Effects of estradiol on deoxyribonucleic acid polymerase alpha activity in the Ishikawa human endometrial adenocarcinoma cell line

The effects of estradiol on DNA polymerase alpha activity were investigated in an estrogen-responsive human endometrial cancer cell line (Ishikawa) derived from a well differentiated endometrial adenocarcinoma. These cells are known to respond to estradiol by increasing progesterone receptor levels, alkaline phosphatase activity, and cell density. Four- to 5-fold increases in DNA polymerase alpha activity occurred when estradiol was added to cultures of Ishikawa cells in medium containing charcoal-treated fetal bovine serum. Maximal stimulation was achieved at 18 h during incubations with 10(-8) M estradiol, but significant effects also were found with 10(-9) and 10(-6) M. These effects were almost completely counteracted by a 100-fold excess of 4-hydroxytamoxifen. At 10(-6) M, the antiestrogen had no influence on the basal levels of DNA polymerase alpha. Medroxyprogesterone acetate (10(-6) M) was ineffective as either an enhancer of enzymatic activity or an antiestrogen when tested in combination with 10(-8) M estradiol, even in the presence of appreciable levels of specific progesterone binders. The responsiveness of the Ishikawa cells to estrogen contrasts with the lack of effects of estradiol on DNA polymerase alpha activity in another human endometrial adenocarcinoma cell line (HEC-50).     

The regulation of apoptosis by activin and transforming growth factor-beta in early neoplastic and tumorigenic ovarian surface epithelium

Most ovarian neoplasms arise from the ovarian surface epithelium (OSE), and multiple growth factors have been implicated to influence the transformation from OSE. The present study was performed to investigate the role of activin and transforming growth factor-beta (TGFbeta) in normal and neoplastic OSE cells. An immortalized OSE cell line (IOSE-29) was generated from normal OSE by transfecting simian virus 40 large T antigen and was rendered tumorigenic after subsequent transfection with the E-cadherin gene (IOSE-29EC). The activin/inhibin subunits and activin receptors were expressed at both messenger ribonucleic acids and protein levels in these cells, suggesting that activin may have an autocrine role in neoplastic OSE cells. Treatments with activin (1-100 ng/mL) resulted in a significant decrease in cell proliferation in both IOSE-29 and IOSE-29EC cells, although we have shown that it stimulated the growth of ovarian cancer cells and had no effect on normal OSE. This inhibitory effect was attenuated with cotreatment with follistatin. Treatment with TGFbeta (0.1-10 ng/mL) also significantly decreased the proliferation of normal, IOSE-29, and IOSE-29EC cells in a dose-dependent manner. In addition, treatments with both activin and TGFbeta resulted in an increase in DNA fragmentation in IOSE-29EC cells in a dose-dependent manner. This apoptotic effect of activin was attenuated by cotreatment with follistatin. Treatment with TGFbeta (1 and 10 ng/mL) resulted in a significant decrease in Bcl-2 protein (up to 50%) in IOSE-29EC, whereas no difference was observed in Bax protein levels. Therefore, down-regulated Bcl-2 by TGFbeta may eventually induce apoptosis in IOSE-29EC cells. In contrast, no difference was observed in Bax and Bcl-2 protein expression after treatment with activin. In conclusion, the present study indicates that activin and TGFbeta inhibited growth and induced apoptosis in early neoplastic (IOSE-29) and tumorigenic OSE (IOSE-29EC) cells. Furthermore, antiapoptotic Bcl-2 protein was down-regulated by TGFbeta, whereas no difference was produced in Bax protein by activin or TGFbeta treatment or in Bcl-2 protein by activin. These results suggest that activin and TGFbeta may play a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic stage of ovarian cancer.     

Follistatin-related gene expression, but not follistatin expression, is decreased in human endometrial adenocarcinoma

OBJECTIVE: Activin A is a multifunctional growth and cell differentiation factor produced by normal endometrium, and secreted in high amounts by endometrial adenocarcinoma. In the present study we evaluated the expression of two inhibitory activin A ligands, follistatin and follistatin-related gene (FLRG), in endometrial adenocarcinoma and in age-matched healthy human endometrium. DESIGN AND METHODS: Atropic menopausal (n=13) and tumoral (n=9 adenocarcinoma) tissues were processed to evaluate mRNA expression levels (by semiquantitative RT-PCR) and peptide localization (by immunohistochemistry). Differences were evaluated by the unpaired t-test and assumed to be statistically significant when P<0.05. RESULTS: Both control and tumoral endometrial samples express and localize follistatin and FLRG. However, whereas follistatin mRNA expression did not differ significantly, FLRG was significantly lower in endometrial adenocarcinoma than in healthy endometrial specimens (P<0.0001). With respect to the localization of proteins, follistatin was immunolocalized in endometrial epithelial and vascular cells both in tumoral and healthy endometrium without any significant difference in intensity. Nuclear and cytoplasmic FLRG immunolocalization was seen in glands, and only nuclear immunolocalization was found in stroma and vessels of healthy endometrium. FLRG was weakly immunostained in endometrial adenocarcinoma. CONCLUSIONS: Whilst follistatin expression is unchanged, FLRG is down-regulated in endometrial carcinoma. As activin A is a differentiation factor of human endometrium, the present findings support an imbalance between increased activin A and decreased FLRG expression in endometrial cancer, so that the failure of the activin A pathway through FLRG may be pivotal in endometrial tumorigenesis.     

Phorbol esters differentially regulate the expression of insulin-like growth factor-binding proteins in endometrial carcinoma cells.

We have examined the effects of protein kinase-C (PKC) activation on expression of the six known insulin-like growth factor-binding proteins (IGFBPs) by human endometrial carcinoma cells. Each of six known IGFBPs was expressed in one or more of the three cell lines examined. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cells resulted in changes in cell morphology, growth inhibition, activation of PKC, and an increase in expression of IGFBP-1. PMA had no effect on these parameters in the Ishikawa cell line, which did not express IGFBP-1. In HEC-50 cells, the effect of PMA was blocked by the concomitant addition of the PKC inhibitor staurosporin and the simultaneous addition of cycloheximide. PMA also resulted in an increase in IGFBP-3 in HEC-50 cells and an increase in IGFBP-6 expression in HEC-1B cells. In contrast, IGFBP-3 expression was down-regulated by PMA in HEC-1B and Ishikawa cells. The abundance of IGFBP-2 and IGFBP-5 mRNAs was also reduced in HEC-1B and Ishikawa cells, respectively. IGFBP-4 was expressed only in HEC-50 cells and was not affected by PMA treatment. These data establish a role for the PKC pathway in regulation of expression of IGFBP-1, -2, -3, and -5 in endometrial adenocarcinoma cells and illustrate the complexity of cell type-specific expression of the IGFBPs.     

Expression of activin A and follistatin core proteins by human prostate tumor cell lines.

Activin and follistatin (FS) messenger RNA and protein are expressed and localized to human prostate tissue from men with high grade cancer and to human prostate tumor cell lines LNCaP, DU145, and PC3. Although activin A induces apoptosis and inhibits cell proliferation in LNCaP cells, PC3 cells are insensitive to the effect of exogenous addition of activin A. The results of this study show that activin A and FS are produced and can be measured by specific enzyme-linked immunosorbent assays in PC3 cells and media but are not detectable in LNCaP cells. Over 10 days in culture, the production of activin A by PC3 cells declines and is inversely correlated (r = -0.779) to FS288 production, which steadily increases and is significantly elevated compared with Day 1 of culture. The presence of FS288 and FS315 proteins was confirmed by immunocytochemistry and showed that only PC3 cells produced the FS288 isoform. Western blotting of PC3 cell media confirmed the presence of the FS288 isoform. Blockade of FS288 activity with a neutralizing antibody rendered PC3 cells responsive to activin A, as measured by inhibition of proliferation. Collectively, these results suggest that PC3 tumor cells are insensitive to activin A because they produce measurable amounts of activin ligand and FS288 protein, which is capable of blocking the autocrine response of these cells to activin A.     

NOD样受体家族蛋白3在中老年子宫内膜样腺癌组织中的表达及意义

目的探讨NOD样受体家族蛋白3（NOD-like receptor protein, NLRP3）在中老年患者子宫内膜样腺癌组织中的表达及临床意义。 方法选取2017年1月至2019年12月在浙江医院和浙江省肿瘤医院行手术切除的子宫内膜样腺癌患者35例,以及同期因子宫脱垂行全子宫切除术患者12例。采用实时荧光定量PCR、免疫印迹、免疫组化等方法检测比较不同子宫内膜组织中NLRP3 mRNA和蛋白的表达水平。同时应用子宫内膜样腺癌细胞系ishikawa和HEC-1A行体外细胞试验,观察NLRP3对子宫内膜样腺癌细胞增殖、侵袭及迁移能力的影响。两组间的比较采用t检验。 结果免疫组化检测：NLRP3在子宫内膜样腺癌组织中的表达水平明显高于正常子宫内膜组织,而且随FIGO分期及病理分级的增高而增高。实时荧光定量PCR检测：子宫内膜样腺癌组织中NLRP3 mRNA和NLRP3蛋白的表达水平均显著高于正常子宫内膜组织（t=3.769、4.316,P<0.05）。蛋白质印迹法：子宫内膜样腺癌组织中NLRP3蛋白表达明显强于正常子宫内膜组织。体外细胞试验：NLRP3 mRNA和NLRP3蛋白在ishikawa细胞和HEC-1A细胞中均有表达,而且两者在HEC-1A细胞中的表达水平均明显高于ishikawa细胞（t=2.373、2.559,P<0.05）;利用siRNA沉默子宫内膜样腺癌ishikawa细胞和HEC-1A细胞的NLRP3后,从第3天开始其增殖能力明显受限,并持续至细胞增殖实验结束的第7天;Transwell实验：沉默NLRP3后ishikawa细胞和HEC-1A细胞的迁移和侵袭数量均明显下降（t=7.343、6.571,3.859、3.289;P<0.05）。 结论NLRP3的表达与子宫内膜样腺癌细胞的增殖、侵袭和迁移能力密切相关。     

Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell proliferation.

Administration of activin A, a member of the transforming growth factor-beta superfamily inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo. However, a role of endogenous activin A in local growth modulation has not been established in any system. The aim of this study was to examine the production of activin A in the human hepatoma cell line HLF and to explore a possible autocrine role of activin as a cell growth inhibitor by blocking production of endogenous activin using antisense oligodeoxynucleotides. Administration of exogenous activin A suppressed HLF cell growth, and immunoreactive activin A was shown to be produced in the cells at confluency by Western blotting analysis. Cells were exposed to phosphorothioate-modified oligodeoxynucleotides, synthesized with antisense or randomly shuffled base sequences of activin betaA subunit messenger RNA, under serum-free conditions. Uptake of the oligodeoxynucleotides into the cells was confirmed by use of fluorescein isothiocyanate-labeled oligodeoxynucleotides. Administration of antisense oligodeoxynucleotides reduced activin A production as confirmed by both competitive PCR and Western blotting. Activin betaA antisense oligodeoxynucleotides significantly increased cell proliferation compared with controls. These findings are consistent with the existence of an autocrine role of activin A as an inhibitor of hepatocyte proliferation.     

Functional Relevance of Activin A in the Intestinal Epithelium

Background: Activin A modulates inflammation and repair in various tissues. The aim of this study was to characterize the effects of activin A on intestinal epithelial cell function and to evaluate a potential role in intestinal epithelial wound repair and inflammation. Methods: The expression of activin A and its receptors ActRI and ActRII in intestinal epithelial IEC-6 cells and in tissues from IBD patients and non-inflamed controls was evaluated using RT-PCR and immunohistochemistry. Functional effects of activin A on intestinal epithelial cell migration and proliferation were assessed using an in vitro wounding model and colorimetric MTT assays. Results: Expression of the activin β A subunit and the activin receptors ActRI and ActRII in IEC-6 cells was demonstrated using RT-PCR. Activin A (50 ng/ml) caused a significant, on average 2.8-fold enhancement of epithelial cell migration and a significant on average 4.1-fold inhibition of IEC-6 cell proliferation. Expression of ActRI and ActRII was observed in all intestinal tissues from patients with IBD and in all controls. In contrast, no expression of the activin β A-subunit was observed in controls, while β A expression was found in intestinal tissues from IBD patients. Conclusions: Activin A may play an important role in the modulation of intestinal epithelial cell function, thus providing a new approach to modulate intestinal wound repair.     

Functional relevance of activin A in the intestinal epithelium

BACKGROUND: Activin A modulates inflammation and repair in various tissues. The aim of this study was to characterize the effects of activin A on intestinal epithelial cell function and to evaluate a potential role in intestinal epithelial wound repair and inflammation. METHODS: The expression of activin A and its receptors ActRI and ActRII in intestinal epithelial IEC-6 cells and in tissues from IBD patients and non-inflamed controls was evaluated using RT-PCR and immunohistochemistry. Functional effects of activin A on intestinal epithelial cell migration and proliferation were assessed using an in vitro wounding model and colorimetric MTT assays. RESULTS: Expression of the activin betaA subunit and the activin receptors ActRI and ActRII in IEC-6 cells was demonstrated using RT-PCR. Activin A (50 ng/ml) caused a significant, on average 2.8-fold enhancement of epithelial cell migration and a significant on average 4.1-fold inhibition of IEC-6 cell proliferation. Expression of ActRI and ActRII was observed in all intestinal tissues from patients with IBD and in all controls. In contrast, no expression of the activin betaA-subunit was observed in controls, while betaA expression was found in intestinal tissues from IBD patients. CONCLUSIONS: Activin A may play an important role in the modulation of intestinal epithelial cell function, thus providing a new approach to modulate intestinal wound repair.     

Expression of activin A/erythroid differentiation factor in murine bone marrow stromal cells.

Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow.     

Human adiponectin inhibits cell growth and induces apoptosis in human endometrial carcinoma cells, HEC-1-A and RL95 2

Obesity is one of the well-established risk factors for endometrial cancer. Recent clinical studies have demonstrated that circulating adiponectin concentrations are inversely correlated with the incidence of endometrial carcinoma. Such epidemiological findings are consistent with the paradoxical observations that adiponectin levels are reduced in obesity. This study investigated the direct effects of adiponectin on two endometrial carcinoma cell lines, HEC-1-A and RL95-2. These cell lines express both variants of adiponectin receptors, adipo-R1 and adipo-R2. Adiponectin treatment leads to suppression of cell proliferation in both cell types, which is primarily due to the significant increase of cell populations at G(1)/G(0)-phase and to the induction of apoptosis. The inhibition of growth in these two cell lines appears to be mediated by different signaling pathways. Although adiponectin treatment markedly increases the phosphorylation (Thr172) of AMP-activated protein kinase alpha in both HEC-1-A and RL95-2 within 30 min, prolonged exposure (48 h) leads to inactivation of Akt as well as reduction of cyclin D1 protein expression in HEC-1-A cells. In contrast, similar treatment of RL95-2 cells with adiponectin, while having no effects on Akt activity and cyclin D1 expression, causes a decrease in cyclin E2 expression and the activity of mitogen-activated kinase (p42/44). We conclude that adiponectin exerts direct anti-proliferative effects on HEC-1-A and RL95-2 cells by inducing cell cycle arrest and apoptosis. Depending on the genotypes of the endometrial cancer cells, the inhibitory effects of adiponectin are associated with the reduction of different pro-growth regulators of cell cycle and signaling proteins. Our study thus provides a cellular mechanism underlying the linkages between endometrial cancer and obesity.     

Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LbetaT2 pituitary gonadotrope cell model

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:     

Effect of activin A and inhibin A on expression of the inhibin/activin beta-B-subunit and gonadotropin receptors in granulosa cells of the hen

Activin A has been shown to be abundant in the theca layer of the large pre-ovulatory follicles of the hen whereas inhibin A is produced in the granulosa layer. The purpose of this study was to investigate the effects of activin A and inhibin A on granulosa cell expression of inhibin beta-B-subunit, FSH receptor (FSHR), and LH receptor (LHR). Granulosa cells were isolated from the F1, F3+F4, and small yellow follicles (SYF; 6-12 mm diameter) of laying hens and pooled according to size. The cells were dispersed and plated in the presence of 0, 10, or 50 ng/ml recombinant human activin A (n=5 replicate cultures). RNA was subsequently extracted from the cells and Northern blots performed. Cell proliferation was determined for all treatments. An identical set of experiments was performed in which the granulosa cells were treated with recombinant human inhibin A (n=4 replicate cultures). Treatment with activin A at 50 ng/ml significantly (p<0.05) increased expression of beta-B-subunit for granulosa cells from all follicles. This dose also significantly increased expression of FSHR in granulosa cells from all follicles (p<0.05) and increased expression of LHR in cells from F1 and F3+F4 follicles (p<0.01) with no significant effect on cells from the SYF. Overall, activin A treatment significantly (p<0.05) decreased cell proliferation at the 50 ng/ml dose. Inhibin A had no significant effect on expression of beta-B-subunit, FSHR or LHR at any dose. There was a moderate stimulatory effect of inhibin A on granulosa cell proliferation. These results suggest that activin A may have an important role in regulating granulosa cell responsiveness to gonadotropins while also modulating follicle development by attenuating cell proliferation.     

Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro.

The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution-hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5' end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard. The results show that 5 micrograms FSH/l for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18.5-fold higher than controls. LH (0.2-100 micrograms/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner. It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.     

人子宫内膜腺癌细胞系的建立

目的构建人子宫内膜腺癌细胞系。方法将1例高分化子宫内膜腺癌手术切除的肿瘤组织制成单细胞悬液,用含10%胎牛血清的DMEM/F-12培养基进行原代培养,倒置显微镜下观察细胞形态,并用免疫荧光染色法检测其角蛋白。结果体外培养细胞传至50代以上,传代后细胞单层贴壁,散在生长,密度增加时见堆积现象,失去接触抑制,细胞质中角蛋白表达阳性。结论成功建立了人子宫内膜腺癌细胞系。     

HPS-1对人肺腺癌A549细胞凋亡诱导作用的机制研究

目的 评价HPS-1对人肺腺癌A549细胞凋亡蛋白Caspase 3、8、9以及Bax、Bcl-2mRNA表达的影响,探究HPS-1抗肺肿瘤可能机制.方法 人肺腺癌A549细胞经低、中、高剂量HPS-1处理不同时间后,噻唑蓝(MTT)法检测各组细胞的增殖情况,流式细胞术(FCM)法检测各组细胞凋亡率及细胞周期比例,比色法检测各组细胞凋亡蛋白Caspase-3、8、9的表达,RT-PCR法检测各组细胞Bax、Bcl-2 mRNA的表达.结果 MTT结果显示,低、中、高剂量HPS-1对人肺腺癌A549细胞的增殖具有明显抑制作用,且具有时间、剂量依赖性;FCM法检测发现,低、中、高剂量HPS-1对人肺腺癌A549细胞的凋亡具有促进作用,且与剂量呈正相关;比色法检测各组细胞发现,HPS-1促进凋亡蛋白Caspase-3、8、9的上调,且HPS-1高剂量组凋亡蛋白上调更明显.结论 HPS-1具有抑制人肺腺癌A549细胞增殖、诱导其凋亡作用,且该作用可能与HPS-1阻滞细胞周期G1期,上调凋亡蛋白Caspase-3、8、9及Bax mRNA表达,下调Bcl-2 mRNA表达有关.