| PurposeTo investigate the radiosensitive effect of the wild type p53 gene on Xuanwei Lung Cancer (XWLC-05), we established the model for Xuanwei Lung Cancer cell with normal p53 function.At the present of different dose, we investigate relationship between the radioationsensitivty of theXunwei Lung Cancer (XWLC-05) and the status of the wild type p53 protein,apoptosis and cell cycle.Materials and methods1. PC53-SN3 plasmid containing wild type p53 gene and the vector plasmid PCMV-Neo-Bam were electrotransformed,amplied and endonuclease identified analyzed2. PC53-SN3 plasmid and the vector plasmid PCMV-Neo-Bam were transfected into lung cancer line XWLC-05 by means of LipofectamineTM2000 mediated transfection method.3. The protein expression levels of p53 and p21 in cells before and after treated with Adramycin 200ng/ml were evaluated using Western blotting analysis in order to determine the p53 function status of the cells.4. The clonogenic assay was performed to determine the survival fraction. The parameters Do,Dq,SF2and N for the single-hit multitarget model and the parametersα,β,SF2 for the linear-quadratic model were calculated.5. Cell cycle progression and apoptotic cells were assessed by Flow Cytometric Analyses at 48 hours after exposure to X ray of 2Gy.At the same time, the group after exposure to X ray of 0 Gy was control group.Results1. The PC53-SN3 plasimid had the wild type p53 gene. PC53-SN3 and PCMV-Neo-Bam plasmid meeted our study requirement..2. PC53-SN3 and PCMV-Neo-Bam plasmid were successfully transfected into the Xuanwei lung cancer cell lines XWLC-05. The PC53-SN3 plasmid transfection efficacy was 3-5 per2×105cells. The PCMV-Neo-Bam plasmid transfection efficacy was 10～15 per2×105 cells. The transfected cells were called XWLC-05-wtp53 and XWLC-05-pNeo, respectively.3. Western blotting analysis demonstrated that XWLC-05 cell lines displayed abnormal p53 function. P53and P21 protein levels increased after wild-type-p53.gene-transfected cells were treated with Adramycin, suggesting those cells obtained normalp53 function.4. Using the single-hit multitarget model and the linear-quadratic model to the fit surviving fraction after irradiation, the XWLC-05. XWLC-05-pNeo and XWLC-05-wtp53 cells have the Do parameters of 2.397Gy,2.295Gy andl.863Gy, respectively; the Dq parameters of 1.035Gy,1.013Gy and 0.502Gy; the a parameters of 0.190 Gy,0.192Gy and 0.330Gy- respectively; the 0 parameters of 0.032Gy,0.036Gy and 0.041 Gy respectively; theα/βparameters of 5.938 Gy,5.333Gy and 8.049Gy-respectively;the SF2 parameters of 0.584.0.569 and 0.422 in the single-hit multitarget, respectively; the SF2 parameters of 0.602.0.589 and 0.439 n the linear-quadratic model respectively,the XWLC-05-pNeo and XWLC-05-wtp53 cells have the SER parameters of 1.044 and 1.287.Compared with XWLC-05 cells with p53 dysfunction D0,Dq,SF2 decreased and a increased were seen in wild-type p53 transfected cells after radiation.The PCMV-Neo-Bam plasimd had no effect on radiosensitivity.5. Flow Cytometric analyses showed that the greatest percentages of apoptotic cells were detected when cells were exposed to X ray of 2Gy,13.8%,10.5% and 2.59% in XWLC-05-wtp53,XWLC-05-pNeo and XWLC-05 cells, respectively. The amount of apoptotic cells decreased when radiation dose increased. XWLC-05 and XWLC-05-pNeo induced G2 arrest after irradiation, while the rate of apoptosis was decreased. P53 transfection induced G1 arrest after irradiation, while the rate of apoptosis was increased.Conclusions1. XWLC-05 cell lines displayed abnormal p53 function, while obtained normal p53 function after being transfected with wild type p53 gene.2. Wild-type p53 gene could enhance the radiosensitivity of Xuanwei cell lines XWLC-05. The PCMV-Neo-Bam plasimd had no effect on radiosensitivity.3. Flow Cytometric analyses showed that the greatest percentages of apoptotic cells were detected when cells were exposed to X ray of 2Gy. XWLC-05 and XWLC-05-pNeo induced G2 arrest after irradiation. P53 transfection induced G1 arrest after irradiation.
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