Human Endometrial Adenocarcinoma Cell Line Research Articles

Aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling in endometriosis.

Are STAT3 signaling molecules differentially expressed in endometriosis? Levels of phospho-STAT3 and HIF1A, its downstream signaling molecule, are significantly higher in eutopic endometrium from women with endometriosis when compared with women without the disease. Endometriosis is an estrogen-dependent inflammatory condition. Interleukin 6 (IL-6) is an inflammatory survival cytokine known to induce prolonged activation of STAT3 via association with the IL-6 receptor. Cross-sectional measurements of STAT3 and HIF1A protein levels in eutopic endometrium from women with endometriosis versus those without. Levels of phospho-STAT3 (pSTAT3) and HIF1A were examined in the endometrium of patients with and without endometriosis as well as in a non-human primate animal model using western blot and immunohistochemical analysis. Levels of pSTAT3 were significantly higher in the eutopic endometrium from women with endometriosis when compared with women without the disease in both the proliferative and secretory phases. HIF1A is known to be stabilized by STAT3 and IL-6. Our immunohistochemistry results show abundant HIF1A expression within the eutopic endometrial epithelial cells of women with endometriosis. Furthermore, pSTAT3 and HIF1A proteins are co-localized in endometriosis. This aberrant activation of pSTAT3 and HIF1A is confirmed by sequential analysis of eutopic endometrium using a baboon animal model of induced endometriosis. Lastly, we confirmed this IL-6 induction of both STAT3 phosphorylation and HIF1A mRNA expression in Ishikawa human endometrial adenocarcinoma cell line. Ishikawa cancer cell line was used to study a benign disease. The peritoneal fluid contains various inflammatory cytokines in addition to IL-6 and so it is possible that other cytokines may affect the activity and expression of STAT3 signaling molecules. Our results imply that aberrant activation of STAT3 signaling plays an important role in the pathogenesis of endometriosis. Our findings could progress in our understanding of the etiology and pathophysiology of endometriosis and potential therapeutic interventions by targeted pharmacological. This work was supported by NIH R01 HD067721 (to S.L.Y and B.A.L) and NIH R01 HD057873 and American Cancer Society Research Grant RSG-12-084-01-TBG (to J.-W.J.). There are no conflicts of interest.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca2+ entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca2+ emptying triggered by the inhibition of the sarco(endo)plasmic Ca2+-ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca2+, indicating that both events are upstream steps for Ca2+ entry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca2+ channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

In vitro cytotoxicity of 4'-OH-tamoxifen and estradiol in human endometrial adenocarcinoma cells HEC-1A and HEC-1B.

Tamoxifen (TAM), used to treat estrogen receptor (ER)-positive breast cancer, is a well known estrogen antagonist in the breast, but a partial estrogen agonist in the endometrium. In addition, TAM metabolites, such as 4'-hydroxy-tamoxifen (4-OH-TAM), have been shown to be more potent than the parent compound. The objective of this study was to determine the effects of 4-OH-TAM and estradiol (E2) on two human endometrial adenocarcinoma cell lines, HEC-1B and HEC-1A. When HEC-1B cells were treated with lower concentrations (10-1,000 nM) of 4-OH-TAM or E2 for 1-3 days, no significant difference in the percentage of cell survival was observed among the varying concentrations. At higher 4-OH-TAM or E2 concentrations (1-100 µM), HEC-1B and HEC-1A cells responded similarly with an obvious decrease in cell growth noted at 10 and 100 µM 4-OH-TAM and 100 µM E2. In order to address the observed cell death, DNA laddering was performed at various time intervals with 4-OH-TAM (10 µM) or E2 (10 or 50 µM). DNA gel electrophoresis failed to show the typical laddering pattern (180-200 bp) observed in apoptosis. Furthermore, western blot analysis of caspase-8 and -3 failed to demonstrate caspase activity. These results suggest that apoptosis was not the underlying cellular mechanism of cell death. Due to the lack of apoptotic markers, a cytotoxic (cell death) effect was differentiated from a cytostatic (growth inhibition) effect confirming that cell death had occurred. In summary, micromolar concentrations of 4-OH-TAM induced a non-apoptotic cytotoxic effect in the endometrium; however further studies are needed to elucidate the cytotoxic pathway being utilized.

MiR-200a is involved in proliferation and apoptosis in the human endometrial adenocarcinoma cell line HEC-1B by targeting the tumor suppressor PTEN

Abnormal cell proliferation is a main driver of tumor formation and development, which involves the deletion, mutation, and downregulation of tumor suppressor genes. One study recently demonstrated that miR-200a plays an oncogenic role by inhibiting phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression. In the human endometrial adenocarcinoma cell line HEC-1B, suppression of miR-200a expression inhibited cell proliferation and promoted apoptosis, whereas its over-expression had no effect on proliferation and apoptosis. Furthermore, inhibition or over-expression of miR-200a increased or reduced the expression of PTEN, respectively, with no change in PTEN mRNA levels. These effects were achieved by directly targeting miR-200a to the 3' untranslated region of the PTEN mRNA to inhibit its translation. Taken together, we propose that in HEC-1B cells, miR-200a functions as an oncogene, affecting proliferation and apoptosis by regulating the expression of the tumor suppressor PTEN at the translational level.

Response-specific progestin resistance in a newly characterized Ishikawa human endometrial cancer subcell line resulting from long-term exposure to medroxyprogesterone acetate

Progestins, particularly medroxyprogesterone acetate (MPA), have for a long time been used as conservative treatment for young patients with clinical stage I, grade I endometrial carcinoma. However, more than 30% of patients with endometrial adenocarcinoma display resistance to endocrine therapies at the time of presentation and most cancer patients that initially respond to progestin treatment will at some point develop resistance, resulting in tumor progression. The cellular mechanisms underlying acquired resistance to progestin are poorly understood. In order to investigate the molecular mechanisms whereby human endometrial adenocarcinoma develops resistance to progestin therapy, we have undertaken to develop human endometrial adenocarcinoma cell lines that are resistant to the growth-inhibitory effects of progestins in vitro. A progestin-resistant subcell line of Ishikawa cells was developed from Ishikawa human endometrial adenocarcinoma cells by stepwise selection in increasing concentrations of the synthetic progestin, MPA, over ten months. The doubling time of the progestin-resistant cells (34.18±3.15 h) grown routinely in the medium containing 10 μM MPA was not significantly different from the doubling time of the parent Ishikawa cells (35.14±2.68 h) grown in the absence of MPA (t=-0.331, P=0.762). Moreover, the effect of treatment with MPA shifted from suppression of growth and invasiveness, as observed in the parent Ishikawa cells, to stimulation of growth and invasiveness in the progestin-resistant Ishikawa cells. The positive rates of estrogen receptor a (ERα) and progesterone receptor B (PRB) of the progestin-resistant Ishikawa cells were significantly reduced, whilst the positive rate of ERβ was significantly enhanced compared to the parent Ishikawa cells. These differences were statistically significant (P<0.05). Our results indicate that long-term treatment with MPA in Ishikawa cells may give rise to a resistance effect to MPA. When the resistant subtype is acquired, treatment with MPA enhances cancer cell proliferation and invasiveness. The imbalance of ER and PR subtypes may contribute to the mechanisms involved in progestin resistance. Determination of the subtypes of ER and PR may provide important additional information on the hormone sensitivity of endometrial carcinoma.

Antiproliferative and apoptotic effect of epigallocatechin-3-gallate on Ishikawa cells is accompanied by sex steroid receptor downregulation

Endometrial cancer is a significant malignancy in developed countries. Unopposed estrogen stimulation is considered as an important risk factor for endometrial cancer. Epigallocathechin-3-gallate (EGCG), biological active component of green tea, inhibits cancer cell proliferation. However, it is unknown whether EGCG has anticancer effects on endometrial cancer and what the molecular mechanism(s) are. We investigated the anticancer effects of EGCG on a human endometrial adenocarcinoma cell line (Ishikawa cells) with or without 17β-estradiol (E2) treatment. Cell proliferation assay was performed using 3-(4,5-dimethylthiaxol-2-yi)-2,5-diphenyltetraxolium bromide (MTT). The cell cycle was determined by flow cytometry and real-time analysis of cyclin and cdk genes. The apoptosis was measured by Annexin V-PI staining and real-time analysis of bcl-2, Bax and caspase genes. The MAPK signal, Akt and caspase-3 were determined by immunoblotting. Decreased estrogen and progesterone receptor expression was observed in EGCG-treated Ishikawa cells, and decreased MAPK signals and phospho-Akt were observed as well. EGCG caused the arrest of cells in the G0/G1 phase of the cell cycle. This compound interfered with Akt activation and MAPK signals, and increased apoptosis signals leading to a controlled caspases, Bcl-2, Bax genes and protein expression. Taken together, EGCG inhibits cell proliferation and induces apoptosis through Akt and MAPK signals. These findings suggest that EGCG may exert growth-inhibitory and apoptosis-inducing effects on endometrial cancer cells, accompanied by decreased estrogen and progesterone receptor. EGCG may have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in endometrial cancer.

Decreased carbonyl reductase 1 expression promotes malignant behaviours by induction of epithelial mesenchymal transition and its clinical significance

Human carbonyl reductase 1 (CBR1) is an enzyme that catalyse the reduction of many compounds by using NADPH-dependent oxydoreductase activity. Although CBR1 is known to regulate the tumour progression, the molecular mechanisms of CBR1 in cancer progression and the clinical significance of CBR1 status remain unclear. Here, we investigated the molecular mechanisms by which CBR1 affects cancer cell behaviour in vitro and the clinical significance of CBR1 using immunohistochemical analyses in endometrial cancer. Here, the role of CBR1 in cancer cell invasion and metastasis, and its molecular mechanisms were investigated by transfection of sense and antisense CBR1 cDNAs into a human endometrial adenocarcinoma cell line. The relationship between CBR1 expression analysed by immunohistochemistry and prognosis such as progression free survival (PFS) and overall survival (OS) was examined in endometrial cancer tissues from FIGO stage I–IV (n=109). Suppression of CBR1 by antisense CBR1 cDNA increased cancer cell invasion, and suppressed E-cadherin expression and capacity for cellular aggregation. In contrast, over-expression of CBR1 by sense CBR1 cDNA increased E-cadherin expression and capacity for cellular aggregation, and suppressed cancer cell invasion. The expression of transcriptional suppressors of E-cadherin, Snail and ZEB1, were increased by CBR1 suppression, but suppressed by CBR1 over-expression. Immunohistochemical analyses showed that decreased CBR1 expression is significantly related with poor PFS and OS compared with strong CBR1 expression. In multivariate analyses, decreased CBR1 expression was an independent prognostic factor for PFS and OS. CBR1 regulates cancer cell invasion in endometrial adenocarcinomas by regulating the epithelial mesenchymal transition. A decreased CBR1 expression can be a useful marker of an unfavourable clinical outcome in patients with endometrial cancer.

Role of prostaglandin E2 in the experimental L-carnitine-induced endometriosis-like inflammatory model

Purpose Ectopic implantation of endometrium leading to endometriosis has been correlated with various immune and non-immune parameters. Inflammatory cytokines including IL-2, IFN-γ, TNF-α, increase in serum and peritoneal fluid of endometriotic patients, while elevated levels of prostaglandins PGE1 and PGE2 have been accused of facilitating the inflammation process. Using the experimental model of L-carnitine (L-Cn)-induced endometriosis, the aim of this study was to explore the role of PGE1 and PGE2 in endometriosis development and define whether L-Cn had a direct effect on endometrial cells. Methods During treatment with L-Cn, experimental mice were injected with indomethacin or specific PGE1 and PGE2 analogs and their effect on the L-Cn-induced inflammatory state was examined. Primary uterine cells or human endometrial adenocarcinoma cell lines were used to assess the direct or indirect effect of L-Cn. Results The seven-day treatment of virgin females with L-Cn induced obvious endometrial abnormalities with accumulation of CD90 and CD11 cells. Administration of indomethacin alleviated some of the L-Cn-induced effects, but could not inverse the phenotype. Interestingly, 11-deoxy-PGE1 aggravated the L-Cn-induced effects, whereas 17-phenyl-trimor-PGE2 reversed them. In vitro experiments showed that the effect of L-Cn depended on the nature of endometrioma cells. L-Cn could aggravate the inflammatory state of Ishikawa-inoculated mice, but could alleviate symptoms from MFE-319 inoculated females. Conculsions Although PGE2 appeared to play a key role in the L-Cn-induced uterus inflammation, the actual role of L-Cn depends on the nature of endometrioma cells and can aggravate or protect from inflammation according to the induced regulatory pathways.

Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.

Functional and Physical Interaction Between the Mismatch Repair and the FA-BRCA Pathways.

AbstractAbstract 3370 Introduction:Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, an increased risk for cancer and leukemia, and congenital abnormalities. Thirteen FA genes have been identified that when mutated result in hypersensitivity to DNA crosslinking agents. Therefore, components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). The monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI, is considered the hallmark of FA pathway activation. It has been reported that FANCJ interacts with the mismatch repair (MMR) complex MutLα and we have previously shown that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation of FANCD2 and FANCI. Interestingly, mismatch repair deficiency is also associated with leukemia in humans and a defect in hematopoietic repopulation in mouse models, suggesting a possible functional overlap between the FA-BRCA and MMR pathways. Methods:Cell lines used: HeLa, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agents mitomycin C (MMC), cisplatin (CDDP), or the alkylating agent temozolomide (TMZ). FANCD2 foci formation was assessed using immunofluorescence. Immunoprecipitation was used to demonstrate interactions between FANCD2, MSH2, and MLH1. Survival assays were performed by crystal violet staining and extraction. Chromosome breakage analysis was performed using metaphase spreads. FANCD2 RNAi flies, spel1-/- flies (MSH2-deficient), and double mutants were treated with diepoxybutane (DEB) and assessed for survival and mutagenicity. Mismatch binding EMSAs were performed using Cy5-labeled matched and mismatched 29-mers. Result:FANCD2 foci formation and chromatin loading is greatly diminished in MSH2-deficient cells, while cells lacking MLH1 show no effect, indicating a requirement for MSH2 in the activation of the FA-BRCA pathway and a possible downstream role for MLH1 in ICL repair. Human and mouse cells lacking MSH2 or human cells lacking MLH1 display increased sensitivity to mitomycin C and cisplatin as compared to their corrected counterparts as well as increased radial formation upon treatment with MMC. Studies in both human cell lines and Drosophila mutants indicate an epistatic relationship between FANCD2 and MSH2 with regards to survival, chromosome breakage, and mutagenicity after treatment with crosslinking agents. MSH2 is required for the interaction between FANCD2 and MLH1, but surprisingly, presence of MLH1 appears to enhance the interaction between FANCD2 and MSH2. Furthermore, the interaction between MSH2 and MLH1 after treatment with MMC is reduced in multiple FA cell lines. FA cell lines also display hypersensitivity to alkylating agents such as temozolomide. These results have led us to examine FA cell lines for a defect in mismatch repair through mismatch binding EMSAs and plasmid-based assays. Conclusion:These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway. Conversely, FA proteins appear to be required for interactions between mismatch repair factors and may also be involved in the repair of DNA mismatches, suggesting significant crosstalk between the FA and MMR pathways. Understanding this complex interplay could lead to the development of new therapies for the treatment of patients both with FA and cancer. Disclosures:No relevant conflicts of interest to declare.

Death-associated protein kinase is essential for the survival of various types of uterine cancer cells

We recently showed that targeted knockdown of death-associated protein kinase (DAPK) expression induces apoptosis in the human endometrial adenocarcinoma cell line HHUA. To investigate the possibility that DAPK may represent a molecular target for anticancer therapies for advanced uterine cancers, we examined the effects of DAPK siRNA transfections on the viability of five different human uterine cancer cell lines. The five uterine cell lines comprised three differentiated endometrial adenocarcinomas, one leiomyosarcoma and one carcinosarcoma. Cell death assays showed that the DAPK siRNA transfection significantly increased the cell death in all five uterine cancer cells examined. Ribonuclease protection assays did not show any remarkable changes in the bcl-2 family gene expressions after the DAPK siRNA transfection in HHUA cells. Since DAPK-mutant mice were reported to be fertile and do not show lethality, DAPK may play a central role in the immortalization and carcinogenesis of uterine cancer cells, possibly without bcl-2 family-related apoptotic regulation. These results indicate that DAPK can be a convincing candidate for molecularly targeted anticancer therapies for patients with various types of advanced uterine cancers, including carcinosarcoma and leiomyosarcoma.

Effects of estradiol-17 beta and progesterone on cell proliferation and differentiation of the human endometrial carcinoma cell line (HHUA) in vitro.

The effects of estradiol-17β and progesterone on cell proliferation and differentiation in the human endometrial adenocarcinoma cell line (HHUA) were studied in vitro at a range of concentration from 10-9 to 10-4 M. Cell proliferation of the HHUA line was found to be promoted by the administration of estradiol-17β at concentrations of 10-9 -10-8 M, while it was suppressed at more than 10-7 M. On the other hand, progesterone showed a dose-dependent suppressive effect on cell growth. The cultured cells showed no remarkable morphological changes in the es-tradiol-17β (10-9 -10-6 M)-treated groups, while they arranged themselves in a papillary or gland-like pattern in the progesterone (10-8 -10-6 M)-treated groups in the in vitro monolayer culture system. The control or estradiol-17β-treated cultures showed compact cell aggregation, while progesterone-treated ones showed gland-like structures and cyst formations in the rotation culture system. Based on this data, we concluded that estradiol-17β promotes the proliferation of endometrial carcinoma cells, while progesterone, on the contrary, suppresses their proliferation and induces differentiation.

HOXA10 regulates endometrial GABAAπ receptor expression and membrane translocation

Expression of the GABA(A) pi receptor has been described previously in the human endometrium in both luminal epithelium and stroma. Its expression is increased during decidualization in rodents and in the implantation window of human endometrium. Here we localized GABA pi subunit receptor protein in human endometrium and identified regulators of gene expression and activation. GABA(A) pi was localized to the cell surface, and expression increased during the window of embryo implantation in human endometrium. The well-differentiated human endometrial adenocarcinoma cell line Ishikawa was treated with progesterone and transfected with pcDNA-HOXA10, HOXA10 siRNA, or respective controls. GABA(A) pi receptor mRNA expression was evaluated by real-time RT-PCR. Protein expression and localization were evaluated using immunofluorescence. GABA(A) pi receptor mRNA expression was increased significantly after either progesterone treatment or HOXA10 transfection. Coadministration of progesterone along with HOXA10 transfection had no additional effect on the expression of GABA(A) pi receptor mRNA over either agent alone. Blocking HOXA10 expression with siRNA prevented progesterone-induced GABA(A) pi receptor mRNA expression. Additionally, either HOXA10 or progesterone independently caused increased translocation of the GABA receptor from the cytoplasm to the cell membrane. Translocation in response to progesterone was blocked with HOXA10 siRNA. Progesterone-induced GABA(A) pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of HOXA10 expression. Modification of subtype composition and translocation of the GABA receptor ion channel likely modulate endometrial receptivity. Whereas HOXA10 typically enhances the expression of progesterone-responsive genes, here HOXA10 expression leads to production of a less progestin-responsive GABA receptor subtype, likely buffering the effects of luteal phase progesterone on GABA receptor activity.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

A Role for Mismatch Repair Factors in the FA-BRCA Pathway.

Abstract 1095Poster Board I-117 IntroductionFanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI. MethodsCell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot. ResultsThrough chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls. oConclusionsThese data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer. DisclosuresNo relevant conflicts of interest to declare.

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

The Genomic Response of a Human Uterine Endometrial Adenocarcinoma Cell Line to 17α-Ethynyl Estradiol

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p </= 0.0001). The annotation available for the genes affected indicates that EE exposure results in changes in multiple molecular pathways affecting various biological processes, particularly associated with development, morphogenesis, organogenesis, cell proliferation, cell organization, and biogenesis. All of these processes are also affected by estrogen exposure in the uterus of the rat. Comparison of the response to EE in both the rat uterus and the Ishikawa cells showed that 71 genes are regulated in a similar manner in vivo as well as in vitro. Further, some of the genes that show a robust response to estrogen exposure in Ishikawa cells are well known to be estrogen responsive, in various in vivo studies, such as PGR, MMP7, IGFBP3, IGFBP5, SOX4, MYC, EGR1, FOS, CKB, and CCND2, among others. These results indicate that transcript profiling can serve as a viable tool to select reliable in vitro systems to evaluate potential estrogenic activities of target chemicals and to identify genes that are relevant for the estrogen response.

Selective Gene Transfer to Endometrial Cancer Cells by a Polymer Against Matrix Metalloproteinase 2 (MMP-2)

A novel cancer-cell-specific gene delivery vector with high transfection efficiency was designed and tested with an in vitro coculture consisting of the human endometrial adenocarcinoma cell line, HEC-1A cells, and normal endometrial stromal cells. For the cancer-cell targeting, polyethylenimine (PEI), a cationic polymer that can be easily combined with anionic DNA to form a particulate complex, polyplex, being capable of transferring a gene into a variety of cells, was covalently conjugated with antibodies against matrix metalloproteinase 2 (MMP-2), a typical surface-marker protein on cancer cells known for its close correlation with angiogenesis and invasion in many types of cancer, using the heterofunctional cross-linker, n-succinimidyl 3-(2-pyridyldithio)-propionamide. Biophysical properties and transfection efficiencies of anti-MMP-2-conjugated PEI were analyzed by means of dynamic light scattering, laser Doppler anemometry, and flow cytometry. Our results reveal that (1) the PEI-anti-MMP-2 antibody conjugate maintains physical parameters, including sizes and surface charges, which appear to be favorable for gene transfer and (2) when the pEGFP-N3 plasmid complexes of the PEI-anti-MMP-2 antibody conjugate are applied to the coculture consisting of HEC-1A cells and human stromal cells, a high level of green fluorescent protein expression occurs in HEC-1A cells over stromal cells, suggesting a specific gene transfer targeting cancer cells. Therefore, targeting invading cancer cells with the PEI-anti-MMP-2 antibody conjugate could be promising in endometrial cancer treatment, and this gene delivery system deserves further optimization in the context of targeted therapeutic gene delivery.

Histone deacetylase inhibitor-induced glycodelin enhances the initial step of implantation

The complex molecular pathways governing implantation are unclear and ethical limitations limit studies in humans. Reversible histone acetylation regulates gene transcription and histone deacetylase inhibitors (HDACI) induce specific genes. Suberoylanilide hydroxamic acid (SAHA), a HDACI recently approved as an anti-cancer drug, induces the morphological and functional differentiation of human endometrial gland cells through up-regulation of glycodelin, a secretory phase dominant protein. We investigated whether SAHA improves implantation in an in vitro implantation assay using the human endometrial adenocarcinoma cell line, Ishikawa and the choriocarcinoma cell line, JAR. In an in vitro implantation assay, JAR spheroids attached and adhered to Ishikawa cells in a time dependent manner. Glycodelin induction, following treatment with ovarian steroid hormones or SAHA, enhanced implantation. The improvement in implantation was also obtained when glycodelin was overexpressed without stimulation and was almost completely abrogated by glycodelin gene silencing. This study demonstrates that glycodelin is a key regulatory protein of implantation and suggests that SAHA may have a capacity to supplant steroid derivatives in the treatment of infertility.

Stomatin-like protein 2 is overexpressed and related to cell growth in human endometrial adenocarcinoma

Stomatin-like protein 2 (SLP-2) is a novel and unusual stomatin homologue of unknown functions. It was first identified to be overexpressed and involved in regulating cell growth and cell adhesion in human esophageal squamous cell carcinoma. We show herein the involvement of SLP-2 in human endometrial adenocarcinoma, and the effects of SLP-2 on endometrial adenocarcinoma cell growth. The expression of SLP-2 was evaluated in human endometrial adenocarcinoma by semi-quantitative RT-PCR, Westernblotting and immunohistochemistry. Sense and antisense SLP-2 eukaryotic expression plasmids were transfected into the human endometrial adenocarcinoma cell line HEC-1B. MTT assay and flow cytometry assay were performed to investigate the roles of the SLP-2 gene. SLP-2 was overexpressed in endometrial adenocarcinoma compared with their normal counterparts (P<or=0.05). Immunohistochemistry showed that SLP-2 was mainly localized in the cytoplasm with some distribution on the membrane. HEC-1B cells transfected with antisense SLP-2 showed decreased cell growth, whereas the cell growth increased with the sense transfection. SLP-2 was first identified as a novel cancer-related gene overexpressed in human endometrial adenocarcinoma. Cell growth changes with the sense and antisense transfection revealed that SLP-2 might be important in endometrial tumorigenesis.