miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.     

Human endometrial epithelial cell lines for studying steroid and cytokine actions

Summary Recent studies suggest that the proliferation and expression of HLA-DR molecules in endometrial epithelium may be regulated by systemic steroids and local cytokines. To test the interacting influences of cytokines and steroids on the expression of HLA-DR and proliferation of epithelial cells, an endometrial cell model is required that is sensitive to both signals. In this study, we characterize cells of carcinoma cell lines of endometrial lineage for their responsiveness to cytokines and steroids. Independently developed for its response to steroid hormones from a well-differentiated adenocarcinoma of human endometrium, EnCa101AE cell line is further cloned for the expression of progesterone receptor. Immunohistochemical localization using monoclonal antibodies demonstrates that both EnCa101AE cell line and cloned ECC1 cells are purely epithelial, as evidenced by the expression of cytokeratin and epithelial membrane antigen, express estrogen receptors, and concomitantly exhibit IFN-gamma receptor. Experiments using radioiodinated IL-1 reveal that these cell lines also possess high affinity receptors for IL-1. As indicated by the induction of HLA-DR molecules, and alterations in morphologic characteristics, these cell lines are sensitive to both IFN-gamma and IL-1 action. The class II molecules (HLA-DR, HLA-DP, and HLA-DQ) are differentially induced by IFN-gamma treatment in carcinoma cell lines, with HLA-DR being the prevailing induced molecule. IFN-gamma inhibits and estradiol-17β promotes growth of ECC1 cells in a dose-and time-dependent manner. These findings indicate that the interacting effect(s) of the cytokines and steroid hormones on endometrial epithelium may be studied in these unique steroid-and cytokine-sensitive epithelial cell lines.     

Aberrant up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer

The LAMB3 and LAMC2 genes encode the laminin-5 β3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.     

Genistein affects histone modifications on Dickkopf-related protein 1 (DKK1) gene in SW480 human colon cancer cell line

Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist, DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand, genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.     

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells

Summary Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEG-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 μM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [³H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate. This investigation was supported by PHS grant HD 07197, awarded by the National Institute of Child Health and Human Development and PHS grant CA 15648, awarded by the National Cancer Institute.     

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

Utility of liquid-based cytology in endometrial pathology: diagnosis of endometrial carcinoma

Objective: The purpose of this study was to examine the utility of SurePath‐liquid‐based cytology (LBC) compared to conventional cytological preparations (CCP) in the identification of endometrial carcinoma. Methods: During a 13‐month period, direct endometrial samples were collected from 120 patients using the Uterobrush. The material comprised 30 cases each of endometrial carcinoma, proliferative endometrium, secretory endometrium and atrophic endometrium. The following points were investigated:(i) the frequency of cell clumps in endometrial carcinoma; (ii) the area of cell nuclei; (iii) overlapping nuclei. Results: (i) Comparison of the frequency of cell clumps with irregular protrusion pattern and papillo‐tubular pattern showed no statistically significant difference in either type of cell clump between CCP and LBC. (ii) Comparison of the nuclear area of cells showed a sequential decrease from endometrial carcinoma to secretory endometrium, to proliferative endometrium and to atrophic endometrium, which was significant in CCP and LBC. (iii) Nuclear area was significantly lower with LBC compared with CCP in endometrial carcinoma, secretory endometrium and proliferative endometrium but not atrophic endometrium. (iv) Comparison of the degree of overlapping nuclei showed a sequential decrease from endometrial carcinoma to proliferative endometrium, to secretory endometrium and to atrophic endometrium, which was significant in both CCP and LBC. (v) Comparison of the degree of overlapping nuclei between CCP and LBC showed no significant difference for normal types of endometrium, but LBC had significantly higher values (P < 0.0001) in endometrial carcinoma than in CCP. Conclusions: The results of this study revealed that applying diagnostic criteria used in CCP to LBC was easy to achieve, because LBC had excellent cytoarchitectural preservation and cells were well presented. Although we have not examined all cytological features of malignancy and have not considered atypical hyperplasia, we believe that this method may be a useful tool in the diagnosis of endometrial cytology.     

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.     

The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.     

Analysis of interferon‐γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal‐conceptus interface and the role of interferon‐γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs

It has long been known that pig conceptuses produce interferon‐γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal‐conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin‐1β did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy‐ and cell‐type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.     

Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30

Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy.