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[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene]
Li-ming Gui1, Li-hui Wei, Ying-mei Zhang
1Department of Gynecology, Peking University People's Hospital, Beijing 100044, P. R. China. weilh19@china.com
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.
Insights
The mutant [12Asp] K-ras4B gene can cause neoplastic transformation in NIH3T3 cells, both in vitro and in vivo. This transformation relies on the Raf signaling pathway, highlighting its role in Ras-driven carcinogenesis.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Context:
- The Ras gene is crucial for intracellular signal transduction pathways.
- K-ras mutations are prevalent in endometrial carcinoma.
- The specific role of Ras gene mutations in endometrial cancer carcinogenesis remains unclear.
Purpose:
- To explore the carcinogenesis and molecular mechanism of the mutant-type [12Asp] K-ras4B gene in endometrial carcinoma.
- To investigate the neoplastic transformation potential of [12Asp] K-ras4B in NIH3T3 cells.
- To elucidate the involvement of the Raf signaling pathway in Ras-induced cell transformation.
Summary:
- Successfully constructed the eukaryotic expression plasmid pcDI-[12Asp] K-ras4B.
- [12Asp] K-ras4B mutant induced neoplastic transformation in NIH3T3 cells in vitro and in vivo.
- Inhibition of Raf signaling pathways (RafS621A) restrained proliferation, while enhancement (RafCAAX) promoted growth, indicating pathway dependence.
Impact:
- [12Asp] K-ras4B gene alone can induce neoplastic transformation.
- Ras-mediated neoplastic transformation is dependent on the Raf signaling pathway.
- Provides insights into the molecular mechanisms of Ras-driven carcinogenesis in endometrial carcinoma.