[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

OBJECTIVE: 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. METHODS: (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. RESULTS: (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. CONCLUSIONS: (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).

RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice.

In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role during early follicular development. However, the role of GDF9 during later stages of follicular development is uncertain. In the present study, a long double-stranded (ds) RNA interference approach was used to investigate the possible role of GDF9 in mediating oocyte regulation of cumulus expansion. Fully grown mouse oocytes injected with Gdf9 dsRNA, Bmp15 dsRNA, or injection buffer were cultured for 24 h and processed for measurement of Gdf9 and Bmp15 mRNA levels using real-time reverse transcription-polymerase chain reaction (RT-PCR) and for measurement of GDF9 protein levels using Western blot analysis and immunofluorescence. Injection with Gdf9 dsRNA knocked down Gdf9, but not Bmp15, mRNA expression in oocytes, and vice versa. Furthermore, GDF9 protein levels were reduced in the Gdf9 dsRNA-injected oocytes. To investigate the role of GDF9 in cumulus expansion, two endpoints were used to evaluate cumulus expansion: Has2 and Ptgs2 mRNA levels were measured in cumulus cells using real-time RT-PCR, and assessment of cumulus expansion was undertaken morphologically. After 24 h of culture in the presence of 0.5 IU/ml of FSH, cumulus shells cocultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, whereas cumulus shells cocultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, Has2 and Ptgs2 mRNA levels after 8 h of coculture were lower in cumulus cells cocultured with Gdf9 dsRNA-injected oocytes than in those cocultured with buffer-injected oocytes. The present results strongly support the concept that GDF9 is a key mediator of oocyte-enabled cumulus expansion in mice.

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

BACKGROUND & OBJECTIVES: Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. METHODS: (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. RESULTS: (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). CONCLUSIONS: (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.