Gui Li-ming, Wei Li-hui, Xu Ming-xu, Wang Jian-liu, Zhong Ying-cheng, Li Xiao-ping, Tu Zheng, Sun Peng-ming, Ma Da-long
Department of Gynecology, Peking University People's Hospital, Beijing 100044, China.
Zhonghua Fu Chan Ke Za Zhi. 2004 Jan;39(1):30-4.
To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line.
(1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins.
(1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively.
(1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).
研究突变型[(12)Asp]K-ras4B基因对子宫内膜癌HEC-1A细胞系中雌激素受体（ER）α和β的表达及其作为转录因子的转录活性的影响。
(1)采用蛋白质免疫印迹法检测[(12)Asp]K-ras4B对ERα和β表达的影响。(2)构建含荧光素酶报告基因和雌激素反应元件（ERE）的真核表达质粒pGL3-荧光素酶-ERE，并与pEGFP-N1共转染NIH3T3和HEC-1A细胞系，检测[(12)Asp]K-ras4B对雌二醇调控的ER转录的影响。此外，将它们转染到pSV5-HER0（含全长野生型ERα cDNA）和pCMV-rafS621A（抑制raf激酶）质粒中，以检测[(12)Asp]K-ras4B/raf信号通路对ER蛋白转录活性的影响。
(1) pcDI转染的对照细胞中ERs的蛋白水平较低，而在pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞中，ERα和ERβ的蛋白水平分别增加了3.6倍（97±25，349±67，P<0.01）和1.9倍（128±37，349±30，P<0.05）。(2) 在pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞中，转染rafS621A质粒前后ERα和ERβ水平的比值分别为2.4:1（724±45，310±46，P<0.05）和1.8:1（493±20，284±20，P<0.01）；在HEC-1A细胞中，这些比值分别为2.1:1（566±22，279±30，P<0.01）和2.4:1（405±33，165±15，P<0.01）。(3) 在低血清（2%）培养条件下，与未加雌二醇（E₂）相比，pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞中，雌二醇（E₂）刺激荧光素酶活性增加了13倍（130±42，1681±242，P<0.01），HEC-1A细胞中增加了19倍（141±39，2644±331，P<0.001）。(4) 在转染pSV5-HER0的pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞和HEC-1A细胞中，与未转染细胞相比，转染细胞中的ER转录活性显著增加。荧光素酶活性分别增加了8倍（1048±91，8099±452，P<0.01）和6倍（2148±259，12705±2670，P<0.001）。rafS621A突变体对HEC-1A细胞和pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞中的荧光素酶活性有抑制作用。pcDI-[(12)Asp]K-ras4B转染的NIH3T3细胞和HEC-1A细胞转染前后荧光素酶活性的比值分别为7.8:1（1184±168，152±27，P<0.05）和6.4:1（1949±212，304±60，P<0.01）。
(1) [(12)Asp]K-ras4B可增强ERα和β蛋白的表达。这可能与[(12)Asp]K-ras4B/raf信号通路有关。(2) 突变型[(12)Asp]K-ras4B基因对HEC-1A细胞中ERs转录活性的影响似乎需要E₂。
