Endometrial Adenocarcinoma Cell Line Research Articles

Identification and Characterization of a Novel Luciferase-Like Protein in the Human Female Reproductive Tract

A novel cDNA was cloned from human endometrium, matching a human gene with the interim name KIAA1463. An mRNA identified by 5'-rapid amplification of cDNA ends was found to be 3349 nt in length. PCR analysis also identified another transcript of 6626 nt, with an open reading frame encoding a 900 amino acid protein. A fold recognition program identified similarity to firefly luciferase containing an AMP-binding motif; hence, we refer to the predicted protein as the AMP binding/luciferase-like protein (ALLP). ALLP mRNA and protein were expressed throughout the female reproductive tract with the highest levels found in the ovary and uterus. In situ hybridization and immunohistochemistry showed predominant localization of the ALLP mRNA/protein in endometrial glandular epithelium and within the theca and granulosa cells in the ovary. In the endometrium expression of ALLP, mRNA and protein were higher during d 16-21 of the secretory phase of the cycle. Western blot analysis showed decreased expression of ALLP in the postmenopausal endometrium, and hormone replacement therapy increased the expression of ALLP. Endometrial adenocarcinoma cell lines expressed more ALLP, compared with cultured primary endometrial cells or normal endometrial tissue. The ubiquitous expression of ALLP in reproductive and nonreproductive tissues suggests that this protein, which is probably regulated by ovarian steroids, plays an important metabolic role and may be involved in such processes as implantation and tumorigenesis.

Differential effects of raloxifene and tamoxifen on the expression of estrogen receptors and antigen Ki-67 in human endometrial adenocarcinoma cell line

Tamoxifen and raloxifene are widely used in clinical practice. It has been found that tamoxifen treatment increases the risk of development of endometrial cancer. The effects of tamoxifen and raloxifene on endometrium might be caused by different estrogen receptor expression. The aim of the present study was immunohistochemical evaluation of the effects of tamoxifen and raloxifene on estrogen receptors, and Ki-67 antigen expression in the human endometrial adenocarcinoma Ishikawa cell line. Tamoxifen in concentrations of 10 microM and 20 microM increased ERalpha expression without any effect on ERbeta. All used concentrations of tamoxifen and raloxifene (0.1 nM, 1 nM, 10 nM, 1 micro M, 10 microM and 20 microM) had no effect on expression of ERbeta. Tamoxifen, but not raloxifene, increased Ki-67 antigen expression in the Ishikawa cell line. Tamoxifen, in contrast to raloxifene, increased proliferation of endometrial adenocarcinoma cells as well as exerted the shift of ERalpha/ERbeta ratio. Thus, it could be responsible for increased carcinogenic effect during tamoxifen treatment.

A HOXA10 Estrogen Response Element (ERE) is Differentially Regulated by 17 Beta-estradiol and Diethylstilbestrol (DES)

The molecular mechanisms by which estrogens regulate developmental gene expression are poorly understood. While 17 beta-estradiol is normally present at high concentrations in pregnancy, exposure to the estrogen diethylstilbestrol (DES) in utero induces developmental anomalies of the female reproductive tract. HOX gene expression is altered by DES, leading to abnormal Müllerian duct differentiation. The mechanism of ligand-specific regulation of HOX gene expression by estrogens has not been characterized. To elucidate the molecular mechanism underlying ligand-specific estrogen regulation of HOXA10 expression, we characterized regulatory regions of the human HOXA10 gene. We identified an estrogen response element (ERE) in the human HOXA10 gene that mediated differential ligand-specific estrogen-responsive transcriptional activation. Deletional analysis and reporter expression assays identified two EREs, ERE1 and ERE2, each of which drove estrogen-responsive reporter expression in the Ishikawa human uterine endometrial adenocarcinoma cell line. ERE1 drove reporter expression maximally. This ERE bound ERα and ERβ, and formed a complex that included SRC-1, but not CBP, N-CoR or SMRT. HOXA10 ERE1 drove luciferase reporter activity to eightfold the level driven by the consensus ERE in response to estradiol in Ishikawa cells. While most EREs demonstrate similar transcriptional activity in response to DES or estradiol, here estradiol induced four- to sevenfold greater reporter activity than did DES from HOXA10 ERE1. DES did not alter ER or SRC-1 binding to HOXA10 ERE1. HOXA10 ERE1 therefore demonstrated ligand specificity distinct from the consensus ERE, and unrelated to changes in ER or coactivator/corepressor binding. The ligand specificity of the HOXA10 ERE may explain the molecular mechanism by which DES leads to reproductive anomalies; differential ligand-specific activation of HOX genes may be a molecular mechanism by which DES signaling leads to inappropriate HOX expression and to developmental patterning distinct from that induced by estradiol.

Estrogen bioassay of ginseng extract and ArginMax, a nutritional supplement for the enhancement of female sexual function.

To determine whether ArginMax (The Daily Wellness Co., Sunnyvale, CA) or the Panax ginseng extract it contains has any estrogenic activity. ArginMax for Women, a nutritional supplement for optimization of sexual health, contains L-arginine, ginseng, ginkgo, damiana, multivitamins, and minerals. A human endometrial adenocarcinoma cell line, Ishikawa, which contains an alkaline phosphatase (AP) enzyme sensitive to estrogen stimulation, was used in a bioassay to determine whether Panax ginseng or ArginMax contained estrogenic components. Neither ArginMax nor Panax ginseng stimulated AP at any of the concentrations tested. No estrogenic activity was evident in the sample of Panax ginseng extract tested or in a sample of ArginMax containing this extract in combination with other ingredients.

Activin A inhibits growth-inhibitory signals by TGF-β1 in differentiated human endometrial adenocarcinoma cells

Although human endometrial adenocarcinoma tissues express high levels of activin A, the pathophysiological functions of endometrial activin A remain unclear. Human endometrial cancer cells express both activin receptor and TGF-beta receptor, and TGF-beta1 utilizes the same intracellular signaling molecules as activin A. Using three differentiated human endometrial adenocarcinoma cell lines, we investigated whether there are interactions between TGF-beta1- and activin A-mediated signaling. Flow cytometric analysis revealed cell surface expression of two types of TGF-beta receptor subunits and four types of activin receptor subunits. TGF-beta1 inhibited cell proliferation in three endometrial adenocarcinoma cell lines. Activin A did not affect the growth of the three endometrial cell lines, and pre-incubation with activin A dose-dependently reduced TGF-beta1-mediated inhibition of cell growth. These results suggest that in endometrial adenocarcinoma cells, the intracellular signals underlying TGF-beta1-mediated inhibition of growth can themselves be inhibited by activin A. Therefore, the increased expression of activin A may be involved in carcinogenesis by reducing TGF-beta-mediated signals inhibiting cell growth in human endometrial adenocarcinoma tissues.

Selective estrogen receptor modulator regulated proteins in endometrial cancer cells

Tamoxifen is the primary hormonal therapy for breast cancer and is also used as a breast cancer chemopreventative agent. A major problem with tamoxifen therapy is undesirable endometrial proliferation. To identify proteins associated with the growth stimulatory effects of tamoxifen in an ER-positive model, the present study profiled total cellular and secreted proteins regulated by estradiol and selective estrogen receptor modifiers (SERMs) in the Ishikawa endometrial adenocarcinoma cell line using two-dimensional gel electrophoresis. Following 24 h incubation with 10 −8 M estradiol, 10 −7 M 4-hydroxytamoxifen, or 10 −7 M EM-652 (Acolbifene), nine proteins exhibited significant increase in expression. The proteins identified were heat shock protein 90-α, and -β, heterogeneous nuclear ribonucleoprotein F, RNA polymerase II-mediating protein, cytoskeletal keratin 8, cytoskeletal keratin 18, ubiquitin-conjugating enzyme E2-18 kDa and nucleoside diphosphate kinase B. These protein profiles may serve as novel indices of SERM response and may also provide insight into novel mechanisms of SERM-mediated growth.

Expression and function of activin receptors in human endometrial adenocarcinoma cells

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

A novel phenoxazine derivative suppresses proliferation of human endometrial adenocarcinoma cell lines, inducing G2M accumulation and apoptosis

We examined the effects of a novel phenoxazine, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx), which was produced by the reaction of 2-amino-5-methyl-phenol with bovine hemoglobin on the proliferation of human endometrial adenocarcinoma cell lines, EN and KLE cells, and on induction of apoptosis and G2M arrest in these cells. Phx inhibited proliferation of these cell lines in a dose- and time-dependent manner, i.e., the inhibition rate of proliferation of EN and KLE cells was 43% and 40%, respectively, in the presence of 50 micro M Phx, and 75% and nearly 100%, in the presence of 100 micro M Phx, after 2 days. When these endometrial adenocarcinoma cells were incubated with a medium containing 100 micro M Phx for 24 h, accumulation of EN and KLE cells in the S and G2M phase and that of apoptotic cells were demonstrated by flow cytometry. Apoptosis of these cells caused by Phx was unlikely to be associated with p53, Bax, and Bcl-2, because the levels of these proteins were not altered regardless of the presence or absence of Phx. The present results suggest that Phx demonstrates antitumor activity against human endometrial adenocarcinoma cell lines EN and KLE cells, by inducing both cell cycle accumulation at S and G2M and apoptosis associated with p53, Bcl-2 and Bax insensitive pathways.

Endometrial HOXA10 expression after controlled ovarian hyperstimulation with recombinant follicle-stimulating hormone

Objective To determine the effects of controlled ovarian hyperstimulation and resultant high levels of E 2 on endometrial HOXA10 expression (a marker of endometrial receptivity). Design Prospective study. Setting University academic medical center. Patient(s) Twenty-five women undergoing controlled ovarian hyperstimulation with recombinant FSH and 30 fertile controls. Intervention(s) Endometrium was obtained by Pipelle endometrial biopsy on cycle days 21–25. In addition, Ishikawa cells (a well-differentiated endometrial adenocarcinoma cell line) were treated with either E 2, recombinant FSH, GnRH agonist, or GnRH antagonist. RNA was extracted and analyzed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Main outcome measure(s) HOXA10 expression. Result(s) Endometrial HOXA10 expression in women undergoing controlled ovarian hyperstimulation (COH) with recombinant FSH was not different from that in fertile controls. Estradiol increased HOXA10 expression in Ishikawa cells in a dose-dependent manner from 10 −9 to 10 −7 M. Neither recombinant FSH, GnRH agonist, nor GnRH antagonist altered HOXA10 expression in these cells. Conclusion(s) Controlled ovarian hyperstimulation did not inhibit endometrial HOXA10 expression in vivo. In addition, in vitro endometrial cell HOXA10 expression was not altered by either recombinant FSH, GnRH agonist, or GnRH antagonist. COH is unlikely to adversely impact endometrial receptivity.

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100 nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of “tissue specificity” and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.

The activation of an extracellular signal-regulated kinase by oestradiol interferes with the effects of trastuzumab on HER2 signalling in endometrial adenocarcinoma cell lines

Cellular response to oestradiol stimuli is mediated both by oestrogen receptor (ER) binding to oestrogen response elements (EREs) and by non-nuclear actions like activation of mitogen-activated protein kinase (MAPK) signal transduction. Therefore, oestradiol stimuli might be able to interfere with the action of antitumoral substances directed against receptor tyrosine kinase signalling. We investigated the effect of oestradiol on the inhibition of HER2 signalling by trastuzumab (Herceptin™) in two human endometrial adenocarcinoma cell lines. Activation of the extracellular signal-regulated kinase (ERK-1/2), a major mediator of HER2 signalling, was measured by means of western blotting experiments and ERE activation was determined in transient reporter-gene assays. In endometrial Ishikawa and HEC-1A adenocarcinoma cells, HER2 signalling was inhibited by trastuzumab only in the absence of oestradiol. We were able to demonstrate that oestradiol counteracted the inhibitory effects of trastuzumab by rapid phosphorylation of ERK-1/2, a kinase downstream of the HER2 receptor. The pure anti-oestrogen ICI 182,780 was able to restore both the trastuzumab-triggered inhibition of the ERK-1/2 pathway and the antiproliferative action of this substance in Ishikawa cells. Our data suggest that combinations of trastuzumab with anti-oestrogens may be effective in the treatment of endometrial cancers with a positive ER and HER2 receptor status.

Hox proteins activate the IGFBP-1 promoter and suppress the function of hPR in human endometrial cells.

Previous studies have shown that progestin activates the transcription of IGFBP-1 (insulin-like growth factor binding protein-1). Four regions in the IGFBP-1 promotor have been identified to enhance the transcription. Two of the regions, located at -73 to -65 bp and -319 to -311 bp formed identical DNA-protein complexes with the nuclear extracts of endometrial stromal/decidual cells. To identify the binding protein(s) in endometrial cells that interact with these two regions, we have used the TGTCAATTA repeats (-319 to -11 bp of the IGFBP-1 promoter) to screen the human decidual cDNA library by yeast one-hybrid system. We found that Hox A10, HoxA11, HoxB2, HoxB4, and HoxD11 interacted with the TGTCAATTA repeats in yeast cells. Among these hox genes, the full-length coding region of HoxA10, HoxA11, and HoxB4 were used for functional analysis in three types of endometrial cells, undifferentiated endometrial stromal cells, decidual cells (differentiated stromal cells) and endometrial adenocarcinoma cell line (HEC1-B). All these endometrial cells produce IGFBP-1. Transient transfection assay showed that HoxA10 expression vector increased the promoter activity (the IGFBP-1 proximal promoter containing TGC/TCAATTA and two functional PRE sites) in endometrial stromal cells and in HEC-1B cells, but not in decidual cells. HoxB4 enhanced the promoter activity only in decidual cells, while HoxA11 had no apparent effect in all three types of cells. To evaluate whether Hox proteins would interact with progesterone receptor (hPR), cells were transfected with the promoter construct, Hox and hPR expression vectors. hPR alone activated the IGFBP-1 promoter activity, but expression of Hox gene suppressed the activation. Hox proteins also suppressed the hPR enhanced promoter activities of MMTV (containing consensus-PRE sites) and glycodelin (GdA, containing Sp1 site which mediates the hPR function). These data showed that Hox genes selectively activate the transcription of the IGFBP-1 and GdA genes in different types of endometrial cells. Hox genes, however, suppress the hPR enhanced activities. In addition, we found that HoxB4 expression was induced by estrogen and progestin. Other investigators have shown that HoxA10 and 11 were stimulated by progestin. These findings show that Hox proteins are molecular mediators of the steroid hormones during endometrial cell development.

DDT and its metabolites alter gene expression in human uterine cell lines through estrogen receptor-independent mechanisms.

Endocrine-disrupting organochlorines, such as the pesticide dichlorodiphenyltrichloroethane (DDT), bind to and activate estrogen receptors (ERs), thereby eliciting estrogen-like effects. Although ERs function predominantly through activation of transcription via estrogen-responsive elements, both ERs, alpha and ss, can interact with various transcription factors such as activator protein-1 (AP-1). Additionally, estrogens may regulate early signaling events, suggesting that the biological effects of environmental estrogens may not be mediated through classic ER (alpha and ss) activity alone. We hypothesized that known environmental estrogens, such as DDT and its metabolites, activate AP-1-mediated gene transactivation through both ER-dependent and ER-independent means. Using two Ishikawa human endometrial adenocarcinoma cell line variants that we confirmed to be estrogen responsive [Ishikawa(+)] and estrogen unresponsive [Ishikawa(-)], we generated stably transfected AP-1 luciferase cell lines to identify the role of an estrogen-responsive mechanism in AP-1-mediated gene expression by various stimuli. Our results demonstrate that DDT and dichlorodiphenyldichloroethane (DDD) were the most potent activators of AP-1 activity; 2,2-bis(p-chlorophenyl) acetic acid failed to activate. Although stimulated in both Ishikawa(+) and Ishikawa(-) cells by DDT and its congeners, AP-1 activation was more pronounced in the estrogen-unresponsive Ishikawa(-) cells. In addition, DDT, DDD, and dichlorodiphenyldichloroethylene (DDE) could also stimulate AP-1 activity in the estrogen-unresponsive human embryonic kidney 293 cells using a different promoter context. Thus, our data demonstrate that DDT and its metabolites activate the AP-1 transcription factor independent of ER (alpha or ss) status.

Establishment and characterization of a poorly differentiated lethal human endometrial carcinoma cell line (NOU-1) with karyotype 46,XX

Immortal gynecologic cell lines, especially those of endometrial origin are diverse with multiple chromosomal alterations, making the study of origin and molecular genetic characteristics of such neoplasms difficult, if not impossible. We have established a new epithelial, poorly differentiated endometrial adenocarcinoma cell line (NOU-1) with a 46,XX chromosome complement. To confirm the tumor characteristics, we injected this cell line subcutaneously into the nude mouse. The tumor grown in vivo had the identical histology and the same 46,XX chromosome complement as the original tumor excised from the patient. The cells from the original tumor and those in the nude mouse shared the same characteristics with respect to histopathology, immunohistochemistry, steroid hormone receptor content, and growth characteristics. We report on the only established cell line of a highly aggressive, poorly differentiated endometrial adenocarcinoma with 46,XX chromosome complement where both estrogen and progesterone receptors are absent.

Effekte von Kinase-Inhibitoren auf die Östradiolwirkung in der endometrialen Adenokarzinomzelllinie Ishikawa

Purpose: Interactions between signal transduction cascades involving tyrosine kinase and its receptor and the cellular response to stimulation with estradiol have received a great deal of study. We studied the effect of inhibitors of receptor tyrosine kinases and cytoplasmatic kinases on the cellular response to estradiol in an endometrial adenocarcinoma cell line that expresses estrogen receptors-alpha. Material and Methods: Estrogen effects were measured with a transient reporter gene assay. Ishikawa endometrial adenocarcinoma cells were cultured without serum and transfected with the vector pERE-TA-SEAP via lipofectamines. The vector contains the secreted alkaline phosphatase (SEAP) reporter gene under the control of an estrogen response element (ERE). After transfection the cells were treated with estradiol, the EGFR inhibitor AG1478, and the MEK inhibitors PD98059 and U0126 for 48 hours. The activity of the EREs was quantified with luminometric measurement of the SEAP concentration in the culture supernatant. Cellular proliferation was measured with an ELISA that quantified the uptake of BrDU into replicating DNA. Results: Physiologic estradiol stimuli caused activation of the ERE. Simultaneous treatment with the EGFR inhibitor AG1478 significantly increased the estradiol-induced activation of ERE. In contrast, both MEK inhibitors reduced both ERE activation and estradiol-induced cellular proliferation. Conclusion: Treatment with the EGFR inhibitor AG1478 increased the response of endometrial adenocarcinoma cells to estrogen stimuli. MEK inhibitors can block MAP kinase signal transduction and inhibit cellular estradiol effects. This suggests that inhibitors of cytoplasmatic kinases may be more useful than EGFR inhibitors in the treatment of endometrial cancers that express estrogen receptors.

Hydrosalpinx fluid diminishes endometrial cell HOXA10 expression

Objective: To determine the effect of hydrosalpinx fluid on the expression of HOXA10, an essential regulator of endometrial receptivity. Design: In vitro study. Setting: Academic medical center. Patient(s): Patients with unilateral or bilateral hydrosalpinx. Intervention(s): Hydrosalpinx fluid was aspirated from 10 patients at laparoscopy. The fluid was serially diluted in minimum essential medium. Ishikawa cells (an endometrial adenocarcinoma cell line, representative of endometrial epithelium) were incubated with this fluid at concentrations of 10% and 50% for 48 hours. Cells were also incubated in undiluted minimum essential medium (MEM) and in 10% serum as controls. After incubation, the cells were lysed in Trizol, and total RNA was extracted and analyzed by Northern blot using a 32P-labeled HOXA10 riboprobe. A 32P-labeled G3PDH probe was used as a control for loading. Main Outcome Measure(s): HOXA10 mRNA expression. Result(s): HOXA10 mRNA expression in endometrial cells decreased with increasing concentrations of hydrosalpinx fluid. Densitometric analysis of the northern blot revealed that HOXA10 mRNA expression was different from control at both concentrations ( P<.007). Conclusion(s): HOXA10 is necessary for implantation in the murine model. HOXA10 expression is diminished by hydrosalpinx fluid. This effect on HOXA10 is a potential molecular mechanism by which implantation rates are diminished in women with hydrosalpinges.

The Ishikawa Cells from Birth to the Present

More than 20 years have passed since the Ishikawa cell line, a well-differentiated human endometrial adenocarcinoma cell line, was established. Because this cell line bears estrogen and progesterone receptors, the cells have been used in numerous basic research areas such as reproductive biology and molecular science, and has been distributed to more than a hundred institutes. However, even the Ishikawa cells, after long-term culture, tend to transform into undifferentiated cells. In addition, it has been reported that estrogen and progesterone receptors disappeared from the cells that I distributed. I therefore attempted to establish well-differentiated cells from the parent Ishikawa cells and to produce a new and good quality supply of this cell line. I believe that it is very important for the investigator who established a cell line to be responsible for maintaining the quality of the cells. That is why I have not deposited this cell line in any cell bank. I would like to take this opportunity to report the history of Ishikawa cells from establishment to the present.

Estradiol up-regulates estrogen receptor messenger ribonucleic acid in endometrial carcinoma (Ishikawa) cells by stabilizing the message.

Estrogens up-regulate expression of the estrogen receptor alpha (ER) gene in most mammalian tissues studied. Using the ovariectomized ewe as a model, we determined that estradiol (E(2)) acted post-transcriptionally to increase endometrial ER mRNA concentrations by enhancing the stability of the message. The purpose of this study was to determine whether a similar E(2) effect occurs in Ishikawa cells, a well-differentiated human endometrial adenocarcinoma cell line. The presence and function of ER protein in Ishikawa cells was demonstrated by transactivation of a transfected plasmid (ERE(2)tkCAT) in response to 10(-)(9) M E(2), resulting in a 550% increase in reporter gene RNA. Ishikawa cells also responded to E(2) by up-regulating their ER mRNA concentration an average of 100% between 7 and 24 h of treatment. The effect of E(2) on ER mRNA stability was measured after blocking transcription with actinomycin D to find that the half-life increased from 6 to 10 h in control and E(2)-treated cells respectively. These results are consistent with cell-free studies which showed significant enhancement of the half-life of radiolabeled ER 3' untranslated region (3'UTR) RNA in extracts from E(2)-treated cells versus those from control cells. Thus, Ishikawa cells provide a relevant model system for the study of E(2)-regulated endometrial gene expression.

Regulation of endometrial adenocarcinoma cell proliferation by Activin-A and its modulation by 17β-estradiol

A role for activins in regulating cellular transformation is suggested by the α-inhibin knockout mouse in which development of gonadal tumors is associated with elevated activin levels. It was the purpose of the current study to determine whether activin had similar actions on endometrial cell lines, specifically on a well differentiated estrogen-responsive endometrial adenocarcinoma cell line (ISH) and estrogen-unresponsive cells (HEC-50) obtained from a poorly differentiated endometrial adenocarcinoma. Activin was secreted by both adenocarcinoma cell lines. Using reverse transcription-PCR, messenger RNA type I and type II activin receptor subtypes were detected in both cell lines: expression of IB and IIB was approximately three- to fourfold greater in ISH cells than in HEC-50 cells, while activin receptor IA and IIA messenger RNA levels were approximately equal in both cell lines. Activin treatment (30–300 ng/ml) caused a dose- and time-dependent inhibition of ISH cells proliferation and resulted in a significant decrease in Bcl-2 protein and mRNA levels. No difference was observed in Bax expression. There was no significant effect of activin when the cultures of ISH cells were exposed to 17β-estradiol. In contrast, activin showed a weak, but significant, mitogenic effect on HEC-50 cells without modifications in Bax and Bcl-2 mRNA and protein levels. The results demonstrate that activin is a regulator of endometrial cancer cell growth. 17β-Estradiol may promote resistance of estrogen-responsive endometrial cancer cells to the growth-retarding effects of activin and one of the mechanisms might be a down-regulation of the activin receptors.

琵琶湖‐淀川水系河川水のバイオアッセイによる包括的毒性評価

The present study was carried out to evaluate comprehensively the estrogenicity, immunotoxicity and mutagenicity of environmental water concentrates prepared by the XAD-2 resin column concentration method. This investigation focused on water from the river basin of Lake Biwa-Yodo River. Estrogenicity was detected by the yeast two-hybrid system and the measurement of alkaline phosphatase activity in human endometrial adenocarcinoma cell line (Ishikawa cells). Although the two methods differed in sensitivity and magnitude of estrogenic activity for lake water and river water containing endocrine-disrupting chemicals (EDCs), estrogenicity was detected in all sampling points except the Katsura River (Miyamae bridge). These results suggested the co-existence of materials with EDCs in environmental water, which inhibited the detection of estrogenic activity. Immunotoxicity was evaluated by the mouse splenic lymphocyte mitogenesis test using lipopolysaccharide and concanavalin A as specific mitogens for B cells and T cells, respectively. Some of the water samples showed specific depression effect on B cell and T cell mitogenesis. Furthermore, the Salmonella/microsome assay (Ames test) with TA98 and TA100 strain series was used to determine the mutagenicity. In all the sampling points, the direct/indirect-frameshift mutagenicity was detected. These results indicated that the yeast two-hybrid system and the measurement of alkaline phosphatase activity in Ishikawa cells, the lymphocyte mitogenesis test and the Ames test are important for evaluating comprehensively each endpoint because there is no relevance among these different toxicities.