Expression Of Estrogen-responsive Genes Research Articles

The Organophosphate Chlorpyrifos Interferes with the Responses to 17β-Estradiol in the Digestive Gland of the Marine Mussel Mytilus galloprovincialis

BackgroundMany pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17β-estradiol (E2) were investigated.Methodology/Principal FindingsMussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E2 (6.75 ng/g dw). Responses were evaluated in CHP, E2 and CHP/E2 treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E2 induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E2 and CHP/E2, induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E2, only 2 genes between E2 and CHP/E2. Moreover, 36 genes were uniquely modulated by CHP/E2. Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments.ConclusionsThe results show complex interactions between CHP and E2 in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens.

Tocotrienols and breast cancer: the evidence to date

Breast cancer is the second most frequent cancer affecting women worldwide after lung cancer. The toxicity factor associated with synthetic drugs has turned the attention toward natural compounds as the primary focus of interest as anticancer agents. Vitamin E derivatives consisting of the well-established tocopherols and their analogs namely tocotrienols have been extensively studied due to their remarkable biological properties. While tocopherols have failed to offer protection, tocotrienols, in particular, α-, δ-, and γ-tocotrienols alone and in combination have demonstrated anticancer properties. The discovery of the antiangiogenic, antiproliferative, and apoptotic effects of tocotrienols, as well as their role as an inducer of immunological functions, not only reveals a new horizon as a potent antitumor agent but also reinforces the notion that tocotrienols are indeed more than antioxidants. On the basis of a transcriptomic platform, we have recently demonstrated a novel mechanism for tocotrienol activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicate a high affinity of specific forms of tocotrienols for ERβ, but not for ERα. Moreover, we have demonstrated that specific tocotrienols increase ERβ translocation into the nucleus which, in turn, activates the expression of estrogen-responsive genes (MIC-1, EGR-1 and Cathepsin D) in breast cancer cells only expressing ERβ cells (MDA-MB-231) and in cells expressing both ER isoforms (MCF-7). The binding of specific tocotrienol forms to ERβ is associated with the alteration of cell morphology, caspase-3 activation, DNA fragmentation, and apoptosis. Furthermore, a recently concluded clinical trial seems to suggest that tocotrienols in combination with tamoxifen may have the potential to extend breast cancer-specific survival.

The Role of Calcium in the Activation of Estrogen Receptor-Alpha

Environmental estrogen mimics, including metalloestrogens that can activate estrogen receptor-alpha (ERα), may contribute to breast cancer risk. However, the underlying mechanisms through which these molecular mimics activate the ERα are generally poorly understood. With concern to this important question, we investigated whether intracellular calcium may mediate the cross-talk between signaling pathways that activate ERα and the ligand-binding domain of ERα. MCF-7 cells treated with EGF, ATP, extracellular calcium, or caffeine to increase intracellular calcium triggered a rapid recruitment of ERα to estrogen-responsive promoters and stimulated expression of estrogen-responsive genes including pS2, complement C3, and progesterone receptor. Induction was blocked by an antiestrogen but also by the chelation of intracellular calcium. Treatment with extracellular calcium also increased the growth of MCF-7 cells through an ER-dependent mechanism. We found that EGF and extracellular calcium activated the C-terminus of ERα and the activation was blocked by the antiestrogen. Mechanistic investigations identified four potential sites on the solvent-accessible surface of the ERα ligand-binding domain as important for calcium activation of the receptor. Taken together, our results suggest that calcium mediates the cross-talk between ERα-activating signaling pathways and the ligand-binding domain of ERα providing a potential explanation for the ability of certain environmental metalloestrogens to activate the receptor.

Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland

Hexavalent Chromium Cr(VI) is an important contaminant considered as a model oxidative toxicant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. On the other hand, in mammals the reduced form Cr(III) is considered an essential microelement, involved in regulation of lipid and carbohydrate metabolism; moreover, recent evidence suggests that Cr may have endocrine effects. In this work, the effects of Cr(VI) were investigated in the digestive gland of the marine bivalve Mytilus galloprovincialis. Mussels were exposed to 0.1–1–10–100 μg Cr(VI) L −1 animal −1 for 96 h. At 100 μg L −1, a large increase in total Cr tissue content was observed; in these conditions, the lysosomal membranes were completely destabilized, whereas other lysosomal biomarkers (neutral lipids-NL and lipofuscin-LF), as well as different enzyme activities and gene expression were unaffected, this indicating severe stress conditions in the tissue. On the other hand, at lower concentrations, changes in other histochemical, biochemical and molecular endpoints were observed. In particular, at both 1 and 10 μg L −1, lysosomal destabilization was associated with significant NL and LF accumulation; however, no changes in catalase and GSH transferase (GST) activities were observed. At the same concentrations, GSSG reductase (GSR) activity was significantly increased, this probably reflecting the recycling of GSSG produced in the GSH-mediated intracellular reduction of Cr(VI). Increased activities of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were also observed, indicating that Cr(VI) could affect carbohydrate metabolism. Cr(VI) induced downregulation or no effects on the expression of metallothioneins MT10 and MT20, except for an increase in MT20 transcription in males. Moreover, significant up-regulation of the Mytilus estrogen receptor MeER2 and serotonin receptor (5-HTR) were observed in both sexes. The results demonstrate that exposure to Cr(VI) in the low ppb range did not result in strong toxicity or oxidative stress conditions in mussel digestive gland. On the other hand, our data support the hypothesis that low concentrations of the metal can exert pleiotropic effects on mussel physiology, from modulation of lipid and carbohydrate metabolism, to effects on the expression of estrogen-responsive genes.

Effects of coumestrol administration to pregnant and lactating mice on intestinal alkaline phosphatase activity

The present study was conducted to clarify the effects of coumestrol administration on Ca metabolism during pregnancy and in lactating mice. From 6.5 to 16.5 days post coitus (dpc), pregnant mice were administered coumestrol at 200 µg/kg body weight/day. The duodenum, jejunum and blood samples were obtained at 17.5 dpc or 10 days after parturition (dap). Coumestrol administration decreased alkaline phosphatase (ALP) activity and mRNA expression of IAP and estrogen responsive genes, c-fos and vascular endothelial growth factor (VEGF), in the duodenum and jejunum of pre-delivery mice. In lactating mice, the ALP activity and mRNA expression of IAP were not changed, although coumestrol administration decreased mRNA expression of c-fos in the duodeum and VEGF in the jejunum. Coumestrol did not affect serum Ca and the expression of vitamin D receptor protein in the duodenum and jejunum. Thus, coumestrol administration during pregnancy may decrease the mRNA expression of IAP and the ALP activity in the intestine of the pre-delivery mice through ERα, but coumestrol had little effect on intestinal ALP activity at 10 days after parturition.

Modulation of some estrogen-responsive genes in the vena cava of ovariectomised Wistar rats by griffonianone C, an isoflavone derived from Millettia griffoniana Baill. (Fabaceae)

In the present study, we investigated whether griffonianone C (Griff C), extracted from root bark of Millettia griffoniana, changes the expression of several estrogen-responsive genes in the vena cava of ovariectomised rats. For this purpose, we subcutaneously administered Griff C (2, 10, or 20 mg/kg/d BW), 17β-estradiol (E2: 10 μg/kg/d BW) as positive control, and a vehicle control respectively for three days. Relative expression levels of estrogen receptor α (ERα), progesterone receptor (PR), cyclooxygenase2 (Cox-2), vascular endothelial growth factor (VEGF), VEGF-receptor 2, angiotensin converting enzyme (ACE), endothelial NO synthase (eNOS), proliferating cell nuclear antigen (PCNA) and Ki67 mRNA extracted from the vena cava of these rats were quantified by real-time PCR. Results showed that Griff C up-regulated the expression of PR, ACE, ERα, VEGF, VEGFR2 and Ki67. However, the results of Cox-2, PCNA, and eNOS expression did not reach significance in the E2 and Griff C treated samples. These results show that griffonianone C regulated a few of the analysed genes in a similar fashion than estradiol; however, others showed a different pattern. This suggests that some of the biological effects attributed to M. griffoniana are mediated via ER pathway others may be mediated via other pathways.

Unexpected Hormonal Activity of a Catechol Equine Estrogen Metabolite Reveals Reversible Glutathione Conjugation

4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in widely prescribed hormone replacement formulations. 4-OHEN is autoxidized to an electrophilic o-quinone that has been shown to redox cycle, generating ROS, and to covalently modify proteins and DNA and thus potentially to act as a chemical carcinogen. To establish the ability of 4-OHEN to act as a hormonal carcinogen at the estrogen receptor (ER), estrogen responsive gene expression and proliferation were studied in ER(+) breast cancer cells. Recruitment by 4-OHEN of ER to estrogen responsive elements (ERE) of DNA in MCF-7 cells was also studied and observed. 4-OHEN was a potent estrogen, with additional weak activity associated with binding to the arylhydrocarbon receptor (AhR). The potency of 4-OHEN toward classical ERalpha mediated activity was unexpected given the reported rapid autoxidation and trapping of the resultant quinone by GSH. Addition of thiols to cell cultures did not attenuate the estrogenic activity of 4-OHEN, and preformed thiol conjugates added to cell incubations only marginally reduced ERE-luciferase induction. On reaction of the 4OHEN-GSH conjugate with NADPH, 4-OHEN was observed to be regenerated at a rate dependent upon NADPH concentration, indicating that intracellular nonenzymatic and enzymatic regeneration of 4-OHEN accounts for the observed estrogenic activity of 4-OHEN. 4-OHEN is therefore capable of inducing chemical and hormonal pathways that may contribute to estrogen-dependent carcinogenesis, and trapping by cellular thiols does not provide a mechanism of termination of these pathways.

Cyclin D1 regulates hepatic estrogen and androgen metabolism

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.

Oral p-Tert-Octylphenol Exposures Induce Minimal Toxic or Estrogenic Effects in Adult Female Sprague-Dawley Rats

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17α-ethinyl estradiol (EE, 10μg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

Relationship Between Plasma Estradiol Levels and Estrogen-Responsive Gene Expression in Estrogen Receptor–Positive Breast Cancer in Postmenopausal Women

PURPOSE To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)-positive breast cancers in postmenopausal women. Materials and METHODS Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = -0.179; P = .05; and r = -0.389; P = .0005, respectively). CONCLUSION Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer.

Xenoestrogens – Impact on Reproduction

Event Abstract Back to Event Xenoestrogens – Impact on Reproduction Paraskevi Papaioannidou1* 1 Aristotle University of Thessaloniki, Department of Pharmacology, Greece Xenoestrogens bind to estrogen receptors α and β and increase the expression of estrogen responsive genes. Although they bind to estrogen receptors with much weaker affinity than estradiol, they may act as estrogen receptor agonists or mixed agonists/antagonists and may disrupt the endocrine system by modulating the hypothalamic-pituitary-gonadal axis. Phytoestrogens have become a major component in the typical Western diet over the last few decades, mainly due to introduction of soy in Western diet. Although soy has been related with beneficial effect in menopause and protective effects in cancer and other diseases, concern has been raised about potential adverse effects due to estrogen disrupting activity and possible effect on human fertility. Common types of phytoestrogens in human diet include flavones, lignans, isoflavones and coumestans. The compounds with the most potent estrogenic activity are genistein (found in soy), resveratrol (found in grapes) and coumestrol (found in clover). Phytoestrogens have been related to low sperm counts and disturbed sperm function, as well as to gonadal dysgenesis when consumed during pregnancy. The use of soy milk as an alternative to breast or cow milk is of particular concern, since the most vulnerable periods for estrogenic insult are thought to be the pre- and neonatal periods, when irreversible damage can be inflicted on the developing germinal epithelium. Environmental xenoestrogens include polychlorinated insecticides like DDT, polychlorinated biphenyls (PCBs), parabens, phthalates, nonylphenol, octylphenol, dioxin and other substances found in common household products, as well as in environment highly contaminated with organochlorines from chemical industries that use great amounts of chlorine. Animal species living in such an environment – birds, fish and small mammals – are unable to reproduce successfully due to high body burdens of various xenoestrogens. There is a possible link between estrogenic pollutants and falling sperm counts in men. Environmental xenoestrogens, like dioxin and PCB’s have been linked to higher levels of endometriosis in female monkeys and great increase in ectopic pregnancies in humans. Environmental protection policy is necessary, and further studies must be carried out to assess the risk of exposure to xenoestrogens, and the effect of our modern life and diet on future fertility and health. Keywords: xenoestrogens, Phytoestrogens, Reproduction, estrogen disrupters Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Xenobiotics and reproduction Citation: Papaioannidou P (2010). Xenoestrogens – Impact on Reproduction. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00178 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Paraskevi Papaioannidou, Aristotle University of Thessaloniki, Department of Pharmacology, Thessaloniki, Greece, ppap@auth.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Paraskevi Papaioannidou Google Paraskevi Papaioannidou Google Scholar Paraskevi Papaioannidou PubMed Paraskevi Papaioannidou Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Genistein: Impact on Reproduction

Event Abstract Back to Event Genistein: Impact on Reproduction Fotios Tsanakalis1 and Paraskevi Papaioannidou1* 1 Aristotle University of Thessaloniki, Department of Pharmacology, Greece Genistein is an isoflavone with estrogenic activity, found in dietary plants. It is the phytoestrogen with the highest estrogenic activity found in soy. Genistein binds to estrogen receptors α and β and increases the expression of estrogen responsive genes. Although it binds to estrogen receptors with much weaker affinity than estradiol, it may act as an estrogen receptor agonist or mixed agonist/antagonist and may disrupt the endocrine system. There is evidence that prenatal exposure of rats to genistein is followed by an increase in mammary gland cancer rates, while neonatal exposure induces uterine adenocarcinoma in later life. Perinatal exposure of male rats to genistein has been related to testicular dysgenesis syndrome and modulation of normal masculinization. In addition, genistein has been found to hinder the process of acrosomal exocytosis and the binding of spermatozoa to the zona pellucida, thus affecting male fertility. Indirect adverse effects on testes have also been described, mediated through the modulation of the hypothalamic-pituitary-gonadal axis. In female mice, perinatal exposure to genistein has been related to altered ovarian function, disrupted estrous cyclicity, and subfertility, while neonatal exposure resulted in disorders of follicular development. In spite of its beneficial effect in menopause and its potential protective effect in cancer, genistein disrupts the endocrine system and may affect the reproductive system. Soy consumption and exposure to high doses of genistein should be a matter of concern during crucial life periods such as pregnancy, lactation and early development. Keywords: Genistein, estrogen disruptors, Fertility, soy Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotics and reproduction Citation: Tsanakalis F and Papaioannidou P (2010). Genistein: Impact on Reproduction. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00208 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Paraskevi Papaioannidou, Aristotle University of Thessaloniki, Department of Pharmacology, Thessaloniki, Greece, ppap@auth.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Fotios Tsanakalis Paraskevi Papaioannidou Google Fotios Tsanakalis Paraskevi Papaioannidou Google Scholar Fotios Tsanakalis Paraskevi Papaioannidou PubMed Fotios Tsanakalis Paraskevi Papaioannidou Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Relationship Between Plasma Estradiol Levels and Estrogen-Responsive Gene Expression in Estrogen Receptor–Positive Breast Cancer in Postmenopausal Women

Relationship Between Plasma Estradiol Levels and Estrogen-Responsive Gene Expression in Estrogen Receptor–Positive Breast Cancer in Postmenopausal Women

Inhibition of SIRT1 deacetylase suppresses estrogen receptor signaling

Estrogen receptor alpha (ERalpha) mediates estrogen-dependent gene transcription, which plays a critical role in mammary gland development, reproduction and homeostasis. Histone acetyltransferases and class I and class II histone deacetylases (HDACs) cause posttranscriptional modification of histone proteins that participate in ERalpha signaling. Here, we report that human SIRT1, a class III HDAC, regulates ERalpha expression. Inhibition of SIRT1 activity by sirtinol suppresses ERalpha expression through disruption of basal transcriptional complexes at the ERalpha promoter. This effect leads to inhibition of estrogen-responsive gene expression. Our in vitro observations were further extended that SIRT1 knockout reduces ERalpha protein in mouse mammary gland. Finally, ERalpha-mediated estrogen response genes are also decreased in mouse embryonic fibroblasts derived from SIRT1-knockout mice. These results suggest that inhibition of SIRT1 deacetylase activity by either pharmacological inhibitors or genetic depletion impairs ERalpha-mediated signaling pathways.

Abstract A74: Estrogen receptor α-regulated growth and transcription is dependent on X-box binding protein-1 in breast cancer cells

Abstract This study was designed to investigate the impact of X-Box Binding protein 1 (Xbp1) mRNA knockdown on the estrogen-responsive growth and gene expression in an estrogen receptor α (ERα) positive breast cancer cell line model. Xbp1 is a basic leucine zipper-containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum. MCF7 cells, a model of ERα positive breast cancer, express high levels of Xbp1 and are dependent on estrogen for growth and ERα regulated transcription. Previous studies have revealed that further overexpression of Xbp1 in ERα positive breast cancer cells leads to estrogen independent ERα regulated transcription and anti-estrogen resistance, suggesting that Xbp1 expression influences ERα regulated transcription. Unknown is whether ERα requires Xbp1 as a cofactor for transcription. Here we show that Xbp1 mRNA knockdown by specific siRNAs prevents estrogen responsive growth of MCF7 cells as well as ERα regulated gene expression, while non-specific control siRNAs had little effect. These results, together with the high expression of Xbp1 seen in ERα positive breast cancers, but not in normal mammary ductal cells, suggests that high levels of Xbp1 enable ERα tumor cells to use estrogen as a growth factor. Thus, high levels of Xbp1 expression could represent an early transformation step leading to ERα positive breast cancer, and could explain why normal ERα positive breast ductal cells have not been established as cell lines. Citation Information: Cancer Res 2009;69(23 Suppl):A74.

Long-range transcriptional control of progesterone receptor gene expression.

Estrogen receptor alpha (ERalpha) binds to specific target DNA sequences, estrogen response elements (EREs), to regulate estrogen-responsive gene expression. The progesterone receptor (PR) gene has been used extensively as a marker of estrogen responsiveness. Although we previously identified cis elements within 1 kb of the PR-B transcription start site that are associated with ERalpha and help to confer estrogen responsiveness, the identification of ERalpha binding sites far removed from the transcription start site suggested that long-range regulation of this gene may occur. We now show that eight regions of the PR gene from 311 kb upstream to 4 kb downstream of the PR-B transcription start site interact with ERalpha and that coactivator proteins and acetylated histones are selectively associated with these gene regions. Specific PR gene regions confer estrogen responsiveness to a heterologous reporter plasmid, and mutation of EREs within these regions diminishes estrogen-induced transactivation. Importantly, chromosome conformation capture assays reveal ERalpha- and ligand-dependent interactions between proximal and distal PR gene regions. Taken together, our studies suggest that distal regions of the PR gene participate in the dynamic regulation of this gene and that the coordinated action of proximal and distal PR gene regions allows cells to respond to changes in hormone levels with extraordinary versatility and sensitivity.

Thioredoxin and thioredoxin reductase influence estrogen receptor alpha-mediated gene expression in human breast cancer cells.

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERalpha, Trx, and TrxR interact and ERalpha and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17beta-estradiol, Trx, and TrxR alter hydrogen peroxide (H(2)O(2)) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H(2)O(2) levels and transcription factor activity, aid ERalpha in regulating the expression of estrogen-responsive genes in target cells.

Comparison of six different sewage treatment processes—Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O 3/m 3), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.

Membrane‐bound melatonin receptor MT1 down‐regulates estrogen responsive genes in breast cancer cells

Melatonin possesses anti-estrogenic effects on estrogen receptor expressing (ER+) breast cancer cells in culture by reducing cell cycle progression and cell proliferation. There is increasing agreement that on a cellular level the effects of melatonin are primarily induced by the membrane-bound receptor MT1. The participation of a second, nuclear receptor of the group of ligand-dependent transcription factors, called RZRalpha, is under debate. In this study we used a number of breast cancer cell lines differing in their expression of the estrogen receptor and the two known melatonin receptors. In MCF-7 breast cancer cells transfected with a vector carrying the MT1 gene (MCF-7Mel1a) binding of CREB-protein to the cAMP-responsive element of the breast cancer suppressing gene BRCA-1 was more strongly reduced by treatment with melatonin than in the parental cells. Expression of estrogen responsive genes was determined in serum-starved cells, cells stimulated for 16 hr with estradiol and cells subsequently treated with melatonin. Expression of BRCA-1, p53, p21(WAF) and c-myc were up-regulated by estradiol. Treatment of the stimulated cells with melatonin counteracted the increase induced by estradiol almost completely. The more MT1 a cell line expressed, the stronger was the reduction of the expression of the estradiol-induced genes. There was no correlation between the expression of the nuclear receptor RZRalpha and the effects of melatonin on these genes.

Apurinic/apyrimidinic endonuclease 1 alters estrogen receptor activity and estrogen-responsive gene expression.

Apurinic/apyrimidinic endonuclease 1 or redox factor-1 (Ape1/Ref-1) is a pleiotropic cellular protein involved in DNA repair and, through its redox activity, enhances the binding of a select group of transcription factors to their cognate recognition sequences in DNA. Thus, we were intrigued when we identified Ape1/Ref-1 and a number of DNA repair and oxidative stress proteins in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Because Ape1/Ref-1 interacts with a number of transcription factors and influences their activity, we determined whether it might also influence ERalpha activity. We found that endogenously expressed Ape1/Ref-1 and ERalpha from MCF-7 human breast cancer cells interact and that Ape1/Ref-1 enhances the interaction of ERalpha with estrogen-response elements (EREs) in DNA. More importantly, Ape1/Ref-1 alters expression of the endogenous, estrogen-responsive progesterone receptor and pS2 genes in MCF-7 cells and associates with ERE-containing regions of these genes in native chromatin. Interestingly, knocking down Ape1/Ref-1 expression or inhibiting its redox activity with the small molecule inhibitor E3330 enhances estrogen responsiveness of the progesterone receptor and pS2 genes but does not alter the expression of the constitutively active 36B4 gene. Additionally, the reduced form of Ape1/Ref-1 increases and E3330 limits ERalpha-ERE complex formation in vitro and in native chromatin. Our studies demonstrate that Ape1/Ref-1 mediates its gene-specific effects, in part, by associating with endogenous, estrogen-responsive genes and that the redox activity of Ape1/Ref-1 is instrumental in altering estrogen-responsive gene expression.