Study On The Hyper-radiosensitivity Induced In Normal Human Skin Fibroblast Cells By Low Levels Of Carbon Ion Radiation, And Corresponding Mechanisms

Objective: All living organisms are exposed to ionizing radiation (IR) on a day-to-day basis. In addition to diagnostic and therapeutic medical radiation exposures, most radiation exposures today are low levels, with primary sources being background radiation, cosmic rays, radioactive waste, radon decay, nuclear powers. Recent advances in studies on biological effects of low-dose ionizing radiation have brought forward some unexpected phenomena, which deviates from extrapolation of empirical linear fits of human data from relatively high-dose exposure. The phenomenon of low-dose hyper-radiosensitivity is one of them, which denotes an effect in which cells die from excessive sensitivity to low doses (<0.5 Gy) of ionizing radiation (low-dose hypersensitivity, HRS), but become resistant to higher doses (induced radioresistance, IRR). Low-dose HRS/IRR was mainly studied in low-LET irradiation with the endpoint of cell death, which has been proved the involvement of ATM mechanism. The unique physical and biological properties of carbon ions, favorable dose distributions with a steep dose fall-off at the field borders and more precise dose localization make it a potentially potent way for tumor radiotherapy in near future. In this way, the heavy charged particle beam gives a lower radiation dose at the entrance region, which is absorbed by normal tissues, while gives a higher dose (Bragg peak) deeper in the medium with a higher RBE obtained in the Bragg peak region that can focus on a target volume of tumor even if it is seated deeply in a body. The aim of this study was to find out whether HRS/IRR occurred under low levels of carbon radiation, and the corresponding mechanism, which may provide some experimental data for protection in the process of carbon ion radiation therapy in near future.Methods: Survival and mutation were measured by clonogenic assay and hprt mutation assay respectively. ATM Ser1981 activation was detected by Western blotting and immunofluorescent staining. Pretreatment of specific ATM inhibitor (10?M KU55933) and activator (20?g/mL chloroquine) prior to carbon ion radiation were adopted to explore the involvement of ATM. The roles of ATM were also investigated in its function concerning G2/M checkpoint and DNA DSB repair. G2/M checkpoint function was tested with mitotic ratio by immunofluorescent staining of phosphor-histone H3 (Ser 10) and G2/M percentage by flow cytometric assay. The related genes, like Chk2 and p53 were detected by Western blotting. DNA DSB repair efficiency was measured using?-H2AX foci assay, while the two pathways for DNA DSB repair, HRR and NHEJ, Rad51 and DNA-PKcs Thr2609 were tested by the representative proteins with Western blotting and immunofluorescent staining assay. The differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation of low dose (HRS) and high dose (IRR) were detected by microarray technique.Results: HRS/IRR was observed with survival and mutation in normal human skin fibroblast, GM cells exposed to low levels of carbon ions, while impaired in AT cells with intrinsic ATM deficiency or normal cells modified with specific ATM activator or inhibitor before irradiation. The dose-response pattern of ATM kinase activation was concordant with the transition from HRS to IRR. The ATM-dependent 'early' G2 checkpoint arrest and DNA DSB repair efficiency could explain the difference between HRS and IRR. Chk2 and p53 were both related to the mechanism of HRS/IRR by regulation of G2/M checkpoint . The low efficiency of DNA DSB repair can explain some of the mechanisms. And two main pathways of DNA DSB repair may also be involved in the mechanism. Besides ATM, other proteins concerning to checkpoint regulation, DNA repair, cell proliferation and cell signaling are supposed to play roles in the mechanism, as shown by microarray observations.Conclusion:These data demonstrate that the HRS/IRR by carbon ion radiation is an ATM dependent phenomenon in the cellular response to DNA damage, while other mechanisms are needed to be explored.