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[Cancer Research 63, 7490-7496, November 1, 2003]
© 2003 American Association for Cancer Research

Endocrinology

Distinct Molecular Conformations of the Estrogen Receptor {alpha} Complex Exploited by Environmental Estrogens1

David Bentrem2, Jennifer E. Fox2, Sandra Timm Pearce, Hong Liu, Sam Pappas, David Kupfer, James W. Zapf and V. Craig Jordan3

Robert H. Lurie Comprehensive Cancer Center [D. B., J. E. F., S. T. P., H. L., S. P., V. C. J.] and Department of Surgery [D. B., S. P.], Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 [D. K.]; and Incyte, San Diego, California 92121 [J. W. Z.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have advanced the view that estrogens activate the estrogen receptor (ER) {alpha} complex differently. A group of planar (estradiol, genistein, and coumestrol) and nonplanar (methoxychlor and its mono- and didemethylated phenolic metabolites) environmental estrogens, which are all full estrogens in MCF-7 breast cancer cell proliferation assays, was shown to segregate discretely into planar and nonplanar groups. These groups were delineated using a novel assay of mutant ER cDNAs stably transfected into MDA-MB-231 cells and the activation of the transforming growth factor {alpha} target gene in situ that putatively describes the external shape of the ER complex. Planar compounds activate estrogen action through the two traditional activation functions (AFs), AF1 and AF2, in the ER. In contrast, nonplanar compounds can activate estrogen action through AF1 and the amino acids Asp-351 and Asp-538, which are exposed when helix 12 silences AF2. The observation that class I (planar) and class II (nonplanar) compounds have different mechanisms of estrogen action may have important implications for tissue selective modulation of the ER.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potency of natural and synthetic estrogens is traditionally defined by the stimulation of estrogen target tissues. Estrogens increase uterine growth, cause vaginal cornification, regulate the menstrual and estrous cycles, and act as promoters during the initiation and growth of breast cancer.

The discovery and characterization of two discrete ERs,4 ER{alpha} and ERß, in target tissues provide an elegant solution to explain the site-specific effects of estrogens around the body (1, 2, 3, 4, 5) . Simply stated, estrogens bind to the unoccupied ER in a target tissue, and the ER is then activated by changes in the three-dimensional shape of the estrogen-ER complex. The complex dimerizes and binds to specific sites in the promoter region of estrogen-responsive genes. Transcription of target genes is initiated by coactivators and other regulatory proteins that bind to the external surface of the immobilized ER complex. The transcription complex either binds directly to DNA at an ER element or is tethered independently at AP-1 or SP-1 sites (6 , 7) .

Although the ER signal transduction pathway (including ER{alpha} and ERß) confers precise target site specificity, the ER is remarkably promiscuous. A broad range of natural and synthetic estrogens, which are either planar or nonplanar, can activate gene transcription appropriately in target tissues and cause hormone-responsive cancer growth (8 , 9) .

Progress in understanding the mechanics of estrogen action has occurred through the discovery that antiestrogenic ligands produce a spectrum of distinct shapes that potentially control the intrinsic activity of the ER complex (10 , 11) . The LBD of ER{alpha} or ERß has been crystallized with several estrogens and antiestrogens (12, 13, 14, 15, 16) , illustrating the different structures that could cause antiestrogenic action.

For activation, an estrogen is sealed within the hydrophobic LBD by a specific region of the protein referred to as helix 12. This process of sealing permits coactivator binding at a specific site on the external surface referred to as AF2 (Refs. 7 and 17 ; Fig. 1ACitation ). Estrogen action can now be initiated by the transformed receptor complex. In contrast, the bulky side chain of antiestrogens prevents helix 12 from sealing the ligand into the binding pocket of the receptor, and helix 12 is repositioned to prevent coactivator binding at AF2. However, it is now clear that antiestrogens can cause the ER to adopt numerous conformations that alter the position of helix 12 (12, 13, 14, 15, 16) .



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  		Fig. 1. A, the external location of the AF2 binding site in the LBD of the planar E2-ER complex. The surface amino acid Asp-351 (green) is not involved because it is nested beneath helix 12 (yellow) that seals the ligand in the hydrophobic pocket. B, the putative binding site AF2b is formed in the tamoxifen-ER complex, when helix 12 is repositioned, and the charged amino acid Asp-351 is exposed. The amino acid Asp-538 (red) on helix 12 is critical for the estrogen-like activity of the tamoxifen-ER complex. The site referred to as AF2b is distinct from the AF2a site (48) . Both AF1 and AF2 are independent and can synergize (A), whereas AF1 is only functional if Asp-351 and Asp-538 remain intact (B). These two extremes of structure are the basis for the classification of planar (class I) estrogens using the conformation depicted in A and nonplanar (class II) estrogens using the conformation in B (18) .

 
In an earlier publication, we used a functional assay system that targeted the TGF-{alpha} gene to demonstrate that the phenyl ring of an estrogenic triphenylethylene might also cause helix 12 to move and block AF2. We suggested that if an estrogen-ER complex adopted a conformation with both AF1 and AF2 working in synergy, the complex would likely result from a planar ligand (E2 or diethystilbestrol), with an external surface represented as in Fig. 1ACitation (18) . In contrast, a complex represented by Fig. 1BCitation would occur with a nonplanar estrogen that forces helix 12 to relocate rather than seal the ligand into the binding site. In the original article (18) , we used only a mutation at D351G in the ER to identify an estrogen that was nonplanar and relocated helix 12. We now have refined the functional assay to provide additional evidence for the putative novel shape of a second category of estrogens. Based on mutagenesis studies, estrogens in this category require each of three components, AF1 and AF2b, which comprises at least two amino acids, Asp-351 and Asp-538 (Fig. 1BCitation ; Refs. 19, 20, 21 ).

In this paper, we have confirmed and extended our initial study (18) by examining select environmental estrogens. Phytoestrogens tend to be planar, whereas estrogenic metabolites of the insecticide MXY are mono- and bisphenolic compounds with a flexible nonplanar structure, so these two groups of compounds were selected to analyze receptor complex conformation at a target gene. MXY metabolites were studied because the compounds are known environmental estrogens, but the ER{alpha} and ERß complexes have distinct estrogenic and antiestrogenic properties, respectively (22 , 23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Compounds and Source.
MXY and its mono- and didemethylated derivatives were obtained from or synthesized by Y. Hu and D. Kupfer, University of Massachusetts Medical School (24 , 25) . E2, 4-hydroxytamoxifen, Gen, and Coum were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Lines and Tissue Culture.
The characterization and growth conditions used for the MDA-MB-231 breast cancer cell lines stably transfected with the wild-type ER (S30 cells), D351G mutant (JM6 cells), AF1 deletion mutant ({Delta}G16 cells), or D538A mutant have been reported previously (19, 20, 21 , 26) . All cells were grown in phenol red-free MEM supplemented with 5% 3x dextran-coated, charcoal-treated calf serum, 2 mM glutamine, 6 ng/ml bovine insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino acids (estrogen-free medium), and 500 µg/ml G418.

Northern Blot Analysis.
Analysis of TGF-{alpha} mRNA expression was assessed by Northern blots as described previously (27) . Cells were seeded into T-75 flasks and treated for 24 h with compounds as indicated in the figure legends. Total RNA was isolated from S30 and JM6 cells using TRIzol reagent (Invitrogen, Carlsbad, CA). The human TGF-{alpha} cDNA probe was derived from an EcoRI digestion of a TGF-{alpha}-containing plasmid (a generous gift from Dr. R. Derynck; Genentech, San Francisco, CA). Bands were quantified densitometrically using ImageQuaNT analysis (Sunnyvale, CA). Experiments were repeated at least three times.

Proliferation Assay.
MCF-7 cells (obtained from Dr. Dean Edwards, San Antonio, 1984) were deprived of estrogen by growth in estrogen-free medium for at least 3 days before beginning the growth assay (28) . On day 0, cells were seeded into 24-well plates at a concentration of 15,000 cells/well in 1 ml of estrogen-free medium. Cells were then incubated overnight, and medium containing the appropriate compound was added on day 1. All of the compounds used in this study were dissolved in 100% ethanol and added to the medium at a 1:1,000 dilution. The medium-containing compounds were changed on days 3 and 5. On day 6, DNA content was measured according to the method described previously (29) using a fluorometer (Bio-Rad, Hercules, CA).

RT-PCR for Quantification of Human TGF-{alpha} Levels.
Total RNA was extracted from S30 cells (27) and JM6 cells (19) using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The total RNA was then reverse transcribed using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA) and random hexamers as primers. Primers and probes for human TGF-{alpha} were designed as follows: sequences for the forward and reverse primers for human TGF-{alpha} are 5'-GCC-TGT-AAC-ACA-CAT-GCA-GTG-A-3' and 5'-TTT-CCA-AAG-GAC-TGA-CTT-GGA-AG-3', respectively. The TGF-{alpha} probe sequence is 5'-AGG-CCT-CAC-ATA-TAC-GCC-TCC-CTA-GAA-GTG-3' with the fluorescent tag FAM as the reporter and QSY7 as the quencher (MegaBases, Inc., Chicago, IL). The quantity of human glyceraldehyde-3-phosphate dehydrogenase mRNA was also measured in each total cDNA sample for normalization purposes. The probe and primers for human glyceraldehyde-3-phosphate dehydrogenase were purchased from Applied Biosystems. The RT-PCR reactions were performed using the TaqMan PCR Core Reagent Kit (Applied Biosystems). In a total volume of 25 µl, 50 ng of total cDNA, 100 nM probe, and 200 nM primers were used in each reaction. RT-PCR was performed using the ABI-Prism 7700 Sequence Detection System. The PCR conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Statistical Analysis.
The data were analyzed using one-way ANOVA and either a Bonferroni or Dunnett post test using the Prism program (GraphPad Software, Inc., San Diego, CA).

Molecular Modeling.
Structural models of the "antagonist conformation" of the human ER bound to 4-hydroxytamoxifen and the "agonist conformation" of the ER bound to diethylstilbestrol are derived from 3ERT.pdb (13) and ERD.pdb (13) , respectively. The models were prepared for docking first by removing ligands and all water molecules, except the ordered water forming a hydrogen bond with the absorbance of Glu-353, and second by minimizing in the consistent valance force field using Discover (Accelrys, San Diego, CA). Heavy atoms were initially restrained to the crystallographic positions with a quadratic force during minimization. In subsequent rounds of minimization, restraints were progressively reduced to C beta and backbone atoms, backbone atoms, and finally only C alpha atoms. During docking, side chains of residues within 10Å of 4-hydroxytamoxifen or diethylstilbestrol were allowed to move, whereas all other residues and all C alpha atoms were restrained. Furthermore, a hydroxyl on the ligand was restrained to be within 4.0Å of both the ordered water molecule and the absorbance of Glu-353. Docking was performed using Affinity, scored using Energy Analysis, and visualized in Insight 2000 (Accelrys). 4-Hydroxytamoxifen and diethylstilbestrol docked into the antagonist and agonist forms of the ER in positions nearly identical to those seen in the crystal structures.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estrogen-Like Activity in MCF-7 Cells.
Each of the test compounds (Fig. 2A)Citation was evaluated in the cell proliferation assay (Fig. 2B)Citation . The EC50 values based on the MCF-7 growth assays were as follows: E2, 3 x 10-12 M; Coum, 3 x 10-10 M; Gen, 3 x 10-9 M; and MXY, 3 x 10-7 M. Compared with MXY, MDM and DDM had increased estrogenic potency with an approximate EC50 of 2 x 10-9 M.



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  		Fig. 2. A, the structure of planar and nonplanar estrogen-like compounds used in the experimental studies. B, growth of MCF-7 breast cancer cells in the presence of increasing concentrations of test compounds. Each concentration was tested in triplicate, and assays were repeated at least twice.

 
Agonist and Antagonist Actions of the Wild Type and D351G ER at the TGF-{alpha} Gene.
The concentration-dependent agonist action of the test compounds was initially measured using wild-type and mutant D351G stable transfectants, but a representative concentration of each compound (10-6 M) was compared repeatedly to illustrate comparative effects. The compounds, with the exception of MXY, were all fully estrogenic at the wild-type ER (Fig. 3A)Citation , but both MDM and DDM were only weakly estrogen-like (Fig. 3B)Citation when the D351G mutant ER was used. RT-PCR was used to confirm these results (data not shown). The fact that MXY and its two metabolites, MDM and DDM, were not full agonists suggests that the compounds were inactive, partial agonists, or antagonists at the D351G mutant ER.



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  		Fig. 3. Northern blot analysis of TGF-{alpha} RNA. MDA-MB-231 cells stably transfected with the wild-type ER (A) or the D351G mutant ER (B) were treated with test compounds (1 µM) for 24 h. Bar graphs show quantification of TGF-{alpha} mRNA levels normalized to ß-actin as a loading control. Experiments were repeated at least three times, and the error bars represent the SD (*, P < 0.05 and P < 0.01 for A and B, respectively, compared with control).

 
MXY did not block the actions of E2 or phytoestrogens at the TGF-{alpha} gene (data not shown). In contrast, DDM blocked the estrogen-like actions of E2, Coum, and Gen using Northern blot analysis (Fig. 4A)Citation . RT-PCR was used to confirm these data and to illustrate the antagonist activity of MDM (Fig. 4C)Citation . DDM produced a concentration-related decrease in E2-stimulated TGF-{alpha} mRNA in cells containing the D351G mutation using RT-PCR (Fig. 4D)Citation . The compounds, notably DDM, exhibited weak partial agonist activity only at the highest concentration tested (10-6 M).



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  		Fig. 4. A, the antiestrogenic action of MDM and DDM in MDA-MB-231 cells stably transfected with the D351G mutant ER. E2 (100 pM), Gen (1 µM), or Coum (1 µM), with or without DDM (1 µM), was incubated for 24 h before Northern blot analysis of TGF-{alpha} RNA. B, the Northern blot results were quantified with TGF-{alpha} RNA normalized to ß-actin as a loading control, and the error bars represent the SD (*, P < 0.01 compared with control; +, P < 0.001 when E2 is compared with E2+DDM, Coum is compared with Coum+DDM, or Gen is compared with Gen+DDM). C and D, cells were treated for 24 h with 1 µM MDM or the concentrations of compounds described above. TGF-{alpha} mRNA levels were measured using RT-PCR to confirm the Northern blot results. All experiments were performed in triplicate, and the error bars represent the SD (*, P < 0.01 compared with control; +, P < 0.001 compared with E2).

 
Examination of the Asp-538 Residue and the AF1 Domain of the ER.
The agonist and antagonist actions of MDM and DDM were measured using two stable transfectants expressing either an AF1 deletion mutation in the ER ({Delta}G16) or an amino acid substitution (D538A) of a key residue of helix 12. Expression of the estrogen-responsive gene TGF-{alpha} was measured by RT-PCR, confirming that both ER mutants responded to the natural agonist E2 (Fig. 5)Citation . In both the AF1 mutant and the D538A mutant, neither DDM nor MDM showed significant agonist activity over control levels. However, DDM showed antagonist activity by blocking the agonist actions of E2 at concentrations of 10-8 and 10-10 M in the AF1 mutant cell line (Fig. 5A)Citation . Similarly, DDM and MDM both acted as E2 antagonists in the D538A cell line (Fig. 5B)Citation .



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  		Fig. 5. The estrogenic and antiestrogenic actions of MDM or DDM liganded to the AF1 deletion mutant (A, {Delta}G16) or the D538A mutant (B). MDA-MB-231 cells stably transfected with either ER mutant were incubated for 24 h with E2 (10-10 or 10-8 M), DDM (1 µM), or DDM+E2 as indicated (A) or with E2 (10-9 M), MDM (1 µM), MDM+E2, DDM (1 µM), or MDM+E2 as indicated (B). TGF-{alpha} mRNA levels were determined using RT-PCR. All experiments were performed in triplicate, and the error bars represent the SD (*, P < 0.01 compared with control; +, P < 0.01 compared with E2 10-8 M; ++, P < 0.01 compared with E2 10-10 M; #, P < 0.01 compared with E2).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate that the phytoestrogens Gen and Coum and the didemethylated metabolites of the insecticide MXY are all potent estrogens that enhance the proliferation of breast cancer cells. In contrast to antiestrogens, estrogens are generally believed to produce their effects through similar perturbations of the ER (12 , 13) , by helix 12 sealing the ligand within the LBD (Fig. 1A)Citation . This maneuver permits coactivator binding at AF2 (13) and synergy between AF1 and AF2, resulting in amplification of the intrinsic activity of the ER complex (30) .

Evidence from both breast cancer cell growth assays (Fig. 2B)Citation and TGF-{alpha} assays with the wild-type ER (Fig. 3A)Citation suggests that an excellent signal transduction pathway can be established for each ligand-ER complex. It is possible, based on X-ray crystallographic data, to dock either Gen (Fig. 6B)Citation or the didemethylated metabolite of MXY (Fig. 6C)Citation in the ER-diethylstilbestrol complex (Fig. 6A)Citation . Gen is easily docked within the diethylstilbestrol conformation of the ER, but the didemethylated metabolite of MXY could bind in the diethylstilbestrol conformation with the two phenolic hydroxyls of DDM stretching to bind in the positions normally occupied by the phenolic hydroxyls of diethylstilbestrol (Fig. 6C)Citation . This solution would suggest that planar phytoestrogens and DDM bind to the ER in the same way. However, this simple solution may be incorrect. The classical ligand binding model appears to be too simplistic, and a more sophisticated solution for individualized ligand-ER signal transduction is required.



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  		Fig. 6. Computer modeling of Gen or DDM docked in the published X-ray crystallographic solutions of diethylstilbestrol or 4-hydroxytamoxifen. Gen (B) and DDM (C) were docked into the ER-diethylstilbestrol crystal structure (A). D, alternatively, DDM was docked into the ER-4-hydroxytamoxifen crystal structure. Experimental data support solution D for DDM binding to the ER.

 
Gould et al. (31) previously noted that the estrogenic environmental contaminant bisphenol A interacts with ER{alpha} in a manner distinct from that of E2. Gaido et al. (22 , 23) additionally observed that the metabolites of MXY interact with ER{alpha} and ERß to produce estrogenic and antiestrogenic complexes, respectively. The estrogenic and antiestrogenic properties of MDM and DDM were compared and contrasted using a C3 luciferase reporter system in HepG2 human liver cancer cells transiently transfected with either ER{alpha} or ERß (23) . Both MDM and DDM were estrogens with no antiestrogenic activity when complexed with ER{alpha}. In contrast, the ligands had only modest estrogen-like properties, and DDM was more effective as an antiestrogen than MDM when liganded with ERß. Indeed, the reported actions of DDM at ERß (23) were almost identical to those observed in the present report with DDM at the D351G mutant ER (Fig. 3D)Citation , i.e., weak agonist action with predominately antiestrogenic activity. We do not wish to imply, however, that there are similarities in the E domain of ERß and ER{alpha} (D351G). Alignment of human ER{alpha} and human ERß (32) indicated only a 59% identity between the two receptors in the E domain, and Asp-351 in ER{alpha} corresponds to Asp-258 of ERß. The fact that DDM is antiestrogenic at ERß is consistent with the idea that a multipoint model could also be needed for the estrogenic action of ERß in the helix 12 altered mode. The DDM-ERß complex would be an antiestrogen because AF1 is impaired (33) , not because the E domains of ERß and ER{alpha} (D351G) are structurally similar. Our data provide additional information to extend the previous observations in ER{alpha} (22 , 23 , 31) and illustrate the complexity of the estrogen signal transduction pathway.

DDM does not have the antiestrogenic side chain of traditional antiestrogens such as raloxifene (12) . Pike et al. (14) suggested that helix 12 seems to be unstable in ERß. Therefore, there was potential for DDM to adopt an ER antagonist conformation in which AF2 is silenced. Alternatively, there is the possibility that DDM does not require the alkylaminoethoxy side chain that is characteristic of nonsteroidal antiestrogens (34 , 35) to produce an antiestrogenic conformation in ERß. The R,R-enantiomer of THC is an agonist when liganded with ER{alpha}, but an antagonist with ERß (36 , 37) . The compound has no alkylaminoethoxy side chain, but X-ray crystallography demonstrates that the ligand can reorient helix 12 in ERß to prevent coactivator binding. This process has been termed "passive antagonism" (16) . In contrast, THC can form an active complex that binds coactivator at AF2 that can synergize with an active AF1 in ER{alpha} (33) . However, unlike THC, DDM does have a bulky phenolic ring that has the potential to occupy an important part of the space that the dimethylaminoethoxyphenyl side chain of 4-hydroxytamoxifen occupies in the ER LBD (Fig. 6D)Citation .

Subtle differences were observed in TGF-{alpha} levels depending on the ligand and cell line used. Overall, it is clear that Asp-351 is required for the agonist activity of MDM and DDM at the wild-type ER, whereas MXY displayed no agonist or antagonist activity (Fig. 3Citation ; data not shown). Both MDM and DDM exhibited partial antagonist activity at the D351G mutant ER (Fig. 4)Citation . These differences could be explained by the fact that MXY has a low affinity for ER{alpha} (38 , 39) , and its estrogenic activity is derived primarily from one of its metabolites, DDM (22 , 23) . In addition, MXY produced a growth curve that is shifted to the right, when compared with DDM and MDM (Fig. 2B)Citation . We suggest that MXY is unable to bind to the ER and therefore produces no estrogenic or antiestrogenic activity at the TGF-{alpha} gene site. In the {Delta}G16 cells, DDM was a complete antagonist (Fig. 5)Citation . In contrast, DDM was a partial antagonist in the D538A cells, and MDM was a complete antagonist. The subtle differences in the substituent groups of MDM and DDM may require different parts of the ER for optimal antagonist activity.

A compound that binds to the ER and activates AF2 can be classified as a class I estrogen and would exploit the traditional receptor conformation (Fig. 1A)Citation . In contrast, a compound that binds to the ER and requires synergy of AF1 and AF2b would be a class II estrogen because the novel conformation of the receptor would induce estrogen action by alternate methods of ER complex activation (18) . The assay systems that we have used interrogate the estrogen-ER complex with strategic mutations at select surface amino acids known to play a role in the estrogenic activity of the tamoxifen-ER complex. In the classic "antiestrogenic conformation" (13) , amino acids Asp-351 (19) and Asp-538 (Ref. 21 ; Fig. 1BCitation ) are both exposed and essential to define the AF2b site that confers allosteric estrogen-like properties when AF1 is present (20 , 40) . Mutation of Asp-351 or Asp-538 specifically identifies compounds that produce an ER complex with an external shape depicted in Fig. 1BCitation . We show that the planar phytoestrogens Coum and Gen, as well as E2 and diethylstilbestrol, are class I estrogens, but we provide experimental data to demonstrate that the nonplanar xenoestrogens, such as MXY, MDM, and DDM (Fig. 2A)Citation , are class II estrogens (18) . In the case of class II estrogens, when any of the three points of the ER (AF1, Asp-351, or Asp-538) that are required for estrogen action in the AF2-silenced complex are mutated (Fig. 1B)Citation , there is no signal transduction, rendering the complex inactive as an estrogen (Figs. 3Citation 4Citation 5)Citation . These data support the conclusion that DDM produces a better fit in the tamoxifen-ER complex (Fig. 6D)Citation than the diethylstilbestrol-ER complex (Fig. 6CCitation ; Ref. 13 ), because each of the principal mutation sites (AF1, Asp-351, and Asp-538) also silences the estrogen-like activity of the tamoxifen-ER complex (19, 20, 21 , 40) . A class I estrogen has independent AFs and can still produce estrogen action even if any of these three principal sites (AF1, Asp-351, or Asp-538) are mutated.

Using our engineered functional assay system in vitro, we could find no evidence to support the idea that Gen adopts an antiestrogenic conformation when a Gen-ER{alpha} complex is formed. It is possible that part of the uncertainties concerning the protective or chemopreventive properties of phytoestogens has occurred because Gen produces an unusual complex when it binds to ERß (14) . In this structure, helix 12 is in an unusual location in that it assumes a position that is an intermediate between the typical agonist and antagonist positions. If this conformation occurs in vivo, then it is possible that the Gen-ERß complex favors an antiestrogenic conformation because AF2 is partly silenced and AF1 is nonfunctional in ERß (33) . This could also explain why MDM and DDM are antiestrogens with ERß (23) .

It is interesting to note that the MCF-7 proliferation assay used in our study is about 100 times more sensitive than the TGF-{alpha} gene induction assay. Rivas et al. (41) also found that the MCF-7 proliferation assay is extremely sensitive for detection of the estrogenic effects of bisphenolic compounds, but there is a 10–100-fold decrease in potency when a gene target site (progesterone receptor or pS2) is monitored. We believe that the extreme sensitivity of breast cancer cell proliferation to estrogen-like compounds, especially the phytoestrogens Gen and Coum in the "environment," has important implications for the use of health foods by breast cancer patients. Clearly, an increase in the ingestion of natural phytoestrogens has the potential to increase breast cancer growth selectively. This is despite the fact that Gen has been suggested to provide some anticancer protection in laboratory models (42, 43, 44) and was found to produce different transcriptional activation profiles at diverse ER elements in the promoters of select genes (45) . A recent laboratory report (46) concerning the growth-promoting properties of Gen on ER-positive breast cancer in an athymic mouse model illustrates a concern for the breast cancer patient.

In conclusion, we provide experimental evidence to demonstrate that compounds that are classified simply as estrogens in one assay system, i.e., breast cancer cell proliferation assays, do not necessarily produce their estrogen-like actions through the same ER conformation. In broad terms, planar estrogens can be classified as class I estrogens with a fully active AF1 and AF2, but nonplanar compounds that are estrogenic can be classified as class II estrogens with an interaction of AF1 and a complex AF2b site (Asp-351 and Asp-538) on the surface of the ER. Indeed, it is possible that a compound such as DDM, which expresses estrogenic properties in breast cancer cell proliferation assays but displays some weak estrogen-like and antiestrogenic actions in RT-PCR assays at the TGF-{alpha} gene (Fig. 4C)Citation , achieves this through an equilibrium mixture of DDM-ER conformations. The actual consequences of the shapes of class II estrogens may only occur in a special cellular context in which excess cell surface signaling (as in the case of MDA-MB-231 cells) produces a phosphorylation cascade that induces varying degrees of intrinsic activity for the ER complex at the target site. We are currently dissecting the actual phosphorylation routes in various breast cancer cell lines, and this will be the subject of a future report. We, and others (45 , 47) , suggest that the reclassification of environmental and synthetic estrogens into their respective classes may provide important insights into target site specificity, which would be useful in determining the potential carcinogenicity of environmental estrogens and for identifying new selective ER modulators.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the Lynn Sage Cancer Research Foundation, the Avon Foundation, the NIH Specialized Programs of Research Excellence in Breast Cancer (Grant P50 CA89018-02) and the Judy Dluge Foundation. Back

2 Both authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, 8258 Olson, 303 East Chicago Avenue, Chicago, IL 60611. Phone: (312) 908-4148; Fax: (312) 908-1372; E-mail: vcjordan{at}northwestern.edu Back

4 The abbreviations used are: ER, estrogen receptor; LBD, ligand-binding domain; AF, activation function; TGF-{alpha}, transforming growth factor {alpha}; RT-PCR, real-time PCR; E2, estradiol; Coum, coumestrol; Gen, genistein; MXY, methoxychlor; MDM, monodemethylated methoxychlor; DDM, didemethylated methoxychlor; THC, tetrahydrochrysene. Back

Received 5/29/03. Revised 8/25/03. Accepted 8/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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