Establishment of a lung cancer cell line A549 highly expressing DNA oxidative damage related repair gene hOGG1 / 第二军医大学学报

ABSTRACT

Objective: To establish a human lung cancer cell line A549 highly expressing human 8-oxoguanine-DNA glycosylase (hOGG1) by co-transfecting pcDNA 3.1 (+)/Myc-HisA-hOGG1 and PGL3 promoter, and to observe the biological behavior of the transfected cells. Methods: PcDNA3.1 (+)/Myc-His A-hOGG1 and PGL3 promoter were steadily co-transfected into A549 cells via mediation of Fu GENE 6 (transfected group); untransfected cells served as blank control and cells transfected with PGL3 + pcDNA3.1 (+)/Myc-HisA served as negative control. The hOGG1 mRNA expression in A549 cells was detected by Bio-luciferase activity and the hOGG1 protein expression by Western blotting analysis. Cells in the three groups were exposed to hyperoxia condition, and the morphological changes were observed by phase-contrast microscope. Comet cell rate and olive tail moment of cells were tested after different exposure periods. After exposure, the cells were incubated for 0, 60, 120, and 180 min, and the same indices were determined by modified comet assay; changes of DNA oxidative damage markers 8-hydroxy-desoxoguanosine (8-OHdG) was tested at the same time. Results: The bio-luciferase activity was stable in the co-transfected cells. Western blotting analysis showed that the expression of hOGG1 protein in co-transfected A549-T cells was significantly higher than those in the other two groups, indicating the successful establishment of hOGG1 highly expressing cells. Under the same hyperoxia condition, the morphological changes of transfected cells were greatly alleviated, and the Comet cell rate and olive tail moment of cells were obviously lower than those of the other two groups(P< 0.05). Transfected A549-T cells had significantly increased ability of DNA repair and shorter repair time compared with cells in the other two groups (P<0.05). Furthermore, the level of 8-OHdG in transfected A549-T cells was significantly lower than those of the other two groups under the same hyperoxia condition, indicating a significantly higher ability of DNA damage resisting and repair (P<0.05). Conclusion: High hOGG1 expression can decrease the cell sensitivity to DNA damage and improve the repair ability of cells.