This article is based on a presentation at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research held 7-8 October 1999 in Research Triangle Park, North Carolina, USA.
Address correspondence to C.L. Walker, University of Texas M.D. Anderson Cancer Center, Dept. of Carcinogenesis, Science Park-Research Division, Park Road 1C, Smithville, TX 78957 USA. Telephone: (512) 237-9550. Fax: (512) 237-2475. E-mail: cwalker@odin.mdacc.tmc.edu
This work was supported in part by National Institute of Environmental Health Sciences (NIEHS) grant ES08263 and National Cancer Institute CA72253 (to CLW), NIEHS Center grant ES07784, and National Institutes of Health grant CA16672.
Care and handling of animals were performed according to National Institutes of Health guidelines in facilities accredited by the Association for the Accreditation of Laboratory Animal Care, and all protocols involving the use of animals were approved by the Institutional Animal Care and Use Committee at the M.D. Anderson Cancer Center.
Received 23 February 2000; accepted 7 June 2000.
Uterine leiomyomas are benign neoplasms of uterine smooth muscle that are present, although not always symptomatic, in the majority of adult women (
1). Although they occur at a frequency arguably greater than that of any other type of neoplasm, the etiology of these tumors is unknown, and the factors that regulate their growth and clinical severity have not been well established. Leiomyomas are morphologically similar at the cellular level to the normal myometrium from which they arise, except for their aberrant growth. However, this growth is not completely unregulated, and in fact, it appears to be primarily under the control of steroid hormones. It is a well-known and clinically used fact that the growth of leiomyomas can be arrested, and furthermore reversed by the administration of gonadotropin-releasing hormone agonists, which create a hypoestrogenic hormonal milieu (
2,3). Unfortunately, the long-term use of this treatment is not feasible due to adverse effects of estrogen ablation in other tissues, and upon cessation of treatment, leiomyomas rapidly return to pretreatment size (
4). Additionally, leiomyomas typically present clinically during the reproductive years, sometimes increase in size during pregnancy, and regress after menopause. Together, these observations demonstrate the dependence of leiomyoma on ovarian hormones to sustain dysregulated growth.
Experimental data also support the role of ovarian hormones, particularly estrogens, as mediators of leiomyoma development. Several studies have demonstrated an increase in estrogen receptor (ER) levels in leiomyomas (5-7) and a decrease in estrogen metabolism (8), suggesting that increased local hormone levels might contribute to pathogenesis. Endometrial glandular hyperplasia is seen at the margins of submucosal leiomyomas, consistent with the existence of a localized hyperestrogenic environment in the proximity of the tumors (9,10). Moreover, an elevated transcriptional response to estrogen exposure (11) suggests that leiomyomas may have an increased response to estrogen stimulation. Epidemiologically, the risk factors for leiomyoma development are similar to those for mammary and endometrial carcinoma and are consistent with unopposed estrogen stimulation (12). However, although it is clear that these tumors are dependent on ovarian hormones, there is little mechanistic understanding of hormonal control of either normal or neoplastic smooth muscle.
Our understanding of leiomyoma etiology and treatment can be significantly enhanced by the use of experimental animal models that recapitulate the human disease. We have previously characterized an in vitro/in vivo animal model for uterine leiomyoma, the Eker rat. Female Eker rats spontaneously develop uterine leiomyoma at a frequency of approximately 65% by 16 months of age. A panel of cell lines established from these tumors has been evaluated for growth characteristics and tumorigenicity. Four of five of these cell lines express ER and progesterone receptor (PR), and respond to ovarian hormones (13,14). The prototypical cell line of this panel, Eker rat leiomyoma tumor-derived (ELT) 3, can be stimulated to proliferate in a dose-responsive manner by 17ß-estradiol (E2) in culture. ELT3 cells form tumors when injected into nude mice, and E2 administration stimulates the growth of these xenografts in vivo (14). This in vitro/in vivo model has been used to evaluate the effects of endogenous hormones, their mechanisms of action, and the potential for hormone receptor-mediated effects by exogenous receptor ligands.
Examination of hormonal regulation of uterine smooth muscle
in vivo has revealed that myometrial tissues from young, sexually mature Eker rats proliferate in response to cyclic variations in levels of estrogen and progesterone, but this proliferation is balanced by concomitant cyclic changes in apoptosis (
15). Although hormone cyclicity remains relatively constant throughout the animal's reproductive life, the levels of both proliferation and apoptosis rapidly decline soon after sexual maturity, with the net result being a retention of tissue homeostasis but a relative insensitivity to hormonal fluctuation. In contrast, leiomyomas have significantly lower levels of apoptosis than normal myometrium, and proliferation occurs even under hormonal conditions that accompany reproductive senescence. This increase in proliferation and reduction in apoptosis correlates with a loss of tissue homeostasis and a net increase in cell number. These data suggest that leiomyoma cells may become hypersensitive to steroid hormone stimulation, perhaps by reacquiring the responsive phenotype of young or pregnant animals (
16) (Figure 1).
Figure 1. A model of uterine myometrial homeostasis over a lifetime. Data from the Eker model show that the highly responsive phenotype of the young, sexually mature myometrium diminishes over the reproductive lifetime. This process may be interrupted by cycles of pregnancy when both hormone levels and tissue responsiveness are high. The tumor regains the responsive phenotype of youth, partly due to reduced levels of apoptosis, resulting in the capacity for sustained proliferation even under conditions of low plasma hormone levels.
If leiomyomas are indeed especially sensitive to the effects of steroid hormone receptor activation, they represent a potential target for endocrine disruptors, a class of environmental compounds that have come under recent scrutiny. The most studied of such compounds are those that mediate transcriptional activation and cellular proliferation via their ability to bind and activate the ER. In many reproductive tissues, activated ER modulates transcription of estrogen-responsive genes and typically has a net effect of stimulating proliferation. Numerous studies have demonstrated that exogenous ER ligands can mimic the effects of the principal endogenous hormone, E2, both in vitro and in vivo. In some cases, these compounds may compete with E2 for binding to the receptor and antagonize its effects. Although it is clear that some xenobiotics interact with the ER at the biochemical level, whether these endocrine-disrupting compounds contribute to disease or neoplasia in humans is a subject of considerable controversy (17,18). The majority of studies examining the potential for endocrine disruption in human tissues by exogenous ER ligands have focused on the uterine endometrium and the breast; the uterine myometrium has been largely overlooked as a target tissue.
It is well recognized that the ultimate effect of the exogenous ligand is extraordinarily difficult to predict. First, receptor binding cannot be predicted from a compound's structure, and many exogenous compounds that bind the receptor bear little resemblance to the natural ligand (Figure 2). Second, the ability of the exogenous ligands to reach the target tissue, their effective concentration in situ, the level of in vivo metabolism, and the endogenous hormonal milieu in which the exposure occurs are just a few of the factors that may control the magnitude of the in vivo effect. Even more important, perhaps, is that the effects of ER ligands are both qualitatively and quantitatively tissue specific (20,21); even the direction of the effect (agonism vs antagonism) is impossible to predict from receptor binding. Thus, a variety of additional tests may be necessary to determine whether a substance carries the potential for either risk or benefit to a given tissue.
Figure 2. Despite dissimilar structures (19), each of these xenobiotic compounds can compete with E2 for binding to ER. The principal natural ligand, E2; a synthetic estrogen, DES; a dietary flavonoid, genistein; a pesticide, HPTE; and the primary active in vivo metabolite of the therapeutic antiestrogen tamoxifen, hydroxytamoxifen (OH-TAM), are shown. OH-TAM is often used in in vitro studies instead of tamoxifen due to its enhanced activity.
The ability of exogenous compounds to bind the ER, and thus affect normal or neoplastic myometrial tissues, need not be viewed only in terms of adverse effects. In fact, the vulnerability of leiomyoma to hormonal control could be exploited to develop therapies that could complement or eliminate surgical intervention. Estrogen antagonism, for example, could induce remission or inhibit leiomyoma growth, at least long enough to allow the natural regression of the tumor during menopause. To develop such therapies, it would be necessary first to determine the magnitude of hormonal modulation that would be effective, and second, to discover a method of achieving this effect without adverse impact on other tissues. Significant progress has been made toward these goals for some hormonally responsive tissues, resulting in the development of selective estrogen receptor modulators (SERMs) that bind ER and exhibit tissue-specific agonist or antagonist activity. In general, these compounds are used for their ability to act as estrogen antagonists in the uterus and in mammary tissues while acting as agonists in the bone and cardiovascular system (20,22). Tamoxifen is a member of this class of compounds and is currently used to treat and prevent estrogen-responsive breast cancer [reviewed in (23)]. Although effective, tamoxifen has the unwanted side effect of endometrial stimulation and has been associated with an increased risk for endometrial cancer (24,25). Second-generation SERMs, such as raloxifene, have been developed to avoid this complication and to achieve greater agonist activity in other tissues such as bone. Few studies have examined the effects of SERMs in the uterine myometrium (26); the effects have often been assumed to be similar to those in the endometrium. Evaluating the effects of supplemental hormone treatment or therapeutic SERMs on the myometrium could reveal valuable information, given the increasing number of women now using either hormone replacement therapy or a therapeutic estrogen antagonist for disease treatment or relief of menopausal symptoms.
Significantly, exogenous estrogens may not be limited to pharmaceuticals administered under the control of a physician. Many potential endocrine disruptors exist in foods and over-the-counter dietary supplements. By controlling the diet or using herbal remedies, individuals may be able to alter the body's normal hormonal milieu and do so to either benefit or detriment. Because it is impossible to know a priori which effect exogenous ligands will have in a given tissue, available data on the effects of these compounds in other tissues cannot be generalized to determine their activity in the myometrium.
Herein we present a summary of data using the Eker rat model to determine the impact of exogenous ER ligands on uterine leiomyoma. We have established that various natural and synthetic compounds have the potential to act as endocrine disruptors in the myometrium either as agonists or antagonists. Furthermore, we have investigated the possibility that SERMs can be used to modulate the incidence or growth of leiomyoma by antagonizing the effects of estrogen in the myometrium. These data summarize our current understanding of the hormonal modulation of smooth muscle growth and the effects of exogenous ER ligands on tumors of the uterine myometrium.
Several
in vitro assays have been employed to determine the potential for exogenous receptor ligands to affect proliferation in the uterine myometrium. Although these ligands are diverse in their origins and chemical structures, receptor dissociation assays show that they bind to the ER with affinities that, for some ligands, are similar to or even exceed that of E
2 (
27-29). ER ligands used in these studies come from three distinct groups: phytoestrogens, organochlorine pesticides, and pharmacological agents. Several classes of ER ligands previously have been described as having activity in other hormonally responsive tissues, but the uterine myometrium has not been evaluated as a target for these endocrine-active compounds. Phytoestrogens, such as those found in soy products and herbal preparations, are reported to have protective benefits against the risk of certain hormone-related cancers and to alleviate discomfort during menstruation or menopause (
30-32). Organochlorine pesticides are a group of proposed estrogen agonists associated, in some studies, with an increased incidence of breast cancer (
33,34). Finally, the SERMs tamoxifen and raloxifene (and its analogs) are pharmacological agents that act as tissue-specific partial agonists that may be of therapeutic benefit in the treatment of uterine leiomyoma.
We have shown that a number of ER ligands induce proliferation of leiomyoma cells. Figure 3 shows modulation of proliferation of ELT3 cells by several doses of representative ligands from each of the three groups of compounds studied, along with a prototypical agonist of the ER, diethylstilbestrol (DES). DES is a well-characterized estrogenic compound associated with reproductive tract abnormalities and adenocarcinoma of the vagina in women exposed to DES in utero (35,36). In vivo animal studies with DES demonstrate a profound estrogen agonism. For example, the uteri of DES-treated Eker rats are dramatically enlarged, with the myometrium exhibiting increased proliferation compared with vehicle-treated control animals (29). In contrast, both in vitro and in vivo studies have shown that the SERM tamoxifen does not induce proliferation of myometrial tissue, although this compound binds to the ER with high affinity (29) and has agonist activity in the uterine endometrium (14,37).
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Figure 3. Xenobiotics affect proliferation of ELT3 uterine leiomyoma cells. Cells are grown in estrogen-free media for 6-8 days in triplicate wells. Change in cell number is expressed as a percentage of the difference between untreated cells (vehicle control) and those grown in 10 nM E2 within the same experiment. Error bars indicate SEM of triplicate wells. Asterisks (*) denote significant differences in cell number from value of the vehicle control at p < 0.02 as determined by ANOVA.
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Genistein is a soy-derived isoflavone that has been shown to have biphasic effects on cell growth in vitro (38). At moderate concentrations, genistein induces leiomyoma cell growth (29) (Figure 3), but at high concentrations (> 100 µM) genistein has an inhibitory effect on cell growth (29). This inhibition does not appear to be mediated through the ER, as it also occurs in ER-negative cell lines. Some of genistein's antiproliferative effects may be due to its inhibitor of tyrosine kinase (39). We have found that, like genistein, several other phytoestrogens have proliferative effects on leiomyoma cell growth. Both naringenin, a flavonoid present in some citrus fruits, and coumestrol, a coumestan found in some grains, stimulate growth of leiomyoma cells at doses in the micromolar range (29).
Of the organochlorine pesticides studied, several increase leiomyoma cell proliferation. HPTE (2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane) stimulates the proliferation of ELT3 cells at concentrations as low as 100 nM (40) (Figure 3). HPTE is the most estrogenic metabolite of the organochlorine pesticide methoxychlor, a pesticide still in common use (41-43). Kepone, a pesticide banned in the United States, in part because of its estrogenic activity, also stimulates leiomyoma cell growth at micromolar concentrations, as does the alpha isomer (but not the beta isomer) of endosulfan (40). Endosulfan-
significantly increases leiomyoma cell number at 10 µM, whereas the beta isomer has no effect on cell growth. The proliferative effects of these pesticides appear to be largely mediated by the ER, as the potent antiestrogen ICI 182,780 significantly inhibits their agonistic effects. In contrast to these phytoestrogens and pesticides, the SERMs hydroxytamoxifen (the active in vivo metabolite of tamoxifen), raloxifene, and various chemical agonists of raloxifene do not induce proliferation of leiomyoma cells in vitro (29).
In parallel with observations from proliferation assays, the results of reporter gene assays used to measure the ability of xenoestrogens to transactivate gene expression have revealed a distinct profile of agonism versus antagonism by these compounds. To determine whether xenoestrogens can activate ER-mediated gene transcription in a myometrial cell background, we transiently transfected leiomyoma cells with a classical estrogen-responsive promoter element (ERE), vitellogenin-ERE, linked to a luciferase reporter gene. Table 1 shows the dose of test ligands required to achieve at least 25% of the normalized luciferase activity of 10 nM E2. As expected, all the ligands that were agonists in the proliferation assay also exhibited transcriptional activity. Additionally, several compounds that do not induce proliferation are able to activate transcription of the reporter gene. For example, all of the organochlorine pesticides are able to induce transcription, including those that do not stimulate proliferation, such as endosulfan-ß, toxaphene, methoxychlor, and dieldrin. Both isomers of endosulfan stimulate transcription despite exhibiting negligible binding to the ER (29). However, the SERMs hydroxytamoxifen and the two raloxifene analogs (LY117018 and LY317783), all antagonists for cell proliferation, do not exhibit transcriptional activity of the vitellogenin-ERE in a myometrial cell background.
An additional, often-used test of estrogen agonism is the ability to induce an endogenous estrogen-responsive gene such as PR gene. The phytoestrogens genistein, coumestrol, and naringenin all induce expression of the PR as assayed by Northern blot analysis (29), and all of the organochlorine pesticides stimulate PR mRNA expression at least one concentration of ligand as assayed by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique (40). In addition, hydroxytamoxifen, LY117018, and LY317783, although unable to stimulate transcription of the vitellogenin-ERE, were able to upregulate PR expression on the mRNA level (Figure 4). In fact, all of the compounds tested for induction of PR expression, except the pure antiestrogen ICI 182,780, upregulated PR mRNA. Although the disagreement between the results of PR induction and reporter gene assays in this system may be, on its face, surprising, it is consistent with the gene-specific and context-specific action of ER ligands in other systems (46-48). Furthermore, unlike a reporter gene assay in which an ERE-driven exogenous gene is transiently expressed in an artificial construct, the endogenous PR promoter is complexed with nucleosomes and is an integral part of the native chromatin. In this system, the induction of PR is not predictive of estrogen agonism at the cellular level.
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Figure 4. Induction of PR mRNA by SERMs. Total RNA was isolated from ELT3 cells treated for 48 hr using 10 nM E2, OH-TAM, LY117018, or LY317783; or 1 mM ICI 182,780, a pure antiestrogen. Doses were chosen based on binding affinity and data from proliferation studies to obtain a dose with maximum effect and minimum toxicity. LY117018 and LY317783 are raloxifenelike compounds for which the pharmacology has been previously described (44,45). LY117018 has the same chemical structure as raloxifene except for the substitution of a pyrrolidine for the terminal pyridine ring. LY317783 (also LY326315) also resembles raloxifene but has a napthalene rather than a benzothiaphene nucleus. A quantitative RT-PCR technique (40) was employed using increasing amounts of a competitive template to determine the amount of endogenous PR mRNA present in each sample. Using known amounts of competitive template added over a series of reactions and the intensities of the competitor PR/endogenous band, linear regression measured the level of endogenous PR transcript. Levels of glyceraldehyde phosphate dehydrogenase (GAPDH) transcript were similarly determined. Bars represent the ratio of PR to GAPDH values +/- SEM for multiple PCR reactions. Asterisks (*) denote significant differences from vehicle control at p  0.05 as determined by ANOVA.
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These data demonstrate that reporter gene transactivation may be overly sensitive for predicting myometrial cell proliferation but can act as a powerful screening tool for xenobiotics that have the capacity for estrogen agonism in the myometrium at the molecular level. We have taken advantage of this system to evaluate a number of herbal preparations consisting of crude ethanol extracts from various plants that have been purported to have estrogenic or antiestrogenic effects in other tissues [reviewed in (
49)]. Some herbal preparations can mimic the impact of E
2 on the hypothalamic/pituitary axis, as evidenced by a decrease in serum luteinizing hormone (LH) levels or by an increase in uterine weight, indicating that the compounds participate in endocrine signaling in the intact animal (
50). Additionally, some extracts contain elements that compete for ER binding (
50-52), suggesting that the mechanism of this effect could be a direct one via the ER. Figure 5 illustrates the induction of vitellogenin-ERE reporter gene activity by extracts of licorice and milk thistle, and at a low level by hops but not by extract of blue cohosh. These results are not entirely predicted by the results of other assays. For example, licorice, which is a potent transactivator, is not able to affect serum LH levels when administered
in vivo. In contrast, milk thistle, which was able to transactivate the reporter gene, was not able to compete with E
2 for receptor binding, as could each of the other compounds shown (
52). This effect is reminiscent of the ability of the endosulfans to induce reporter gene expression in the absence of measurable direct interaction with the receptor. The mechanism of this transactivation remains to be explained.
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Figure 5. Reporter gene transactivation by herbal extracts. Extracts of dried or fresh herbs were prepared by cold extraction using ethanol and distilled water in a ratio (w/w) of 1:1.5 to 1:4 parts plant material. Plant extracts tested were hops (Humulus lupulus), blue cohosh (Caulophyllum thalictroides), licorice root (Glycyrrhiza uralensis), and milk thistle (Silybum marianum). ELT3 cells were transiently cotransfected with the firefly luciferase reporter gene linked to the vitellogenin-ERE, along with human ER and a constitutive ß-galactosidase reporter. Triplicate wells of transfected cells were washed and incubated for 40 hr in serum-free, phenol red-free medium in the presence of test compound or vehicle control, then harvested and assayed for reporter gene activity normalized to activity of the constitutive reporter. Fold induction in treated samples is calculated as normalized reporter gene activity (mean of three wells) divided by activity in basal medium (vehicle control). A representative experiment is shown.
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The ability of ER ligands to exhibit tissue-specific activity has recently been exploited in the development of SERMs with tissue-specific therapeutic profiles. However, the impact of SERMs on the uterine myometrium or leiomyomas had not been previously evaluated; thus an analysis of the effects of the SERMs tamoxifen and raloxifene on these target tissues was undertaken in the Eker rat model.
In vitro studies using the ELT cell lines were performed to examine the effects of tamoxifen, raloxifene, and the pure antiestrogen ICI 182,780. Estrogen-induced increases in cell proliferation were inhibited by all three compounds in ER-positive cell lines, whereas ER-negative cell lines were refractory to the modulatory effects of E
2, SERMs, and ICI 182,780 (
53). Interestingly, while although tamoxifen was able to inhibit cell proliferation, it could not induce apoptosis, even though the apoptotic pathway in ELT3 cells was shown to be intact by serum starvation (
54). These data support the concept of a signaling defect in uterine leiomyoma consistent with the observation of an altered apoptotic response to steroid hormone regulation in uterine leiomyoma
in vivo. The presence of this defect appears to make these cells refractory to normal
in vivo apoptotic stimuli, such as withdrawal of steroid hormones, and contributes to tumor growth under conditions in which normal myometrial cells undergo apoptosis.
SERMs have also been shown recently to have therapeutic potential for uterine leiomyoma. Spontaneous uterine leiomyomas develop in approximately 65% of female rats carrying the Eker mutation by 16 months of age. Treatment of female Eker rats from 12 to 16 months of age with tamoxifen or the raloxifene analog LY326315 decreases tumor incidence by approximately 50% in treated animals relative to vehicle-treated controls (55). Tumors present in treated animals after 2-4 months of treatment were smaller and had a lower mitotic index than the tumors in vehicle-treated controls. Therefore, it appears that SERMs may represent a novel therapeutic strategy for treatment of women with this disease.
The Eker model has illuminated some key features of myometrial cell regulation that may be targets for endocrine disruption. Specifically, normal myometrial cells are subject to regulation by steroid hormones at the level of proliferation and apoptosis, particularly in early reproductive maturity. Leiomyomas, which appear later in the reproductive life span, have an inappropriate reacquisition of steroid hormone responsiveness but fail to undergo commensurate apoptosis. This defect may make uterine leiomyomas more sensitive to the influences of both endogenous and exogenous hormonal influences. However, because the effects of ER ligands are highly tissue specific, empirical data are needed to determine which exogenous compounds influence leiomyoma growth or disrupt the homeostasis of the normal myometrium.
The results from in vivo and in vitro assays using experimental model systems such as the Eker rat can help elucidate the mechanism of estrogen agonism in the myometrium. For example, the ability of exogenous compounds to increase levels of PR mRNA was not predictive of their ability to act as estrogen agonists at the level of cell proliferation or in vivo. Although PR induction is commonly used as an indicator of estrogen agonism, these results are consistent with similar results in other tissues (24,56) and suggest that induction of PR is a promiscuous effect of ER ligands that is not predictive of agonist activity. For instance, each of the SERMs examined in this system could induce PR, although the results of all other analyses, including in vivo studies, showed that these compounds antagonize estrogen action rather than mimic it. Nonetheless, PR is an important component of endocrine signaling in hormone-responsive tissues, and PR has been shown by several investigators to be overexpressed in uterine leiomyoma in women (57-59). The biological significance of PR induction by exogenous ER ligands is not known.
In contrast to induction of PR, molecular agonism at the vitellogenin-ERE correlates with the capacity for estrogen agonism in uterine myometrium. Furthermore, results from this assay have helped define the characteristics of estrogen agonism in myometrial tissues. Tissue and promoter specificity of ER ligands may be due partly to the ability of ligands to activate transcription via activation function (AF-1) or via both AF-1 and AF-2 domains of the ER. Data from previous reporter gene studies suggest that in the uterine myometrium, agonist activity is mediated via activation of AF-2 of the ER (29). Although activation of the AF-1 domain is generally considered a constitutive function of both partial and full agonists, activation of the AF-2 domain has been shown to be ligand, promoter, and cell-type specific (46). The classical vitellogenin-ERE requires AF-2 for transcriptional activity, and tamoxifen cannot activate transcription via this element (47). The inability of SERMs, which are antagonists for leiomyoma cell growth, to stimulate transcription of vitellogenin-ERE in a myotivation of the ER correlates with agonist activity for this cell type (29). Like uterine leiomyoma cells, breast cancer cells require AF-2 activation for agonism of the classical ERE. In contrast, in uterine endometrial cells, the partial agonist hydroxytamoxifen may stimulate AF-1-requiring promoters such as C3 and the collagenase promoter containing an AP1 site (60,61). These data indicate that the uterine myometrium has a profile of estrogen responsiveness more similar to that of mammary tissue than to that of other uterine compartments, and that assays that can differentiate AF-1 and AF-2 activities can effectively predict which compounds are agonists in this tissue.
Despite the utility of a reporter assay for screening, the most selective indicator of estrogen agonism in the myometrium is ELT3 cell proliferation (Figure 6). This assay consistently recognizes exogenous ER ligands that are agonists at the molecular level, but can eliminate some ligands that are toxic after persistent exposure to a stimulatory dose or have opposing effects not mediated by the ER. This assay reflects in vivo activity in this model, and it may be uniquely able to identify compounds effective within the limits of physiologic dose. Although the proliferation assay appears to be the most selective assay for predicting agonism in the myometrium, this is not to say that effects other than cell proliferation are inconsequential. Molecular agonism, even in the absence of proliferation in vitro, may effect an alteration of endocrine signaling that may contribute to the development and/or growth of uterine leiomyoma.
Figure 6. Diagrammatic representation of the hierarchical agonist response to endocrine disruptors in a uterine myometrial cell background. Agonist responses range from promiscuous (i.e., all compounds eliciting a positive response) in the induction of PR to selective (i.e., fewest compounds scoring as positive) in the cell proliferation assay.
These data indicate that concern over the role of endocrine disruptors in leiomyoma development may be warranted. In this model system, exogenous ER ligands ranging from industrial pollutants to dietary constituents have been shown to have the potential for proliferative influence on myometrial tissues. This type of stimulation could affect normal quiescent myometrial tissue but may also preferentially exacerbate the growth of existing leiomyomas that are more sensitive to hormonal stimulation. Conversely, the hormonal sensitivity of these tumors renders them subject to regulation by endocrine modulators in a therapeutic strategy. The potential for using SERMs to modulate the growth of leiomyomas in humans is unexplored, despite the possibility that these compounds could be potent regulators of tumor growth while eliminating many of the undesirable side effects of estrogen ablation. The use of this model system will continue to edify the impact of exogenous ER ligands on leiomyomas and to test strategies for modulation of tumor progression.
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Last Updated: October 3, 2000
