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Objectives: Although a potential role for estrogen receptors (ER) in pancreatic tumors has been debated for many years, the importance of the receptors in these neoplasms remains unknown. Even the expression of the 2 ER isoforms, ER-alpha and ER-beta, in histological subtypes of pancreatic neoplasms is controversial. The aim of the present study was to systematically review the available literature about ER expression in pancreatic tumors and to discuss the potential importance of estrogen signaling in them., Methods: We performed a comprehensive literature search and analyzed the results regarding ER expression in pancreatic tumors, with special emphasis on the specificity of the antibodies used for immunohistochemistry., Results: Many articles have been published investigating the expression of ERs in pancreatic tumors, but the results are inconsistent. Moreover, most studies used antibodies that detected only ER-alpha, not ER-beta. Thus, the expression pattern of ER-beta in pancreatic neoplasm remains especially unclear., Conclusions: The lack of detailed studies evaluating the expression of both ER-alpha and ER-beta receptors using isoform-specific antibodies likely contributes to the inconsistency of published results concerning ER expression in pancreatic tumors. Available published evidence suggests that a thorough reexamination of the potential role of ERs in pancreatic neoplasms is warranted.

The estrogen receptor alpha (ERalpha), progesterone receptor (PR), and erbB2 (Her2 in humans) are important prognostic markers of human breast cancer, and they are variably expressed in different subtypes of breast cancer. The basal subtype, for example, is negative for ERalpha, PR, and Her2 by immunohistochemistry. We investigated the expression of these signaling molecules in enriched populations of mouse mammary stem cells and luminal cells that were isolated according to their differential expression of CD24 and the alpha6beta1-integrin complex. We found that the basal population, which is enriched in mouse mammary stem cells, did not express ERalpha, PR, or ErbB2/Her2 but did express epidermal growth factor receptor (EGFR)/ErbB1, whereas the subset of cells enriched for luminal cells expressed ERalpha (37% of cells) and PR (40% of cells) but not ErbB2/Her2 or EGFR/ErbB1. Ovariectomy confirmed the importance of estrogen signaling to luminal cell proliferation but had no effect on the size of the mouse mammary stem cell-enriched population. Thus, mouse mammary stem cells were negative for ERalpha, PR, and ErbB2 and appeared to share common properties with poor-prognosis basal breast cancer.

The desmoids are a group of rare clinically diverse, deep-seated fibrous neoplasms. The exact etiology is unknown, but several factors are considered to be positively correlated with their development and growth, i.e., genetic and hormonal factors and trauma. These tumors may be sporadic or associated with a genetic disease such as familial adenomatous polyposis (FAP). Devoid of metastatic potential, they tend to form large, infiltrative masses which, if not completely excised, recur repeatedly. Although surgery is widely accepted as the first-line treatment for extra-abdominal and abdominal wall desmoids, a proportion of cases are successfully palliated with either estrogen antagonists (tamoxifen, toremifene, and raloxifene) or nonsteroidal anti-inflammatory drugs. We describe and compare four methods for evaluating the expression of estrogen receptors alpha/beta and COX-1 and COX-2 in desmoid tumor-derived cells and tissues: immunocytochemistry, immunohistochemistry, RT-PCR, and two-color Western blot detection with the Odyssey infrared imaging system. Through this comparative analysis, Western blot with Odyssey was recognized as the best method to analyze the expression particularly of low expressed proteins in desmoid-derived cells. The use of a specific and reliable assessment method becomes fundamental in the evaluation of the presence and modulation of proteins which are important but weakly expressed in these rare tumors.

Background: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) alpha and beta are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERalpha or ERbeta in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERalpha and ERbeta expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status., Methods: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERalpha and ERbeta, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERalpha and ERbeta with smooth muscle actin, a marker of VSMCs., Results: ERbeta and ERalpha were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERbeta expression exceeded ERalpha expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERbeta expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERalpha expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERalpha but not ERbeta declined with age (P < 0.01) in HT women only. Age had no effect on ERalpha or ERbeta expression in non-HT or premenopausal women., Conclusions: ERbeta is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERbeta expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.

Background: Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERbeta-positive bladder cancer cell line. However, the expression of ERbeta in human bladder cancer has not been thoroughly investigated., Methods: ERalpha and ERbeta expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERalpha and ERbeta protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays., Results: Analyses revealed that only 2 human bladder cancers weakly expressed ERalpha. In contrast, the expression of ERbeta was detected in 141 tumors (63%). ERbeta was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P = .085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERbeta expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERbeta expression. The differences in ERbeta expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P < .001). Metastatic transitional cell carcinomas had ERbeta expression (80%) comparable to that of muscle invasive bladder cancers. Western blot analysis detected ERbeta protein expression in each of the 5 bladder cancer cell lines tested, whereas no or very low levels of ERalpha were found. Quantitative RT-PCR revealed that higher levels of ERbeta than ERalpha mRNA were present in 5637, T-24, TSU-Pr1, and TCC-Sup bladder cancer cells, whereas ER-alpha mRNA levels were greater than ERbeta in RT4 cells. Treatment with 17beta-estradiol modestly increased RT4 cell growth, whereas the antiestrogens, 4-hydroxtamoxifen, raloxifene, or ICI 182,780 inhibited the growth of RT4 cells., Conclusions: ERbeta is the dominant receptor expressed in bladder cancer cell lines and in the majority of human bladder tumors. Moreover, the degree of ERbeta expression increases with increasing stage and grade of differentiation. Antiestrogens have an inhibitory effect on the growth of bladder cancer cells in vitro., (Copyright 2006 American Cancer Society.)

Aim: To quantify ER variants/isoforms for women with breast cancer in Taiwan, an area with remarkably low breast cancer rates, but with an early onset and poor prognosis., Methods: Real-time PCR technology was exploited to quantify mRNAs of ERalpha, ERalphaE3Delta, ERalphaE5Delta, ERalphaE7Delta and ERbeta, in 49 breast cancer and paired adjacent normal tissues. Clinical parameters were assembled and tested for assocation with the ER expression., Results: Comparison of cancer and matched normal samples showed significantly decreased ERbeta (p < 0.001) in cancer tissues, and constant amounts of ERalpha and their variants. The results revealed significantly lower ERalphaE7Delta/ERalpha (p = 0.030) and ERbeta/ERalpha (p = 0.035) ratios in patients with lymph node (LN) metastasis than in those without LN metastasis., Conclusion: Our data suggests that ERalphaE7Delta and ERbeta may regulate ERalpha in normal human breast tissue.

There is scarce information about the factors associated with estrogen receptors (ER) at menopause. In 113 volunteers pre- and post-menopausal healthy women, grouped as with and without obesity, estrogen receptors-alpha and -beta, and progesterone receptor (PR) were measured by immunohistochemistry in skin punch biopsies obtained from the external gluteal area. In pre-menopausal women, biopsies and a blood sample were performed between days 7 and 14 of the cycle. Serum hormone levels were measured by immunoradiometric assay or radioimmunoassay. After menopause, ER and PR amounts decreased significantly. At pre-menopause, obese women had lower PR levels than non obese (P<.006). In the post-menopausal group, obese women showed higher ER-alpha (P<.03) and ER-beta (P<.02) levels than the non obese group. In the analysis of factors associated with the amount of steroid receptors for the total group, log[ER-alpha], log[ER-beta], and log[PR] were associated with age (P<.002, <.005, and <.004, respectively). The log[ER-alpha] was also associated with log[FSH] (P<.0008); meanwhile, the log[PR] showed a marginal correlation with log[FSH]. In pre-menopausal women no factor associated with any of the three receptors was found. In post-menopausal women log[ER-alpha] was associated with log[estrone] and log[DHEAS] (P<.003 and <.02, respectively). log[PR] was associated with BMI (P<.002), years since menopause (P<.05), and log[DHEAS] (P<.003). We concluded that ER and PR diminish sharply at post-menopause. At this stage the amount of receptors depends on several factors such as BMI, years since menopause, and androgen precursors.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Teleost choriogenins, precursors of the inner layer subunits of egg envelope, have been recently introduced as sensitive biomarkers for exposure to estrogenic compounds. In this study, two full-length cDNAs-ojChgH and ojChgL which encode the choriogenin H and L forms, respectively, were cloned from the marine medaka, Oryzias javanicus. The deduced protein sequences of ojChgH and ojChgL are highly similar to the corresponding homologues in the freshwater medaka (O. latipes) with identities of 77.2 and 87.6%, respectively. Phylogenetic analysis indicated that ojChgH and ojChgL are members of two different classes of liver-specific ZP-domain containing proteins (ZPB and ZPC, respectively). Computer analysis of ca. 2 kb of the 5'-flanking sequences of ojChgH and ojChgL revealed that both genes contain a number of putative estrogen response elements (EREs) and/or half-site EREs. In vivo mRNA expression patterns of the genes were examined by quantitative real-time RT-PCR. ojChgH is expressed exclusively in the liver while ojChgL is co-expressed in the liver (major) and ovary (minor). Exposure of fish to waterborne 17beta-estradiol (E2) at environmentally relevant concentrations (1, 5, 10 and 100 ng/L) resulted in dose-dependent induction of both genes in the liver, with higher sensitivity and magnitude of induction in males than in females. In the male liver, induction of ojChgH is more sensitive to E2 than that of ojChgL and two other estrogen-responsive genes, estrogen receptor alpha (ojERalpha) and vitellogenin (ojVTG). The lowest-observed-effect concentration (LOEC) of E2 on induction of hepatic ojChgH mRNA is 1 ng/L. In the ovary, expression of ojChgL is non-responsive to E2 treatment. In conclusion, the present study suggested that induction of hepatic ojChgH mRNA in male fish may be a highly sensitive biomarker for exposure to environmental estrogens.

Purpose: Cap43 is known as a nickel- and calcium-inducible gene. In the present study, we examined whether 17beta-estradiol (E2) could affect the expression of Cap43 in breast cancer., Experimental Design: Real-time PCR, immunoblotting, and immunocytochemistry were used to examine the expression of Cap43 and estrogen receptor-alpha (ER-alpha) in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cell lines were transfected with ER-alpha cDNA to establish cells overexpressing ER-alpha. Immunohistochemistry was used to evaluate the expression of the Cap43 protein in breast cancer patients (n = 96), and the relationship between Cap43 expression and clinicopathologic findings was examined., Results: Of the eight cell lines, four expressed higher levels of Cap43 with very low levels of ER-alpha, whereas the other four expressed lower levels of Cap43 with high ER-alpha levels. Treatment with E2 decreased the expression of Cap43 dose-dependently in ER-alpha-positive cell lines but not in ER-alpha-negative lines. Administration of antiestrogens, tamoxifen and ICI 182780, abrogated the E2-induced down-regulation of Cap43. Overexpression of ER-alpha in both ER-alpha-negative cell lines, SK-BR-3 and MDA-MB-231, resulted in down-regulation of Cap43. Immunostaining studies showed a significant correlation between Cap43 expression and the histologic grade of tumors (P = 0.0387). Furthermore, Cap43 expression was inversely correlated with the expression of ER-alpha (P = 0.0374)., Conclusions: E2-induced down-regulation of Cap43 seems to be mediated through ER-alpha-dependent pathways in breast cancer cells both in culture and in patients. Cap43 has potential as a molecular marker to determine the therapeutic efficacy of antiestrogenic anticancer agents in breast cancer.

In order to elucidate the relative importance of oestrogen receptor (ER)alpha, ERbeta and an ERbeta variant (ERbeta2/betacx) in the response of breast cancers to tamoxifen, tumour levels of each receptor were assessed in 36 patients before and after 3 months of neoadjuvant treatment with tamoxifen (20 mg daily). All patients were postmenopausal women presenting with large ERalpha-positive breast cancers. Clinical response to treatment was assessed by tumour volume changes as determined from sequential ultrasounds and pathological response by comparison of the tumour morphology before and after treatment. Of 33 cases, 23 (70%) were classified as having a clinical response and 16 (48%) as having a response pathologically. All tumours stained positively for ERalpha and ERbeta and 15 out of 33 (45%) for ERbeta2/betacx. There were no significant differences in quantitative expression of any receptor between tumours that subsequently responded and that did not, whether response was assessed clinically or pathologically. Tamoxifen treatment was associated with a decrease in ERalpha, but an increase was the most frequent change (17 out of 33) in ERbeta, and no consistent change was evident in staining of the ERbeta2/betacx variant. In summary, ERbeta1 and ERbeta2/betacx variant protein are detected in ERalpha-positive breast tumours but their expression is not associated with a response to tamoxifen. Differential changes in ERalpha and ERbeta were seen with treatment.

Background & Objective: Estrogen receptor-related receptor alpha (ERRalpha), a member of the subfamily of orphan nuclear receptors, could compete with estrogen receptor alpha (ERalpha) to bind the same target genes and interfere in ER signal pathway. Therefore, it might be associated to the tumorigenesis of endometrial carcinoma. This study was to explore the regulatory effect of 17beta-estradiol (17beta-E(2)) on ERRalpha expression, and to elucidate the relationship between ERRalpha and ER signal pathway in endometrial carcinoma cell lines., Methods: ERalpha-positive cell line Ishikawa and ERalpha-negative cell line HEC-IA were treated with different concentrations of 17beta-E(2) (1x10(-10) mol/L, 1x10(-8) mol/L, and 1x10(-6) mol/L) for 24 h and 48 h, respectively. The levels of ERRalpha mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. 17beta-E(2) (1x10(-8) mol/L) and complete ER inhibitor ICI182,780 (1x10(-6) mol/L) were given concomitantly to observe the change of ERRalpha expression., Results: The levels of ERRalpha mRNA and protein in Ishikawa cells were down-regulated after stimulated for 24 h and 48 h by different concentrations of 17beta-E(2). The maximal effect was observed at the concentration of 1x10(-8) mol/L. When 17beta-E(2) and ICI182,780 were given simultaneously to Ishikawa cells, this down-regulation was blocked. However, the level of ERRalpha mRNA, not protein, in HEC-IA cells was up-regulated after stimulated by different concentrations of 17beta-E(2) for 24 h. After stimulated by 17beta-E(2) for 48 h, the level of ERRalpha protein was up-regulated, which could not be blocked by ICI182,780., Conclusions: 17beta-E(2) can down-regulate the expression of ERRalpha in Ishikawa cells, which is mediated by ERalpha. 17beta-E(2) can up-regulate the expression of ERRalpha in HEC-IA cells, but this regulation cannot be blocked by ICI182,780.

An estrogen dependency has been suggested for the growth of fibroadenomas: however, thus far, none of the steroid hormone receptors acting on breast tissues has been demonstrated in the stroma of breast fibroepithelial lesions. In this study, the expression of estrogen receptor (ER)-alpha and -beta was investigated by immunohistochemistry in 33 fibroadenomas and in 30 benign, three borderline and seven malignant phyllodes tumors, all with spindle cell growth and in one distant metastasis. In addition, the presence of ER-beta mRNA and its variants was evaluated by RT-PCR in microdissected stroma. The possible correlation between hormone receptor expression and differentiation processes of stromal cells was investigated by smooth muscle actin and calponin immunostaining. ER-beta was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-beta was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-beta-positive stromal cells showed expression of smooth muscle actin and/or calponin, as demonstrated by double immunostaining. In addition, the mean age at diagnosis was significantly lower in patients with ER-beta-positive vs ER-beta-negative fibroadenomas. In contrast, in phyllodes tumors, ER-beta expression was higher in older patients. In conclusion, (i) only ER-beta is detected in the stroma of fibroadenomas and phyllodes tumors; (ii) its expression correlates with the expression of smooth muscle markers and suggests a role of ER-beta in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-beta-positive stroma cells, may further indicate a hormone-receptor mechanism involved in regulating the growth of fibroadenomas. Conversely, the older age of patients with ER-beta-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.

Background: High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER) to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified., Methods: The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg). Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S), a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H) and a polyclonal antibody raised against the amino terminus of ER beta., Results: ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa) were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg., Conclusion: This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage.

Phytoestrogens are increasingly consumed in artificially high doses as herbal preparations and nutritional supplements. The flavanone 8-prenylnaringenin (8PN) is a potent phytoestrogen, but its benefits and risks after long-term application are poorly identified. Therefore, we tested two doses of 8PN and 17beta-estradiol-3-benzoate (E2B) (effective doses: 6.8 and 68.4 mg/kg body weight (BW) of 8PN, and 0.17 and 0.7 mg/kg BW of 17beta-estradiol (E2)) and compared their effects on uterine weight, pituitary hormones (LH, FSH and prolactin) and the expression of estrogen-regulated genes and of estrogen receptor (ER)alpha and ERbeta in the hypothalamus, pituitary and uterus. Both doses of E2 and the high dose of 8PN suppressed serum LH and FSH, and stimulated serum prolactin levels, uterine weight, and progesterone receptor, insulin-like growth factor I and complement protein C3 mRNA transcripts. In the preoptic and the mediobasal areas of the hypothalamus, all treatments had negligible effects on ERalpha and ERbeta and gonadotropin-releasing hormone (GnRH) receptor gene expression, while ERbeta and GnRH receptor transcripts in the anterior pituitary were reduced under both E2 doses and the high 8PN dose. The mRNA concentrations of the LHalpha and -beta subunits in the pituitary were suppressed by E2 and 8PN. In summary, 8PN had very similar though milder effects than E2 on all tested parameters. Inhibition of climacteric complaints by E2 takes place in the hypothalamus, where it inhibits the overactive GnRH pulse generator. Hence, 8PN may be used to inhibit climacteric symptoms effectively. Human pharmacologic studies will show whether the stimulatory effect on the uterus that was found in the present animal model would require the concomitant administration of progestins to prevent endometrial overstimulation.

The pleiotropic effects of estrogen are mediated via stimulation of two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Although a number of studies have identified expression of one or both subtypes in estrogen target tissues, fewer studies have correlated ER expression with a functional role of these proteins in regulating cellular excitability. In the present study, we have combined cellular fluorescence, immunocytochemistry, and molecular expression techniques with single-channel patch-clamp studies to determine which ER mediates estrogen-stimulated potassium channel activity in human coronary artery smooth muscle cells (HCASMC). We had demonstrated previously that estrogen stimulates activity of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel in HCASMC via a nongenomic mechanism. We now demonstrate expression of both ERalpha and ERbeta subtypes in HCASMC. Functionally, however, expression of ERalpha antisense plasmid abolished the acute effect of estrogen on these channels, whereas estrogen retained its ability to stimulate BK(Ca) channels in cells transfected with only green fluorescence protein. In contrast, overexpression of ERalpha enhanced the stimulatory action of estrogen in HCASMC. Transfection with ERalpha antisense/sense plasmid did not alter ERbeta expression. These findings indicate that the ERalpha isoform mediates estrogen-induced stimulation of BK(Ca) channels in HCASMC and thereby provide evidence for a receptor-dependent signaling mechanism that can mediate estrogen-induced inhibition of cellular excitability.

Agonists to A3 adenosine receptor (A3AR) have been reported to inhibit cell growth and/or induce apoptosis in various tumors. We tested the effect of a novel A3AR agonist generically known as LJ-529 in breast cancer cells. Anchorage-dependent cell growth and in vivo tumor growth were attenuated by LJ-529, independently of its estrogen receptor (ER) alpha status. In addition, apoptosis was induced as evidenced by the activation of caspase-3 and c-poly(ADP)ribose polymerase. Furthermore, the Wnt signaling pathway was down-regulated and p27(kip) was induced by LJ-529. In ER-positive cells, the expression of ER was down-regulated by LJ-529, which might have additionally contributed to attenuated cell proliferation. In ER-negative, c-ErbB2-overexpressing SK-BR-3 cells, the expression of c-ErbB2 and its downstream extracellular signal-regulated kinase pathway were down-regulated by LJ-529. However, such effect of LJ-529 acted independently of its receptor because no A3AR was detected by reverse transcription-PCR in all four cell lines tested. In conclusion, our novel findings open the possibility of LJ-529 as an effective therapeutic agent against both ER-positive and ER-negative breast cancers, particularly against the more aggressive ER-negative, c-ErbB2-overexpressing types.

Although it is widely accepted the existence of putative estrogen receptors (ERs) localized at extranuclear domains in the brain, their molecular identity is still unclear. We have previously demonstrated in a murine septal cell line the existence of a membrane-related ER (mER) that participates in estrogen-mediated neuroprotection. To investigate the molecular structure of mER, we have used a battery of antibodies raised against different domains of the classical ERalpha to immunoblot with plasma membrane fractions from septal SN56 and hippocampal HT22 cell lines, and microsomal fractions of mouse septal and hippocampal tissues. The results confirmed that mER is the homologue of its intracellular counterpart ERalpha, suggesting the possibility that both nuclear and extranuclear receptors may share a common origin.

Purpose: S100A4 and the estrogen-inducible osteopontin are alone capable of inducing angiogenesis and metastasis in rodent models for breast cancer. The present study assesses the relationship of S100A4 and osteopontin with vessel density and estrogen receptor alpha (ERalpha) in primary tumors and with survival of patients to ascertain their involvement in metastatic breast cancer., Experimental Design: Primary tumors from 312 patients treated for minimally invasive human breast cancer were immunocytochemically stained and then assessed for the significance of their association with each other using Fisher's exact test or with patient survival over 18 years of follow-up using Kaplan-Meier plots and Wilcoxon-Gehan statistics., Results: Antibodies to S100A4 significantly stained endothelial cells of vessels adjacent to S100A4-staining groups of carcinoma cells, and antibodies to osteopontin significantly stained groups of carcinoma cells staining for ERalpha (P < 0.0001). There was a significant association of tumors staining for S100A4 with those with high vessel density (P = 0.021) and of tumors staining for osteopontin with those staining for ERalpha (P = 0.034). The association of staining for S100A4, osteopontin, or vessel density with patient death was significant (P < 0.0001, P = 0.005, and P = 0.014, respectively). The difference in cumulative proportion surviving between S100A4-positive patients with higher or lower vessel density increased up to about 12 years, but thereafter decreased to virtually zero after 18 years of follow-up. Patients with both S100A4-positive and osteopontin-positive primary tumors showed a statistically significant reduction in survival time over those with either one alone (P < 0.019), although in multivariate regression analysis, only staining for S100A4 was significant (P < 0.001)., Conclusions: It is suggested that in human breast cancer, S100A4 exerts some of its effects through angiogenesis, and that osteopontin is dependent on ERalpha for its expression.

Dysfunction of the host immune system in cancer patients can be due to a number of reasons including suppression of tumour associated antigen reactive lymphocytes by regulatory T (Treg) cells. In this study, we used flow cytometry to determine the phenotype and relative abundance of the tumour infiltrating lymphocytes (TILs) from 47 enzymatically dissociated tumour specimens from patients with infiltrating ductal carcinoma (IDC) of the breast. The expression of both effector and regulatory markers on the TILs were determined by using a panel of monoclonal antibodies. Analysis revealed CD8(+) T cells (23.4+/-2.1%) were predominant in TILs, followed by CD4(+) T cells (12.6+/-1.7%) and CD56(+) natural killer cells (6.4+/-0.7%). The CD4(+)/CD8(+) ratio was 0.8+/-0.9%. Of the CD8(+) cells, there was a higher number (68.4+/-3.5%) that expressed the effector phenotype, namely, CD8(+)CD28(+) and about 46% of this subset expressed the activation marker, CD25. Thus, a lower number of infiltrating CD8(+) T cells (31.6+/-2.8%) expressed the marker for the suppressor phenotype, CD8(+)CD28(-). Of the CD4(+) T cells, 59.6+/-3.9% expressed the marker for the regulatory phenotype, CD4(+)CD25(+). About 43.6+/-3.8% CD4(+)CD25(+) subset co-expressed both the CD152 and FOXP3, the Treg-associated molecules. A positive correlation was found between the presence of CD4(+)CD25(+) subset and age (> or =50 years old) (r=0.51; p=0.045). However, no significant correlation between tumour stage and CD4(+)CD25(+) T cells was found. In addition, we also found that the CD4(+)CD25(-) subset correlated with the expression of the nuclear oestrogen receptor (ER)-alpha in the tumour cells (r=0.45; p=0.040). In conclusion, we detected the presence of cells expressing the markers for Tregs (CD4(+)CD25(+)) and suppressor (CD8(+)CD28(-)) in the tumour microenvironment. This is the first report of the relative abundance of Treg co-expressing CD152 and FOXP3 in breast carcinoma.

Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor alpha (ERalpha) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERalpha mutant strain that retains some of the protein with transactivation activity. Here, we use an ERalpha-/- mouse strain in which no ERalpha transcript can be detected to analyze mammary gland development in the complete absence of ERalpha signaling. The ERalpha-/- females show no development beyond a rudimentary ductal system. By grafting ERalpha-/- epithelium or stroma in combination with ERalpha WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with ERalpha-/- mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERalpha signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When ERalpha-/- mammary epithelial cells are in close vicinity to ERalpha WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium.

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor alpha and beta (ERalpha and ERbeta). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERalpha and ERbeta in human secondary lymphoid tissues. We observed that, in tonsils, ERbeta is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERalpha is expressed only in activated germinal centres but not in the follicular zone. ERbeta is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERbeta is abundant while ERalpha is not detectable. Our results indicate that ERbeta is the predominant type of ER in mature lymphocytes. We suggest that ERalpha and ERbeta have distinct roles in secondary lymphoid tissues and that further studies with ERbeta-specific agonists will help to elucidate the role of ERbeta in these tissues.

Breast cancer is one of the most common malignancies affecting women in the Western world and one in seven women is predicted to develop invasive breast cancer in their lifetime. Breast cancer arises following the accumulation of a series of somatic changes often including deregulation of key signal transduction pathways. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be activated in breast cancer and overexpression of PI3K is sufficient to confer a malignant phenotype. Activation of the PI3K pathway serves to repress forkhead box class O (FoxO) transcription factor-mediated growth arrest and apoptosis. In this study, we used small interfering RNA (siRNA) to knockdown PI3K in three breast cancer cell lines representing different stages of cancer development. Transfection of PI3K siRNA in breast cancer cells resulted in a significant decrease in cell viability and induction of apoptosis irrespective of their estrogen receptor alpha (ERalpha) or ErbB2 status. PI3K depletion also resulted in a significant G(1) phase cell cycle arrest in ERalpha-positive breast cancer cells. Further, our data showed that PI3K knockdown resulted in a significant activation of FoxO; interestingly, a simultaneous knockdown of FoxO1a rescued the cells from apoptosis. Furthermore, the downstream effects of FoxO activation were found to be inhibition of cyclin-dependent kinase 4, cyclin-dependent kinase 6, and cyclin D1, and accumulation of p27/Kip1. Thus, we suggest that (a) PI3K plays a critical role in breast cancer development and (b) gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K could be developed for the management of breast cancer.

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Until recently, studies dealing with the uterus of the pregnant cow focus primarily on the placentome or on early and late pregnancy. Thus, there is a paucity of information about many aspects of the interplacentomal uterine wall including adherent foetal membranes. Corresponding tissue specimens were collected at the slaughterhouse and in animals undergoing premature caesarean section. Two specimens per month of pregnancy were assessed immunohistochemically for progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, Ki-67 protein and TUNEL procedure was performed. The latter two methods were employed in three animals each per months 1 and 2, 3 and 4, 7 and 8 and in six animals undergoing caesarean section at days 274 and 275 post insemination or during spontaneous labour. Results indicate that proliferation and apoptosis are of minor importance for tissue homeostasis since both can histochemically be detected only sporadically. Thus, at the sites investigated here, cellular hypertrophy plays an important role for tissue growth during pregnancy. Progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, however, exhibit cell type and pregnancy stage specific distribution patterns within the tissues assessed. Progesterone receptor immunoreactive scores remained fairly unchanged during pregnancy. Oestrogen receptor alpha scores, however, generally decreased and glucocorticoid receptors increased with ongoing gestation. Progesterone receptors and oestrogen receptor alpha were present in endometrial stroma and in myometrial smooth muscle cells during whole pregnancy. Oestrogen receptor alpha was detectable during whole pregnancy also in uterine glands. Progesterone receptors were, however, present at a very low level at the latter site only during months 1-3 and 6-9. Oestrogen receptor alpha and glucocorticoid receptors may also mediate uterine blood flow since they were present in the tunica media of uterine blood vessels. Results of the present study indicate, that progesterone and its receptor play an important role during whole gestation, mainly for uterine quiescence. Glucocorticoids and their receptors - possibly in cooperation with oestrogens and decreasing amounts of the oestrogen receptor alpha - should trigger processes initiating parturition, such as endometrial prostaglandin production. Further studies - including the periparturient period - should help to understand the exact role of the extraplacental compartment of the uterine wall for the initiation and progress of parturition.

Objective: In the following study, we investigated treatment-related changes in mammary gland histomorphology and structure after the administration of soy to adult virgin ovariectomized (OVX) female rats. Additionally, mammary receptor regulation was extensively evaluated by immunohistochemical analysis, and tissue proliferative activity analyzed by cell nuclear proliferating antigen expression (Ki67)., Design: OVX rats were treated, for 6 weeks, with either the vehicle, the soy extract (SSE 100 mg/kg/d PO), or 17beta-estradiol (0.5 mg/kg/d PO); a sham control group (SHAM) was also included in the study. When killed, mammary glands were collected and subsequently processed for light microscopy or immunohistochemistry. Immunoreactivity was quantified by a scoring system that took into account both the percentage of positive cells and the intensity of the staining., Results: The 17beta-estradiol--treated rats had stimulated mammary glands compared with OVX rats, with an average lobulo-alveolar development not different from the SHAM controls. Only a partial regression of the glandular atrophy was observed in OVX rats receiving 100 mg/kg/d SSE, with a histological appearance between that of the OVX and SHAM controls. No significant changes were observed among experimental groups in the median ERalpha scores of the epithelial compartment (score of 3 in all groups); in the stromal compartment, a tendency toward decreased expression was seen with 17beta-estradiol rats compared with OVX controls (scores of 2 and 5, respectively). A significant reduction in ERbeta immunostaining was observed in the mammary glands of SSE-treated rats, in both epithelium and stroma (scores of 4 and 3, respectively), compared with those of OVX controls (score of 8 in both compartments). The ERbeta receptor status was not significantly affected by 17beta-estradiol. Compared with OVX rats (score of 1), PR expression was up-regulated by 17beta-estradiol (score of 6), whereas an ovariectomy-like pattern was observed after the administration of SSE (score of 0). Ki67 immunoreactivity in the epithelium and stroma was increased by the administration of 17beta-estradiol (scores of 4 and 5, respectively) and was unchanged after SSE treatment (scores of 0 and 2, respectively), compared with OVX controls (scores of 1 and 2, respectively)., Conclusions: The differences observed in the histological pattern, hormonal receptor status regulation, and Ki67 modulation suggest a different role for phytoestrogens and 17beta-estradiol in postmenopausal rodent mammary glands.

A first step to elucidating the roles that steroids may play in the processes of ovarian development and early follicular growth is to identify the cell types that are likely to be receptive to steroids. Thus, cell types expressing receptors for oestrogen (alpha and beta form; ERalpha and ERbeta respectively), androgen (AR) and progesterone (PR) were determined by in situ hybridisation and immunohistochemistry in ovine ovarian tissues collected during ovarian development and follicular formation (days 26-75 of fetal life) as well as during the early stages of follicular growth. Expression of ERbeta was observed early during ovarian development and continued to be expressed throughout follicular formation and also during the early stages of follicular growth. ERbeta was identified in germ cells as well as in the granulosa cells. At the large preantral stage of follicular growth, expression of ERalpha was also consistently observed in granulosa cells. AR was first consistently observed at day 55 of fetal life in stroma cells throughout the ovary. Within the follicle, expression was observed in granulosa and thecal cells from the type-2 to -3 stage of follicular growth. PR mRNA did not appear to be expressed during ovarian development (days 26-75 of gestation). However, PR (mRNA and protein) was observed in the theca of type-3 (small preantral) and larger follicles, with mRNA -- but not protein -- observed in granulosa cells of some type-4 and 5 follicles. Expression of ERbeta, ERalpha and AR, as well as PR, was also observed in the surface epithelium and ovarian stroma of the fetal, neonatal and adult ovary. Thus, in sheep, steroid hormones have the potential to regulate the function of a number of different ovarian cell types during development, follicular formation and early follicular growth.

The induction of vascular endothelial growth factor (VEGF) expression by 17beta-estradiol (E(2)) in many target cells, including epithelial cells, fibroblasts and smooth muscle cells, suggests a role for this hormone in the modulation of angiogenesis and vascular permeability. We have already described a cyclic increase in Flk-1/KDR-expressing capillaries in the human endometrium during the proliferative and mid-secretory phases, strongly suggestive of an E(2) effect on Flk-1/KDR expression in the endometrial capillaries. However, it is unclear whether these processes are due to a direct effect of E(2) on endothelial cells. Using immunohistochemistry, we report an increase in Flk-1/KDR expression in endometrial capillaries of ovariectomized mice treated with E(2), or both E(2) and progesterone. This process is mediated through estrogen receptor (ER) activation. In vitro experiments using quantitative RT-PCR analysis demonstrate that Flk-1/KDR expression was not regulated by E(2) in human endothelial cells from the microcirculation (HMEC-1) or macrocirculation (HUVEC), even in endothelial cells overexpressing ERalpha or ERbeta after ER-mediated adenovirus infection. In contrast, Flk-1/KDR expression was up-regulated by VEGF itself, in a time- and dose-dependent manner, with the maximal response at 10 ng/ml. Thus, we suggest that E(2) up-regulates Flk-1/KDR expression in vivo in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is currently unknown whether or not the endothelial origin might account for differences in the E(2)-modulation of VEGF receptor expression, particularly in relation to the vascular bed of sex steroid-responsive tissues.

Introduction: Failure to detect co-expression of estrogen receptor-alpha (ERalpha) and proliferation 'markers' such as Ki67 in human mammary epithelium led to the view that estrogen acts indirectly to stimulate mammary epithelial proliferation. The mitotic index was so low in prior studies, however, that transient co-expression of ERalpha and Ki67 during the cell cycle could have been below detection limits., Methods: Immunohistochemistry was used on mammary tissue sections from estrogen treated rhesus monkeys to investigate co-expression of ERalpha and the proliferation antigen Ki67. Using the same methods, we investigated the cell localization of proteins involved in estrogen-induced proliferation, including cyclin D1, stromal cell-derived factor (SDF)-1, and MYC., Results: ERalpha was co-expressed with the proliferation marker Ki67 as well as with SDF-1, MYC and cyclin D1 in mammary epithelial cells from estrogen-treated monkeys., Conclusion: ERalpha is expressed in proliferating mammary epithelial cells together with the estrogen-induced proteins MYC, cyclin D1 and SDF-1, consistent with a direct mitogenic action by estrogen in primate mammary epithelium.

In the past decade, interest and knowledge in the role of estrogen in male reproduction and fertility has gained significant momentum. More recently, the cellular distribution and activity of estrogen receptors (alpha and beta)(ER) and aromatase (estrogen synthesis) has been reported in the penis, making the penis the latest "frontier" in the study of estrogen in male reproduction. ER and aromatase are broadly and abundantly expressed in various penile compartments and cell types (erectile tissues, urethral epithelia, vascular and neuronal cells), suggesting the complexity and significance of the estrogen-ER system in penile events. Unraveling this complexity is important and will require utilization of the various resources that are now at our disposal including, animal models and human lacking or deficient in ER and aromatase and the use of advanced and sensitive techniques. Some of the obvious areas that require our attention include: 1) a comprehensive mapping of ER-alpha and -beta cellular expression in the different penile compartments and subpopulations of cells, 2) delineation of the specific roles of estrogen in the different subpopulations of cells, 3) establishing the relationship of the estrogen-ER system with the androgen-androgen receptor system, if any, and 4) characterizing the specific penile phenotypes in human and animals lacking or deficient in estrogen and ER. Some data generated thus far, although preliminary, appear to challenge the long held dogma that, overall, androgens have a regulatory monopoly of penile development and function.

Background: Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors., Results: We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERalpha in a panel of ERalpha+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors., Conclusion: Our results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.

Recently, it has been shown that 17beta estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the alpha oestrogen receptor (ERalpha) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERalpha interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERalpha is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERalpha. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.

Tetramethrin, a synthetic pyrethroid insecticide, is used globally for agriculture, and thus potential environmental exposure to tetramethrin is a concern. Environmental chemicals that are hormonally active (particularly estrogen or androgen) may adversely affect the reproductive and endocrine systems. However, little is known about the estrogenic and androgenic activities of tetramethrin. In this study, uterine CaBP-9k gene expression assay and a uterotrophic assay were conducted for estrogenic activity assessment of tetramethrin, and a Hershberger assay was conducted for androgenic activity. Estrogen receptor (ERalpha and ERbeta) protein levels were also measured in tetramethrin-treated rat uteri. Northern blot analysis showed reduction in uterine CaBP-9k mRNA levels in response to tetramethrin, as well as when rats were given both tetramethrin and 17beta-estradiol (E2). In the uterotrophic assay using 18-d-old female Sprague-Dawley rats, subcutaneous treatment with tetramethrin (5 to 800 mg/kg/day) for 3 d led to a statistically significant decrease in absolute and relative uterine wet weights at all doses tested. Moreover, tetramethrin blocked the effect of E2 on uterine weights. In addition, tetramethrin reduced absolute and relative vaginal wet weights, and also inhibited the increases of vaginal weights produced by E2. Tetramethrin showed no androgenic on antiandrogenic activities in the Hershberger assay. These results suggest that tetramethrin might exert endocrine-disrupting effects on female rats through antiestrogenic action.

Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions.

Transcriptional activation of estrogen receptor alpha (ERalpha) is regulated by the ligand-dependent activation function 2 and the constitutively active N-terminal activation function 1. To identify ERalpha N-terminal-specific coregulators, we screened a breast cDNA library by T7 phage display and isolated histone deacetylase 4 (HDAC4). HDAC4 interacts with the ERalpha N terminus both in vitro and in vivo. Presence of the ERalpha DNA binding domain and hinge region reduce HDAC4 recruitment whereas full-length ERalpha enhances recruitment. HDAC4 interaction is selective for the ERalpha and not ERbeta N terminus and occurs in the nucleus. We demonstrate in vivo that HDAC4 is recruited by the N terminus to the promoter of an endogenous estrogen responsive gene. HDAC4 suppresses transcriptional activation of ERalpha by estrogen and selective ER modulators (SERMs) such as tamoxifen in a cell type-dependent manner. Consistently, silencing of HDAC4 promotes the agonist effect of SERMs (tamoxifen and raloxifene) in a cell type-specific manner. These findings indicate a role for HDAC4 in regulating ERalpha activity as a novel N-terminal coregulator and uncover a mechanism by which certain cell types regulate SERM behavior.

A fast, label-free, and multiplexed method based on piezoresistive cantilevers is reported for the detection of specific protein conformations at the nanoscale level. The ligand-binding domain of the human oestrogen receptor (ERalpha-LBD) is used as the experimental model system, and ERalpha-LBD with or without oestradiol (E2) is detected using the conformation-specific peptides alpha/betaI (Ser-Ser-Asn-His-Gln-Ser-Ser-Arg-Leu-Ile-Glu-Leu-Leu-Ser-Arg, which recognizes E2-bound ER) and alpha/betaII (Ser-Ala-Pro-Arg-Ala-Thr-Ile-Ser-His-Tyr-Leu-Met-Gly-Gly, which recognizes E2-free ER). Target-specific signals are obtained in situ at protein concentrations of 2.5-20 nM. The in-build electrical readout of the piezoresistive cantilevers provides a convenient alternative to the conventional optical detection, and the presented method offers the possibility of detecting protein conformational changes using miniaturized microarrays.

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.

Estrogens have been implicated to play certain but yet undefined roles in the normal and neoplastic growth of prostate gland. Studies of perinatal exposure in rodents demonstrate that effects of perinatal estrogenization are permanent and carcinogenic in prostate gland. In the Noble (Nb) rat model, prostatic dysplasia and neoplastic lesions can be induced by a chronic treatment with both testosterone and estrogen at adulthood. However, by this conventional protocol, neoplastic lesions are mostly confined to the lateral (LP) and ventral (VP) prostates, while gross prostatic tumors are rarely induced. Based on these two experimental models, we developed a modified treatment protocol for the enhancement of prostate cancer induction in Nb rat model by combining neonatal estrogen exposure of male offspring followed by the hormonal treatment at adulthood (NeoE + T-E2). Using this modified protocol, we were able to induce more extensive development of neoplastic lesions in all three prostatic lobes and also gross tumors at relatively high incidence within 6-9 months. Western blottings and immunohistochemistry showed that ERalpha expression was increased in the hypertrophic peri-acinar and -ductal smooth muscle cells while ERbeta and AR expressions are markedly decreased in dysplastic and neoplastic lesions in NeoE + T-E2-treated prostates. Immunohistochemistry showed that expression of three tumor suppressors (BRCA2, PTEN, and Rap1) and tubulin-alpha are markedly decreased in dysplastic and neoplastic lesions. In addition, loss of expression of smooth muscle differentiation markers (desmin, alpha-actin, and vinculin) and defects of basement membranes were also seen in the reactive stroma. These results suggest that exposure to high levels of estrogens, either endogenous or exogenous, in early life could play a role in the development of prostate cancer in later life.

Background: Gender differences have been described in major mental illnesses (MMI). The dorsolateral prefrontal cortex (DLPFC) and hippocampus are estrogen-sensitive brain regions structurally and functionally altered in patients with MMI. We hypothesized that gender-specific alterations in DLPFC and hippocampus estrogen receptor alpha (ERalpha) mRNA levels may exist in MMI patients., Methods: We used Northern blot analysis to survey the expression of ERalpha mRNA transcripts in brain and body, detected by our human ERalpha riboprobe and in situ hybridization, to examine the expression pattern and quantify ERalpha mRNA levels in DLPFC and anterior hippocampus of patients with major depressive disorder (MDD), schizophrenia, and bipolar disorder compared with normal control subjects., Results: Northern blotting revealed brain-region-specific differences in expression levels of a 5 kb ERalpha mRNA transcript. By in situ hybridization, ERalpha mRNA was detected in all layers of DLPFC and all hippocampal subfields in all subjects. We detected greater DLPFC ERalpha mRNA expression in male compared with female MDD subjects and reduced ERalpha mRNA levels in the dentate gyrus of schizophrenics compared with control subjects., Conclusions: Our results suggest that alterations in ERalpha mRNA levels exist in distinct telencephalic regions in male and female MDD patients, and in both genders in schizophrenia.

Many common craniofacial pain conditions are more prevalent in women than men and may be related to the phase of the menstrual cycle. Long-term effects of oestrogen in the nervous system are produced by receptor-mediated [oestrogen receptor alpha (ERalpha) and beta (ERbeta) isoforms] mechanisms; however, it is not known if the distribution of ER-positive neurons in the trigeminal system is similar in males and females. Quantitative immunocytochemistry was used to compare the distribution of ERalpha-labelled neurons in the trigeminal brainstem complex (TBC) and ganglion of male and female rats at different stages of the oestrous cycle. A high density of ERalpha-labelled neurons was seen in the superficial laminae (I-III) throughout the trigeminal subnucleus caudalis (Vc) and the upper cervical dorsal horn. Counts of ERalpha-positive neurons in laminae I-III were similar for prooestrous and dioestrous females, while males had fewer cells. The deeper laminae (IV-V) of the Vc and the cervical dorsal horn had few ERalpha-positive neurons in all groups. At the region surrounding the central canal at caudal levels of the Vc, prooestrous females had more ERalpha-positive neurons than dioestrous females or males. Few labelled cells were seen rostral to the trigeminal subnucleus interpolaris/caudalis transition region (Vi/Vc) in any group. In the trigeminal ganglion, prooestrous and dioestrous females had a moderate (8-10%) number of nuclear-labelled small or medium-sized neurons, while males had fewer labelled cells (4.5%). Qualitatively, the pattern of staining for ERbeta was similar, although weaker, than for ERalpha in the trigeminal dorsal horn or ganglion. These results were consistent with the hypothesis that oestrogen acts through trigeminal ganglion cells and caudal portions of the Vc to modulate sensory and autonomic aspects of craniofacial pain in a sex-related manner.

Objective: Our objective was to assess vascular endothelial function and morphology in resistance vasculature from healthy pre- and postmenopausal women in vitro and to determine potential mechanisms of vascular protection by estrogenic compounds., Methods: Arteries (approximately 220 microm) were dissected from sc fat biopsies obtained from healthy premenopausal and postmenopausal women. Flow-mediated dilatation, agonist-induced endothelium-dependent and -independent relaxation, and myogenic responses to changes in intraluminal pressure were evaluated before and after incubation (3 h) with 17beta-estradiol, propyl pyrazole triol [a selective estrogen receptor-alpha (ERalpha) agonist], raloxifene (a second-generation selective ER modulator), and the phytoestrogen genistein, using pressure myography technique. In addition, endothelial morphology was assessed in arteries from pre- and postmenopausal women, and distribution of ERs within the artery wall from postmenopausal women was evaluated., Results: Functional and morphological disturbances of endothelial function were observed in small arteries from postmenopausal women. Incubation with 17beta-estradiol improved postmenopausal resistance artery function, an effect mimicked by propyl pyrazole triol but not raloxifene or genistein. Immunohistochemical staining revealed similar expression of ERalpha and ERbeta in the smooth muscle of arteries from postmenopausal women; however, ERalpha was dominant in endothelium., Conclusions: The resistance arteries from postmenopausal women show functional and morphological abnormalities. ERalpha may contribute to vascular protection by estrogens in the peripheral resistance circulation in postmenopausal women. Selective ERalpha agonists warrant further investigation as therapeutic agents for prevention of cardiovascular disease in postmenopausal women.

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

Purpose: We examined the immunolocalization of estrogen receptor (ER)alpha and ERbeta in the human fetal prostate., Materials and Methods: Tissue sections from human fetal prostates at 7 to 22 weeks of gestation were stained with antibodies to ERalpha, ERbeta, and cytokeratin 10 and 14., Results: ERalpha expression was not detected until 15 weeks of gestation with sparse staining in the utricle. By 19 weeks increased ERalpha expression was seen in the luminal cells of the ventral urogenital epithelium (UGE), basal cells of the dorsal UGE, utricle, distal periurethral ducts, peripheral stroma and posterior prostatic duct. K14 was detected in basal cells of the UGE and in several posterior acini. At 22 weeks ERalpha expression was more intense in all of these areas. ERbeta was expressed throughout the UGE, ejaculatory ducts, müllerian ducts and entire stroma at 7 weeks. Intense ERbeta staining was observed in these areas and in the prostatic buds by 8 weeks with persistent intense staining through 22 weeks., Conclusions: To our knowledge we report the first immunolocalization of ERalpha in the human fetal prostate and the earliest demonstration of ERbeta expression in the prostate at 7 weeks of gestation. ERbeta expression is intense during ductal morphogenesis, suggesting a role in normal glandular growth and proliferation. The induction of squamous metaplasia in the UGE, distal periurethral ducts and utricle is associated with ERalpha expression in these areas, while the induction of squamous metaplasia in peripheral prostatic acini is associated with peripheral stromal ERalpha expression. This study suggests estrogen signaling pathways in the human fetal prostate via ERalpha that involve epithelial-epithelial and epithelial-stromal interactions.

Purpose: We examined expression of 14-3-3sigma, a regulator of cell proliferation, and evaluated its clinical significance in endometrioid endometrial carcinoma., Experimental Design: One hundred three endometrioid endometrial adenocarcinoma cases were examined using immunohistochemistry with archival specimens. We correlated this finding with various clinicopathologic variables, including the status of estrogen receptor, progesterone receptor, and MIB-1 (Ki-57)., Results: 14-3-3sigma Immunoreactivity was detected in 78 of 103 (75.3%) of carcinoma cases. No statistically significant correlation was detected between status of 14-3-3sigma and any of clinicopathologic variables examined. There was, however, a statistically significant correlation between loss of 14-3-3sigma expression and adverse clinical outcome of the patients (P = 0.0007). In the early stages of cancer (stages I and II), 14-3-3sigma immunoreactivity was absent in 5 of 10 (50.0%) patients who showed recurrence during follow-up, whereas its absence was detected in only 13 of 68 (19.1%) disease-free patients in the same period. In addition, 14-3-3sigma immunoreactivity was absent in 4 of 5 (80.0%) patients who died, whereas its absence was detected in only 14 of 73 (19.2%) patients who had lived during the same period. Patients whose tumors were negative for 14-3-3sigma were at much greater risk to develop recurrent and/or mortal disease (P = 0.0372 and 0.0067). In multivariate analysis using the Cox proportional hazards model, absence of 14-3-3sigma turned out to be statistically independent risk factor in disease-free survival and overall survival even in patients with early-stage disease (P = 0.0321 and 0.0191)., Conclusions: Results of our study showed that loss or absence of 14-3-3sigma determined by immunohistochemistry may be an important tool to identify endometrial carcinoma cases at high risk of recurrence and/or death, who are otherwise not detected by current clinical and pathologic evaluation, especially in the early stages of the disease. In addition, results of 14-3-3sigma immunohistochemistry in the early stage of endometrial carcinoma could contribute to planning postoperative follow-up and adjuvant therapy.

Background: It is important to clarify the biological function of the female sex hormones estrogen and progesterone in periodontal ligament cells, as these hormones may affect periodontal health. We have previously shown that human periodontal ligament cells express estrogen receptor beta (ERbeta) but not ERalpha, whereas human breast cancer cells (MCF7) express both ERalpha and ERbeta. Data on progesterone receptor (PgR) expression in human periodontal ligament cells have not been reported., Objectives: Determine PgR expression in human periodontal ligament and MCF7 cells and to investigate how estrogen affects DNA and collagen synthesis in these two cell types showing different pattern of expression for ERalpha and beta., Methods: Periodontal ligament cells were obtained from the periodontal ligament of premolars extracted for orthodontic reasons and MCF7 cells from the American Type Culture Collection (ATCC). PgR expression was determined by immunocytochemistry. DNA and collagen synthesis was determined by [(3)H]thymidine and L-[(3)H]proline incorporation, respectively., Results: PgR immunoreactivity was observed in nuclei of MCF7 but not periodontal ligament cells. Treatment with estrogen (17beta-estradiol, E(2)) at physiological concentrations for 24 h stimulated DNA synthesis by more than two times in MCF7 cells, whereas there was no effect on periodontal ligament cell DNA synthesis. The ER blocker ICI 182780 fully reversed the stimulatory effect of E(2). Not only short-term (24 h) but also long-term (5 days) treatment with E(2) lacked effect on DNA synthesis in periodontal ligament cells. Neither periodontal ligament cell viability nor collagen synthesis was affected by E(2) treatment. Identical results were observed in periodontal ligament cells from male and female subjects., Conclusions: Human MCF7 but not periodontal ligament cells express PgR, suggesting that progesterone via PgR affects MCF7 but not periodontal ligament cells. Further, estrogen stimulates breast cancer MCF7 cell proliferation, whereas it has no effect on proliferation of periodontal ligament cells, probably reflecting cell type specific ER expression pattern in these two cell types.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids, among them oestrogens are the end products obtained from the irreversible transformation of androgens by aromatase (P450arom). Up today P450arom has been demonstrated in male germ cells of all mammals so far studied (mice, rat, bank vole, bear, monkey). In man Leydig cells and immature germ cells as well as ejaculated spermatozoa express a biologically active aromatase. Moreover germ cells and spermatozoa contain oestrogen receptors (ER-alpha and ER-beta) and it is of note that a truncated form of ER-alpha is present in spermatozoa. These observations clearly suggest that oestrogens are likely concerned in various stages of male germ cell development.

Ovarian clear-cell carcinoma (OCCA) and ovarian endometrioid carcinoma (OEMC) are often associated with endometriosis. This study determined the immunohistochemical expression of steroid hormone receptors and cancer-related genes in OCCA and OEMC specimens containing endometriosis and atypical endometriosis. Pathological progression from endometriosis to atypical endometriosis to carcinoma was identified in 4 of 22 OCCAs and 4 of 16 OEMCs. Using paraffin-embedded tissue samples, expression and changes in expression for estrogen receptors (ERs) and progesterone receptors (PRs) were examined in relation to the observed pathological changes. A gradual reduction in ERalpha and PRA expression was observed with malignant transformation from endometriosis to atypical endometriosis to OCCA. In OEMC, ERalpha expression increased with malignant transformation. In conclusion, reductions in ERalpha and PRA expression were observed with progression from endometriosis to atypical endometriosis in OCCA specimens. Disappearance of hormone dependency might therefore be associated with malignant transformation to OCCA.

Long-term formalin tissue fixation results in antigen masking, probably through aldehydic linkage between proteins and fixative molecules. Immunohistochemistry results depend on the type of the detection procedure and the type of antibody used for the reaction. Considering the difficulty in working with estrogen receptor (ER) antibodies and the lack of standardization of the antigen retrieval methods, we quantified the immunoexpression of ER using the 1D5 antibody and a standard streptavidin-biotin detection procedure retrieving with microwave oven, steamer, pressure cooker, and water bath in a set of SBR grade 2 invasive breast carcinomas. Pressure-cooking provided the best results. No significant differences were observed in using the other methods. Pressure-cooking should be recommended as the method of choice for standardization of the ER immunohistochemical reaction.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.