Analysis of mechanism of estrogen action on estrogen receptor cDNA transfected endometrial cancer cells

雌激素对雌激素受体cDNA转染子宫内膜癌细胞的作用机制分析

• 批准号: 12670176
• 负责人: WATANABE Jun
• 金额: $ 1.98万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670176/
• 关键词: endometrial cancer; estrogen; estrogen receptor; gene trasfection; in vitro; cell probferation

1. Purpose: This study was designed to establish estrogen responsive human endometrial cancer cell lines positive for estrogen receptor (ER) by trasfecting ER expression vector, and to investigate a mechanism of estrogen action.2. Methods: Ishikawa cell line was used for the study. It was originally was positive for ER but it became less responsive to estrogen, as the level of ER was gradually decreased. pSG5-HEGO was used as ER expression vector that contained human ER cDNA, wild type. Superfect method was carried out to transfect the vector into the cells. ER transfected cells were cloned by using Neomycin. The levels of ER expression was measured by immunoblotting or enxyme-linked immunoassay. Analysis of cell proliferation was performed by cell growth curve. Effects of estrogen on regulation of cell cycle regulators were examined by immunostaining and Western blotting.3. Results: ER level of null Ishikawa cells was very low. Thirty six clones were analyzed and three of them expressed high level of ER. Maximal effect of estrogen on cell proliferation was observed at a concentration of 10nM. An 20% increased of growth was detected on Day 6. Levels of Ki-67, Cyclin A, D1, E was also enhanced by estrogen.4. Conclusion: Stable endometrial cancer cell lines that express high ER and are responsive to estrogen were established. They could be useful to investigate the molecular mechanism of estrogen action.

1.目的：本研究旨在通过转染雌激素受体（ER）表达载体，建立雌激素敏感的人子宫内膜癌细胞系，并探讨雌激素的作用机制.方法：采用石川细胞系进行研究。它最初对ER呈阳性，但随着ER水平逐渐降低，它对雌激素的反应变得不那么敏感。以pSG 5-HEGO为ER表达载体，构建了人ER野生型cDNA表达载体。采用Superperfect方法将载体导入细胞。用新霉素克隆ER转染细胞。免疫印迹法或酶联免疫法检测ER表达水平。通过细胞生长曲线进行细胞增殖分析。采用免疫组化染色和Western blotting检测雌激素对细胞周期调控因子的影响.结果：空石川细胞ER水平很低。对36个克隆进行了分析，其中3个克隆表达高水平的ER。在10 nM浓度下观察到雌激素对细胞增殖的最大作用。在第6天检测到生长增加20%。雌激素还能提高Ki-67、Cyclin A、D1、E的表达.结论：建立了稳定的高表达雌激素受体的子宫内膜癌细胞系。它们可能有助于研究雌激素作用的分子机制。

项目成果

Kanai, T., Watanabe, J., Kamata, Y., Hamano, M. and Kuramoto H.: "Stimulator effect of medroxyprogesterone acetate on p27 protein in endometrial cancer cells"Kitasato Med.. 31. 392-397 (2001)

Kanai, T.、Watanabe, J.、Kamata, Y.、Hamano, M. 和 Kuramoto H.：“醋酸甲羟孕酮对子宫内膜癌细胞中 p27 蛋白的刺激作用”Kitasato Med.. 31. 392-397 (2001)

T.Fujisawa: "Immunohistochemical study on VEGF expression in endometrial carcinoma-comparison with p53 expression, angiogenesis, and tumor histologic grade"J.Cancer Res. Clin. Oncol. 127. 668-674 (2001)

T.Fujisawa：“子宫内膜癌中 VEGF 表达的免疫组织化学研究 - 与 p53 表达、血管生成和肿瘤组织学分级的比较”J.Cancer Res。

赤星誠: "子宮内膜癌におけるcdk2およびlci-67発現と関する免疫組織化学的検討"日本臨床細胞学会雑誌. 40. 121-127 (2001)

Makoto Akahoshi：“子宫内膜癌中 CDK2 和 LCI-67 表达的免疫组织化学研究”日本临床细胞学会杂志 40. 121-127 (2001)。

Kyushima, N., Watanabe, J., Hata, H., Jobo, T., Kameya, T. and Kuramoto, H.: "Expression of cyclin A in endometrial adenocarcinoma and its correlation with proliferative activity and clinicopathological variables"J. Cancer. Res. Clin. Oncol.. (in press).

Kyushima, N.、Watanabe, J.、Hata, H.、Jobo, T.、Kameya, T. 和 Kuramoto, H.：“子宫内膜腺癌中细胞周期蛋白 A 的表达及其与增殖活性和临床病理变量的相关性”J。

Kamai T: "Stimulatory effect of medroryprogesterone acetaft on P27 protein in endemetrial cancer cells"Kitasato Med.. 31. 392-397 (2001)

Kamai T：“乙酰甲孕酮对子宫内膜癌细胞中 P27 蛋白的刺激作用”Kitasato Med.. 31. 392-397 (2001)