Jing He1, Genze Shao, Rouli Zhou. 1. Department of Cell Biology and Genetics, Peking University School of Basic Medical Sciences, Beijing 100083, China.
Abstract
OBJECTIVE: To study the biological effects of the novel gene LAPTM4B high expressed in hepatocellular carcinoma on cell proliferation and tumorigenesis of NIH3T3 cells. METHODS: Eukaryotic expression plasmid pcDNA3-TM4B was constructed and transfected into mouse NIH3T3 cell line using Lipofectamin-2000 mediating gene transfer technique. Monoclonal transfected cells with high expression of LAPTM4B gene were identified and selected by RT-PCR, Northern blot and Western blot method. Cell surface morphology was detected by scanning electronic microscopy (SEM). The cell attachment/spreading on various matrices was examined. The cell growth curves were measured by acid phosphatase method. Cell cycle was detected by flow cytometry (FCM). The expression level of Cyclin E protein was examined using Western blot. RESULTS: The cell surface of the LAPTM4B transfected cells showed abundant tube/finger-like microvilli, which were distinguished significantly from the MOCK cells. The cell attachment/spreading of transfected cells increased on fibronectin, matrigel and laminin. The growth rate of transfected cells was faster than that of the MOCK cells. FCM analysis indicated that the amount of the transfected cells in S phase was increased significantly. Cyclin E protein expression of transfected cells was much higher than that of the MOCK cell. The serum-dependence of transfected cells was decreased. The fibrosarcoma was formed at a tumorigenic rate of 50% in NIH mice inoculated with LAPTM4B transfected cells. CONCLUSION: LAPTM4B gene promotes the cell proliferation by involving in the regulation of cell cycle control and causes tumorigenesis of NIH3T3 cells, indicating that it plays important roles in tumorigenesis.
OBJECTIVE: To study the biological effects of the novel gene LAPTM4B high expressed in hepatocellular carcinoma on cell proliferation and tumorigenesis of NIH3T3 cells. METHODS: Eukaryotic expression plasmid pcDNA3-TM4B was constructed and transfected into mouse NIH3T3 cell line using Lipofectamin-2000 mediating gene transfer technique. Monoclonal transfected cells with high expression of LAPTM4B gene were identified and selected by RT-PCR, Northern blot and Western blot method. Cell surface morphology was detected by scanning electronic microscopy (SEM). The cell attachment/spreading on various matrices was examined. The cell growth curves were measured by acid phosphatase method. Cell cycle was detected by flow cytometry (FCM). The expression level of Cyclin E protein was examined using Western blot. RESULTS: The cell surface of the LAPTM4B transfected cells showed abundant tube/finger-like microvilli, which were distinguished significantly from the MOCK cells. The cell attachment/spreading of transfected cells increased on fibronectin, matrigel and laminin. The growth rate of transfected cells was faster than that of the MOCK cells. FCM analysis indicated that the amount of the transfected cells in S phase was increased significantly. Cyclin E protein expression of transfected cells was much higher than that of the MOCK cell. The serum-dependence of transfected cells was decreased. The fibrosarcoma was formed at a tumorigenic rate of 50% in NIH mice inoculated with LAPTM4B transfected cells. CONCLUSION:LAPTM4B gene promotes the cell proliferation by involving in the regulation of cell cycle control and causes tumorigenesis of NIH3T3 cells, indicating that it plays important roles in tumorigenesis.