Sundberg.
Conducted experiments: Mwinyi, Cavaco, and Yurdakok.
Performed data analysis: Mwinyi, Cavaco, Yurdakok, and Mkrtchian.
Wrote or contributed to the writing of the manuscript: Mwinyi, Cavaco, Mkrtchian, and Ingelman-Sundberg.
Footnotes
S.M. and M.I.-S. share the last authorship.
This work was supported by Torsten och Ragnar Söderbergs Stiftelser, the Swedish Research Council, the Lundbeck Foundation, and the Portuguese Foundation for Science and Technology [Grants Serviço de Formação de Recursos Humanos/Bolsa de Pós-Doutoramento/34152/2006, Institute for Biotechnology and Bioengineering/Centre for Molecular and Structural Biomedicine-Laboratório Associado, Fundo Europeu De Desenvolvimento Regional Programa Operacional Ciência e Inovação 2010] (to I.C.).
This work is included in the following thesis: Mwinyi J (2010) CYP2C19 and CYP2C9: New Aspects of Pharmacogenetics and Transcriptional Regulation. Ph.D thesis. Karolinska Institutet, Stockholm, Sweden.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.175075.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- PXR
- pregnane X receptor
- CAR
- constitutive androstane receptor
- EMSA
- electrophoretic mobility-shift assay
- NE
- nuclear extract
- PCR
- polymerase chain reaction
- ChIP
- chromatin immunoprecipitation
- EE
- 17β-estradiol
- ETE
- 17α-ethinylestradiol
- 4-OHT
- 4-hydroxytamoxifen
- R
- raloxifene
- OC
- oral contraceptives
- SERM
- selective estrogen receptor modulator
- HNF
- hepatocyte nuclear factor
- ERE
- estrogen responsive element
- ER
- estrogen receptor
- ICI 182,780
- 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol
- kb
- kilobase
- bp
- base pair
- TBP
- TATA-box binding protein.
- Received September 14, 2010.
- Accepted April 11, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics