Estrogen Receptor mRNA Levels Research Articles

Microarray-based determination of ER, PR and HER2 receptor status: validation and comparison with IHC assessments.

Abstract Abstract #3007 Background
 In breast cancer patients the level of expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 is predictive for prognosis and/or treatment response. However, differences in assessment methods and interpretation can substantially affect the accuracy and reproducibility of the results. Previously, we have determined the association between immunohistochemistry (IHC) and mRNA levels for ER, PR and HER2, and have confirmed the accuracy of microarray readout on >400 samples. In the current study we describe the use of this microarray based readout on prospectively collected samples. We compared these readouts with multiple IHC and fluorescent in situ hybridization (FISH) assessments generated in various hospitals and a CLIA-certified reference laboratory and developed a microarray based test called TargetPrint™.
 Methods
 Gene expression data for ER, PR and HER2 were obtained by analysis of 100 breast carcinomas that have been collected prospectively within the RASTER study. Samples were stratified as receptor positive or negative using thresholds for ER, PR and HER2 mRNA levels. IHC assessment was performed (1) according to local standards of the hospital from where the sample originated, (2) by the central laboratory of the Netherlands Cancer Institute, and (3) at an independent reference laboratory using FDA-approved procedures and ASCO/CAP guidelines. A tumor was classified positive for ER and PR when ≥10% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In case of 2+ samples, a FISH was performed to assess final HER2 amplification status. The cohort used in this study was pre-selected to include about two-third ER and PR positive samples and one-third HER2 positive samples.
 Results
 Multiple microarray readouts were highly reproducible (Pearson correlation 0.991) and resulted in 67, 61 and 39 percent positive samples for ER, PR and HER2, respectively. Comparison of microarray results with IHC (including FISH for HER2) performed at the three centers indicated highly similar results for receptor readout with a concordance of 92, 93 and 92% for ER; 84, 81 and 86% for PR; and 93, 95 and 94% for HER2. Overall misclassification rates between microarray and IHC readout were low for ER (0.08) and HER2 (0.06) and quite low for PR (0.14), and were comparable to the misclassification rates between the three IHC methods.
 Conclusion
 A microarray-based assessment of ER, PR and HER2 in relation to mRNA levels gives results comparable to multiple IHC methods and FISH and provides an objective and more quantitative assessment of tumor receptor status than IHC alone. Using TargetPrint™ for microarray readouts for hormone and HER2 receptor in addition to standard IHC will improve molecular characterization of breast cancer tissue. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3007.

A role for GATA-3 in control of estrogen receptor alpha expression.

Abstract Abstract #3050 Expression of the GATA-3 binding protein correlates with estrogen receptor (ER) alpha expression in primary and cultured breast cancer cells. While the significance of this correlation and role of GATA-3 in breast cancer remains unclear, GATA-3 may serve as a good prognostic indicator in breast tumors. Recently, a reciprocal interaction between GATA-3 and ER has been demonstrated. GATA-3 may regulate ER levels through binding to an enhancer region upstream of the ER gene while ER can associate with and regulate GATA-3 expression. Meta-analyses of several cancer microarrays indicate that GATA-3 is co-expressed with ER and other ER responsive genes, further suggesting a role of GATA-3 in the ER signaling pathway. Our laboratory has been investigating the role of the RNA binding protein HuR/ELAV1 in control of mRNA stability in breast cancer cells. We have previously reported that ER mRNA stability is increased through binding of HuR to the 3'UTR. Microarray analysis of those transcripts associated with HuR in MCF7 cells showed that GATA-3 mRNA associates with HuR compared to an IgG control. Biotin pulldown assays using a biotinylated GATA-3 RNA probe confirmed that HuR binds to the GATA-3 3'untranslated region (UTR). Inhibition of HuR using siRNA probes decreased GATA-3 mRNA and protein expression, further indicating a role for HuR in regulation of GATA-3 mRNA stability. siRNA mediated inhibition of GATA-3 expression significantly reduced ER mRNA and protein levels. Surprisingly, we found that GATA-3 bound to the ER mRNA by binding to two out of four regions within the ER 3'UTR, suggesting that while GATA-3 may play a role in upregulation of ER mRNA transcription through the enhancer interaction, GATA-3 may stabilize ER mRNA once transcribed by binding to the ER 3'UTR. Studies are ongoing to fully characterize this role of GATA-3 in control of ER mRNA stability. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3050.

Influence of Evolution in Tumor Biobanking on the Interpretation of Translational Research

Translational cancer research increasingly relies on human tissue biospecimens and this has coincided with a shift in tissue biobanking approach. Newer biobanks (post year 2000) deploy standard operating procedures to reduce variability around biospecimen collection. Because current translational research is based on pre-2000 and post-2000 era biospecimens, we consider whether the collection era may influence gene expression data. We compared the range of breast tumor collection times from pre-2000 and post-2000 era biobanks and compared estrogen receptor (ER) protein expression with collection time. We then collected 10 breast tumor biospecimens under a standardized protocol and examined whether the expression of c-myc and ER was influenced by storage on ice < or = 24 hours. The range of collection times achieved at a pre-2000 versus post-2000 era biobank differed. Thirty-two percent of biospecimens were cryopreserved within 30 minutes at the pre-2000 era biobank versus 76% at the post-2000 era biobank. Collection time and ER protein level was inversely correlated (r = -0.19, P = 0.025; n = 137). We observed a wide range in initial c-myc and ER mRNA levels (50- versus 130-fold). Although mRNA levels of both genes declined with increasing collection time, the rate of change differed because c-myc was significantly reduced after 24 hours (mean reduction to 79% of initial) versus ER (94% of initial). The overall shift in biobanking around the year 2000 is reflected in the ranges of collection times associated with pre-2000 and post-2000 era biobanks. Because collection time can differentially alter gene expression, the biospecimen collection era should be considered in gene expression studies.

Effects of cyclopenta[ c]phenanthrene and its derivatives on zona radiata protein, ERα, and CYP1A mRNA expression in liver of rainbow trout ( Oncorhynchus mykiss Walbaum)

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) have been detected in the environment, the ability of CP-PAH to induce cellular and tissue responses remains poorly characterized. In this study, xenoestrogen-associated responses (mRNA levels of estrogen receptor α, ERα, and zona radiata protein, Zrp) and xenobiotic effects (CYP1A mRNA) have been investigated in liver of juvenile rainbow trout after short-term treatment (8 and 24 h) with following compounds administered singly: cyclopenta[ c]phenanthrene (CP[ c]Ph); its derivatives, 5A-CP[ c]Ph; 5A6M-CP[ c]Ph; 5A9M-CP[ c]Ph; B[ c]Ph, a structurally similar polycyclic aromatic hydrocarbon; B[ a]P, a model CYP1A inducer; and zearalenone (ZEA), naturally occurring ligand for ER. The CYP1A mRNA expression after 24 h of exposure with CP[ c]Ph or its derivatives, except 5A9M-CP[ c]Ph, was 3–9-fold higher compared to controls ( P < 0.05), but it was less than that caused by B[ a]P (65-fold up regulation; P < 0.01). Moreover, neither of the CP-PAH compounds modulated liver ERα or Zrp mRNA levels as compared to effects associated with ZEA. Interestingly, a treatment with this ER-ligand, caused moderate but significant increase of CYP1A mRNA expression (about 2.5-fold; P < 0.05). The finding that ZEA is capable of acting as either estrogenic and xenobiotic compound, should be further explored in a more detailed and differently designed experiment.

The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Ilyanassa obsoleta

Molluscs exposed to endocrine-disrupting chemicals (EDCs) have exhibited changes in reproductive tract development that are typically associated with androgen or estrogen signaling in vertebrates. However, a role for androgens and estrogens in molluscan reproductive endocrinology has yet to be established. In this study, we investigated putative roles for steroidal androgens and estrogens in recrudescence of the eastern mud snail Ilyanassa obsoleta. Our objectives were to: (1) identify associations among concentrations of testosterone and 17β-estradiol, sex, and reproductive status in mud snails that suggest these hormones are involved in recrudescence; and (2) determine whether mud snails express NR3C4-like (androgen receptor) and NR3A-like (estrogen receptor) mRNAs in a manner indicative of a role in recrudescence. Temporal changes in testosterone levels in males were consistent with a positive role in recrudescence. Such a trend was not evident in females or for 17β-estradiol in either sex. Efforts to identify an androgen receptor from the mud snail using targeted, degenerate RT-PCR were unsuccessful. However, an estrogen receptor (ER) cDNA was identified that is highly similar to known ERs of other molluscs. Studies with the ER of other molluscs have shown that this protein does not actually bind estrogens. We therefore considered the possibility that the mud snail ER may regulate reproductive maturation as a ligand-independent transcription factor based upon its tissue abundance. Males expressed greater levels of ER mRNA than did females over the entire reproductive cycle, and this difference was most evident during recrudescence. ER mRNA levels were significantly elevated during recrudescence in males but not females. In conclusion, testosterone may have a role in male reproductive tract recrudescence; however, this putative activity is independent of a NR3C4-type androgen receptor. The ER also may function in male recrudescence, though apparently independent of 17β-estradiol. The retinoid signaling pathway is discussed as a possible alternative hormone/receptor-mediated signaling pathway that regulates male recrudescence.

Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis – a cohort study

BackgroundThe estrogen receptors α and β (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes.Methods23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression.ResultsNo ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value < 0.01 were genotyped in a second cohort where no association with obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009–0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction.ConclusionESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation between ESR1 mRNA levels and lipolytic responsiveness to adrenoceptor agonists implies that low adipose tissue ESR1 levels attenuate catecholamine resistance in sc fat cells of obese women hereby contributing to loss of sc and gain of visceral fat. There is no evidence for a genetic impact of ESR1 on lipolysis or lipogenesis.

Molecular Basis for Estrogen Receptor Deficiency in BRCA1-Linked Breast Cancer

BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Testicular Expression of Steroidogenic Enzyme Genes Is Related to a Transient Increase in Serum 19-nortestosterone during Neonatal Development in Pigs

Cytochrome P450 aromatase is responsible for the biosynthesis of estrogen. It is also responsible for the endogenous production of 19-nortestosterone (nandrolone), an anabolic androgen unique to pigs. Plasma concentrations of 19-nortestosterone are highest between two and four weeks after birth in male pigs. In the present study, the physiology of 19-nortestosterone was investigated by measuring the mRNA levels of steroidogenic enzymes, estrogen receptors and androgen receptor in the tissues of growing pigs. The expression of aromatase, 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase in the testes of male piglets increased between birth and two weeks of age, and then decreased progressively. Similar developmental expressional patterns were observed for 17α- hydroxylase and 3β-hydroxysteroid dehydrogenase in the ovaries of female piglets, but without significant aromatase expression. The major form of aromatase expressed in the testes of piglets was identified as type I. Expression of estrogen receptor-α and -β and androgen receptor genes was also detected in both testes and ovaries. A transient elevation of androgen receptor mRNA in male piglets at two weeks of age was also observed in testes. Significant expression of the androgen receptor gene, but not of estrogen receptor-α and -β genes, was also demonstrated in adipose tissue and muscle. We conclude that the observed increase in the testicular expression of aromatase in male pigs could account for the production of large amounts of 19-nortestosterone at between two and four weeks of age in males. Androgen receptor and 19-nortestosterone appeared to be important for testicular development and might contribute to sexual dimorphism in body composition and muscle development in juvenile pigs. (Key Words : Cytochrome P450 Aromatase, Pig Testis, 19- Nortestosterone)

Promoter Methylation Regulates Estrogen Receptor 2 in Human Endometrium and Endometriosis1

Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.

Laboratory exposure to 17β-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis

To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes—estrogen receptor ( ER) and vitellogenin ( VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17β-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner—confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1–3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.

Embryonic exposure to the fungicide vinclozolin causes virilization of females and alteration of progesterone receptor expression in vivo: an experimental study in mice

BackgroundVinclozolin is a fungicide that has been reported to have anti-androgenic effects in rats. We have found that in utero exposure to natural or synthetic progesterones can induce hypospadias in mice, and that the synthetic progesterone medroxyprogesterone acetate (MPA) feminizes male and virilizes female genital tubercles. In the current work, we selected a relatively low dose of vinclozolin to examine its in utero effects on the development of the genital tubercle, both at the morphological and molecular levels.MethodsWe gave pregnant dams vinclozolin by oral gavage from gestational days 13 through 17. We assessed the fetal genital tubercles from exposed fetuses at E19 to determine location of the urethral opening. After determination of gonadal sex, either genital tubercles were harvested for mRNA quantitation, or urethras were injected with a plastic resin for casting. We analyzed quantified mRNA levels between treated and untreated animals for mRNA levels of estrogen receptors α and β, progesterone receptor, and androgen receptor using nonparametric tests or ANOVA. To determine effects on urethral length (males have long urethras compared to females), we measured the lengths of the casts and performed ANOVA analysis on these data.ResultsOur morphological results indicated that vinclozolin has morphological effects similar to those of MPA, feminizing males (hypospadias) and masculinizing females (longer urethras). Because these results reflected our MPA results, we investigated the effects of in utero vinclozolin exposure on the mRNA expression levels of androgen, estrogen α and β, and progesterone receptors. At the molecular level, vinclozolin down-regulated estrogen receptor α mRNA in females and up-regulated progesterone receptor mRNA. Vinclozolin-exposed males exhibited up-regulated estrogen receptor α and progesterone receptor mRNA, effects we have also seen with exposure to the synthetic estrogen, ethinyl estradiol.ConclusionThe results suggest that vinclozolin virilizes females and directly or indirectly affects progesterone receptor expression. It also affects estrogen receptor expression in a sex-based manner. We found no in vivo effect of vinclozolin on androgen receptor expression. We propose that vinclozolin, which has been designated an anti-androgen, may also exert its effects by involving additional steroid-signaling pathways.

Adrenal incidentaloma in pregnancy: Clinical, molecular and immunohistochemical findings

Adrenal incidentalomas detected during pregnancy are very rare, and the natural history of these tumors during gestation is unknown. We report a case of a pregnant woman with an adrenal mass discovered serendipitously, who was followed-up during gestation and underwent adrenalectomy shortly after delivery. This allowed the evaluation of both the clinical outcome and the molecular/immunohistochemical correlates. Estrogens may indeed influence the function and proliferation of human adrenal cells, and a state of circulating estrogen excess can represent an in vivo model to test their effect on the adrenals. No evidence of adrenal change in morphology and function was found in our patient throughout pregnancy, as shown by adrenal ultrasound imaging and adrenal hormone measurements. Four months after delivery, the patient underwent laparoscopic right adrenalectomy, and pathologic analysis revealed a 2.7 cm benign adrenocortical adenoma. The diameter of the adrenal mass at ultrasonography correlated highly with post-partum mass diameter measured by abdominal computed tomography (CT). Quantitative expression of both ERalpha and ERbeta by real-time RT-PCR analysis and Western blotting findings did not differ among adenoma, normal adjacent adrenal and normal adrenal control tissues. This case of an adrenal incidentaloma discovered during pregnancy shows that a close observation with endocrine investigations and ultrasonography could be an appropriate approach, delaying the decision of surgical intervention after delivery. Estrogen receptor mRNA levels in the adrenal mass similar to those observed in normal adrenals suggest that estrogen oversecretion during pregnancy was not a risk factor for tumor progression.

Oestrogen receptor-related receptor alpha (ERRα) and oestrogen receptors (ERα and ERβ) exhibit different gene expression in human colorectal tumour progression

We have investigated the presence of oestrogen receptor-related (ERR) mRNA in human colorectal tumour tissues and adjacent normal mucosa by reverse transcriptase and nested-polymerase chain reaction. ERRα was found in 100% of the patients and ERRγ in approximately 30% while ERRβ was not detected at all. The multiplex PCR analysis showed elevated levels of ERRα mRNA in tumour tissue compartment as compared to normal mucosa, whereas ERRγ mRNA was found in lower levels but in both tissue compartments. In contrast, oestrogen receptor (ERα and ERβ) mRNA levels were shown to be decreased in tumour tissues. A positive correlation was observed between ERα and ERβ and between ERα and ERRα, respectively, in normal mucosa but not in tumour tissue. ERRα expression in tumour tissues significantly increased from TNM stages II to IV, whereas both ERs progressively declined. These findings suggest that ERRα, as well as the two ERs, might play a critical role in the progression of the colorectal cancer.

Endometrial Endothelial Cell Steroid Receptor Expression and Steroid Effects on Gene Expression

Controversy exists regarding the expression of specific steroid receptor proteins and mRNA in human microvascular endometrial endothelial cells (HEECs). Thus, we studied steroid receptor expression in early passaged HEEC cultures and freshly isolated HEECs. Analysis of estrogen receptor (ER) and progesterone receptor (PR) mRNA levels was carried out with real-time quantitative RT-PCR, and the repertoire of genes activated by their respective steroid ligands was assessed by mRNA microarray analyses of 18,400 genes and expressed sequence tags. We observed that cultured and freshly isolated HEECs each express ER-beta mRNA but not ER-alpha. In addition, PR mRNA was also detectable in both HEEC sources. Microarray analysis demonstrated that treatment of HEEC cultures with either estradiol or medroxyprogesterone acetate produced differential effects on a wide variety of genes, and cluster analysis demonstrated that many of the genes are involved in intracellular signaling and enzymatic pathways. Thus, quantitative RT-PCR and microarray analyses demonstrate that HEECs express ER-beta and PR mRNA and that gene expression by HEECs is differentially regulated by treatment with estrogen or progestin.

Developmental expression of estrogen receptor (ER) alpha and ERbeta in the hamster ovary: regulation by follicle-stimulating hormone.

Perinatal expression of estrogen receptor (ER) protein and mRNA and the influence of FSH on this process were examined by immunofluorescence and RT-PCR using ovaries from fetal (d 13-15 of gestation) and postnatal [postnatal d 1-15 (P1-P15)] hamsters and from 8-d-old hamsters exposed in utero to an anti-FSH serum on d 12 of gestation and saline or equine chorionic gonadotropin (eCG) on P1. A few somatic cells expressing ERalpha immunoreactivity appeared first on d 14 of gestation and increased markedly by P8-P15 in the interstitial cells and granulosa cells of primordial follicles. In contrast, appreciable ERbeta immunoreactivity was localized on d 13 of gestation, and more cells expressed ERbeta immunoreactivity by P1-P8. By P7, ERbeta immunoreactivity was present in cells adjacent to the oocytes, and by P8, ERbeta was preferentially localized in the granulosa cells. Receptor immunoreactivities decreased markedly in P8 ovaries exposed in utero to the FSH antiserum but were reversed with postnatal eCG replacement. Oocytes and somatic cells expressed ERalpha and ERbeta mRNA, and levels of ER mRNA in the ovary increased by P7-P8, corresponding to the appearance of primordial follicles. Thereafter, only ERbeta mRNA levels increased progressively with postnatal ovary development. Similar to ER protein, mRNA levels decreased significantly in FSH antiserum-treated ovaries but were restored by eCG. These results indicate that both ER subtypes are expressed in undifferentiated somatic cells and the oocytes during perinatal ovary development in the hamster; however, ERbeta expression segregates with the differentiation of granulosa cells. Furthermore, ER expression and differentiation of somatic cells to granulosa cells depend on perinatal FSH action.

Differential regulation of nuclear receptors, neuropeptides and peptide hormones in the hypothalamus and pituitary of food restricted rats

Food restriction is associated with a number of endocrine disturbances. We validated the experimental conditions for several house-keeping genes and determined the effects of 12 day 50% food restriction on hypothalamic and pituitary transcription of genes involved in different neuroendocrine systems, using real-time quantitative polymerase chain reaction (PCR). A total of 7 nuclear receptors and 12 neuropeptides and peptide hormones were investigated in the dorsal and ventral hypothalamus and the pituitary gland in rats. In the hypothalamus, food restriction reduced mRNA levels of estrogen receptor α (ERα), progesterone receptor, glucocorticoid receptor, thyroid hormone receptor α and β, pro-opiomelanocortin (POMC), growth hormone-releasing factor (GHRF), corticotropin-releasing factor (CRF), thyrotropin-releasing factor (TRF), somatostatin, and increased that of neuropeptide Y (NPY). In the pituitary, the treatment reduced growth hormone (GH), luteinizing hormone β (LHβ) and thyrotropin β, but increased ERα mRNA levels. The study provides a map of how food restriction affects the regulation of a number of transcripts involved in neuroendocrine control.

Suppression of cell proliferation and regulation of estrogen receptor alpha signaling pathway by arsenic trioxide on human breast cancer MCF-7 cells.

In recent years, breast cancers have aroused much concern. Together with a growing incidence all over the world, the development of drug resistance to tamoxifen, the most commonly prescribed chemotherapeutic drug for breast cancer patients, has highlighted the importance of developing a new chemotherapeutic drug in combating breast cancer. With the aim of treating breast cancers, the anti-tumor effects of arsenic trioxide in MCF-7 cells have been studied. MCF-7 cells are estrogen responsive cells which mimic breast cancers at the early stage. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and direct cell counting were used to measure cell proliferation. The mechanisms of action were elucidated through the measurement of estrogen receptor (ER) binding, mRNA and protein levels of ERalpha and its activity. We have demonstrated that arsenic trioxide was capable of reducing cell survival in MCF-7 cells via the suppression of the estrogen-induced growth stimulatory effects in MCF-7 cells. Arsenic trioxide was shown to suppress the action of estrogen through the regulation of the ERalpha signaling pathway. Arsenic trioxide could down-regulate ERalpha mRNA and protein levels without competing with estrogen for ERalpha binding. Arsenic trioxide also inhibited the transcription activity mediated by the ERalpha signaling pathway and ultimately it down-regulated c-myc protein expression and inhibited cell entry to S phase under estrogen's stimulation. In conclusion, arsenic trioxide could inhibit the growth of MCF-7 cells by reducing the growth stimulatory effect of estrogen. As estrogen is a primary risk factor in promoting the growth of breast tumor cells, the anti-estrogenicity exhibited by arsenic trioxide sheds light on the therapy of breast cancer.

Trastuzumab plus tamoxifen: anti-proliferative and molecular interactions in breast carcinoma.

HER2 overexpression has been associated with anti-estrogen resistance in human breast cancer, and it has been suggested that the combined treatment of an anti-HER2 antibody plus tamoxifen has enhanced anti-cancer efficacy in breast cancer. The detailed anti-proliferative interactions between trastuzumab and tamoxifen were analyzed with the isobologram and Chou and Talalay methods, which assess the presence of synergy, addition or antagonism. We used the breast cancer cell lines that are estrogen receptor (ER)-positive and HER2-positive. We also analyzed the molecular changes on the HER2 and (ER) signaling pathways that are induced by trastuzumab plus tamoxifen. In terms of cancer cell proliferation, the simultaneous combination of trastuzumab and tamoxifen on BT-474 cells was more growth inhibitory (44%) than the treatment with trastuzumab (24%) or tamoxifen (31%) alone. Isobologram analysis of simultaneous trastuzumab plus tamoxifen exposure showed, however, that there were antagonistic interactions at an effect level of 30% (IC30). Using Chou and Talalay analysis we also observed antagonistic interactions at lower levels of cell kill, although there were additive effects at highest levels of cell kill. Trastuzumab followed by tamoxifen showed antagonism at all effects levels. Tamoxifen followed by trastuzumab showed antagonism at lower levels of cell kill, and additivity at higher levels of cell kill. Similar interactions were observed using T47D cells. The molecular effects of the combined treatment with trastuzumab plus tamoxifen on the levels of HER2 and ER signaling showed that, with respect to HER2 protein levels, trastuzumab downregulated HER2 by 27%, tamoxifen upregulated HER2 by 40%, and the combination of trastuzumab plus tamoxifen did not induce changes in HER2 respect to control. With respect to HER2 mRNA, trastuzumab upregulated HER2 mRNA to 367%, tamoxifen to 166%, and the combination to 401%. With respect to HER2 phosphorylation, trastuzumab upregulated HER2 phosphorylation to 352%, tamoxifen to 202% and the combination to 633%. Epidermal growth factor receptor levels were not changed by trastuzumab or tamoxifen alone, and were upregulated to 138% by the combination. The protein levels and activity of extracellular recptor kinase were not modified by trastuzumab, tamoxifen or the combination. Finally, estrogen receptor protein and mRNA levels were downregulated to about 50% by trastuzumab, tamoxifen or the combination. Taken together, our results show that in ER-positive breast cancer cells overexpressing HER2, trastuzumab plus tamoxifen have antagonistic interactions when used in combination, and that this antagonism may be related with an increase in HER2 signaling pathways that occurs when tamoxifen is added to trastuzumab.

Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment.

Endocrine-disrupting compounds (EDC) are predominantly investigated with respect to their ability to mimic or block estrogenic actions. However, it is well-known that EDC can act as agonists or antagonists of androgen- and estrogen-response systems. For that reason, there is an obvious need for bioassays providing the possibility of detecting (anti-)estrogenic and (anti-)androgenic effects. The retinol-binding protein (RBP) seems to be a useful molecular biomarker for assessing all modes of action of EDC, because it is regulated by sex steroid hormones. This study was conducted to establish RBP as a biomarker for determination of (anti-)estrogenic and (anti-)androgenic effects of EDC using a Xenopus laevis primary hepatocyte culture system. It could be shown that RBP mRNA expression in X. laevis hepatocytes was stimulated by estrogens in a dose-dependent manner whereas a combination of estrogen and androgen or estrogen and anti-estrogen treatment suppressed estrogenic stimulating effects. Androgens testosterone and dihydrotestosterone were able to reduce RBP mRNA expression and the anti-androgen vinclozolin could abolish the mRNA synthesis-suppressing activity of the androgen dihydrotestosterone. These results clearly demonstrated that RBP mRNA expression patterns in Xenopus laevis hepatocytes have different modes of (anti-)estrogenic and (anti-)androgenic action and can be used for examination of suspected EDC. Moreover, water samples from sewage-treatment plant effluents were applied to liver cells and expression levels of RBP and estrogen receptor mRNA (a known estrogenic biomarker) were detected. These samples had high estrogenicity but caused low to moderate induction of RBP mRNA synthesis, leading to the conclusion that RBP levels represent the sum of all possible effects (estrogenic and other effects) of EDC in environmental samples.