Contrasting Effects Of KrüPpel-Like Factor4on X-Ray　Induced DNA Breaks In Mouse Astrocytes

Purpose: External radiotherapy of brain tumors causes radiation injury to normalbrain tissue. Astrocytes are the most common cell types in the brain and they play aprincipal role in its repair. The effects of Krüppel-like factor4(KLF4) in the responseto DNA damage induced by X-ray on astrocytes were unclear.Materials and methods: astrocytes were isolated from the murine brain (GFAP+),Inhibition of KLF4expression was gained with interfered KLF4lentivirus.double-strand DNA breaks (DSBs) were determined with a neutral comet assay and theγH2AX method, single-strand DNA breaks(SSBs) were determined with a basic cometassay.Results: In total,91%of the cells were astrocytes, as determined byimmunolabeling with anti-GFAP rabbit antibodies. Figure was one of the representativeresults.A significant effect was observed on the astrocytes after irradiation. Comparingthe irradiated group with the control, the highest KLF4expression level (1.8-foldincrease) was observed1h after irradiation, as shown by the respective real-timequantitative RT-PCR.The astrocytes grown on coverslips were irradiated with4Gy of X-rays. After1h,the immunofluorescence was stained with KLF4, γH2AX, and nucleus. More than85%of KLF4colocalized with γH2AX in the nucleus of the astrocytes.An siRNA-mediated knockdown in astrocytes was performed to determine theeffect of KLF4on DNA damage. The lentivirus transduced the astrocytes with highefficiency. The cells with lentivirus-transduced shKLF4had half the KLF4expressionlevel compared with those treated with the negative control lentivirus at both the RNAand the protein level.Phosphorylation of the H2A histone variant H2AX at Ser-139to form γH2AX is arapid response to the formation of DSBs. Astrocytes with low KLF4levels showed low γH2AX expression compared with the control cells. After irradiation (IR) with4Gy ofX-rays, γH2AX expression increased in both the shKLF4and the control lentivirustreatment groups. However, the lower γH2AX expression corresponded to lower KLF4levels at all time points post irradiation.The tail moment (percentage of DNA in the tail multiplied by tail length) of theneutral comet showed the severity of DNA DSBs in the astrocytes. The experimentconfirmed the result of the γH2AX experiment, i.e., lower DSBs corresponded to lowerKLF4expression at all time points post IR.The comet assay with alkaline lysis buffer and basic electrophoresis solutionshowed SSBs in the DNA. In contrast to DSBs, disrupted KLF4expression showed asignificantly increased tail moment of astrocytes compared with the control cells,regardless of irradiation and time point post IR.Atm and DNA-PKcs couldn’t be found in non-irradiated astrocytes, but they wereinduced half hour after irradiated of4Gy X-ray and low level of Klf4showed moreproteins Atm and DNA-PKcs. Ku70and Brca1didn’t show clear change afternormalized to GAPDH. Xrcc4had opposite trend to Atm and DNA-PKcs. It had highestlevel on non-irradiated astrocytes with shNC transfected and became lower when Klf4was interferedConclusions: The data suggests that once astrocytes around a brain tumor areirradiated during tumor radiation therapy, KLF4is overexpressed, which induces moreDSBs and reduces SSBs in astrocytes. This causes the adverse effects of radiationtherapy in the treatment of brain tumors.