Study On Effect Of ATM On Telomerase Activity And HTERT Expression Of AT Cells

Objective: Using gene transfection technique,we established the ATM+-AT cell strains in which we aimed to observe the effect of exogenous ATM protein on telomerase activity and hTERT expression and compared that with endogenous ATM. We also aimed to study on the effect of dose and time factors on telomerase activity and hTERT expression. Accordingly we investigate the regulation of hTERT expression to telomerase activity.Methods: (1) PEBS7-YZ5 plasmids containing ATM gene cDNA were transfected into AT cells by electroporation. Hygromicin is used to select the cells expressing ATM protein stably. RT-PCR was used to detect transcription of ATM and Western blot was used to detect expression of ATM protein in order to verify the ATM gene transfection.(2) After exposed to 0, 1, 3 and 5 Gy of 60Coγ-rays, TRAP and HPLC were used to quantitative analysis of telomerase activity in AT,PEBS7-AT,ATM+-AT and GM cells, respectively.(3) After exposed to 3 Gy of 60Coγ-rays for 2, 24, 48 and 72 hours, TRAP and HPLC were also used to quantitative analysis of telomerase activity in AT,PEBS7-AT,ATM+-AT and GM cells, respectively.(4) After exposed to 0, 1, 3 and 5 Gy of 60Coγ-rays, RT-PCR was used to detect hTERT mRNA expression and Western blot was used to examine hTERT protein in AT,PEBS7-AT,ATM+-AT and GM cells, respectively.(5) After exposed to 3 Gy of 60Coγ-rays for 2, 24, 48 and 72 hours, RT-PCR was used to detect hTERT mRNA expression and Western blot was used to examine hTERT protein in AT,PEBS7-AT,ATM+-AT and GM cells, respectively.Results:(1)PEBS7-YZ5 plasmids were transfected into AT cells successfully. ATM+-AT cell strains expressing ATM protein stably were obtained after selected by hygromicin. RT-PCR detected fragment of ATM cDNA. Western blot detected blot of ATM protein.(2) Except for GM cell, there were telomerase activity in AT, PEBS7-AT and ATM+-AT cells without ionizing radiation. whereas, the telomerase activity in ATM+-AT cell was significantly lower than that of AT and PEBS7-AT cells(P<...