ERbeta Expression Research Articles

Estrogen receptor beta (ERbeta) is expressed in brain astrocytic tumors and declines with dedifferentiation of the neoplasm.

Estrogen receptor beta (ERbeta) is the second identified receptor mediating the effects of estrogen on target tissues. The role of ERbeta in cancer pathobiology is largely unknown, because specific antibodies have not been available until recently. Initial studies have shown that ERbeta expression declines in breast, ovarian, prostatic, and colon carcinomas. Tamoxifen, a synthetic anti-estrogen compound that is a mixed agonist/antagonist of estrogen receptor alpha (ERalpha) and a pure antagonist of ERbeta, has moderate beneficial effects in human astrocytic neoplasms. However, most published studies agree that glial tumors do not express ERalpha. The purpose of this study was to explore the expression of ERbeta in astrocytic neoplasms. ERbeta expression was monitored immunohistochemically in 56 cases of astrocytomas of all grades (grade I-IV) and in adjacent non-neoplastic brain tissue. Moderate or strong nuclear immunopositivity was obtained in non-neoplastic astrocytes and in low-grade astrocytomas, whereas the majority of high-grade tumors were immunonegative or displayed weak immunoreactivity. The progressive decline in ERbeta expression paralleled the increase in tumor grade. In as much as ERbeta is possibly the only ER expressed in astrocytes, its decreased expression may play an important role in astrocytic tumor initiation and in the potential response of glial neoplasms to tamoxifen.

Differential regulation of estrogen receptor subtypes alpha and beta in human aortic smooth muscle cells by oligonucleotides and estradiol.

We investigated the mechanisms regulating estrogen receptor (ER) expression in human aortic smooth muscle cells (HASMCs) and the mechanisms by which estradiol inhibits HASMC growth. The autologous down-regulation pathway involves binding of liganded ER to the ER gene, thus suppressing transcription. Blockade of this pathway with sense and AS-OLIGOs to ERs up-regulated the expression of ERalpha but not ERbeta. Activation of the autologous down-regulation pathway with ER agonists down-regulated the expression of ERalpha but not ERbeta. The proteasomal degradation pathway entails ubiquination of liganded ER, followed by proteasome-mediated degradation. Blockade of the proteasomal degradation pathway increased the expression of ERbeta. Up-regulation of ERalpha by AS-OLIGOs did not increase the antimitogenic effects of estradiol on HASMCs; the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent inhibitors of HASMC growth, compared with estradiol; and blockade of metabolism of estradiol to hydroxyestradiols and methoxyestradiols abrogated the inhibitory effects of estradiol on HASMC growth. We conclude that, in HASMCs: 1) the expression of ERalpha is regulated by the autologous downregulation pathway; 2) the expression of ERbeta is governed by the proteasomal degradation pathway; and 3) the antigrowth effects of estradiol are not mediated by ERalpha, but rather by metabolism of estradiol to methoxyestradiols.

Regulation of estrogen receptor-beta expression in the female rat hypothalamus: differential effects of dexamethasone and estradiol.

Estrogen and glucocorticoids interact in multiple aspects of endocrine regulation by exerting opposing influences on the expression of selective genes. In rats, estrogen receptor (ER)-beta is the predominant form of ER present in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, suggesting its involvement in neuroendocrine regulation. To date, the hormonal regulatory profile of the ERbeta gene in the rat central nervous system has not been closely elucidated. In the present study, we first examined the effects of dexamethasone (DEX) and estradiol benzoate (EB) on the ERbeta protein expression in the PVN and SON of ovariectomized female rats. In the SON and parvocellular and magnocellular parts of the PVN, the number of ERbeta immunoreactive nuclei significantly increased after DEX treatment, compared with the control group, whereas EB treatment caused a significant decrease. The effect of EB was consistent across other brain nuclei such as the anteroventral periventricular nucleus and medial preoptic nucleus. To determine the molecular level at which DEX and EB control ERbeta expression, we examined the effects of these steroids on ERbeta mRNA levels using real-time RT-PCR. EB significantly decreased the expression of ERbeta mRNA in the PVN (P = 0.0006) and SON (P < 0.01). In contrast, DEX did not change ERbeta mRNA levels. These results indicate that glucocorticoids and estrogen exert opposing regulatory influences on the ERbeta gene expression. This may represent a mechanism by which these steroids can alter the cellular sensitivity of ERbeta-expressing neurons to subsequent steroidal activation.

Expression of ERα, ERβ and Ki-67 in primary tumors and lymph node metastases in breast cancer

The success of current hormonal therapies used in breast cancer patients often depends on estrogen receptor (ER) status. The simultaneous pathological assessment of primary breast tumors (PTs) and metastases to regional lymph nodes (MRLNs) could improve the effectiveness of this treatment, but is rarely performed. Consequently, we studied by immunohistochemistry the expression of ERalpha (ERalpha), ERbeta (ERbeta), and the proliferation marker (Ki-67) in PTs and matching MRLNs. The expression of each ERalpha, ERbeta, and Ki-67 positively correlated between PTs and MRLNs (r=0.751, p<0.0001; r=0.391, p<0.03; and r=0.707, p<0.0001, respectively). A negative correlation between the expression of ERalpha and Ki-67 was found in PTs (r=-0.678, p<0.001) and MRLNs (r=-0.501, p<0.005). A negative correlation between ERalpha and Ki-67 was also observed in PTs vs. MRLNs (r=-0.619, p<0.0001). Moreover, the expression of ERalpha and Ki-67 was not associated with nodal status, contrary to ERbeta expression, which correlated with negative axillary node status. The results indicated that the associations among ERalpha, ERbeta, and Ki-67 are maintained in PTs and MRLNs. Thus, simultaneous assessment of selected markers in PT and MRLN might help in understanding the mechanisms of breast cancer progression as well as result in the development of new principles for diagnosis and individual therapy planning of estrogen dependent cancer.

Augmented endothelial nitric oxide synthase (eNOS) protein expression in human pregnant myometrium: possible involvement of eNOS promoter activation by estrogen via both estrogen receptor (ER)alpha and ERbeta.

The aim of the present study was to investigate the possible contribution of estrogen to pregnancy-associated modulation of nitric oxide production in the human myometrium during pregnancy. Both endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) proteins were clearly expressed in the non-pregnant myometrium and were elevated in the first trimester of pregnancy. Oral contraceptive pills augmented eNOS, but not iNOS, protein expression in the non-pregnant human myometrium. In cultured human myometrial cells, estrogen receptor (ER)alpha and ERbeta expression was extremely low. Therefore, we used either ERalpha or ERbeta expression vector to investigate the effect of 17beta-estradiol treatment on eNOS promoter activity using eNOS promoter/luciferase vector in cultured human myometrial cells. 17beta-estradiol treatment significantly augmented eNOS promoter activity in cells co-transfected with either ERalpha or ERbeta, and this augmentation was dose-dependently suppressed by ICI 182780, an estrogen antagonist. These data suggest the possibility that both ERalpha and ERbeta are involved in the estrogen-associated regulation of eNOS gene expression in the human myometrium.

Estrogen receptor beta localization in the lizard (Podarcis s. sicula) testis.

There is increasing evidence that 17beta-estradiol is necessary for normal male fertility. The aim of the present study was to characterize estrogen receptor beta (ERbeta) expression in a non-mammalian vertebrate model, the lizard (Podarcis s. sicula) testis. Immunocytochemical analysis shows that ERbeta proteins are present among germ cells in the nucleus of the spermatogonia, in primary spermatocytes and spermatids. Western blot analysis with antibodies against the ERbeta gene product revealed an isoform with a specific weight of 55 kDa. In conclusion, the widespread expression of ERbeta in the Podarcis s. sicula testis is consistent with a role for estrogens in modulating spermatogenesis in the male.

Detection of oestrogen receptors (ER) alpha and beta in conjunctiva, lacrimal gland, and tarsal plates.

Lack of Oestrogen and androgen may be of importance in the pathogenesis of keratoconjunctivitis sicca (KCS). The signal of Oestrogens is transmitted via specific Oestrogen receptors (ER). It was the purpose of this study to evaluate the expression of ER alpha and ER beta in tear-producing tissues. Reverse transcriptase-polymerase chain reaction (RT-PCR, ER alpha + beta) and immunohistochemical evaluation (ER alpha only) were performed for ER detection and localization in tissue samples of bulbar conjunctiva (20 samples of 20 patients undergoing cataract surgery), tarsal plates (14 samples of 12 patients undergoing eye lid surgery), and lacrimal glands (20 samples of 13 cornea donors). Messenger RNA ER alpha was identified via RT-PCR in all tissue samples with variable expression, ER beta predominantly in lacrimal gland tissue. Immunohistochemical staining for ER alpha was negative in most cases, probably due to the thermolability of ERs and very small sample sizes. The detection of ER alpha and ER beta mRNA expression supports the concept of a receptor-based effect of Oestrogen in these tissues contributing to KCS. This may encourage therapeutical efforts including topical Oestrogen administration.

Acquisition of luteolytic capacity: changes in prostaglandin F2alpha regulation of steroid hormone receptors and estradiol biosynthesis in pig corpora lutea.

The pig corpora lutea (CL) acquires luteolytic capacity at about Day 13 of the estrous cycle, after which luteolysis occurs in response to prostaglandin F2alpha (PGF2alpha) treatment. We postulated that differences in transcription factors such as the steroid hormone receptors may be responsible for the differences in PGF2alpha-induced gene expression after acquisition of luteolytic capacity. In these studies, we evaluated the effect of PGF2alpha on luteal expression of receptors for progesterone (nuclear and membrane progesterone receptor [PR]), estradiol (ERalpha and ERbeta), glucocorticoid, androgens, and two enzymes in estradiol synthesis (P450-17alpha and aromatase). Two experiments were conducted to examine the early (0.5 h, experiment I) and late (10 h, experiment II) effects of PGF(2alpha) on the expression of these receptors in CL with (Day 17) or without (Day 9) luteolytic capacity. PGF2alpha decreased ERalpha mRNA (35%) and increased ERbeta mRNA (558%) and protein (376%) only in Day 17 CL and not Day 9. The estradiol biosynthetic pathway was upregulated by PGF2alpha in Day 17 CL but not Day 9 CL, with a dramatic increase in aromatase mRNA and luteal estradiol content. Nuclear PR was not affected by PGF2alpha, but was greater (176%) in Day 9 CL, while a putative membrane PR was greater (156%) in Day 17 than Day 9 CL. There were no detectable changes in glucocorticoid or androgen receptor mRNA. Thus, luteolytic capacity is associated with upregulation of estradiol biosynthesis, which in conjunction with increased ERbeta expression and altered PR expression may promote luteolysis in the pig CL.

Potential action of androstenedione on the proliferation and apoptosis of stromal endometrial cells

BackgroundHyperandrogenic conditions have been associated with a high prevalence of endometrial pathologies related to cell survival. However, the action of androgens on proliferation and apoptosis in endometrial cells is poorly understood. Therefore, the aim of the present study was to evaluate the effect of androstenedione on cell proliferation, cell death and expression of estrogen receptor (ER) isoforms and proteins related to apoptosis in endometrial cells using two in vitro experimental approaches.MethodsThe endometrial tissue was obtained from 20 eumenorrheic women [28.7 (25 – 35) years] during the early secretory phase. We analyzed cell proliferation (immunohistochemistry of Ki-67 and spectrophotometric assay); apoptosis (DNA fragmentation (TUNEL) and Annexin V-FITC binding); ER-alpha, ER-beta bcl-2 and bax mRNA abundance (RT-PCR) in explants and isolated endometrial epithelial (EEC) and stromal cells (ESC) incubated with androstenedione 1 micro mol/l (A4) or A4 plus hydroxyflutamide 10 micro mol/l (F) for 24 h.ResultsIn explants, A4 induced an increase of cell proliferation and a decrease on apoptosis in the stromal compartment (p < 0.05). In isolated ESC, proliferation augmented with A4 (p < 0.05), whereas, no significant modifications in the expression of ER-alpha, ER-beta bcl-2 and bax nor in the apoptotic index were observed. In EEC, A4 increase the ER-beta mRNA abundance (p < 0.05) and a decrease of the bcl-2/bax ratio (p < 0.05), without an increase in the apoptotic index. Hydroxyflutamide reverted the effect of androstenedione on the parameters described.ConclusionsThese results indicate that androstenedione may modulate cell survival, expression of ER-beta and proteins related to apoptosis, suggesting a potential mechanism that associates the effect of hyperandrogenemia on the endometrial tissue.

Expression of estrogen receptor beta in human colorectal cancer.

To determine the expression of estrogen receptor (ER) beta in Chinese colorectal carcinoma (CRC) patients. ERbeta expression in CRC was investigated by immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections from 40 CRCs, 10 colonic adenomas, and 10 normal colon mucosa biopsies. The percentage of positive cells was recorded, mRNA expression of ERalpha and ERbeta in 12 CRC tissues and paired normal colon tissues were detected by RT-PCR. Positive ER immunoreactivity was present in part of normal epithelium of biopsy (2/10), adenomas (3/10), and the sections of CRC tissue, most of them were nuclear positive. In CRCs, nuclear ERbeta immunoreactivity was detected in over 10% of the cancer cells in 57.5% of the cases and was always associated with cytoplasmic immunoreactivity. There was no statistical significance between ERbeta positive and negative groups in regard to depth of invasion and nodal metastases. Of the 12 CRC tissues and paired normal colon tissues, the expression rate of ERalpha mRNA in CRC tissue and corresponding normal colon tissue was 25% and 16.6%, respectively. ERbeta mRNA was expressed in 83.3% CRC tissue and 91.7% paired normal colon tissue, respectively. There was no significant difference in ERbeta mRNA level between CRC tissues and paired normal colon tissues. A large number of CRCs are positive for ERbeta, which can also be detected in normal colonic epithelia. There is a different localization of ERbeta immunoreactivity among normal colon mucosae, adenomas and CRCs. ERalpha and ERbeta mRNA can be detected both in CRC tissue and in corresponding normal colon tissue. A post-transcriptional mechanism may account for the decrease of ERbeta protein expression in CRC tissues.

Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest.

Studies indicate that estrogen receptor (ER) alpha mediates breast cancer-promoting effects of estrogens. The role of ERbeta in breast cancer is unknown. Elucidating the role of ERbeta in the pathogenesis of breast cancer is important because many human breast tumors express both ERalpha and ERbeta. We show that adenovirus-mediated expression of ERbeta changes the phenotype of ERalpha-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERalpha. In contrast, introducing ERbeta into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERbeta inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21(Cip1) and p27(Kip1), which leads to a G(2) cell cycle arrest. These results demonstrate that ERalpha and ERbeta produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERbeta-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

Expression of estrogen receptors alpha and beta in the fetal baboon testis and epididymis.

Although studies in transgenic mice suggest that estrogen is important for development of the testis, very little is known about the potential role of estrogen in maturation of the primate fetal testis. Therefore, as a first step to determine whether estrogen regulates maturation of the fetal primate testis, we used immunocytochemistry to determine estrogen receptor (ER) alpha and beta expression in the fetal baboon testis. Second, we established methods to quantify ERbeta mRNA levels by competitive reverse transcription-polymerase chain reaction in Sertoli cells isolated by laser capture microdissection (LCM) from the fetal baboon testis. ERbeta protein expression was abundant in the nuclei of Sertoli, peritubular, and interstitial cells in baboon fetuses at mid (Day 100) and late (Day 165) gestation (term is 184 days). ERbeta mRNA level was 0.03 attomole/femtomole 18S rRNA in Sertoli cell nuclei and associated cytoplasm isolated by LCM. ERalpha was expressed in low level in seminiferous tubules and in moderate level in peritubular cells on Day 165. Germ cells expressed very little ERalpha or ERbeta protein, whereas the baboon fetal epididymis exhibited extensive ERalpha and ERbeta immunostaining at mid- and late gestation. In contrast to the robust expression of ERbeta, androgen receptor protein was not demonstrable within the cells of the seminiferous cords but was abundantly expressed in epididymal epithelial cells of the fetal baboon. In summary, the results of this study show that the fetal baboon testis and epididymis expressed the ERalpha and ERbeta, and we suggest that our nonhuman primate baboon model can be used to study the potential role of estrogen on maturation of the fetal testis.

Immunolocalization of estrogen receptor beta in the hypothalamic paraventricular nucleus of female mice during pregnancy, lactation and postnatal development

Previous studies have shown that estrogen receptor beta (ERbeta) is the predominant estrogen receptor in the hypothalamic paraventricular nucleus (PVN) of mouse, mediating estrogen regulation of the neuroendocrine activities of the PVN, but the exact roles that ERbeta plays in the PVN remain unclear. In this study, we used immunocytochemistry to investigate the expression of ERbeta in the maternal PVN of mice during pregnancy (pregnant days 8, 10, 12, 15 and 18), lactation (postpartum days 1, 4 and 8) as well as in the PVN of the females from postnatal days 1, 3, 5, 7, 9, 15, 30 and 70. We found out that ERbeta was predominantly localized in the magnocellular divisions of PVN. In the pregnant female brain, generally, the ERbeta was lower than that of the postnatal development, the lowest level was found at gestational days 10–12; then from gestational day 18 to postpartum day 1, it increased to higher levels, followed by a decrease from postpartum day 4. During the postnatal development, the highest level of ERbeta was found at early postnatal days (before postnatal day 15), thereafter, it decreased to a lower level. The above results indicate that circulating sex steroids may differentially regulate the expression of ERbeta in the PVN of mice. It also suggests that this receptor may play important roles in the regulation of parturition and in the development, food intake and body weight increases of the newborns by acting on the neuropeptides, which were also detected in the PVN.

The different effects of endocrine-disrupting chemicals on estrogen receptor-mediated transcription through interaction with coactivator TRAP220 in uterine tissue.

An endocrine-disrupting chemical (EDC) can alter endocrine functions through a variety of mechanisms, including nuclear receptor-mediated changes in protein synthesis, interference with membrane receptor binding, steroidogenesis or synthesis of other hormones. Although major chemicals have been shown to disrupt estrogenic actions mainly through their binding to estrogen receptor (ER) or androgen receptor, it is not clear how EDCs affect endocrine functions in vivo. We present evidence that the EDCs bisphenol A and phthalate activate ER-mediated transcription through interaction with TRAP220. Moreover, bisphenol A had positive effects on the interaction between ER-beta and TRAP220 and on the expression of ER-beta and TRAP220 compared with phthalate and estradiol in uterine tIssue. These data suggested that some EDCs might alter endocrine function through the change of the receptor and coactivator levels in uterine tIssue and through the different effect on the interaction between ERs and coactivator TRAP220.

Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary.

Net estrogen sensitivity in target tissues critically depends on the regulated expression of full-length and alternately processed estrogen receptor (ER) isoforms. However, the molecular mechanisms for the control of pituitary responsiveness to estrogen remain partially unknown. In the present communication, we report the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ERalpha and ERbeta isoforms, as well as those for the truncated ERalpha product (TERP), and the variant ERbeta2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERbeta and ERbeta2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ERalpha. Administration of estradiol benzoate (as potent agonist for alpha and beta forms of ER) or the selective ERalpha agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERbeta ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERbeta agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ERalpha agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ERalpha ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted through ERalpha. Differential regulation of ER isoforms may represent a relevant system for the self-tuning of estrogen responsiveness in female pituitary.

Osmotic regulation of estrogen receptor-beta expression in magnocellular vasopressin neurons requires lamina terminalis.

Estrogen receptor-beta (ER-beta) expression in rat magnocellular vasopressin (VP) neurons of the supraoptic and paraventricular nuclei (SON and PVN, respectively) becomes undetectable after 72 h of 2% NaCl consumption. To test the hypothesis that osmosensitive mechanisms that originate in the region of the organum vasculosum lamina terminalis (OVLT) control ER-beta expression in the SON and PVN, animals were water deprived after electrolytic lesions were performed on the area anterior to the ventral third ventricle (AV3V). Such lesions prevent osmotic stimulation of VP release. Four weeks after surgery, male rats [lesioned (n = 16) or sham (n = 14)] were water deprived for 48 h or allowed water ad libitum. Water deprivation eliminated ER-beta-immunoreactivity (-ir) in SON and magnocellular PVN of sham-lesioned animals. Fos-ir was evident in these neurons, and plasma osmolality (Posm) and hematocrit (Ht) were significantly elevated compared with the sham-hydrated rats (Posm, 304 +/- 1 vs. 318 +/- 2 mosmol/kgH2O; P < 0.001; Ht, 49.6 +/- 0.6 vs. 55.0 +/- 0.9%; P < 0.001). ER-beta expression was comparable in sham-hydrated, AV3V-hydrated, and 6 of 8 AV3V-dehydrated rats despite significant increases in Posm in both groups (AV3V hydrated, 312 +/- 2; AV3V dehydrated, 380 +/- 10 mosmol/kgH2O; P < 0.001). OVLT was not ablated in the AV3V-dehydrated rats in which ER-beta was depleted. Fos-ir was low or undetectable in SON in the AV3V-hydrated animals despite elevated Posm values. In AV3V-dehydrated rats, Fos-ir was significantly less than in sham-dehydrated animals but was significantly increased compared with the sham-hydrated group. This could reflect activation by nonosmotic parameters that do not inhibit ER-beta expression. These data support the hypothesis that inhibition of ER-beta expression in the SON by osmotic stimulation is mediated by osmoreceptive neurons in the lamina terminalis.

K-ras mutation in tamoxifen-related endometrial polyps.

K-ras mutation is thought to occur at an early stage of neoplastic progression in the endometrium. The authors investigated mutations in codon 12 of K-ras in tamoxifen (TAM)-related endometrial polyps. DNA was extracted from 11 frozen endometrial polyps from TAM-treated patients with breast carcinoma. Mutations were detected using the mutant allele-specific amplification method. The results subsequently were analyzed for correlations with immunohistochemical data that were obtained using antibodies against estrogen receptors (ERs; alpha and beta forms), progesterone receptors (PRs; A and B forms), and Ki-67. Mutations in codon 12 of K-ras were observed in 7 of 11 TAM-related endometrial polyps. Expression levels of ER-alpha and PR-B were high in the glandular epithelium and low in the stroma. PR-A expression was high in both the glandular epithelium and the stroma. In the glandular epithelium, expression of ER-beta appeared to be lower than expression of ER-alpha. The Ki-67 index in the glandular epithelium ranged from 2 to 38, whereas the index ranged from 0 to 4 in the stroma (P < 0.01). The incidence of mutations in codon 12 of K-ras in TAM-related endometrial polyps (64%) was greater than the incidence of these same mutations in sporadic endometrial hyperplasias (4.5-23%). High expression levels of ER-alpha, PR-A, and PR-B in the glandular epithelium were observed in all polyps, regardless of K-ras codon 12 mutation status and Ki-67 index. The authors' findings may support the hypothesis that the polyp-carcinoma sequence partly indicates the development of endometrial carcinoma in postmenopausal women who have been treated with TAM.

Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling.

Antiestrogens include agents such as tamoxifen, toremifene, raloxifene, and fulvestrant. Currently, tamoxifen is the only drug approved for use in breast cancer chemoprevention, and it remains the treatment of choice for most women with hormone receptor positive, invasive breast carcinoma. While antiestrogens have been available since the early 1970s, we still do not fully understand their mechanisms of action and resistance. Essentially, two forms of antiestrogen resistance occur: de novo resistance and acquired resistance. Absence of estrogen receptor (ER) expression is the most common de novo resistance mechanism, whereas a complete loss of ER expression is not common in acquired resistance. Antiestrogen unresponsiveness appears to be the major acquired resistance phenotype, with a switch to an antiestrogen-stimulated growth being a minor phenotype. Since antiestrogens compete with estrogens for binding to ER, clinical response to antiestrogens may be affected by exogenous estrogenic exposures. Such exposures include estrogenic hormone replacement therapies and dietary and environmental exposures that directly or indirectly increase a tumor's estrogenic environment. Whether antiestrogen resistance can be conferred by a switch from predominantly ERalpha to ERbeta expression remains unanswered, but predicting response to antiestrogen therapy requires only measurement of ERalpha expression. The role of altered receptor coactivator or corepressor expression in antiestrogen resistance also is unclear, and understanding their roles may be confounded by their ubiquitous expression and functional redundancy. We have proposed a gene network approach to exploring the mechanistic aspects of antiestrogen resistance. Using transcriptome and proteome analyses, we have begun to identify candidate genes that comprise one component of a larger, putative gene network. These candidate genes include NFkappaB, interferon regulatory factor-1, nucleophosmin, and the X-box binding protein-1. The network also may involve signaling through ras and MAPK, implicating crosstalk with growth factors and cytokines. Ultimately, signaling affects the expression/function of the proliferation and/or apoptotic machineries.

Ligand-induced regulation of ERalpha and ERbeta is indicative of human breast cancer cell proliferation.

Two estrogen receptors (ER), ERalpha and ERbeta, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ERbeta protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ERalpha and ERbeta protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ERalpha and ERbeta, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ERbeta in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ERbeta expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ERalpha in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ERalpha expression. Although a change in the ERalpha/ERbeta ratio may play a role in the effect seen in cell proliferation, this study indicates that ERbeta is a poor prognosticator of cell proliferation in breast cancer and that ERalpha is a positive prognosticator of responsiveness to antiestrogen treatment.

Validation of real-time RT-PCR for analysis of human breast cancer cell lines resistant or sensitive to treatment with antiestrogens.

Using a quantitative real-time RT-PCR technique we have compared the expression of a number of genes in two different human breast cancer model systems for development of acquired resistance to antiestrogens. The model system developed at the Danish Cancer Society comprises the cell lines MCF-7, MCF-7/TAMR-1, MCF-7/182R-6 and MCF-7/182R-7, and the model system developed at the Lombardi Cancer Research Center consists of the cell lines MCF-7/LCC1, MCF-7/LCC2 and MCF-7/LCC9. The findings on the well-known parameters estrogen receptor (ER)alpha, progesterone receptor (PR) and epidermal growth factor receptor (EGFR) are in good agreement with previous reports, thus documenting the usefulness of the real-time RT-PCR technique for multiparametric RNA analysis. The gene expression levels in the two model systems were found to be quite similar in relation to ERalpha, AIB1 (amplified in breast cancer-1), breast cancer antiestrogen resistance gene 1 (BCAR1) and ErbB-2 mRNA expression, whereas significant differences were observed on the expression of ERbeta, multidrug resistance gene 1 (MDR1), PR and EGFR. Furthermore, the presented data suggest that ERbeta, AIB1, BCAR1, CYP19 and MDR1 are unlikely to be causally involved in development of antiestrogen resistance in these breast cancer cell lines.