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Inhibitory effect of mutant cytosine deaminase D314A against human colon cancer cells
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Abstract:
Objective:To construct a mutant :D314A: of Escherichia coli cytosine deaminase (ECCD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. :Methods: :Eukaryotic expression plasmid containing ECCD gene (pcDNA3.1CDwt) was constructed, and the mutant pcDNA3.1CDD314A plasmid, with aspartic acid (D) at position 314 of ECCD gene substituted by alanine (A) (ECCDD314A), was established by sitedirected mutation. ECCDwt and ECCDD314A were transfected into human colon cancer cell line LoVo via LipofectamineTM 2000, and positive LoVoCDwt and LoVoCDD314A cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of ECCD and ECCDD314A genes on LoVo cells were evaluated by MTT assay. :Results: :The mutant :D314A: was confirmed by sequence analysis. ECCD and ECCDD314A mRNA were expressed after transfected into LoVo cells. The IC50 of LovoCDD314A cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVoCDwt cells (\[689.76±0.45\] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVoCDwt cells and LovoCDD314A cells were (48.5±049)% and (17.3±0.40)% (P=0.000), respectively. :Conclusion：:Mutatant ECCD gene (ECCDD314A) has a significantly increased antitumor effect on LoVo cells compared with wild type EGCD gene, and it may become a new candidate gene for tumor gene therapy.
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Supported by the Social Development Foundation from Science and Technology Bureau of Nanjing (No. 200605010)
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