Distinct functional differences of human progesterone receptors A and B on gene expression and growth regulation in two endometrial carcinoma cell lines

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59).Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells.We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.     

Progesterone-regulated gene expression in the primate endometrium

Progesterone action is essential for maturation of the endometrium to a receptive state for implantation in humans and nonhuman primates. The orchestration of progesterone-regulated gene expression is also temporally controlled during the secretory phase based on the limited window for implantation. The genes and gene networks affected by progesterone are likely to involve both activation and repression. Our laboratory has used the rhesus monkey as a model to study the regulation of genes known or suspected to be involved in endometrial maturation. In addition, we have used subtractive hybridization and differential display techniques to identify novel or unsuspected genes that are regulated by progesterone during endometrial maturation. Our studies have led us to propose a working model of progesterone action during the primate secretory phase that includes waves of gene activation and repression that culminate in a receptive endometrium.     

Steroid-modulated proliferation of human endometrial carcinoma cell lines: any role for insulin-like growth factor signaling?

OBJECTIVES: Estrogen-stimulated proliferation of the normal and malignant human endometrium is balanced by the differentiating properties of progesterone. This study evaluated the role of insulin-like growth factor (IGF) signaling in steroid-induced modulation of endometrial cancer cell proliferation. METHODS: We used the human endometrial, estrogen-responsive ECC-1 and progesterone-responsive PRAB-36 cell lines. Proliferation studies with IGFs in combination with either estrogen or progesterone were conducted. Furthermore, the mRNA and protein expression of insulin-like growth factor-binding proteins (IGFBPs) was evaluated. RESULTS: Using the ECC-1 cell line, we observed that estrogen-induced proliferation is modulated via the IGF-receptor signaling pathway, and that IGF-1-induced stimulation of proliferation does not influence estrogen receptor signaling. Furthermore, expression of the main modulators of IGF action, the IGFBPs, was found to be regulated by estrogen and progesterone in both cell lines. IGFBP-4 was up-regulated by estrogen in the ECC-1 cell line, and IGFBP-3 and IGFBP-6 were down-regulated by progesterone in the PRAB-36 cell line. CONCLUSION: Estrogen-induced stimulation of proliferation of ECC-1 endometrial cancer cells is partly achieved via IGF signaling. Furthermore, the IGFBPs are regulated by estrogens as well as progestagens and could potentially play a role in the modulation of endometrial cancer cell proliferation.     

Molecular tools to reestablish progestin control of endometrial cancer cell proliferation

OBJECTIVE: Endometrial cancers often arise in a setting of estrogen stimulation unopposed by the differentiating effects of progesterone. Our laboratory and others have previously shown that progesterone receptor down-regulation or perturbation of progesterone receptor isoform A or B expression is associated with the development of poorly differentiated endometrial cancers that are not growth inhibited by progestins. The purpose of these studies was to reestablish high progesterone receptor isoform A and B gene expressions in such endometrial cancer cells and to examine the effects of progestin treatment on cell growth and metastatic potential after this transformation. STUDY DESIGN: To induce high levels of expression of the progesterone receptor isoforms in KLE and Hec50 endometrial cancer cells, adenoviral vectors encoding the genes for progesterone receptor isoforms A and B were created. The characteristic ability of cancer cells to grow independently of anchorage to the surrounding solid matrix was measured by counting colony formation on soft agar for 8 to 14 days. Cell proliferation in response to a time course of progestin treatment was tested with flow cytometry. RESULTS: After treatment with a control vector without a progesterone receptor--encoding insert, no effect of progestin treatment on cell proliferation was found; after treatment with vectors encoding progesterone receptor isoform A or B, however, progestin treatment resulted in significant inhibition of cell growth. The anchorage-independent cell growth on soft agar assay showed that by 8 to 14 days the number of cell colonies was reduced by 50% relative to control preparations in the presence of progesterone receptor isoform A plus progestin (P <.0001, both Hec50 and KLE cell lines) and by 90% in the presence of progesterone receptor isoform B plus progestin (P <.0001, both Hec50 and KLE cell lines). Progestin treatment also resulted in a time-dependent reduction in cell proliferation as measured by flow cytometry. Although transfection with both progesterone receptor isoforms A and B reduced cell proliferation according to our assays, progesterone receptor isoform B caused a much more dramatic decrease in cell growth (P =.001, Hec50 cells; P <.0001, KLE cells). CONCLUSION: In poorly differentiated endometrial cancer cells that are resistant to progestin therapy, adenovirus-induced expressions of progesterone receptors A and B reestablish progestin control of endometrial cancer cell proliferation.     

Epidermal growth factor receptor signaling enhanced by long-term medroxyprogesterone acetate treatment in endometrial carcinoma

OBJECTIVE: Progestin is an effective endocrine treatment for patients with atypical hyperplasia or with endometrial carcinoma that is estrogen receptor (ER) positive and progesterone receptor (PR) positive. However, long-term progestin treatment may lead to resistance. We have studied the progestin resistance phenotype that frequently develops in endometrial carcinoma. METHODS: Ishikawa endometrial carcinoma cells were cultured for a long period (10 months) in the presence of the synthetic progestin medroxyprogesterone acetate (MPA), thereby generating a subline refractory to the growth-suppressive effects of MPA. RESULTS: The MPA-resistant subline showed growth stimulation rather than inhibition after MPA treatment. Immunocytochemical analysis showed reduced ER alpha and PR-B expression and increased ER beta expression in this subline compared with parental Ishikawa cells. Progestin-resistant Ishikawa cells also showed increased expression of transforming growth factor alpha (TGFalpha), the epidermal growth factor receptor (EGFR), and EGFR tyrosine kinase (EGFR-TK); MPA treatment further stimulated the expression of TGFalpha in these cells. Additionally, progestin-resistant Ishikawa cells were highly sensitive to growth stimulation by TGFalpha and to growth inhibition by the EGFR-TK-specific inhibitor AG1478, and they showed increased dependence on TGFalpha-EGFR signaling. CONCLUSIONS: Our results suggest that prolonged treatment of endometrial carcinoma cells with MPA induces resistance to the growth-suppressive effects of MPA and enhances cancer cell proliferation. The downregulation of ER alpha and PR-B, the upregulation of ER beta, and highly activated TGF-EGFR signaling are thus likely to contribute to progestin resistance in endometrial carcinoma. Therefore, an EGFR-TK-specific inhibitor might be useful in the treatment of progestin-resistant endometrial carcinoma.     

Overexpression of estrogen receptor-alpha in the endometrial carcinoma cell line Ishikawa: inhibition of growth and angiogenic factors

BACKGROUND: A high level of estrogen receptor-alpha (ER-alpha) is believed to be favorable in the prognosis and treatment of endometrial, ovarian, and breast cancer. High levels of ER-alpha have been shown to inhibit the growth and invasive, metastatic potential of breast cancer cell lines. To bring about these inhibitory effects, ER-alpha probably acts through other cellular factors involved in the regulation of cell growth. OBJECTIVE: To investigate the role of high levels ER-alpha in growth inhibition of endometrial cancer cells. METHODS: A human ER-alpha cDNA was stably overexpressed in an endometrial cancer cell line, namely, Ishikawa. ER-alpha-overexpressing, parent, and control Ishikawa cells were grown in vitro and their growth rates were compared by cell count. ER-alpha-overexpressing and parent Ishikawa cells were also grown in vitro as tumors in a chicken chorioallantoic membrane (CAM) model, and tumor growth and angiogenesis was measured. Finally, levels of angiogenesis-modulating factors, nitric oxide synthase (NOS), and vascular endothelial growth factor (VEGF) were examined in relation to ER overexpression. RESULTS: The growth of Ishikawa cells was found inhibited in culture as well as in the CAM model. Angiogenesis of CAM tumors was also found inhibited in ER-overexpressing cells. Angiogenic factor VEGF was inhibited whereas the activity of NOS was found elevated following ER overexpression. CONCLUSION: Our work on the Ishikawa cell line indicates that high levels of ER-alpha in endometrial cancer may inhibit cancer growth by modulating angiogenic factors, thereby limiting the blood supply to the growing tumor. Our results support the earlier data from other groups that have shown a positive correlation between high ER content and better prognosis of endometrial cancers.     

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.     

应用cDNA表达谱芯片研究子宫内膜癌基因的表达谱

目的:应用cDNA表达谱芯片研究不同分化程度人子宫内膜癌组织的基因表达谱,分析子宫内膜癌发生、发展过程中基因表达的变化。方法:用一步法抽提5例高分化和4例低分化人子宫内膜癌组织和36例正常子宫内膜组织的总RNA,在反转录合成cDNA探针的同时以Cy3和Cy5标记肿瘤组和正常对照组,将Cy3和Cy5标记探针混合后与含有13 824个基因的人表达谱芯片杂交。经洗片、扫描、软件处理后,分析子宫内膜癌和正常子宫内膜对照之间基因表达谱的差异。对高分化和低分化子宫内膜癌基因表达谱进行聚类分析。结果:子宫内膜癌组织和正常对照基因表达谱比较,差异2倍以上基因共有26条,表达上调和下调的基因分别为2条和24条。聚类分析显示,通过基因表达谱基本可将高分化和低分化子宫内膜癌分开。结论:通过基因表达谱差异研究可以发现与子宫内膜癌发生、发展相关的基因;癌基因表达谱聚类分析可能会帮助判断高危和低危子宫内膜癌,有利于子宫内膜癌患者个体化治疗方案的制定。     

应用抑制性消减杂交筛选卵巢癌紫杉醇耐药差异表达基因

目的:应用抑制性消减杂交技术研究人卵巢癌紫杉醇耐药细胞株OC3/Tax300与其亲本细胞OC3(敏感株)之间基因表达的差异,筛选耐药相关基因,探讨基因表达差异与卵巢癌耐药的相关性。方法:以卵巢癌紫杉醇耐药细胞株OC3/Tax300 mRNA为检测子(tester),以其亲本细胞OC3(敏感株)mRNA为驱赶子(driver),构建cDNA消减文库。随机挑取文库克隆测序,所得结果在GenBank中做同源性比较分析。结果:成功构建了卵巢癌紫杉醇耐药细胞株的特异性cDNA消减文库。从文库中选取阳性克隆,其中76个测序成功,再随机选取36个序列与GenBank数据库进行初步比对,8个可能为新基因,1个为蛋白序列,另27个来源于16个已知基因,这些差异表达基因主要涉及细胞代谢、信号转导、细胞骨架、凋亡、蛋白翻译合成、发育等相关基因。结论:部分基因表达差异与卵巢癌紫杉醇耐药有关。     

氨基导眠能与己酸孕酮治疗子宫内膜癌的作用比较

将子宫内膜癌３９例以双盲法随机分为３组，分别用己酸孕酮、氨基导眠能或两药联合治疗。结果：（１）氨基导眠能使雌二醇（Ｅ２）、孕酮（Ｐ）含量下降，己酸孕酮使血中Ｐ含量增加，联合治疗后Ｐ增加、Ｅ２下降（Ｐ〈０．０５￣０．０１）。（２）己酸孕酮使雌激素受体（ＥＲ）、孕激素受体（ＰＲ）阳性率略下降（Ｐ〉０．０５），氨基导眠能及联合用药后ＥＲ阳性率显著下降（Ｐ〈０．０５）。（３）三种不同方法治疗后均出现癌细胞     

Progesterone-mediated regulation of catechol-O-methyl transferase expression in endometrial cancer cells

The effects of estrogen and progesterone on the expression of estrogen-metabolizing enzymes such as catechol-O-methyl transferase (COMT) are not known. COMT converts genotoxic catecholestrogens to anticarcinogenic methoxyestrogens in the endometrium. The aim of this study is to investigate the effect of progesterone on COMT expression in well-differentiated endometrial cancer cells. The wild-type Ishikawa cell line as well as progesterone receptor A- or progesterone receptor B-transfected Ishikawa cells were used for in vitro studies. The regulation of COMT expression by progesterone was studied using Western blots, Hoechst dye DNA proliferation studies, and wild-type and/or site-directed mutagenesis of COMT promoter 1-luciferase reporter gene. Progesterone upregulated COMT protein expression in Ishikawa cells through progesterone receptor A isoform. COMT promoter activity was differentially regulated by the 3 half-site progesterone response elements in the COMT promoter. High doses of 2-ME2 inhibited Ishikawa cell proliferation. These data suggest that COMT expression is hormonally regulated in well-differentiated human endometrial cancer cells. COMT regulation and 2-ME2 production in the endometrium may affect endometrial carcinogenesis.     

M344 is a novel synthesized histone deacetylase inhibitor that induces growth inhibition, cell cycle arrest, and apoptosis in human endometrial cancer and ovarian cancer cells

OBJECTIVE: Histone deacetylase inhibitors (HDACIs) can inhibit cell proliferation, induce cell cycle arrest, and stimulate apoptosis of cancer cells. METHODS: We investigated the effects of a novel synthesized HDACI, M344, on Ishikawa endometrial cancer cell line, SK-OV-3 ovarian cancer cell line, and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of M344, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. RESULTS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that all endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of M344, although normal endometrial epithelial cells were viable after the treatment with the same doses of M344 that induced growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to M344 decreased the proportion of cells in the S-phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, M344 treatment of these cell lines increased acetylation of H3 and H4 histone tails. CONCLUSIONS: These results raise the possibility that M344 may prove particularly effective in the treatment of endometrial cancers and ovarian cancers.     

Heterogeneity in hormone receptor status in primary and metastatic endometrial cancer

The rationale for endocrine therapy in patients with advanced endometrial carcinoma may be based on the presence of estrogen or progesterone receptors in the primary tumor. A study was designed to evaluate tumor cell heterogeneity of steroid hormone receptors in the primary and metastatic sites in endometrial cancer. Primary endometrial cancer tissue samples from 10 patients and 16 metastatic tumor sites were simultaneously analyzed for estrogen and progesterone receptors, using a radioligand biochemical assay. The primary tumor was estrogen receptor (ER) and progesterone receptor (PR) positive in 70 and 60% of the patients, respectively. The metastatic sites were ER positive in 63% and PR positive in 25%. The primary tumor tissue and the metastatic disease showed an identical ER and PR status in only 25 and 19%, respectively. Four patients had multiple metastatic sites analyzed. In two of four patients the PR values, and in three of four patients the ER values, in these metastatic sites were discordant. These data support the concept of tumor cell heterogeneity for steroid hormone receptors in endometrial cancer. To optimize treatment planning, it may be important to biopsy primary, metastatic, and recurrent tumor sites for individual analysis of receptor activity.     

甲基化在子宫内膜癌中的研究进展

甲基化是表观遗传学的重要内容之一,甲基化异常与恶性肿瘤细胞增殖、自噬、凋亡、细胞间黏附、浸润和转移密切相关。子宫内膜癌是女性生殖系统常见的恶性肿瘤,研究发现,基因的异常甲基化与子宫内膜癌发病的高危因素、分型、肿瘤进展、早期诊断、化疗耐药、孕酮抵抗和预后评估有关。特异的甲基化标志物可用于子宫内膜癌的早期诊断和预后评估,调控关键基因的甲基化状态能改善子宫内膜癌细胞对化疗和孕酮的敏感性。综述DNA、RNA和组蛋白甲基化在子宫内膜癌发病机制、早期诊断、药物治疗和预后评估中的研究进展。