Study On The Role Of DNA-PKcs In The Late Repair Of Radiation-induced DNA Double Strand Break

objective: Radio therapy is one of the major treatmentmodalities. To investigate the factors affecting tumor radio sensitivity isof great importance for improving curability of radio therapy. Theintrinsic radio sensitivity of tumor cells has close relation to cell cyclephase. Cell cycle progression will be blocked after radiation, which is oneof the major mechanisms for radio resistance. After being radiated, DNAlesions are produced, and cells with DNA lesions will be arrested in G1and G2phase when they encounter G1and G2checkpoints, respectively.The cell cycle arrest provides time for repairing DNA lesions, so that thedamaged DNA won’t be copied in S phase and won’t be passed todaughter cells in mitosis. The fidelity of mitosis is thus ensured and celldeath is limited. DNA single strand break (SSB) and DNA double strandbreak (DSB) are two major lesions generated by ionizing radiation.Un-repaired SSB and DSB will cause cell death and genetic aberrations.It is recognized that DSB repair is the major DNA lesions leading to celldeath and loss of proliferation capacity. Also, the repair kinetics of DNArepair after radiation showed that there are two components in DSBrepair-the fast component and the slow component. The half time of fastrepair is generally within0.08and1.2hours, which generally happenedin G1phase, whereas the slow component is within2.4to6hours when most of cells are arrested in G2phase. Because the predominant DSBrepair pathways in G1is NHEJ (non-homologous end joining), and NHEJis generally fast and effective, it is thought the fast component is fulfilledby NHEJ. The mechanism of slow repair is not as clear as fast repair.Both NHEJ and HRR（homologous recombination repair）works in G2arrest. The slow component of DSB repair is important in determiningcell fate after radiation. To clarify the roles of HRR and NHEJ in slowrepair is important for enhancing radio sensitivity through regulatingfunctions of key proteins in DSB repair. The catalytic subunit of DNAdependent protein kinase (DNA-PKcs) is a major protein kinase in NHEJand belongs to PI3-Kinase-like-Kinases (phosphatidylinositol kinaserelated protein, PIKK) family which also includes ataxia telangiectexia(ATM) and ATM and Rad3-related (ATR).The kinase activity ofDNA-PKcs is essential for NHEJ. It also participate the regulation ofG2-M together with ATM and ATR. The current study sought toinvestigate the possibility of blocking slow DSB repair after radiationthrough inhibiting the function of DNA-PKcs. The role of DNA-PKcsdependent NHEJ in slow repair will be checked and results of the currentstudy will provide supports to design strategies improving tumor radiosensitivity. Method:1.The cell cycles of HNE-1were determined by flowcytometer (FCM) after radiation at different time points.2.Immunofluorescence staining was used to analyze the number of DSB of HNE-1after radiation.3. Colony formation assays were used to measurethe inhibition of NU7441on clonal survival of HNE-1after radiation.4.ASAS9.1statistical software was used for all statistical analysis. Usingtwo-factor factorial design analysis of variance for mean comparisonsbetween groups.Results:1.The flow cytometry analysis showed that thecell population distribution0.5hours after2Gy radiation in G1、G2/Mand S phase were32.67%、8.00%and59.33%, respectively. At1hourafter radiation, the corresponding percentages are32.05%、8.00%and59.95%for G1、G2/M and S phase, respectively. At4hours afterradiation, the data changed to14.62%、19.44%and65.94%, respectively.For5Gy radiation, the relative populations at0.5hours after radiation inG1、G2/M and S phase were38.94%、8.00%and53.06%, respectively,and changed to37.49%、8.00%、54.51%at1hours after radiation,14.16%、21.58%and64.26%at4hours after radiation. The results of cellcycle analysis clear showed that the majority of NHE-1cells werearrested in S and G2/M phase at4hours after radiation.2.According tothe fluoresecence staining results, the average number of DSB per cellafter2Gy radiation at0.5hour、1hour and4hour are19.67±3.06、22.00±3.00、12.33±2.08, respectively. The average number of DSB percell after5Gy radiation at0.5hour、1hour and4hour are41.00±2.65、48.67±3.21and29.67±3.51, respectively. In agreement with reportedkinetic of DSB repair process, there are certain number of residual DSB left at4hours after radiation.3. Results from colony formation assaysshowed that PE of HNE-1cell line was65.50%. Nu7441is a specifickinase inhibitor of DNA-PKcs. The survivals of HNE-1after2Gyradiation with0μm、0.1μm、1μm and10μm of NU7441were(64.89±3.11)%、(62.03±1.91)%、(64.89±5.40)%and (63.00±1.14)%,respectively. The survivals after5Gy radiation with0μm、0.1μm、1μmand10μm of NU7441were (3.69±0.49)%、(4.07±1.44)%、(3.82±0.66)%and (4.09±1.14)%, respectively. The application of NU74414hours afterradiation didn’t change the clonogenic survival of HNE-1cells.4.A SAS9.1statistical software was used for all statistical analysis. Usingtwo-factor factorial design analysis of variance for mean comparisonsbetween groups.The four drug concentrations there was no statisticallysignificant difference between the cell survival rate after2Gy irradiation(F=0.37, P=0.776). The four drug concentrations there was no statisticallysignificant difference between the cell survival rate after5Gy irradiation(F=0.01, P=0.999).Conclusions: The results suggested that it wasunlikely that DNA-PKcs participated in the slow repair component. Fourhours after radiation, part of DSB left unrepaired and most of cells are inG2arrest. The inhibition of DNA-PKcs kinase activity at this timewindow did not affect the cell clonogenic survival. We concluded that thekinase activity of DNA-PKcs has little effect in DSB repair in G2arrest.HRR and DNA-PKcs-independent end joining are likely the major repair pathways in this special time inverval. Our study for the first timerevealed that DNA-Pkcs dependent NHEJ is not the major pathways inlate phase of DSB repair and has no contribution to cell survival. Thefinding of current study adds an important part of understanding themechanism of radio sensitivity and provides new views for designingstrategies to improve radio sensitivity.