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Elevated expression of cyclooxygenase 2 (COX2 (PTGS2)) has been reported to occur in human ovarian cancer and to be associated with poor prognosis. We have previously demonstrated that COX2-derived prostaglandin E2 (PGE2) promotes human ovarian cancer cell invasion. We had also demonstrated that epidermal growth factor (EGF) induces human ovarian cancer cell invasion by downregulating the expression of E-cadherin through various signaling pathways. However, it remains unclear whether COX2 and PGE2 are involved in the EGF-induced downregulation of E-cadherin expression and cell invasion in human ovarian cancer cells. In this study, we showed that EGF treatment induces COX2 expression and PGE2 production in SKOV3 and OVCAR5 human ovarian cancer cell lines. Interestingly, COX2 is not required for the EGF-induced downregulation of E-cadherin expression. In addition, EGF treatment activates the phosphatidylinositol-3-kinase (PI3K)/Akt and cAMP response element-binding protein (CREB) signaling pathways, while only the PI3K/Akt pathway is involved in EGF-induced COX2 expression. Moreover, we also showed that EGF-induced cell invasion is attenuated by treatment with a selective COX2 inhibitor, NS-398, as well as PGE2 siRNA. This study demonstrates an important role for COX2 and its derivative, PGE2, in the mediation of the effects of EGF on human ovarian cancer cell invasio. [ABSTRACT FROM AUTHOR]

The etiology of granulosa cell tumors (GCTs) is largely unknown. The primary mode of treatment is surgical, however not all women are cured by surgery alone. Thus, it is important to develop improved treatments through a greater understanding of the molecular mechanisms that contribute to this disease. Recently, it has been shown that a FOXL2 402C>G (C134W) mutation is present in 97% of human adult-type GCTs, suggesting an important role for this mutation in the development of GCTs. We have shown previously that gonadotropin-releasing hormone (GnRH)-I and -II induce apoptosis in cultured normal human granulosa cells. Moreover, it has been reported that FOXL2 can bind to the promoter of the mouse GnRH receptor gene and regulate its transcription. Thus, we hypothesized that C134W mutant FOXL2 could modulate the pro-apoptotic effects of GnRH via aberrant regulation of GnRH receptor levels. Using KGN cells, a human GCT-derived cell line which harbors the FOXL2 402C>G mutation, we show that treatment with GnRH-I and -II induces cell apoptosis, and that small interfering RNA-mediated depletion of GnRH receptor abolishes these effects. Overexpression of wild-type FOXL2 increases both mRNA and protein levels of GnRH receptor and consequently enhances GnRH-induced apoptosis. Importantly, neither the expression levels of GnRH receptor nor GnRH-induced apoptosis were affected by overexpression of the C134W mutant FOXL2. Interestingly, knockdown of endogenous FOXL2 down-regulates GnRHR expression in normal human granulosa cells with wild-type FOXL2, but not in KGN cells. These results suggest that the FOXL2 402C>G mutation may contribute to the development of human adult-type GCTs by reducing the expression of GnRH receptor, thus conferring resistance to GnRH-induced cell apoptosis. [ABSTRACT FROM AUTHOR]

Endocrine-disrupting chemicals ( EDCs) are natural or synthetic compounds present in the environment which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. Most EDCs tend to bind to steroid hormone receptors including the oestrogen receptor ( ER), progesterone receptor ( PR) and androgen receptor ( AR). As EDCs disrupt the actions of endogenous hormones, they may induce abnormal reproduction, stimulation of cancer growth, dysfunction of neuronal and immune system. Although EDCs represent a significant public health concern, there are no standard methods to determine effect of EDCs on human beings. The mechanisms underlying adverse actions of EDC exposure are not clearly understood. In this review, we highlighted the toxicology of EDCs and its effect on human health, including reproductive development in males and females as shown in in vitro and in vivo models. In addition, this review brings attention to the toxicity of EDCs via interaction of genomic and non-genomic signalling pathways through hormone receptors. [ABSTRACT FROM AUTHOR]

Apoptosis in ovarian surface epithelial (OSE) cells is induced by transforming growth factor-beta ( TGF-β). However, high-grade serous ovarian carcinomas (HGC) are refractory to the inhibitory functions of TGF- β; their invasiveness is up-regulated by TGF-β through epithelial- mesenchymal transition (EMT) activation. Serous borderline ovarian tumors (SBOT) have been recognized as distinct entities that give rise to invasive low-grade serous carcinomas (LGC), which have a relatively poor prognosis and are unrelated to HGC. While it is not fully understood how TGF-β plays disparate roles in OSE cells and its malignant derivative HGC, its role in SBOT and LGC remains unknown. Here we demonstrate the effects of TGF-β on cultured SBOT3.1 and LGC-derived MPSC1 cells, which express TGF-β type I and type II receptors. TGF-β treatment induced the invasiveness of SBOT3.1 cells but reduced the invasiveness of MPSC1 cells. The analysis of apoptosis, which was assessed by cleaved caspase-3 and trypan blue exclusion assay, revealed TGF-β-induced apoptosis in MPSC1, but not SBOT3.1 cells. The pro- apoptotic effect of TGF-β on LGC cells was confirmed in another immortalized LGC cell line ILGC. TGF-β treatment led to the activation of Smad3 but not Smad2. The specific TbRI inhibitor SB431542 and TbRI siRNA abolished the SBOT3.1 invasion induced by TGF-β, and it prevented TGF- β-induced apoptosis in MPSC1 cells. In SBOT3.1 cells, TGF-β down- regulated E-cadherin and concurrently up-regulated N-cadherin. TGF-β up- regulated the expression of the transcriptional repressors of E-cadherin, Snail, Slug, Twist and ZEB1. In contrast, co-treatment with SB431542 and TbRI depletion by siRNA abolished the effects of TGF-β on the relative cadherin expression levels and that of Snail, Slug, Twist and ZEB1 as well. This study demonstrates dual TGF-β functions: the induction of SBOT cell invasion by EMT activation and apoptosis promotion in LGC cells. [ABSTRACT FROM AUTHOR]

Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent. [ABSTRACT FROM AUTHOR]

Gestational diabetes mellitus (GDM) has been shown to be associated with high risk of diabetes in offspring. However, the mechanisms involved and the possibilities of transgenerational transmission are still unclear. We intercrossed male and female adult control and first-generation offspring of GDM (F1-GDM) mice to obtain the second-generation (F2) offspring in four groups: C♂-C♀, C♂-GDM♀, GDM♂-C♀, and GDM♂-GDM♀. We found that birth weight significantly increased in F2 offspring through the paternal line with impaired glucose tolerance (IGT). Regardless of birth from F1-GDM with or without IGT, high risk of IGT appeared as early as 3 weeks in F2 offspring and progressed through both parental lineages, especial the paternal line. IGT in male offspring was more obvious than that in females, with parental characteristics and sex-specific transmission. In both F1 and F2 offspring of GDM, the expression of imprinted genes Igf2 and H19 was downregulated in pancreatic islets, caused by abnormal methylation status of the differentially methylated region, which may be one of the mechanisms for impaired islet ultrastructure and function. Furthermore, altered Igf2 and H19 gene expression was found in sperm of adult F1-GDM, regardless of the presence of IGT, indicating that changes of epigenetics in germ cells contributed to transgenerational transmission. [ABSTRACT FROM AUTHOR]

Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-b1 and interleukin-1b, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the avb3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function. [ABSTRACT FROM AUTHOR]

Human endometrium resists embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) is involved in the regulation of endometrial receptivity by intracellular Ca2+. Currently, this protein is known to be mainly expressed in brain, kidneys, and pancreas, but potential role(s) of CaBP-28k in the human uterus during the menstrual cycle remain to be clarified. Thus, in this study we demonstrated the expression of CaBP-28k in the human endometrium in distinct menstrual phases. During the human menstrual cycle, uterine expression levels of CaBP-28k mRNA and protein increased in the proliferative phase and fluctuated in these tissues, compared with that observed in other phases. We assessed the effects of two sex-steroid hormones, 17beta-estradiol (E2) and progesterone (P4), on the expression of CaBP-28k in Ishikawa cells. A significant increase in the expression of CaBP- 28k mRNA was observed at the concentrations of E2 (10(-9 to -7) M). In addition, spatial expression of CaBP-28k protein was detected by immunohistochemistry. CaBP-28k was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells during the proliferative phases (early-, mid-, late-) and early-secretory phase of menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium binding protein, is abundantly expressed in the human endometrium, suggesting that uterine expression of CaBP-28k may be involved in reproductive function during the human menstrual cycle. [ABSTRACT FROM AUTHOR]

Recent studies have shown that genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non-toxic prodrugs to toxic metabolites selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs were capable of migrating to human ovarian cancer cells and examined the potential therapeutic efficacy of the gene-directed enzyme prodrug therapy against ovarian cancer cells in vitro. The expression of cytosine deaminase (CD) or carboxyl esterase (CE) mRNA of GESTECs was confirmed by RT-PCR. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to ovarian cancer cells. GESTECs (HB1.F3.CD or HB1.F3.CE cells) engineered to express a suicide gene (CD or CE) selectively migrated toward ovarian cancer cells. A [3H] thymidine incorporation assay was conducted to measure the proliferative index. Treatment of human epithelial ovarian cancer cell line (SKOV-3, an ovarian adenocarcinoma derived from the ascites of an ovarian cancer patient) with the prodrugs 5-fluorocytosine (5-FC) or camptothecin-11 (CPT-11) in the presence of HB1.F3.CD or HB1.F3.CE cells resulted in the inhibition of ovarian cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD/CE may have a potent advantage to selectively treat ovarian cancers. ( Cancer Sci 2010; 101: 955–962) [ABSTRACT FROM AUTHOR]

Background: Antagonists of growth hormone-releasing hormone (GHRH) are being developed for the treatment of various human cancers. Methods: MTT assay was used to test the proliferation of SKOV3 and CaOV3. The splice variant expression of GHRH receptors was examined by RT-PCR. The expression of protein in signal pathway was examined by Western blotting. siRNA was used to block the effect of EGFR. Results: In this study, we investigated the effects of a new GHRH antagonist JMR-132, in ovarian cancer cell lines SKOV3 and CaOV3 expressing splice variant (SV)1 of GHRH receptors. MTT assay showed that JMR-132 had strong antiproliferative effects on SKOV3 and CaOV3 cells in both a time-dependent and dose-dependent fashion. JMR-132 also induced the activation and increased cleaved caspase3 in a time- and dose-dependent manner in both cell lines. In addition, JMR-132 treatments decreased significantly the epidermal growth factor receptor (EGFR) level and the phosphorylation of Akt (p-Akt), suggesting that JMR-132 inhibits the EGFR-Akt pathway in ovarian cancer cells. More importantly, treatment of SKOV3 and CaOV3 cells with 100 nM JMR-132 attenuated proliferation and the antiapoptotic effect induced by EGF in both cell lines. After the knockdown of the expression of EGFR by siRNA, the antiproliferative effect of JMR-132 was abolished in SKOV3 and CaOV3 cells. Conclusions: The present study demonstrates that the inhibitory effect of the GHRH antagonist JMR-132 on proliferation is due, in part, to an interference with the EGFR-Akt pathway in ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Background: Homeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited. Methods: To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay. Results: Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect. Conclusions: Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation. [ABSTRACT FROM AUTHOR]

Huang Q, Cheung AP, Zhang Y, Huang HF, Auersperg N, Leung PC. Effects of growth differentiation factor 9 on cell cycle regulators and ERK42144 in human granulosa cell proliferation. Am J Physiol Endocrinol Metab 296: E1344-E1353, 2009. First published April 14, 2009; doi:10.1152/ajpendo.90929.2008.-GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-43 1542), ERK42/44 (PD098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [[sup3]H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [[sup3]H]thymidine incorporation, enhanced cell transition from G[sub0]/G[sub1] to S and G[sub2]/M phases (whereas both SB-43 1542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D1 and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15[supINK4B] and p16[supINK4A]. GDF-9 also activated Rb protein (a critical G1 to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D1 and E but not the decrease in p15[supINK4B] and p16[supINK4A] induced by GDF-9. In contrast, SIS3 reversed the decrease in p15[supINK4B] and p16[supINK4A] but not the increase in cyclin D1 and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D1 and E and suppressing p15[supINK4B] and p16[supINK4A] via both Smad-dependent and Smad-independent pathways. [ABSTRACT FROM AUTHOR]

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G0/G1. In contrast to GnRH signaling in pituitary gonadotropes, the involvement of Gαq, protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein Gαi, leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin-induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration/invasion have started to emerge. In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed. [ABSTRACT FROM AUTHOR]

Interrelationships between HOXA7 expression and ovarian cancer progression are investigated by cDNA array and by immunohistochemistry of normal ovaries and 538 epithelial ovarian tumor microarrays. Outcomes of patients and HOXA7 expression were compared by Kaplan-Meier survival curves. HOXA7 mRNA expression was higher in carcinomas than in benign tumors. HOXA7 protein was absent in normal surface epithelium but appeared in metaplastic regions. All carcinomas stained for HOXA7 protein, with staining intensities unrelated to histotype, grade, or patient outcome. There were significant associations of strong HOXA7 staining of stroma and tumor nuclei with the clear cell histotype (stroma: P =. 0022, nuclei: P =. 0003) and of weak/absent staining with serous carcinomas. Tumor E-cadherin expression correlated significantly with HOX7 staining in stroma (P = .0002) but not within tumors. HOXA7 staining of tumor cell nuclei is correlated significantly with improved disease-specific survival (P = .0104), which is suggestive of the biological and potentially clinical importance of subcellular HOXA7 localization. [ABSTRACT FROM AUTHOR]

Gonadotropin-releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at −641/−636 (CATGCC, designated GII-Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG-3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF-κB subunit p65 and the retinoic acid receptors, RARα and RXRα, to the promoter sequences. Over-expression of these protein factors indicated that p65 is a potent repressor, and the RARα/RXRα heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real-time PCR. Treatment of cells with the RARα/RXRα ligands, all- trans retinoic acid and 9- cis-retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG-3 cells, respectively. Taken together, these data demonstrate the differential roles of NF-κB p65 and RARα/RXRα, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell-specific manner. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer is a highly fatal disease for which prevention strategies have been limited; in part because of our poor understanding of the underlying biology of its precursor, the ovarian surface epithelium (OSE). The OSE is a single layer of flat-to-cuboidal mesothelial cells that covers the surface of the ovary. Despite its inconspicuous appearance in vivo, it is believed that OSE cells actively participate in the cyclical ovulatory rupture and repair process. The continuous rupture of the OSE at ovulation and the subsequent proliferation to repair the wound renders the cells susceptible to genetic damage and malignant transformation. As the ovary is a rich source of multiple hormones, and normal OSE and ovarian carcinomas secrete and have receptors for hormones, growth factors and cytokines, these factors are strong candidates to regulate normal OSE physiology and the transformation and progression of ovarian cancers. In particular, alterations of hormone/growth factor production and receptor expression are common in ovarian tumors. This review summarizes the current knowledge in the field of endocrinology and its relationship to the biology and pathology of the OSE. [ABSTRACT FROM AUTHOR]

Homeobox (HOX) genes are important transcriptional regulators in development and in adult tissues. A major obstacle to the understanding of their roles in humans has been the lack of well-defined anti-human HOX antibodies. We generated a thoroughly characterized polyclonal rabbit antibody against human HOXA7 and used it to study the distribution, role, and regulation of HOXA7 in human ovarian folliculogenesis and in granulosa cell tumors. Immunohistochemically, follicles were strongly HOXA7-positive compared with stroma. Oocytes expressed little HOXA7. Granulosa cells were predominantly negative in primordial follicles, had uniformly HOXA7-positive nuclei in primary follicles, and, as follicles matured, the subcellular localization of HOXA7 changed from nuclear to predominantly cytoplasmic. HOXA7 was mainly cytoplasmic in theca interna, but completely absent in theca externa. Granulosa cell tumors were mainly HOXA7 positive and, like in preovulatory follicles and theca interna, staining was predominantly cytoplasmic. The change in HOXA7 expression from negative primordial to positive primary follicles suggested a relationship with granulosa cell proliferation. To test this hypothesis, SV40 Tag-immortalized human granulosa cells (SVOG) were double stained with anti-HOXA7 antibody and with Ki-67 as proliferation marker. HOXA7 expression was highest in mitotic cells. In addition, growth differentiation factor-9 (GDF-9), known to be secreted by oocytes in primary human follicles, up-regulated HOXA7 protein, and stimulated proliferation of SVOG, while TGF-β1 inhibited HOXA7 expression and proliferation. This is the first report on the expression of any HOX gene in human ovarian follicles and granulosa cell tumors. It shows that HOXA7 undergoes cell type- and stage-specific changes during ovarian folliculogenesis, likely regulates granulosa cell proliferation, and in subcellular location differs between proliferating and secretory cells. The increase in HOXA7 protein in response to GDF-9 represents the first demonstration of a possible regulatory role of oocytes in ovarian follicular HOX gene expression. [ABSTRACT FROM AUTHOR]

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin- D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats. [ABSTRACT FROM AUTHOR]

Although Calbindin-D9k (CaBP-9k), a cytosolic calcium binding protein which has calcium binding sites, is expressed in various tissues, i.e., intestine, uterus, and placenta, potential roles of this gene and its protein are not clearly understood. Uterine CaBP-9k may be involved in controlling myometrial activity related with intracellular calcium level and is not under the control of vitamin D despite the presence of vitamin D receptors. But, it is under the control of the sex steroid hormones, estrogen (E2) and progesterone (P4), in female reproductive systems including the uterus and placenta. Thus, in this review, we summarize recent research literature in regards to the expression and regulation of CaBP-9k in mammals and introduce the research data of recent studies by us and others. [ABSTRACT FROM AUTHOR]

To investigate the influence of maternal corticosteroid administration on neutrophil apoptosis in early onset preeclampsia. We investigated five groups: early onset preeclampsia (EOPET, <34 weeks, n = 10); late-onset preeclampsia (LOPET, ≥34 weeks, n = 7); normotensive intrauterine growth restriction (nIUGR, n = 11); normal pregnancy (NPC, n = 22); and non-pregnancy ( n = 10). We examined, by flow cytometry, spontaneous neutrophil apoptosis after 18 h culture (hypodiploid DNA, Annexin V binding, propidium iodide [PI] permeability). For the 10 women with EOPET exposed to betamethasone in the previous 48 h, we found that neutrophil apoptosis was not inappropriately inhibited, in contrast to our previous findings in women not thus exposed. Neither LOPET nor nIUGR differed from normal pregnancy. Betamethasone alters the rate of spontaneous neutrophil apoptosis in EOPET. The anti-inflammatory influence of betamethasone may explain some of the differences between our previous and present findings with respect to neutrophil apoptosis in EOPET. Corticosteroids ameliorate the course of antenatal and postnatal preeclampsia. These results may reflect the mechanisms that underlie the transient improvements seen with antenatal dexamethasone use. [ABSTRACT FROM AUTHOR]

Objective:Ovarian surface epithelium (OSE), the precursor of the epithelial ovarian carcinomas, has limited growth potential in culture. Epidermal growth factor+hydrocortisone (EGF+HC) enhances its growth but induces epitheliomesenchymal transition (EMT). This study was undertaken to define the effects of EGF+HC and their reversibility, to optimize growth-promoting media, and to relate OSE phenotypes in vitro to physiologic states in vivo.Methods:OSE was cultured in media 199/MDCB105 or EBM (Clonetics) with 2% or 10% fetal bovine serum with or without 10 ng/mL EGF, 1.0 μg/mL HC, and 1.0 μg/mL bovine brain extract. Growth rates and growth potentials (population doublings [PD] to senescence) were defined, and growth patterns and expression of keratin and collagen types III and IV were compared with the ovarian cancer cell lines OVCAR3 and SKOV3.Results:EGF+HC increased growth potentials from 12-14 PD to 40-42 PD and reduced PD time from 53 hours to 20 hours. Without EGF+HC, OSE cells remained uniformly epithelial. EGF+HC induced EMT (mesenchymal shapes, reduced keratin, and production of collagenous extracellular matrix), but the EMT response varied greatly among OSE from different women. EMT was reversed over 1-2 weeks by seculture into EGF+HC-free medium in passage 1, but inconsistently thereafter. EGF+HC had no effect on the differentiation of ovarian carcinoma lines.Conclusion:The phenotype of intact OSE in vivo is most closely reproduced in media without EGF+HC. EGF+HC enhances growth but initiates EMT, which likely mimics a repair response. Variations in EGF+HC-induced phenotypes point to the existence of OSE subpopulations with differing responsiveness to growth factors or steroids, which may relate to their susceptibility to malignant transformation. [ABSTRACT FROM PUBLISHER]

A hybrid cell line, IOSE-Ov29, was created through fusion of cells from the human ovarian adenocarcinoma line OVCAR3 and the non-tumorigenic SV40 Tag-transfected human ovarian surface epithelial line IOSE-29. OVCAR3 cells exhibit a differentiated epithelial phenotype, whereas line IOSE-29 expresses mesenchymal characteristics that were acquired in culture by epithelio-mesenchymal transition. Microsatellite analysis, comparative genomic hybridization (CGH), and MFISH showed the genotype of the IOSE-Ov29 cells to contain components of both parent cell lines, but to be predominantly OVCAR3 derived. IOSE-Ov29 resembled OVCAR3 and differed from IOSE-29 as shown by its unlimited life span, tumorigenicity, epithelial morphology, keratin, occludin, E-cadherin and CA125 expression, increased expression of kinases of the PI3K pathway, and loss of cGMP-dependent protein kinase expression. IOSE-29-derived properties included SV40 Tag expression, growth inhibition by activin, collagen type III secretion, increased adhesion and spreading on tissue culture plastic, and increased growth rate. Proliferation of all three lines was stimulated by FSH and ATP and inhibited by GnRH I and GnRH II. Interestingly, IOSE-Ov29 was more anchorage independent than either parent line and was the only line that invaded Matrigel in Boyden chambers and formed invasive branches in collagen gels. The results indicate that IOSE-Ov29 is an IOSE-29/OVCAR3 hybrid, which differs from both parent lines genetically and phenotypically. Unexpectedly, fusion with the non-tumorigenic IOSE-29 cells enhanced malignancy-associated characteristics of OVCAR3, presumably as a result of the expression of IOSE-29-derived mesenchymal properties that are usually acquired by carcinoma cells through epithelio-mesenchymal transition during metastatic progression. [ABSTRACT FROM AUTHOR]

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gone is expressed under the control of a PRE. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases which are activated in response to a diverse array of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. It has been demonstrated that MAPKs are activated by external stimuli including chemotherapeutic agents, growth factors and reproductive hormones in ovarian surface epithelial cells. Thus, the MAPK signaling pathway may play an important role in the regulation of proliferation, survival and apoptosis in response to these external stimuli in ovarian cancer. In this article, an activation of the MAPK signaling cascade by several key reproductive hormones and growth factors in epithelial ovarian cancer is reviewed. [ABSTRACT FROM AUTHOR]

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells. [ABSTRACT FROM AUTHOR]