Mutant D314a Of Escherichia Coli Cytosine Deaminase Enhances Its Antitumor Effect Significantly

Objective:To construct the mutant D314A (substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase)of Escherichia coli cytosine deaminase (EC-CD) and investigate its antitumor effect.Methods:The mutant D314A of EC-CD (EC-CDD314A) was established by site-directed mutation. EC-CDwt and EC-CDD314A were transducted into human colon cancer cell line LoVo via Lipofectamine? 2000 to produce LoVo-CDwt and LoVo-CDD314A cells. Gene expression in these cells was assayed by RT-PCR. The cytotoxicity and bystander effect were evaluated in vitro by MTT assay. Results:The mutant D314A was confirmed by sequencing analysis. LoVo-CDwt and LoVo-CDD314A cells proliferated in medium containing 0.6g/L G418 stably. LoVo-CDwt cells were more sensitive to 5-fluorocytosine (5-FC) compared with the parent cells (P=0.000), while LoVo-CDD314A cells were more susceptible to 5-FC than LoVo-CDwt cells. LoVo-CDD314A cells presented more excellent bystander effect (P=0.000).Conclusion:Mutant D314A of Escherichia coli cytosine deaminase enhances its antitumor effect significantly, and may become a superior suicide gene.