Human Endometrial Adenocarcinoma Cell Line Research Articles

Identification of a Signaling Pathway Involving Progesterone Receptor, Calcitonin, and Tissue Tranglutaminase in Ishikawa Endometrial Cells

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium (Ca(2+)) homeostasis, is transiently induced by steroid hormone progesterone (P) in the uterine epithelia of the rat and human within the window of implantation. Targeted disruption of uterine CT expression markedly impaired implantation in the rat. To gain insight into the molecular events underlying CT action in the endometrium, we performed gene expression profiling in response to CT in a human endometrial adenocarcinoma cell line, Ishikawa. We identified the gene encoding tissue tranglutaminase type II (tTGase), which participates in Ca(2+)-dependent, protein-protein cross-linking, as a downstream target of CT. Interestingly, addition of P alone to Ishikawa cells led to a marked induction in the level of both CT and tTGase, indicating the existence of a pathway involving P receptors, CT, and tTGase in these cells. Other studies revealed that regulation of the tTGase gene by CT occurs via its cell surface receptor and uses both cAMP and Ca(2+) signaling pathways. We also noted that tTGase protein is expressed in human endometrium during the P-dominated midsecretory phase of the menstrual cycle, and it is localized at the basal membrane of glandular epithelium and the surrounding stroma. The spatio-temporal expression of tTGase in human endometrium during the cycle closely overlapped with that of CT. In summary, we have uncovered a novel steroid-regulated signaling cascade in which P induces CT, which, in turn, induces tTGase and potentially plays a critical role in the human endometrium during implantation.

Estrogenic and clastogenic potential of the mycotoxin alternariol in cultured mammalian cells

The mycotoxin alternariol (AOH) is found in food and beverages infected by Alternaria alternata. Because consumption of foodstuffs contaminated with A. alternata has been implicated in an elevated incidence of esophageal carcinogenesis, we have investigated the estrogenic potential, the effect on cell proliferation, and the genotoxic effect of AOH in cultured mammalian cells. AOH replaced E2 from isolated human estrogen receptors α and β and increased the level of alkaline phosphatase (ALP) mRNA and the enzymatic activity of ALP in a human endometrial adenocarcinoma cell line (Ishikawa cells). The estrogenicity of AOH was about 0.01% of that of E2. The effects in Ishikawa cells were reversed by the ER antagonist ICI 182,780. Analysis of cell proliferation by flow cytometry and microscopy of Ishikawa and Chinese hamster V79 cells revealed that AOH inhibited cell proliferation by interference with the cell cycle. The genotoxic potential was assessed by the micronucleus (MN) assay and immunochemical differentiation between MN containing whole chromosomes (kinetochore-positive) and DNA fragments (kinetochore-negative) in Ishikawa and V79 cells. AOH induced kinetochore-negative MN in both cell lines. This is the first report on the estrogenic potential, inhibition of cell proliferation and clastogenicity of AOH in Ishikawa and V79 cells in vitro.

Histone Deacetylase Inhibitors Induce Differentiation of Human Endometrial Adenocarcinoma Cells through Up-Regulation of Glycodelin

Histone reversible acetylation, which is controlled by histone acetyltransferases and deacetylases, plays a fundamental role in gene transcription. Histone deacetylase inhibitors (HDACIs), such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have been characterized not only as anticancer drugs, but also as cytodifferentiation-inducing agents. In human endometrium, postovulatory production of progesterone directs estrogen-primed endometrial glandular cells to differentiate and thereby produce a number of unique bioactive substances, including glycodelin, that are critical for implantation at the secretory phase of the menstrual cycle. In this study, we show that TSA and SAHA, belonging to the hydroxamic acid group of HDACIs, can induce the phenotype of a human endometrial adenocarcinoma cell line, Ishikawa (originally derived from the glandular component of the endometrium), to differentiate to closely resemble normal endometrial epithelium in a time- and dose-dependent manner, as determined by morphological changes, synthesis of glycogen, and expression of secretory phase-specific proteins, including glycodelin. The proliferation- and differentiation-modulating effects elicited by TSA and SAHA at their optimal concentrations were comparable or more potent than those exerted by combined treatment with progesterone and estradiol. Furthermore, the gene silencing of glycodelin by small interference RNA resulted in the blockade of HDACI-induced differentiation in Ishikawa cells, suggesting the requirement for glycodelin for endometrial epithelial differentiation. Our results collectively indicate that TSA and SAHA are potent differentiation inducers for endometrial glandular cells, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting glycodelin.

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.

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.

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.

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.

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.

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.

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.

Ligand activated hPR modulates the glycodelin promoter activity through the Sp1 sites in human endometrial adenocarcinoma cells

Human endometrium produces glycodelin-A (GdA). The GdA mRNA is highly expressed in progestin-sensitized human endometrial glandular epithelial cells. The mechanism of GdA gene expression, however, is not clear. To understand the cell specific GdA gene transcription, our first approach was to identify the cis-element in the GdA promoter using transfection assay in a human endometrial adenocarcinoma cell line (HEC-1B, a cell line originally derived from the glandular component of the endometrium). The GdA promoter (−1900 to +20 bp) was linked to the luciferase reporter gene to construct p1900Luc, along with two shorter promoter constructs, p1100Luc and p304Luc. Deletion analysis showed that the basal promoter activity was derived from the region between −304 to +20 bp. This region contains three putative Sp1 binding sites (Sp1-1, −243 to −238 bp; Sp1-2, −207 to −202 bp; and Sp1-3, −56 to −49 bp). Mutation analysis at the Sp1 sites showed that p304Spm2Luc and p304Spm3Luc reduced the activity by 80%, while p304Spm1-2-3Luc reduced the activity by 95%. Sp1-1 mutation, however, had no effect. These results showed that two of the three Sp1 cis-elements mediate the basal promoter activity of the GdA gene. Electrophoretic gel mobility shift showed that at least two specific binding proteins in the nuclear extracts of HEC-1B cells bound to the oligo containing Sp1-2 or Sp1-3 cis-element. Sp1 antibody reduced the specific binding complex by 70% suggesting that Sp1 transcription factor regulates GdA gene expression. In addition, over expression of Sp1 increased the promoter activity. To determine whether progestin would modulate the promoter activity, HEC-1B cells were transfected with p304Luc and with progesterone receptor (either hPR-A or hPR-B) expression vector. Medroxyprogesterone acetate increased the promoter activity (3-fold) derived from p304Luc but not from the mutant, p304Spm1-2-3Luc. In contrast, the promoter activity was slightly reduced in cells treated with estradiol and co-transfected with estrogen receptor expression vector. These data indicate that ligand-activated PR stimulates GdA gene expression mediated through the functional Sp1 sites.

A Newly Characterized Human Endometrial Adenocarcinoma Cell Line (CAC-1) Differentiates in Response to Retinoic Acid Treatment

A new cell line of poorly differentiated human endometrial adenocarcinoma cells termed “CAC-1” cells has been established. These cells are epithelial, as indicated by positive cytokeratin and negative vimentin staining. They are rounded and possess a high nuclear-to-cytoplasmic ratio, desmosomes, surface microvilli, intercelular lumens, and pleomorphic nuclei containing multiple nucleoli. These cells have been in long-term culture for 2 years. Our previous studies demonstrated that moderately differentiated (RL95-2) cells differentiated in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., 1996; Anticancer Res.16, 17–24). CAC-1 cells exhibited a similar response because they also organized actin filaments and enlarged in response to retinoic acid treatment. Concurrently, retinoic acid treatment caused a 40% decrease in cell detachment in an in vitro detachment assay compared to controls. A slight lag in cell growth was observed when CAC-1 cells were treated with 1 μM 13-cis or all-trans retinoic acid during a 12-day growth curve. In addition, we examined the effects of retinoic acid on protein kinase C-α (PKC-α) and myristoylated alanine-rich C-kinase substrate (MARCKS). Treatment with retinoic acid caused cytoplasmic PKC-α to increase concomitant with a decrease in PKC-α in the membrane. In contrast, MARCKS increased in the membrane in response to retinoic acid treatment. These data indicate that retinoid treatment causes inactivation of PKC-α, allowing MARCKS to relocalize to the membrane, where it can cross-link actin filaments. CAC-1 cells represent an ideal model for investigating the effects of retinoids on differentiation induction concomitant with actin reorganization.

Expression of cyclooxygenase 2 by prostaglandin E(2) in human endometrial adenocarcinoma cell line HEC-1B.

The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE(2) in human endometrial adenocarcinoma cell line HEC-1B. One microM PGE(2) could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE(2) also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE(2)-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE(2). PGE(2) could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE(2)-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE(2), and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE(2)-induced activation of MAP kinase in HEC-1B cells.

Kappa opioids and TGFbeta1 interact in human endometrial cells.

The transforming growth factor beta1 (TGFbeta1) is a major regulator of human endometrial function. Human endometrium possesses specific opioid binding sites, the majority of which belong to the kappa type, for which the prodynorphin-derived opioids are the endogenous ligands. Since these two systems interact in several other tissues we postulated that opioids may affect the production of TGFbeta1 in human endometrium. We have found that kappa opioids exerted a time- and dose-dependent inhibitory effect on TGFbeta1 production from endometrial stromal and epithelial cells and from the Ishikawa human endometrial adenocarcinoma cell line. This effect was reversible by the specific opioid antagonist diprenorphine. To examine if this effect represents a paracrine endometrial response to locally produced kappa opioids we searched for the presence of the endogenous kappa opioid receptor ligands. Indeed, the prodynorphin transcript was detectable on Northern blots from normal and tumoral human endometrial cells; its size was that of the pituitary transcript, i.e. approximately 2.4 kb long. Most immunoreactive dynorphin from human endometrium had a molecular weight of 8 kDa. Finally, immunofluorescence staining of normal and tumoral human endometrial cells revealed the presence of dynorphin-positive cytoplasmic secretory granules. Taken together, our data suggest that in human endometrium, kappa opioids and the TGFbeta1 form a paracrine network which appears to be retained by the Ishikawa human endometrial adenocarcinoma cell line.

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.