Role Of DNA Repair In Radioadaptive Response And Its Molecular Mechanism

Organisms living in an environment filled with vary low dose radiation including residential radon,nuclear power,clinical irradiation,cosmic radiation and other occupational radiation exposure are unavoidably affected by these environmental stress. Many effective cellular defense strategies have evolved in organisms to decrease genotoxic damage.One of them is called radioadaptive response(RAR) induced by low dose radiation.RAR is a biological defense mechanism of which low dose ionizing radiation induces cellular resistance to the genotoxic effects of subsequent challenging irradiation.Up to now,the molecular mechanisms responsible for RAR are not fully known.The characteristics of RAR at cellular level include surviving better and less damage and apoptosis,so DNA repair up-regulation could be involved in the RAR. But there have long been controversies about the role of DNA repair in the RAR.Applied three Chinese hamster ovary(CHO) cell lines of different DNA damage repair capacity; the present thesis exploredγ-rays induced RAR and its relationship with DNA damage repair and further investigated the role of P53 and NF-κB in the induction of RAR in Chang liver cells.Chapter One:radioadaptive response and its relationship with DNA repairObjective:To exploreγ-rays induced radioadaptive response(RAR) in Chinese hamster ovary(CHO) cell lines of different DNA damage repair capacity.Methods:CHO-9 cells and its two repair-deficient strains,EM-C11(DNA single strand break repair deficient) and XR-C1(DNA double strand break repair deficient), were irradiated with a priming dose of 0.08Gy or 0.016Gy,after 4 or 7 hours of interval time,they were irradiated again with a challenging dose of 1 Gy.Then micronucleus induction and plating efficiency of these cells were assayed.The expressions of XRCC1, DNA-PKcs mRNA in CHO cell were analysis by cross-species semi-quantitative RT-PCR under the condition of 4h interval time.Results:Following to 0.08Gy priming dose and 4 hours interval time,only CHO-9 cells showed RAR to 1 Gy challenging irradiation.When the interval time was prolonged to 7 hours,both CHO-9 and EM-C11 showed RAR,but XR-C1 did not.When the cells were pretreated with a lower priming dose of 0.016Gy,after 4 hours interval,all those three different cells showed RAR to the subsequent 1Gy irradiation.The results of RT-PCR showed that there was no significantly difference in the expression of XRCC1 and DNA-PKcs mRNA between the group pretreated with 0.016Gy irradiation and the group irradiated with 1Gy alone.But the mRNA levels of XRCC1,DNA-PKcs of the group pretreated with 0.08Gy irradiation were higher than that of the group irradiated with 1Gy alone,consist with the induction of RAR.Conclusions:RAR is not only related to the priming dose and the time interval between irradiations,but also has close dependence on the ability of DNA damage repair.Chapter two:Roles of NF-κB and P53 in the radioadaptive response of Chang liver cells.Objective:to investigate the subcellular signaling pathway of adaptive response induced by low-dose radiation(LDR),and to explore the role of Nuclear Factor-κB (NF-κB) and P53 in RAR induced by r-rays in Chang liver cells.Methods:Chang liver cells were first irradiated with a priming dose of 0.016Gy, 0.08Gy,or 0.16Gy at a dose rate of 0.016Gy/min,after 4 hours of interval time,they were irradiated again with a challenging dose of 2Gy or 3Gy at a dose rate of 0.8Gy/min.Then micronucleus induction in these cells was assayed.For 0.08Gy priming irradiation,cells were treated with either P53 inhibitor of PFT-α(20uM) or NF-κB inhibitors of, BAY11-7082(3uM) 3h before irradiation,then micronucleus induction,protein expressions of P53,Phospho-P53,and nuclear NF-κB were measured.Results:When pretreated with a priming dose of 0.08Gy,cells showed RAR to challenging irradiation.But unexpectedly the priming irradiation of 0.016Gy or 0.16Gy could not induce RAR in Chang liver cells.The radiosensitivity of Chang liver cells was reduced by the treatment with PFT-α.Under the conditions of 0.08Gy priming dose,4 hours interval time and challenging dose of 2Gy cells didn't show significantly RAR,but when challenging dose was raised to 3Gy,significantly RAR was observed.However,for both challenging doses of 2Gy and 3Gy,the generation of RAR in Chang liver cells was suppressed by the NF-κB inhibitor of BAY11-7082.The result of Western-Blot measurement showed that nuclear NF-κB could be induced by 0.08Gyγ-rays irradiation, furthermore,the expression of nuclear NF-κB in the cells primed with 0.08 Gy irradiation was higher than that in the cells exposed to the challenging irradiation only.In contrast, 0.08Gy irradiation didn't elicit expressions of P53 and phospho-P53(ser15),and the expression phospho-P53 in the primed cells was lower than that in the cells exposed to challenging irradiation alone although the expression of P53 remained no difference between primed cells and non-primed cells.Conclusions:Our results showed that the induction of RAR depends on the priming irradiation dose and it is NF-κB rather than P53 that contributes to LDR induced adaptive response in Chang liver cells.