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We previously showed that growth hormone (GH) receptors (GHR) are expressed in the most commonly studied human prostate cancer (PCa) cell lines and that GHR isoforms undergo differential, cell-type-specific hormonal regulation. We now report that human GH (hGH) can stimulate/modulate insulin-like growth factor (IGF) and β-oestradiol (E(2) ) receptor (ER(β) ) gene expressions in these cells and interact with IGF-I and E(2) to stimulate androgen-dependent LNCaP cell proliferation. We observed a cell type-dependent, differential regulation of IGF axis gene expression by GH: IGF-I was stimulated in the androgen-dependent LNCaP cells; IGF-II was stimulated in androgen-insensitive (AI) PC3 cells; the IGF-I cognate receptor, IGF-IR, was stimulated in LNCaP cells, but inhibited in PC3 cells; IGF-IIR was stimulated in both LNCaP and PC3 cells. GH also stimulated ER(β) gene expression in LNCaP and PC3 cells, but had little or no effect on any of those genes in AI DU145 cells. The potent androgen analogue, mibolerone, also stimulated IGF-I, IGF-IR and ER(β) , but reduced IGF-IIR mRNAs in LNCaP cells. Furthermore, triiodothyronine (T(3) ) and E(2) also stimulated the expression of those four genes in LNCaP cells, but co-administration of GH had almost no effect. Finally, we also studied the effects of GH, IGF-I and E(2) , alone or in combination, on LNCaP cell proliferation. Importantly, we demonstrated, for the first time, that although GH and IGF-I alone had no effect on LNCaP cell proliferation, concomitant administration for 96 h revealed a permissive role of GH on IGF-I-induced proliferation. GH also appeared to exert a synergistic effect on E(2) -stimulated LNCaP cell proliferation. Taken together, these findings indicate that GH via GHRs, most likely in concert with gonadal steroids, T(3) , IGF system axis and probably other hormones and growth factors, potentially plays an important role in the mechanisms underlying tumour cell growth in PCa.

The nuclear receptors encompass a group of regulatory proteins involved in a number of physiological processes. The estrogen receptors (ERs), of which one alpha and one beta form exist in mammals function as transcription factors in response to 17beta-estradiol (E2). In zebrafish there are three gene products of estrogen receptors and they are denoted esr1 (ERalpha), esr2a (ERbeta2) and esr2b (ERbeta1). Total RNA of zebrafish early life stages (<3, 6, 12, 24, 48, 72, 96 and 120 hours post fertilization) and of adult fish (liver, intestine, eye, heart, brain, ovary, testis, gill, swim bladder and kidney) were isolated following in vivo exposures. Using specific primers for each of the three zebrafish ERs the expression levels were quantified using real time PCR methodology. It was shown that in absence of exposure all three estrogen receptors were expressed in adult fish. The levels of expression of two of these three ER genes, the esr1 and esr2a were altered in organs such as liver, intestine, brain and testis in response to ligand (E2, diethylstilbestrol or 4-nonylphenol). During embryogenesis two of the three receptor genes, esr1 and esr2b were expressed, and in presence of ligand the mRNA levels of these two genes increased. The conclusions are i) estrogen receptor genes are expressed during early development ii) altered expression of esr genes in response to ligand is dependent on the cellular context; iii) the estrogenic ligand 4-nonylphenol, a manufactured compound commonly found in sewage of water treatment plants, acts as an agonist of the estrogen receptor during development and has both agonist and antagonist properties in tissues of adult fish. This knowledge of esr gene function in development and in adult life will help to understand mechanisms of interfering mimicking endocrine chemicals in vivo.

Objective: To explore the regulatory effect and mechanism of Ningxin Hongqi Capsule on local ovarian autocrine and paracrine factors in peri-menopausal rats., Methods: SD female rats aged 4 months were allocated in a normal control group (A) and those aged 14 months with vagino-cytologic figure of oestrus elongation were allocated in a senile female rat model group (B). Rats in Group B were subdivided into 5 groups randomly as the B1, B2 and B3 subgroups treated respectively with high, moderate and low dose Ningxin Hongqi Capsule, the B4 subgroup treated with estradiol and the B5 subgroup untreated for control. Rats' ovaries were obtained at the end of the experiment for observing the conditions of ovarian growing follicles and corpus luteum by HE staining, determining expressions of ovarian estradiol receptor (ER), progesterone receptor (PR), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin alpha (INHalpha), activin (ACT) alpha-beta, follistatin (FS), and insulin-like growth factor (IGF-1)., Results: As compared with Group B5, the ovary index, number of growing follicle were higher and levels of FSH and LH were lower in Group B2 and B3, expression of ER was higher in Group B1 and B4, IGF-1 and INHalpha was higher in Group B2 and B3, and ACTalpha-beta and FS were lower (all P < 0.05)., Conclusion: Nirigxin Hongqi Capsule could adjust and balance the local ovarian autocrine and paracrine factors to improve the ovarian function.

The influence of chronic (14 days) administration of 5-HTIA receptors agonist--8-OH-DPAT (0.05 mg/kg, s.c.) and 5-HT(1A) receptors antagonist NAN-190 (0.1 mg/kg, i.p.) alone or in a combination with 17beta-estradiol (0.5 mg on each animal, i.m.) for on depressive behavior and expression of 5-HT(1A)-, 5-HT(2A)-, 17beta- estradiol receptors mRNAs was estimated in hippocampus in adult ovariectomized (OVX) female rats. The model of depression in rats was carried out in Porsolt test. The measurement of expression of 5-HT(1A)-, 5-HT(2A)-, 17beta-estradiol receptors mRNAs in the hippocampus was performed by semiquantitative RT-PCR. In Porsolt test 17beta-estradiol in OVX rats reduced time immobility to some extent. 8-OH-DPAT alone significantly decreased time immobility in OVX rats. Chronic 8-OH-DPAT administration in a combination with 17beta-estradiol in OVX females resulted in potentiated antidepressive effect. Simultaneously, 8-OH-DPAT induced significant increase of 5-HT(1A)-, 5-HT(2A)-receptors mRNAs expression and decrease of 17beta-estradiol receptor mRNA expression in hippocampus in OVX rats as compared to the control. The data obtained indicate a close interaction of the ovary hormonal and serotonergic systems of the brain in mechanisms of depression.

As part of an investigation on the role of estrogen in liver disease, we tested the effects of estradiol-3-benzoate (EB) in the thioacetamide (TAA)-induced rat liver cirrhosis model. Male F344 rats (n = 100) were divided into six groups. Animals of groups 1-4 received TAA (0.03% in drinking water) for 12 weeks, and groups 5 and 6 served as controls without TAA. For the exposure period, EB pellets were implanted subcutaneously to give doses of 0 (groups 1 and 5), 1 (group 2), 10 (group 3), and 100 mug (groups 4 and 6) simultaneously. All animals were sacrificed at week 12. Significant increase of liver cirrhosis, liver weight, collagen content, and lipid peroxidation in the livers was evident in groups 3 and 4 (p < 0.05) compared with group 1. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was significantly elevated in group 4 (p < 0.01), along with expression of alpha-smooth muscle actin (alpha-SMA) and stellate cell activation-associated protein (STAP), as determined by RT-PCR analysis (p < 0.01). However, there were no differences in liver weight, collagen content, lipid peroxidation, 8-OHdG formation, and alpha-SMA and STAP mRNA expression between groups 5 and 6. We conclude that EB treatment enhances TAA-induced cirrhosis, associated with increase of oxidative stress and activation of hepatic stellate cells.

To study the activity of telomerase and the relationship between telomerase and other prognostic variables in cases of invasive ductal breast carcinomas, fifty fine-needle aspiration biopsies (FNABs) obtained from the same number of female patients, diagnosed cytologically and confirmed histologically after surgery, were examined. The same cases were studied immunocytochemically using monoclonal antibodies to telomerase, estradiol receptors (ER) and HER-2 (CB11) and a standard alkaline phosphatase (APAAP) method. Telomerase activity was found in 72% of the carcinomas studied. An association was found between telomerase activity, ER receptors and HER-2 expression (p <0.005). A relationship between telomerase activity, histological grade and lymph node status (LNS) was found as well (p<0.005). The above results seem to be significant prognostic factors and should be taken into consideration in the follow-up of patients after appropriate treatment for breast cancer.

We measured the expression of ERM gene, a nuclear transcription factor belonging to the ets family, in a series of 364 unselected primary breast cancers from patients who underwent locoregional surgery in the Centre Oscar Lambret between May 1989 and December 1991. The expression of ERM was quantified with a real-time one-step reverse transcription-PCR assay based on the 5'-nuclease activity of the TaqDNA polymerase and with an Abi Prism 7700 Sequence Detector System (Applied Biosystems, Courtaboeuf, France). ERM was positively correlated (Spearman test) to epidermal growth factor receptor (EGFR; P < 0.001, r = 0.296) and to histoprognostic grading (P = 0.044, r = 0.112), whereas it was negatively correlated to estradiol receptors (P = 0.019, r = -0.124), HER3 (c-erbB-3; P = 0.01, r = -0.135), and HER4 (c-erbB-4; P = 0.003, r = -0.154). Using the chi2 test, a positive relationship was found between the expression of ERM and EGFR (chi2 = 7.795, P = 0.007). In overall survival studies, Cox univariate analyses demonstrated a prognostic value of ERM (P = 0.006; risk ratio, 2.95) besides the classical prognostic factors histoprognostic grading, node involvement, tumor size, estradiol receptors, progesterone receptors, EGFR, HER3, and HER4. In multivariate analyses, ERM preserved its prognostic value (P = 0.004; risk ratio, 3.779) together with histoprognostic grading, tumor size, estradiol receptors, and progesterone receptors. In relapse-free survival studies, univariate analyses demonstrated that histoprognostic grading, node involvement, tumor size, and HER4 were prognostic factors. These parameters, except histoprognostic grading, retained their prognostic value in multivariate analyses. This study demonstrates for the first time that ERM gene expression is an independent adverse prognostic factor for overall survival in breast cancer patients.

Endometrium biopsy is a useful indicator of endometrium proliferation and is clinically relevant to diagnose cell proliferation and to evaluate response to progestin treatment and to monitor hormone replacement therapy. The aim of our study was to investigate the in vitro effects of progesterone and synthetic progestins on endometrium explants with a particular focus on estradiol receptor (ER) and progesterone receptor (PR) expression which reflects through cell secretion the hormone treatment efficiency. Most widely used progestagens belonging to three distinctive groups were investigated, i.e, medroxyprogesterone acetate (MPA), norethindrone acetate (NOR) and nomegestrol acetate (TX) which are respectively pregnane, 19-nortestosterone and norpregnane derivatives. We used organ culture from human proliferative endometrium, in which tissue integrity, particularly gland/stroma relationships are preserved. Progestins induce epithelial cell secretion and most effects were observed at the highest concentration tested (10(-7) M) and by TX and MPA on homogeneous and on heterogeneous (including also secretory glands) proliferative endometrium respectively. In these conditions, ER as well as PR expression were decreased on both glandular and stromal cells. In contrast, progesterone at 10(-7) M significantly decreased only PR, in glands and in stroma of homogeneous proliferative endometrium, and just in stroma of heterogeneous endometrium. NOR exhibited less effects. At lower concentrations (10(-8) M, 10(-9) M), significantly less effects were observed by synthetic progestins on proliferative endometrium. The experiments show that the different types of progestins do not exhibit in vitro similar effects. Since progestins variably act on proliferative endometrium, the exposure of endometrium explants to progestins may be a useful tool to predict clinical response to hormone therapy (individual "hormonogram") and to monitor endometrium proliferation.

In numerous fish species, dopamine has been found to strongly inhibit gonadotropin release. Among the enzymes that regulate dopamine turnover, tyrosine hydroxylase (TH), the rate-limiting anabolic enzyme, could be a target for endocrine feedback regulation. Since dopamine turnover is stimulated by estradiol in rainbow trout, we have investigated the effect of estradiol on TH and estradiol receptor expression. In situ hybridization was used to quantify mRNA levels in the brain of ovariectomized female rainbow trout implanted or not with estradiol pellets. We demonstrated that preoptic TH and estradiol receptor mRNA levels are greatly decreased by gonadectomy during vitellogenesis. For TH expression, this effect was reversed in part by estradiol supplementation. We have also confirmed the existence of an inhibitory gonadal feedback on FSH secretion, mediated by estradiol. The stimulating effect of estradiol on TH expression found in this study could be a pathway involved in gonadal feedback on gonadotropin release.

According to the classic model of regulation of sex hormone receptors biosynthesis in target tissues, oestrogen stimulates and progesterone inhibits biosynthesis in both receptors. One of the consequences of this model is a direct correlation between oestrogen (ER) and progesterone receptors (PR) in target tissues. Here we investigate a correlation between ER and PR in calf endometrium and breast cancer (BC) tissues of women. A direct correlation was found between receptors in the calf endometrium (r = +0.70; p < 0.01). There were three variants of BC tissues: without correlation, with positive correlation (r = +0.49; p < 0.01), and with non-linear negative correlation between ER and PR. The last variant of samples were detected exclusively in patients operated in spring and fall. The non-linear negative correlation between ER and PR in BC tissues in spring and fall may indicate disregulation of sex hormone receptors biosynthesis under the influence of external factors.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

We have refined the regulated expression of UASGAL1, 10-driven genes in yeast by modifying a vector encoding the beta-estradiol inducible activator, GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and beta-estradiol-regulated expression was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-regulated gene product. Target gene expression regulated by low levels of GEV has several advantages over the standard galactose-inducible expression systems. (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains. In addition, any UASGAL1,10 expression construct can be used without modification of the target gene or many gal4 delta host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.

Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.

An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.

Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17beta-oestradiol (E2), leading to increases in reductive 17beta-hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception alpha (ERalpha). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL-6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose-dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 pM. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERalpha+ cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERalpha by IL-6.

The study of misplaced endometrial cells, which abnormally implant and grow outside the uterine cavity, is of considerable interest for the understanding of the pathophysiology of endometriosis. However, endometriotic cells, particularly epithelial cells, required for primary cell culture are not easily available. We report here the characterization of an endometriotic cell line immortalized after infection of primary endometriotic cell cultures with simian virus 40. Transformed cells express T-antigen, and blot hybridization analysis showed that the viral genome is present as an episome. Cytogenetic analysis revealed a polyploid karyotype with numerical and structural rearrangements involving mainly the same chromosomes (6, 10, 11, 15, and 17). The cell line has been maintained in culture for over 80 passages and was still proliferating without any noticeable change in the biological properties investigated. Transformed endometriotic cells expressed both progesterone and estradiol receptors and were stimulated by these ovarian hormones to secrete monocyte chemotactic protein-1, a factor that may play an important role in the recruitment and activation of peritoneal macrophages. In addition, this response was enhanced in interleukin-1-treated cells. Taken together, these findings support the view that this cell line may be an interesting tool for the study of the pathophysiology of endometriosis.

Estradiol receptors are regarded to predict a likely success of hormonal therapeutic efforts and the prognosis of breast cancer patients. But today its prognostic importance is controversial, discussed as either reflecting intrinsic property of the tumor tissue or better therapeutic accessibility of receptor positive tumors. Moreover, the most important clinical prognosticators--tumor size and axillary lymph node involvement do not seem to be related to the estradiol receptor status. In our investigation, the length of disease free interval is similar in estradiol receptor positive and negative patients and in all sites of distant metastases, but it is significantly reduced if more than 4 axillary lymph nodes are involved. Post recurrence survival is significantly longer in estradiol receptor positive than negative patients and also in patients treated by tamoxifen containing therapies. Its length is independent of the number of axillary lymph node metastases and the type of distant metastases, with a tendency to be longer in estradiol receptor positive than negative patients. In addition, the overall survival is longer for estradiol receptor positive than negative patients and becomes reduced with more than 4 axillary lymph node metastases. Frequency of deaths in estradiol receptor positive patients is half that of negative subjects. Furthermore, the length of overall survival is independent on the type of distant metastases, with tendency to be longer in estradiol receptor positive than negative patients. Longest overall survival could be observed for estradiol receptor positive patients who got therapy regimens containing tamoxifen. The weak prognostic advantages of estradiol receptor positive patients are interpreted by estradiol receptors as intrinsic parameters of breast cancer tissue characterizing more its biological behavior than therapeutic accessibility.

Several strains of laboratory rats have a high background incidence of mammary tumors and develop a persistent, anovulatory estrus condition at about 12 mo of age. The increased tumor incidence is believed to be associated with elevated estradiol (E2) and prolactin during the period of persistent estrus. A pharmacodynamic estrus cycle (PD-EC) model for the Sprague-Dawley rats has been developed in an attempt to analyze the physiological basis of early-onset persistent estrus and to examine the potential sites of interactions in the hypothalamic-pituitary-ovarian axis for endocrine-modulating xenobiotics that accelerate the onset of persistent estrus. This initial estrus cycle model focused solely on cyclical changes in E2 and luteinizing hormone (LH). An LH surge was scheduled when a hypothetical estrus cycle-related protein (EC-RP) under transcriptional control by the E2 receptor reached a critical concentration. In the model, aging-related cumulative hypothalamic E2 exposure impaired the LH surge by reducing the rate of production of the EC-RP. The progressively decreasing intercycle resynthesis rate leads first to longer, variable-length cycles and finally to persistent estrus at about 12 mo of age. This model construct is consistent with early-onset persistent estrus related to neonatal E2 exposures, with acyclicity associated with high-dose E2 exposure in the adult, and with persistent estrus conditions associated with exposures to xenobiotic endocrine modulators that are either weak E2 antagonists or weak E2 agonists. With further development these pharmacodynamic estrus cycle models should be useful in aiding risk assessments for compounds causing mammary-tissue tumors associated with persistent estrus states.

Epidemiological evidence and estrogen replacement studies suggest that estrogen has a protective effect on the cardiovascular system against coronary artery disease. Vascular smooth muscle (VSM) cell replication has been shown to play a causative role in the pathogenesis of atherosclerosis. Therefore, in this study, we investigated the effect of chronic treatment of cultured guinea pig coronary artery VSM cells with physiological concentrations of 17beta-estradiol (E2) on thymidine incorporation, cell proliferation, and bradykinin-stimulated cytosolic calcium concentration ([Ca2+]i). Bradykinin at physiological concentrations causes contraction of endothelium-denuded guinea pig coronary artery rings in a concentration-dependent manner. VSM cells were first treated with low doses of E2 (10 pg/ml) for 1-2 days followed by treatment for 4-6 days with 50 pg/ml of E2, a concentration similar to that found in pregnancy. Using these protocols, we consistently observed the presence of E2-receptor mRNA in VSM cells by a ribonuclease protection assay. Fetal calf serum-stimulated [3H]thymidine incorporation was significantly reduced (P < 0.05) in E2-treated cells compared with untreated control cells. Similarly, E2 treatment significantly inhibited fetal calf serum-stimulated VSM cell proliferation compared with untreated control cells (P < 0.05). We also tested the hypothesis that E2 treatment attenuates agonist-stimulated [Ca2+]i in VSM cells because acute E2 treatment has been shown to produce relaxation of precontracted isolated coronary artery preparations. E2 treatment of VSM cells resulted in a significant decrease in bradykinin-stimulated [Ca2+]i compared with untreated cells (P < 0.05). In conclusion, our data demonstrate that estrogen at physiological concentrations directly regulates coronary VSM cell function.

This is the first report on estrogen-dependent growth of human-derived colon carcinoma cells. Under selected conditions, growth of subconfluent Caco-2 cells is triggered by estradiol. Cell growth is estradiol concentration dependent, with maximal effect occurring at about 0.4 nM. Growth is prevented by two different antiestrogens: the partial agonist, OH-Tamoxifen, and the pore antagonist, ICI 182,780. The growth effect is specific for estradiol since other hormonal steroids tested do not affect cell growth. The amount of estradiol receptor in subconfluent Caco-2 cells, detected by blot with monoclonal antibodies directed against the receptor as well as estradiol binding assays, is similar to that of the classical estradiol-responsive, human mammary cancer-derived MCF-7 cells. Estradiol treatment of subconfluent Caco-2 cells rapidly and reversibly stimulates four important intermediates in a signal transduction pathway that is known to trigger cell proliferation: two members of the large family of c-src-related tyrosine kinases, c-src and c-yes, and two serine/threonine kinases, the mitogen-activated protein (MAP) kinases, erk-1 and erk-2. Tyrosine kinases activated by estradiol are up-stream MAP kinases and Caco-2 cell proliferation. In fact, genistein, a specific tyrosine kinase inhibitor, abolishes the estradiol stimulatory effect on both erk-2 activity and cell proliferation. Our findings show that in subconfluent Caco-2 cells, the estradiol-receptor complex activates the c-src, c-yes/MAP kinase pathway and activates growth. This could have important implications for the understanding of human intestinal carcinogenesis.

Background: We have previously shown that combined administration of testosterone (T) and a low dose of estradiol 17 beta (T+LDE2) for 16 weeks induces an atypical proliferative lesion, termed dysplasia, in the dorsolateral prostates of intact Noble rats (1, 2). The lesion was accompanied by increases in the levels of a moderate affinity, high capacity, estrogen-binding site (type II sites) found exclusively in dorsolateral prostates of these animals (1, 3). In contrast, a proliferative response and type II sites were not observed in the ventral prostates (VP) of the same rats treated with this hormonal regimen. In the current study, rats were treated with a higher dose of E2 (4 x LDE2) but the same dose of T (T+HDE2) for 16 weeks. Our aims were to determine how the VP would respond to the T+HDE2 treatment., Experimental Design: Intact Noble rats were treated with T+HDE2 for 16 weeks. Prostatic tissues were removed for histology, electronmicroscopy, and type II site measurements. Proliferating cells were identified by the histochemical detection of proliferating cell nuclear antigen and colcemid-arrested mitotic figures. Apoptotic cells were recognized by their characteristic histologic and ultrastructural features and by in situ detection of nuclear DNA fragmentation. Data were compared with results previously obtained from VP of rats treated with T+LDE2., Results: The VP of T+HDE2-treated animals contained focal atypical hyperplasia and wide-spread apoptosis. Proliferating cell nuclear Ag-positive-stained epithelial cells and mitotic figures were only present in foci of atypical hyperplasia. Total DNA content of the VP was significantly increased, but the tissue wet weight was not augmented. Nuclear type II sites, never observed in untreated or T+LDE2-treated rats, were detected in the VP of the majority of T+HDE2-treated animals., Conclusions: The administration of a high dose of E2 with T produced a unique lesion in the VP, characterized by simultaneous occurrence of apoptosis and proliferation. The synergy between androgens and estrogens, via type II site induction, likely produces the proliferative response. On the other hand, inhibition of intracellular androgen activation pathways, leading to reduction in cell survival factors, may be the cause for the apoptotic development. Our model, thus, provides a unique opportunity to further study the balance/switch between cell proliferation and apoptosis that is often disturbed during cancer development.

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

We previously demonstrated that simultaneous treatment of Noble (NBL) rats with estradiol (E2) and testosterone (T) for 16 weeks induces a proliferative response selectively in the dorsolateral prostates (DLP) of all treated animals [1, 2]. The unique sensitivity of rat DLP to the conjoint androgen-estrogen-induced mitogenic action may be attributable to the presence of a moderate affinity, high capacity, nuclear estrogen binder (type II sites) found exclusively in this prostatic lobe [2, 3]. Little is known about whether prostatic type II site levels are under hormonal regulation. The aim of this study is to determine whether testicular steroids play a role in regulating the basal and/or induced levels of type II site expression in rat DLP. In the first experiment, rats were castrated and immediately treated with 5 alpha-dihydrotestosterone (DHT) and/or E2 for 6 weeks to determine whether these steroids, separately or jointly, could sustain DLP type II site levels in castrates. Treatments of castrated rats with DHT and DHT+E2 were found to be effective in maintaining DLP type II site levels and gland wet weights at values close to those found in intact untreated controls, while treatments with E2 failed to maintain these parameters at levels observed in intact animals. In the second experiment, intact rats were treated with an androgen (T or DHT) or E2, alone or in combination, for 16 weeks to ascertain which hormonal regimen could induce a higher level of type II site expression in the DLP. Treatments of rats with an androgen (T or DHT) or E2 alone did not change DLP type II site levels even though T treatment caused a slight increase in gland weight, while E2 treatment induced prostatic atrophy. Contrary to single hormone treatments, combined T + E2 and DHT+E2 treatments were effective in inducing a doubling of type II sites and increases in wet weight of the DLPs. These data indicate that testicular androgen is the primary factor responsible for maintaining a basal level of type II site expression in rat DLP, while conjoint androgenic-estrogenic action is needed for the induction of a higher level of type II site expression in the tissue.

Objective: To compare effects of various anti-estrogens on early pregnancy., Methods: Fifty regularly cycling guinea pigs were divided into five groups (five animals/group), of which three groups received 3 mg/kg body weight Nafoxidine, Centchroman, or Tamoxifen, respectively, on the 1st, 2nd, or 3rd day of pregnancy. The rest of the animals were kept as controls, and received either saline or propylene glycol. Autopsies were done on the 8th day of pregnancy in all the groups. Presence or absence of implantation sites was observed by using a stereomicroscope. Plasma levels of 17 beta-estradiol and progesterone and their receptors, as well as enzyme levels in the uterine tissue, were estimated., Results: During normal early pregnancy there was an increase in the concentration of peripheral plasma levels of steroid hormones with respect to enzyme and steroid hormone receptor levels in the uterus. Administration of anti-estrogens showed a decreased trend in all the variables studied, especially in the tamoxifen-treated animals., Conclusions: The results of the study suggest that anti-estrogens, if administered during early pregnancy, can prevent the process of ovum implantation in this species.

In 22 patients with endometrial carcinoma, Stage I-III (mean age 57.8 years) with obesity, initial and reactive hyperinsulinemia (during glucose load) was revealed. Significant correlations between values of "peak" and field-square of the insulin secretion curve and cytoplasmatic receptors to Estradiol and Progesterone in the tumor were found. In a group of the patients with high values of reactive hyperinsulinemia significantly larger amounts of steroid hormone receptors in the tumor were determined as compared to the group of the patients who had low insulinemia values. On considering these data a possible conclusion was reached as to the modifying influence of hyperinsulinemia on sensitivity of endometrial carcinoma to hormone receptor synthesis in the tumor by insulin.

The results of assay of receptorial status of tumor in 152 primary patients with endometrial carcinoma are presented. It was shown that a 10-day course of preoperative Tamoxifen therapy (course dose 0.6 g) was able to increase significantly concentrations of nuclear estradiol receptors and cytoplasmatic progesterone receptors in the tumor. This phenomenon followed morphological changes in the tumor and was established in both hormone dependent type (72.7%) and hormone independent type of endometrial carcinoma (45.5%). The assay of the dynamics of receptorial status of primary endometrial carcinoma under influence of Tamoxifen before surgery might be an informative test for selection of sensitive patients with poor prognosis for prolonged hormone therapy in remission to prevent development of recurrencies.

The concentration of estradiol and progesterone receptors in tumor cytosol from human breast cancer patients represents an important factor for the selection of patients for endocrine therapy and for the prognosis of the disease. This report presents results obtained by measurement of estradiol receptor in 157 breast tumors by the dextran coated charcoal method in media containing sodium molybdate (102 cases) or glycerol (55 cases). Results obtained when these two reagents were present separately in the assay media were similar, at least when the frequencies of estradiol receptor positive cases and the mean values for estradiol receptor concentrations were compared. Concomitant analyses for estradiol and progesterone receptors were done in 25 primary mammary carcinomas by the method of dextran coated charcoal using single saturating doses of hormones and cytosols obtained in a glycerol containing buffer. The results are discussed in relation with the steroid hormone mechanism of action.

The time course of subcellular receptor distribution in porcine endometrial epithelium was studied after intrauterine administration of estradiol alone or in combination with puromycin. In untreated cells, the major proportion of receptor is associated with cytoplasmic membranes. The solubilization of receptor from isolated nuclei is independent of their estradiol content. Smooth cytoplasmic membranes are the site of origin of receptor which is swiftly translocated into the nucleus in a 1:1 ratio with the hormone after exposure of the cells to estradiol. Simultaneously administered puromycin delays receptor synthesis and reveals that the nuclear passage of receptor is terminated by receptor degradation. The synthesis of receptor proceeds in rough endoplasmic membranes. A subsequent finishing and deposition in smooth membranes depends on intact protein synthesis.

Our recent studies demonstrated that the continuous administration of a GnRH agonist (GnRH-Ag; WY 40972) induces abortion in rats by suppressing plasma progesterone (P) levels within 24 h. This fall in P levels is not accompanied by a fall in ovarian venous plasma testosterone (T) or estradiol (E) levels. In this study an attempt was made 1) to determine whether the suppression of P by GnRH-Ag is due to decreased estrogen present in the corpora lutea (CL) and/or a decrease in luteal receptors of E, and 2) to investigate the effects of GnRH-Ag on the nocturnal surges of PRL. Rats were treated continuously on days 7-11 of pregnancy with 5 micrograms/day GnRH-Ag using an osmotic minipump. Ovarian blood samples were obtained on day 8; at autopsy CL were harvested and incubated with medium 199 for 4 h at 37 C under an atmosphere of 95% oxygen-5% carbon-dioxide. Additional rats were killed on day 8 or 10; CL were isolated from the ovary and pooled within the group for the measurement of nuclear and cytosolic E receptors. In other experiments, on days 8, 9, 10, and 11 of pregnancy, blood samples (0.3 ml) were collected via an indwelling intraatrial Silastic cannula at 0330, 0500, or 0600 h for the measurement of PRL and P. While the net synthesis of P by CL in the GnRH-Ag-treated rats decreased to 48 +/- 12 from 224 +/- 47 ng/CL in controls, T and E levels were not different from their respective control values. Steroid levels in ovarian venous plasma reflected a similar response. Nuclear E receptors levels were 82 and 80 in controls and 39 and 41 fmol/mg DNA in the treated group on days 8 and 10, respectively. Nocturnal surges of PRL in plasma were detected at 0330 h in controls as well as in treated rats. However, plasma PRL levels at 0330 h were 101 +/- 24, 120 +/- 22, 196 +/- 40, and 103 +/- 13 ng/ml in controls and 44 +/- 8, 50 +/- 10, 29 +/- 13, and 20 +/- 9 in the GnRH-Ag-treated group on days 8, 9, 10, and 11, respectively. These results suggest that GnRH-Ag has no effect on the ability of the luteal synthesis of T and E and that the antipregnancy effect of GnRH-Ag may be at the level of the CL due to the direct inhibitory effect of GnRH-Ag on the luteal synthesis of P, which, in turn, results in decreased nocturnal surges of PRL and a fall in E receptors in the CL. Alternatively, GnRH-Ag treatment could suppress ovarian or luteal receptors for PRL, which, in turn, lower luteal E receptors, leading to a fall in luteal synthesis and release of P.

Restriction enzyme mapping has been used to study the relationship between methylation at the 5' end of the human estrogen receptor gene (ER) and its various levels of expression in estrogen target tissues, like endometrium, placenta and breast. The methylation patterns were compared with that found in white blood cells. A cluster of CpG sites, including the transcription start site, was undermethylated in all examined tissues. In front and downstream of this area, a 1 Kb region was undermethylated in endometrium where the ER gene is highly expressed, methylated to some extent in placenta and breast, which express the gene to a low degree, and greatly methylated in white blood cells which are thought not to express this gene. These data provide evidence that in the 5' end of the human ER DNA, CpG methylation is tissue-specific, and related to the level of the ER gene expression.

The concentration of the estradiol and progesterone receptors in cytosol increased within a week after ovariectomy in rats, the nucleic concentration of receptors sharply diminishing. The uterus atrophy following ovariectomy in associated with a reduction of both nucleic and cytosol receptors in the organ. Administration of estradiol (1 mg) restored the organ mass and the character of hormonal reception within a week. Progesterone and clomyphen exerted some uterotropic effect. Ovariectomy reduced the creatinkinase activity by 2.9 times and the peroxydase activity by 13 times. Two types of synthesis of female sex hormones receptors seem to exist in the rat uterus: the constitutional type depending on no hormonal effects and the induced one depending on the sex steroids, estradiol in particular.