Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene
Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene作者机构：Department of General Surgery The First Affiliated Hospital of Nanjing Medical University 300 Guangzhou Road Nanjing 210029 Jiangsu Province China.
出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)
年 卷 期：2002年第8卷第2期
页 面：270-275页
核心收录：
学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学]
基 金：the Creation Foundation of Nanjing Medical University No.Cx9905
主 题：Gene Therapy Genetic Vectors Adenoviridae Animals Antimetabolites Bystander Effect Carcinoembryonic Antigen Cell Line Colorectal Neoplasms Cytosine Deaminase Flucytosine Hela Cells Humans Nucleoside Deaminases Promoter Regions (Genetics) Research Support, Non-U.S. Gov't Tumor Cells, Cultured
摘 要：AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micr
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