Study On The High Radiosensitivity Of AT Cell And Its Signal Transduction Mechanism

Objective: Cytokinesis-Block micronucleus method, Conventional chromosome aberraton analysis method and Hypoxanthine phospho-ribosyl transferase gene mutation analysis technique were used to study the radiosensitivity of Ataxia-telangiectasia fibroblast cells at the cell, chromosome and molecular level respectively. Using gene transfection technique,we established the AT cell strains with PEBS7-YZ5 plasmid in which we aimed to study whether ATM controlled the phosphorylation of P53. K562 cells served as a p53 mutation model in which we intended to study whether ATM controlled the phosphorylation of P21 directly without the mediation of P53. Accordingly we study the mechanism of the high radiosensitivity of AT cell from the aspect of signal transduction pathway Methods: (1)Using Cytokinesis-Block micronucleus method, the micronucleus frequencies(MNF) and micronucleus cell frequencies(MNCF) of AT cells (AT5BIVA) with 0, 1, 2, 3 and 4 Gy exposure to (60)~Co γ-rays were observed, compared with those of GM cells (GM0639) from the skin of the normal subject; (2)Using conventional chromosome aberration analysis method, chromosome aberration frequences(CAF) of AT cells with 0,1,2,3 and 4 Gy exposure to (60)~Co γ-rays were observed, compared with that of GM cells; (3)Using Hypoxanthine phospho-ribosyl transferase gene mutation analysis technique, the hprt mutation frequencies (hprtMF) of AT cells irradiated with 0, 1, 2, 3 and 4 Gy of 60Co γ-rays were observed, compared with that of GM cells; (4)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. (5)The Co-immunoprecipitation and Western blot technique were used to study whether ATM controlled the phosphorylation of P53 and identify the interrelationship between ATM and p53 in PEBS7-YZ5-AT cells. (6)K562 cells serve as a p53 mutation model in which we used co-immunoprecipitation and Western blot technique to study the interrelationship between ATM and p21, and identify whether ATM phosphorylated p21 directly without the mediation of P53. Results: (1)After exposed to 1, 2, 3 and 4 Gy of 60Co γ-rays, the MNF and MNCF induced by irradiation were significantly higher in the AT cells compared with those of GM cells(P<0.01). In the two cell types, both MNF and MNCF had a positive correlation with dose, and the linear regression equations of AT and GM cells were: Y=a+bX. The slopes of MNF and MNCF linear regression equations in AT cells are larger than those of GM(P<0.01) ; (2)After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, the CAF induced by irradiation was significantly higher in the AT cells compared with that of GM cells(P<0.05). In the two cell types, CAF had a positive correlation with dose, and the linear regression equations of AT and GM cells were Y=a+bX. The slope of CAF linear regression equations of AT cells is larger than that of GM cells(P<0.05); (3)After exposure to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, hprt MF was significantly higher in AT cells than that in GM cells(P<0.01). In the two cell types, hprt MF had a positive correlation with dose, and their linear regression equations were y=a+bx. The slope of AT hprt MF linear regression equations were larger than that of GM(P<0.05); (4)PEBS7-YZ5 plasmids were transfected into AT cells successfully. 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; (5)After exposed to ionizing radiation, P53 of PEBS7-YZ5-AT cells was phosphorylated. Immunoprecipitation showed that ATM interreacted with p53; (6)P21 of K562 cells was phosphorylated after exposure to 60Co γ-rays. P21 protein was found in the Immunoprecipitation complex by ATM antibody. Conclusion: (1)After exposed to 1, 2, 3 and 4 Gy of 60Co γ-rays, both MNF and MNCF of AT and GM cells had a positive correlation with dose. At the same dose point, the MNF and MNCF of AT cells were significantly higher than those of GM cells (P<0.01). So we concluded that the radiosensitivity of AT cells is significantly higher than that of GM cells at the cell level;(2) After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, CAF of AT and GM cells had a positive correlation with dose. At the same dose point, the CAF of AT cells was significantly higher than that of GM cells(P < 0.01). It showed that theradiation-induced chromosome damage of AT cells is more serious than that of GM cells and the radiosensitivity of AT cells is significantly higher than that of GM cells; (3) After exposed to 0, 1, 2, 3 and 4 Gy of 60Co γ-rays, hprt MF of AT and GM cells had a positive correlation with dose. At the same dose point, the hprt MF of AT cells was significantly higher than that of GM cells(P<0.01). It showed that the radiation-induced genetic locus damage of AT cells is more serious than that of GM cells, and the radiosensitivity of AT cells is significantly higher than that of GM cells; (4)In the AT cell strains transfected with PEBS7-YZ5 plasmid, exogenous ATM mRNA and ATM protein expressed stably; (5)Ionizing radiation can activate exogenous ATM kinase, and activated ATM kinase phosphorylated downstream p53 gene. More P53 was phosphorylated by ATM with the increase of radiation dose;(6) Radiation-induced ATM kinase can phosphorylate P21 directly without P53 mediation. And more P21 was phosphorylated by ATM with the radiation dose increasing.