The Effect Of G2/M Checkpoint Mediated By Cdc25c On Low-does Hyper-radiosensitivity In Human Lung Cancer Cell Line A549

Objective To control the cell cycle checkpoint by down regulation of Cdc25c expression and observe the effect of cell cycle checkpoint on HRS/IRR,and discuss the mechanisms of HRS.Methods Hairpin siRNA templates targeting Cdc25c gene were synthesized and cloned into plasmid vector pGCsi-RAN-U6neo,transfected plasmids into human lung cancer cell line A549,selected the most effective plasmid by RT-PCR and Western blot, which were used to detect the expression of Cdc25c in the level of mRNA and protein. The cell lines including A549# transfected with pGCsi-RAN1 and A549* transfected with pGCsi-RANNC were constructed and irradiated with X-rays at different doses. Cell survival fraction was measured by the colony-forming assay based on ?uorescence-activated cell sorter, and the cell cycle distribution was examined with flow cytometry, and the expression of Cdc25c and P- Cdc25c(ser216) was detected by Western blot.Result The most effective plasmid is pGCsi-RAN1,and the expression of Cdc25c in A549# group was significantly lower than that in A549* group. HRS/IRR was observed in both cell lines while a exaggerated HRS was found in A549# cells. The ratio of G2/M phase cells was increased after exposure to X-ray doses >0.2Gy,while the cell cycle almost had no change at doses of <0.2Gy..A549# cells involved higher ratio of G2/M phase than that in A549* cells in unirradiated group, and which exhibited little difference at doses of <0.3Gy.After exposure to doses >0.3Gy,the ratio of G2/M phase cells was significantly increased and appeared ahead of time. These data indicate that high dose irradiation resulted in G2/M arrest and the threshold dose is different in these two kinds of cells. P- Cdc25c(ser216) protein expression was appeared and Cdc25c protein expression was decreased at doses of 0.3Gy in A549* cells while that is observed at doses of >0.3Gy in A549# cells. These data indicate that the expression of P- Cdc25c(ser216) and Cdc25c was accordance with G2/M checkpoint.Conclusions Low-does hyper-radiosensitivity may be associated with G2/M checkpoint which is mediated by Cdc25c and P- Cdc25c(ser216) protein.