Influence of promoter region ate-mutant of DNA polymerase gene on its transcriptional activity in EC-1 cell

Objective To investigate the influence of promoter region ate-mutant of DNA polymerase β gene(pol β) on its transcr iptional activity in esophageal carcinoma cell line (EC-1).Methods We extracted genomic DNA of normal mimortalized cell 293T and EC-1 cell.The sequences of the wildtype pol β in 293T and mutanttype pol β with mutation at -137nt and -166nt(G→A) sites in EC-1 were obtained with polyme rase chain reaction(PCR) and DNA sequence analysis.Then the luciferase reporter gene recombinant plasm ids pGL 3-neo-293T-pol β/promoter(wild-type) and pGL 3-neo-EC1-pol β/promoter(mutant-type) were built.The recom binant plasmids were transfected into EC-1,Eca9706 or 293T cells and screened by G418.The lucife rase activity of each group was detected.Results The wild-type and mutant-type pol β promoters were inserted in to pGL3-neo-enhancer in right direction.Control group (pGL 3-neo-enhancer),wild-type group and mutant group were transfected into EC-1,Eca9706 and 293T cells,respectively.The differences of luciferase activitie s between the wild and the control group,the mutant and the control group were significant(P< 0.001).There was no significant difference between wild-type and control-type group(P > 0.05) as well as between mutant EC-1,Eca9706 and 293T cells(P < 0.05,P < 0.001,P < 0.001).Conclusion The mutation at -137nt and -166nt(G→A) of DNA pol β promoter in EC-1 can enhance the promoter activity.The promoter activity of the mutant DNA pol β promoter reporter gene vector is significantly higher in esophageal carcinoma cell than in mimorta lized normal cell 293T indicating that there maybe some factors enhancing the mutant DNA pol β promoter activity in esophageal cancer cells.