The Role And Mechanism Of DNA-PKcs In Biologic Bystander Effect Induced By Irradiation Stimulus

Objective: To investigate the effects of DNA-PKcs on radiation-induced biologic bystander effect,and to explore the mechanism of DNA-PKcs regulating radiation-induced biologic bystander effect.Methods: Human glioma cells M059 K and M059 J and human cervical cancer Hela cells were used.M059 K and Hela cells express normal DNA-dependent protein kinase activity,M059 J cells lack DNA-dependent protein kinase activity,and Hela was treated with DNA-PKcs inhibitor NU7026,each cell was divided into normal group,irradiation group and bystander effect group,the irradiation dose was 2Gy,the radiation bystander effect model was constructed as a bystander effect group 1h after 2Gy ? irradiation.The effect of DNA-PKcs on cell growth was detected by cell growth curve in the RIBE;The effect of DNA-PKcs on cell proliferation was observed by cell formation assay in the RIBE;The effects of DNA-PKcs on DNA damage by micronuclear test;DCFH-DA method was used to observe the effect of DNA-PKcs on ROS content in the RIBE;The effect of DNA-PKcs on cell cycle was detected by flow cytometry in the RIBE;Western Blot was used to detect the expression of DNA damage repair protein,H2 AX,53BP1 and CHK2-pT68.Results: 1.After irradiation,M059 K,M059J,Hela and NU7026 treated Hela cells was slowly grown;The formation of cell clones was reduced(P<0.05);The intracellular micronucleus was increased significantly(P<0.05);Intracellular ROS content was increased significantly(P<0.05);The cell cycle was detected by flow cytometry and the cells showed different degrees of G2/M phase arrest;Western Blot results showed that the expression of intracellular ?H2AX,53BP1 and CHK2-pT68 was increased.2.Bystander effect could lead to that the cell growth was slow;The formation of cell clones was reduced(P<0.05);The intracellular micronucleus was significantly increased(P<0.05);The intracellular ROS content increased significantly(P<0.05);The cell cycle was detected by flow cytometry and the cells showed different degrees of G2/M phase arrest;The expression of DNA damage repair protein ?H2AX?53BP1 and CHK2-pT68 was increased.3.In the absence or inactivation of DNA-PKcs,cells were more severely damaged by radiation bystander effect,The formation of cell clones was reduced(P<0.05);The intracellular micronucleus was significantly increased;Intracellular ROS content increased significantly(P<0.05);The cell cycle arrest was significantly prolonged;The expression of DNA damage repair protein 53BP1 was increased,The expression of ?H2AX and CHK2-pT68 was decreased.Conclusions:1.Bystander effect could lead to decreased cell viability;increased the production of intracellular micronucleus;increased intracellular ROS content;G2/M phase arrest of cell cycle;increased expression of DNA damage repair proteins ?H2AX,53BP1 and CHK2-pT68.2.In the absence or inactivation of DNA-PKcs,the frequency of micronucleus in Bystander group was significantly increased,the expression of ?H2AX and CHK2-pT68 were decreased,the expression of 53BP1 was increased,the cells were more severely affected by the radiation-induced bystander effect.3.DNA-PKcs participated in the radiation-induced bystander effect by regulating cell cycle and the expression of ROS,?H2AX,CHK2-pT68.