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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-2456
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3688-3695
Copyright © 2005 by The Endocrine Society

High-Dose Estrogen and Clinical Selective Estrogen Receptor Modulators Induce Growth Arrest, p21, and p53 in Primate Ovarian Surface Epithelial Cells

Jay W. Wright, Richard L. Stouffer and Karin D. Rodland

Division of Reproductive Sciences (J.W.W., R.L.S.), Oregon National Primate Research Center, Beaverton, Oregon 97006; Department of Physiology and Pharmacology (R.L.S.), Oregon Health Sciences University, Portland, Oregon 97201; and Molecular BioSciences Department (K.D.R.), Pacific Northwest National Laboratory, Richland, Washington 99352

Address all correspondence and requests for reprints to: Jay Wright, Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006. E-mail: wrightj{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian cancer is the most lethal gynecological cancer affecting women. Hormone-based therapies are variably successful in treating ovarian cancer, but the reasoning behind these therapies is paradoxical. Clinical reagents such as tamoxifen are considered to inhibit or reverse tumor growth by competitive inhibition of the estrogen receptor (ER); however, high-dose estrogen is as clinically effective as tamoxifen, and it is unlikely that estrogen is acting by blocking ER activity; however, it may be activating a unique function of the ER that is nonmitogenic. For poorly defined reasons, 90% of ovarian cancers derive from the ovarian surface epithelium (OSE). In vivo the ER-positive OSE is exposed to high estrogen levels, reaching micromolar concentrations in dominant ovarian follicles. Using cultured rhesus OSE cells in vitro, we show that these levels of estradiol (1 µg/ml; ~3 µM) block the actions of serum growth factors, activate the G1 phase retinoblastoma checkpoint, and induce p21, an inhibitor of kinases that normally inactivate the retinoblastoma checkpoint. We also show that estradiol increases p53 levels, which may contribute to p21 induction. Supporting the hypothesis that clinical selective ER modulators activate this novel ER function, we find that micromolar doses of tamoxifen and the "pure antiestrogen" ICI 182,780 elicit the same effects as estradiol. We propose that, in the context of proliferation, these data clarify some paradoxical aspects of hormone-based therapy and suggest that a fuller understanding of normal ER function is necessary to improve therapeutic strategies that target the ER.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVARIAN SURFACE epithelium (OSE) is highly prone to malignant transformation. This small population of cells comprises less than a thousandth of the total ovarian volume yet gives rise to nearly 90% of ovarian cancers in women, with a lifetime risk of 1 in 50 (1, 2). The underlying causes of ovarian cancer are poorly understood and largely untested, but OSE transformation may be influenced by unique features of normal ovarian physiology. Models for ovarian cancer etiology propose risk factors that include the repeated wounding and repair after ovulation, the displacement of OSE cells into premalignant cysts, genetics, and hormonal contributions (3, 4, 5, 6).

Estrogen, a major steroidal product of the ovary, has been associated with increased ovarian cancer risk (7), and it can promote tumor growth and cell proliferation in estrogen receptor (ER)-expressing cell lines (8, 9, 10). The proliferative effects of estrogen in cell culture occur at picomolar or nanomolar concentrations typical of serum levels; however, micromolar concentrations of estrogen occur within the ovary, reaching 1–5 µM in the dominant follicle selected to ovulate (11, 12, 13, 14). The OSE surrounding the ovary is ER-positive, yet the effects of micromolar estrogen levels on the OSE have been largely overlooked, and whether these play a role in normal OSE biology or transformation has not been investigated.

The proliferative effects of picomolar and nanomolar estrogen are mediated by the ER and can be countered by excess concentrations of selective ER modulators (SERMs) (15, 16). Although SERMs block estrogen-ER interactions, the SERM-bound ER is not fully inactivated or negated. Normal functionality of the ER can be partially preserved, and novel transcriptional activity may occur (17, 18). The unique conformational properties of ER-SERM complexes can generate a net ER activity that is estrogenic, nonestrogenic, or antiestrogenic, depending on the SERM and the biological context (19, 20). The agonistic and antagonistic effects of SERMs are difficult to predict (15, 21), but a basic goal in drug design has been to create ideally suited SERMs that block some ER activities while promoting others, to improve therapeutic benefit and reduce negative side effects (22, 23). This strategy is hampered by limitations in our understanding of the range of normal ER activities, making distinctions between estrogenic and antiestrogenic effects difficult.

Hormone-based cancer therapies use high doses of tamoxifen, the most widely prescribed SERM (24), or similar compounds to putatively override endogenous estrogen activation of the ER (20); however, it may be inaccurate to attribute the growth-inhibitory effects of tamoxifen to an opposition of ER function. Paradoxically, the clinical response to high doses of estrogen is identical to tamoxifen in regards to tumor flare, rebound rate, and regression (25, 26, 27). The basis for the clinical benefits of high-dose estrogen is unknown, and the effects on normal cell physiology have not been studied, but these data raise the possibility that a qualitatively different, antiproliferative activity of the ER is engaged by high-dose estrogen in vivo that is agonistically induced by tamoxifen or other SERMs. In clinical applications, these SERMs and their metabolites reach micromolar concentrations in ER-positive tissues, comparable to the inhibitory concentrations used in cell culture (28, 29). Consistent with the beneficial effects of high-dose estrogen in cancer therapy, recent data show that micromolar levels of estrogen itself inhibits cell proliferation of ER-positive cells (30, 31).

We previously reported that micromolar estrogen (1 µg/ml) acts in opposition to mitogenic factors, abrogating the proliferative effects of 1% serum in cultured RhOSE cells (31). This concentration of estrogen was noncytotoxic and did not induce apoptosis, even after 5-d exposure, nor did it down-regulate ER protein expression (32). We investigated the effects of estrogen on a number of serum-mediated cell cycle components, including MAPK cascade activation, cyclin D expression, retinoblastoma (Rb) phosphorylation, and DNA synthesis. The inhibition of cell proliferation by estrogen was accompanied by a decrease in phosphorylated Rb (32), and this protection of the Rb checkpoint could account for growth arrest (33). The inhibitory response to estrogen was seen in other ER{alpha}-positive ovarian and breast cancer cells, but not ER{alpha}-negative cells, and did not require ERß expression (32, 34).

Here we examine the basis of Rb activation by estrogen and compare the effects of tamoxifen and the "pure antiestrogen" ICI 182,780. At micromolar concentrations, each of these compounds inhibits RhOSE cell proliferation and elevates p21 expression, a protein directly involved in cell cycle arrest by preventing Rb phosphorylation (35). These compounds also increase levels of p53, a mediator of DNA repair, growth arrest, and cell death (36, 37). We show that these compounds work additively to block proliferation, so that low noninhibitory doses of each compound can, when combined, inhibit proliferation. These data define a novel function of the ER and suggest that the beneficial effects of clinical SERMs in cancer therapy are the result of normal and agonistic ER activity unique to micromolar concentrations of estrogen or other SERMs. The finding that the ER induces p21 and p53 places the ER in a central position to mediate proliferation, growth arrest, and apoptotic potential. Future studies to understand these roles may improve therapeutic strategies to treat breast and ovarian cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

Rhesus ovaries were obtained through the Tissue Distribution Program (Oregon National Primate Research Center), under a protocol approved by the Institutional Animal Care and Use Committee. Surface epithelial cells were isolated and cultured as described previously (31). RhOSE cells were passaged at 1:3, and were not used beyond 10 passages. Normal culture medium was Deficient DMEM; High Glucose (Irvine Scientific, Santa Ana, CA) with 10% fetal bovine serum (Irvine Scientific). Previous work in our laboratory determined that RhOSE cell proliferation rates are similar between 1 and 10% serum, and starvation medium is 0% serum. Media were phenol red-free, and supplemented with penicillin-streptomycin, fungizone (100 U/ml, 100 µg/ml, 0.25 µg/ml; Life Technologies, Inc., Gaithersburg, MD), and tetracycline (10 µg/ml; Sigma-Aldrich, St. Louis, MO).

Cell culture experiments were performed as described (31, 32), using serum deprivation in some protocols where noted. Control medium that promotes cell proliferation contained 1% fetal calf serum, which was charcoal stripped in experiments using SERMs at concentrations less than 500 ng/ml. SERMs were purchased from Calbiochem (San Diego, CA; estradiol), Sigma-Aldrich (tamoxifen), or Tocris Cookson (Bristol, UK; ICI 182,780).

Immunohistochemistry

Immunohistochemical analysis was performed on cells grown in chambered slides (Nalge Nunc International, Rochester, NY). Cells were fixed with an antigen retrieval fixative (38). Primary antibodies were used against the following antigens: cyclin D1 (Zymed Laboratories, San Francisco, CA), INK4a (Upstate Biotechnology, Lake Placid, NY), phospho-Rb and p21 (Cell Signaling Technology, Inc., Beverly, MA), p53 (Calbiochem), and bromodeoxyuridine (BrdU) (Roche Diagnostics Corp., Indianapolis, IN). DNA synthesis was measured as BrdU incorporation, as described (32). For BrdU detection, cells were incubated in 2 N HCl for 30 min after the initial fixation, per instructions of the antibody provider. Secondary antibodies were alkaline-phosphatase-conjugated antimouse or antirabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and ALEXA FLUOR-conjugated antimouse or antirabbit IgG (Molecular Probes, Eugene, OR). Antibodies were used at a concentration of 1:1000, except the ALEXA FLUOR secondaries, which were used at 1:500. Colorimetric reactions were developed using a nitro blue tetrazolium/bromo-chloro-indolyl phosphate premixed solution (Kirkegaard & Perry Laboratories). Cells were visualized using differential interference contrast optics or appropriate fluorescent filters on an inverted Olympus OM microscope, and digitally imaged with an Olympus DP11 camera (Olympus, Melville, NY).

Image analysis

Data were analyzed by examining multiple fields of view within each slide chamber to establish consistent and comprehensive sampling of cell populations. Data are presented as a count of positively labeled cells, with the labeling antibody specified for each experiment. Fluorescent analysis required imaging identical fields under the appropriate fluorescent channels followed by a measure of the color composition (intensity) of paired images using the Adobe Photoshop 7.0.1 Color Picker tool (Adobe Systems, Inc, San Jose, CA).

Replicating data and statistical analysis

Reproducibility of results was assured by a minimum of three replicate trials of each experiment. One-way ANOVA was performed on data sets, followed by unpaired, two-tailed t tests. Significance was defined as P < 0.05. Coefficients of correlation were calculated by Spearman rank order correlation, using SigmaStat, version 2.0, statistical software (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estradiol affects cyclin D expression and Rb phosphorylation

During normal progression through the G1 phase of the cell cycle, cyclin D expression and Rb phosphorylation are temporally and functionally linked, with newly synthesized cyclin D forming active complexes with cyclin-dependent kinase (CDK) 4 and CDK6 to initiate the phosphorylation of Rb, necessary for entry into S phase (39). We wished to examine the nature of estrogen-dependent inhibition of cyclin D expression and Rb phosphorylation by determining whether estrogen either prevents the initiation of these events or triggers their down-regulation. We did this by serum-depriving RhOSE cells, then altering the time at which these cultures were exposed to estrogen in relation to the timing of serum stimulation. Within 24 h of serum deprivation, RhOSE cells are almost entirely negative for cyclin D and phospho-Rb; however, 24 h after stimulation with 1% serum, cyclin D and phospho-Rb reach maximal levels. In one set of experiments, RhOSE cells were serum-deprived for 24 h, then treated with 1 µg/ml estradiol for 4 h and stimulated with serum for 24 additional hours. These experiments examined the ability of estrogen to prevent cyclin D expression and/or Rb phosphorylation by serum. In a second set of experiments RhOSE cells were serum-deprived, then stimulated with serum for 24 h. At this time they were exposed to 1 µg/ml estradiol for 1 or 3 d. These experiments examined the ability of estradiol to down-regulate cyclin D expression and/or Rb phosphorylation, after being up-regulated by serum.

We found 1 µg/ml estradiol did not block the initial up-regulation of cyclin D by serum, and did not significantly reduce the levels of cyclin D in stimulated cultures until after 3 d of exposure to estradiol, at which time cyclin D was detected in approximately one fourth of the cells in untreated control cultures (Fig. 1Go). In contrast, pretreatment with estradiol before serum stimulation attenuated Rb phosphorylation by serum to 40% of that seen in untreated controls. In cultures stimulated by serum before estrogen exposure, the level of phosphorylated Rb fell to 50% and 30% by d 1 and 3, respectively, in response to 1 µg/ml estradiol, compared with cultures not exposed to estradiol.



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  		FIG. 1. Estradiol reduces cyclin D expression and Rb phosphorylation, but at different levels of directness. RhOSE cells were serum-deprived for 24 h, then stimulated with serum in the presence or absence of 1 µg/ml estradiol and assayed for cyclin D expression and Rb phosphorylation. Estradiol was added 4 h before (Pre) or 24 h after (Post) serum stimulation. RhOSE cells were cultured for 1–3 d after the addition of estrogen and were then fixed for immunohistochemical analysis. Cyclin D1 expression and Rb phosphorylation were assayed as the number of positive nuclei and compared with control cells not treated with estrogen. Asterisks denote P < 0.05 relative to controls. Error bars represent the SD. Data represent a minimum of three replicate experiments for each antigen.

 
These results show that estradiol prevents the phosphorylation of Rb in response to serum but has no effect on the initial induction of cyclin D. In addition, when serum stimulation occurs before the addition of estradiol, phospho-Rb and cyclin D are decreased at different rates, suggesting a less direct regulation of cyclin D levels, which may fall as a secondary effect of preventing cell cycle progression.

Exposure to 1 µg/ml estradiol induces p21 and p53, but not INK4a

Because estrogen does not prevent the initiation of cyclin D expression after serum stimulation but does prevent Rb phosphorylation, we examined the potential for estrogen-mediated inhibition of cyclin D/CDK activity. This can be achieved by INK4a, which binds cyclin D and prevents its association with CDK4–6, or by p21, which binds to and inactivates cyclin/CDK complexes. INK4a was not detected in RhOSE cell cultures treated with 1 µg/ml estradiol (data not shown). However, p21 expression was increased by 1 µg/ml estradiol within 24 h (Fig. 2AGo). In the absence of serum, 1 µg/ml estradiol increased the number of p21-positive nuclei to approximately 3-fold of that seen in untreated groups. In the presence of 1% serum, there was a 2-fold increase in p21 levels over that seen in cultures not exposed to estradiol. Serum alone caused an increase in p21 expression, consistent with the observation that the p21 promoter is targeted by many transcription factors, and during a normal mitogenic response mediates a feedback regulation of Rb (40); however, the fold increase in p21 by estrogen is comparable in the presence and absence of serum, indicating serum-independent induction of p21 by estrogen. When RhOSE cells were incubated in the presence of serum, estrogen, and the transcriptional inhibitor actinomycin D, p21 expression fell to levels below those seen in the presence of serum and estrogen, demonstrating that p21 levels are mediated at the transcriptional level, not by translation of extant mRNA.



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  		FIG. 2. Estradiol causes an increase in both p21 and p53 protein in cultured RhOSE cells. Cells were grown in medium containing 0 or 1% serum for 24 h, and then for an additional 24 h in the presence or absence of 1 µg/ml estradiol or in the presence of estradiol and 1 µg/ml actinomycin D. After culture, cells were fixed and processed for immunohistochemical analysis using antibodies against p21 (left) or p53 (right). Data represent the number of positively labeled nuclei counted from five replicate experiments, normalized to the control basal values (cells cultured in the absence of estradiol). Single asterisks denote P < 0.05 relative to controls grown in 0% serum. Double asterisks denote P < 0.05 relative also to controls grown in 1% serum. Error bars represent the SD.

 
Because p21 is regulated by many transcription factors, and is a prime mediator of cell cycle arrest by p53, we examined whether p53 is up-regulated by estradiol. We observed a similar response to estradiol in p53 levels as seen with p21, i.e. 1 µg/ml estradiol caused a 2-fold increase in p53 levels in the presence and absence of serum (Fig. 2BGo). In addition, serum alone increased p53 levels, but to a lesser degree than did 1 µg/ml estradiol. Actinomycin D did not reduce the levels of p53 seen in the presence of serum and estrogen; however, actinomycin D alone triggers high levels of p53 protein expression (41, 42), possibly by enhancing translation of p53 mRNA or by stabilizing p53 protein (43).

Phospho-Rb correlations with cyclin D, p21, and p53

The use of fluorescently labeled secondary antibodies for immunohistochemical visualization of cells allowed us to survey antigen coexpression on a cell-by-cell basis. We sought to determine whether the pattern of Rb phosphorylation had a positive, negative, or neutral correlation with the presence of cyclin D, p21, or p53. We found positive coefficients of correlation for cyclin D1 expression and phospho-Rb in the presence (closed circles) and absence (open circles) of 1 µg/ml estradiol of 0.73 and 0.62 (P < 0.05), respectively (Fig. 3AGo). The coefficient of correlation between p21 and phospho-Rb was negative and significant (–0.31; P < 0.05; Fig. 3BGo), consistent with its role in preventing cyclin/CDK phosphorylation of Rb. p53 also negatively correlated with phospho-Rb, but this correlation was not significant (–0.23; P > 0.05; Fig. 3CGo). These data support a mechanism of Rb activation by transcriptional control of p21 to inhibit cyclin/CDK activity and Rb phosphorylation.



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  		FIG. 3. Fluorescent labeling shows correlations between cyclin D, p21, and p53 levels and Rb phosphorylation. Cells were cultured in medium containing 1% serum, with or without 1 µg/ml estradiol for 24 h. Immunohistochemical analyses were performed using fluorescently labeled secondary antibodies to detect anti-phospho-Rb (green) and -cyclin D1, -p21, or -p53 (red, A, B, and C, respectively). Arrows are included to provide landmarks in each frame and to readily show instances of differential visibility between fluorescent channels. Color sampling of digital images was used to plot the relative fluorescent intensities of the red and green channels associated with each nucleus, to assess positive or negative correlations in antigen expression. In the case of cyclin D expression, cultures were also examined that had not been exposed to 1 µg/ml estradiol (A, open circles). Line plots show the coefficients of correlation (light bar is in the absence of estradiol, A). Scale bar, 10 µm.

 
Tamoxifen and ICI 182,780 inhibit DNA synthesis in cultured RhOSE cells

We wished to determine the effects of other ER ligands on RhOSE cell proliferation by culturing cells in the presence of the partial estrogen antagonist, tamoxifen, and the pure antiestrogen, ICI 182,780 (44). Each had a similar effect as estradiol on DNA synthesis in RhOSE cells (Fig. 4Go). At 20 or 200 ng/ml, tamoxifen and ICI 182,780 had neither a positive nor a negative effect on DNA synthesis; however, at 1000 ng/ml, tamoxifen and ICI 182,780 reduced the number of nuclei actively synthesizing DNA to 40% and 50%, respectively, of the number in untreated groups, compared with a reduction to 45% of untreated controls by 1000 ng/ml estradiol. When the ER-negative breast cell line, HBL-100, was incubated in the presence of 1000 ng/ml tamoxifen or ICI 182,780, no effect on proliferation was observed (data not shown), and these cells were not growth-inhibited by 1000 ng/ml estradiol (32).



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  		FIG. 4. RhOSE cell proliferation is inhibited by tamoxifen and ICI 182,780. Cells were cultured in medium containing 1% serum for 24 h. After the addition of 20–1000 ng/ml tamoxifen (Tam) or ICI 182,780 (ICI), or 1000 ng/ml estradiol (Est), cells were grown for an additional 8 h, at which time BrdU (1 mM) was introduced to treated or untreated control cells, and allowed to continue in culture for 16 more hours. After fixation, cells were labeled using an anti-BrdU antibody, and nuclei containing BrdU-positive nuclei were counted. Data are the result of five independent experiments, normalized to controls (grown in the absence of estradiol, tamoxifen, or ICI 182,780). Asterisks denote P < 0.05 relative to untreated controls. Error bars represent the SD.

 
Tamoxifen and ICI 182,780 modulate cell cycle regulators as does estradiol

Because both tamoxifen and ICI 182,780 had similar effects as estradiol on RhOSE cell proliferation, we wished to determine whether the cell cycle components regulated by estradiol were affected in the same or a distinct manner by tamoxifen and ICI 182,780. When RhOSE cells were cultured in 1% serum, in the presence or absence of 1 µg/ml estradiol, tamoxifen, or ICI 182,780, cyclin D levels were not significantly affected within 24 h of exposure to these compounds; however, all three compounds reduced Rb phosphorylation by comparable amounts (Fig. 5AGo). Similar mechanisms of inhibition of cell proliferation by these ligands are supported by the observation that each of these ligands induced p21 and p53 (Fig. 5BGo) to a degree that was statistically identical to the effects of estradiol.



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  		FIG. 5. Estradiol (Est), tamoxifen (Tam), and ICI 182,780 (ICI) have similar effects on key regulators of the cell cycle. RhOSE cells were cultured in medium containing 1% serum for 24 h before the addition of 1 µg/ml estradiol, tamoxifen, or ICI 182,780. Cultures were maintained for an additional 24 h in the presence or absence (controls) of these compounds. Immunohistochemical analyses were performed for the specified antigens, and positively labeled nuclei were counted. Data show the results of four independent experiments for each antigen detection, normalized to controls (cells grown in the absence of ER ligand). Asterisks denote P < 0.05 relative to controls. Error bars represent the SD.

 
Estradiol, tamoxifen, and ICI 182,780 act additively to inhibit RhOSE cell proliferation

Because the primary mode of action of antiestrogenic SERMs is attributed to competitive binding and inhibition of normal ER function, we wished to determine whether the effects of estradiol, tamoxifen, and ICI 182,780 are consistent with competitive interactions between these ligands. In the presence of 1% serum, RhOSE cells were proliferatively active and incorporated BrdU into newly synthesized DNA. The percentage of nuclei incorporating BrdU was not significantly different when cells were cocultured in the presence of 0.5 µg/ml estradiol, tamoxifen, or ICI 182,780 (Fig. 6Go). However, when cells were cultured with all three ligands, each at 0.5 µg/ml, the number of BrdU-positive nuclei declined to about 30% of that seen in the absence of ligand or in the presence of each ligand alone at a low dose. This decline in DNA synthesis was comparable to that observed in RhOSE cells cultured with 1.5 µg/ml estradiol alone, to 40% of untreated controls, and was statistically indistinguishable from such cultures. These data indicate additive, dose-dependent, interactions between estradiol, tamoxifen, and ICI 182,780, in regard to mediating RhOSE cell proliferation, and are not consistent with competitive, inhibitory receptor binding between these SERMs.



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  		FIG. 6. Noninhibitory doses of estradiol (Est), tamoxifen (Tam), and ICI 182,780 (ICI) act additively to inhibit RhOSE cell proliferation. Cells were cultured in medium containing 1% serum for 24 h, before the introduction of the indicated amount of estradiol, tamoxifen, and/or ICI 182,780. BrdU (1 mM) was added 8 h after addition of estradiol, tamoxifen, and/or ICI 182,780, and cultures were continued for 16 more hours. BrdU incorporation was determined as described. Data are the result of four independent experiments, normalized to controls (grown in the absence of estradiol, tamoxifen, or ICI 182,780). Asterisks denote P < 0.05 relative to controls. Error bars represent the SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present work we report the antiproliferative effects of estradiol, tamoxifen, and ICI 182,780 in RhOSE cell cultures at high physiological (intraovarian) and clinically beneficial concentrations. Naturally occurring levels of estradiol in the dominant ovarian follicle selected to ovulate reach about 1 µg/ml and may influence the ER-positive OSE surrounding the ovary (11, 13). In cancer therapy, tamoxifen is typically administered at 20 mg daily, resulting in 10 µg/g of tissue of tamoxifen and its metabolites in ER-positive tissues, and 1 µM in plasma (28, 45). High-dose estrogen typically is administered at 15 mg daily and also accumulates in ER-positive normal and cancerous breast tissue (46). At high micromolar concentrations, we show that these SERMs elicit inhibitory effects on the cell cycle, and in this context the partial antagonist tamoxifen and the pure antiestrogen ICI 182,780 (47, 48) act the same as estrogen, blocking the mitogenic effects of serum. Our findings indicate that the mechanism of estrogenic inhibition of the cell cycle is by preventing Rb phosphorylation, which can effectively block entry into S-phase (49). This is supported by the observed 3- to 4-fold increase in the expression of the cyclin/CDK inhibitor p21, in response to estrogen, tamoxifen, and ICI 182,780. We demonstrate that inhibition of transcription by actinomycin D prevents estrogen-mediated induction of p21, indicating p21 may be a target for transcriptional activity by the ER or other transcription factors induced by the ER. A cell-by-cell analysis shows that RhOSE cells expressing p21 in response to estrogen do not contain high levels of phospho-Rb and, in complementary findings, cells that retain phospho-Rb do not express p21. This significant negative correlation (–0.31; P < 0.05) supports the proposal that p21 is an effector of estrogen-dependent growth arrest.

We examined changes in p53 levels in response to these SERMs, because steroid receptors can regulate the DNA damage response protein p53, and because p21 is the target of multiple transcription factors, including p53 (50, 51, 52). We found that p53 was elevated 2- to 3-fold in response to estrogen, tamoxifen, and ICI 182,780, and like p21 this increase was seen in the presence and absence of serum. Because p53 can be induced in a transcription-independent manner by actinomycin D and other inhibitors of transcription (42, 43), we were unable to conclude whether the ER directly mediates the transcription of p53, due to these obscuring effects. The ER and p53 can transcriptionally regulate each other (53, 54), but there is also evidence for direct interaction between the ER and p53 proteins (55) and indirect interaction at DNA promoter regions (50), so diverse levels of p53 regulation could be affected by the ER. The role for p53 in estrogen-mediated p21 induction and the means by which estrogen causes an elevation in p53 levels are currently being investigated, and include the compelling possibility that direct interaction with the liganded ER protects p53 from murine double minute 2-mediated degradation, an important topic of therapeutic interest (56).

The observation that tamoxifen and ICI 182,780 prevent RhOSE cell proliferation and induce p21 and p53 in the absence of endogenous estrogen is significant in demonstrating that these compounds actively mediate these effects and are not simply counteracting the effects of estrogen. Others have shown that tamoxifen and ICI 182,780 elevate p21 in cells that are mitogenically stimulated by low, nanomolar levels of estrogen; however, it has been proposed that this is the result of antiestrogenic effects on the ER, to block the down-regulation of p21 (57, 58). Here, we show that the elevation of p21 by these SERMs is not due to the inactivation of normal ER activity, but is a consequence of high doses of these compounds that initiate growth arrest, even in cells exposed to serum factors. These data resolve the additive effects of estrogen, tamoxifen, and ICI 182,780 in preventing cell cycle progression.

Our findings emphasize the need to reevaluate our understanding of normal estrogenic activities and the modes of activity of clinically prescribed SERMs. Understanding the physiological, pathological, and clinical effects of high levels of estrogen and other SERMs is important, but not well studied. The long-term effects of high levels of estrogen are unknown, but may relate to the predisposition of the OSE to transformation, because this minute component of the ovary gives rise to the vast majority (~90%) of ovarian cancers in human. Paradoxically, it is possible that, instead of estrogen contributing to ovarian cancer risk, removal of high estrogen after menopause may abrogate cell cycle inhibition (p21) and DNA repair (p53) mechanisms in the OSE and contribute to the transformation of these cells.

By investigating the effects of high-dose estrogen on the cell cycle, we have shown not only that tamoxifen and ICI 182,780 are not obviously antiestrogenic in regards to cell proliferation, but also that our understanding of true estrogenic function is limited. We show that dose-response is another defining factor in normal ER function, suggesting that some instances of antiestrogenic effects of tamoxifen in cell culture models can be attributed simply to high dose, where tamoxifen is used in micromolar concentrations, 10 or 100 times higher than estradiol (e.g. Ref. 59). In this regard, delineating the qualitative differences between high and low doses of ER agonists is important. Previous work has shown proliferation to occur in postovulatory rabbit (60) and mouse (61) OSE, and could be mediated at least in part by estrogen. In the primate ovary, peak micromolar levels of estrogen occur just before ovulation and decline during the luteal phase. It is possible that peak levels of estrogen are growth inhibiting in vivo but lower levels promote or permit OSE repair in the postovulatory period, consistent with a dual mode of estrogenic action.

The current work provides resolution to some paradoxical actions of estrogens and other SERMs. Redefining tamoxifen and ICI 182,780 as estrogenic in their ability to prevent cell growth suggests that pharmacological strategies for hormone-based therapy should aim at activating, rather than inactivating, the ER. Another target for drug design is suggested by the effects of these SERMs on p53. If p53 is an essential intermediary between the ER and cell cycle inhibition, then determining whether the ER up-regulates p53 by transcriptional or nontranscriptional mechanisms is essential. If the ER modulates p53 by direct protein-protein interactions, then the chemical motif driving this regulation could be a novel pharmacological target for cancer therapy that would be effective even in ER-negative cancers.


   Footnotes
 
This work was supported by National Institutes of Health Grants RR00163, HD-20869 (to R.L.S.), and CA-78722 (to K.D.R.).

First Published Online March 8, 2005

Abbreviations: BrdU, Bromodeoxyuridine; CDK, cyclin-dependent kinase; ER, estrogen receptor; OSE, ovarian surface epithelium; Rb, retinoblastoma; SERM, selective estrogen receptor modulator.

Received December 14, 2004.

Accepted February 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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