CDC25A Influence the Radiosensitivity of Lung Cancer Cell Line
|Keywords||CDC25A Ku70 γH2AX lung cancer cell radiosensitive cellcycle cell proliferation|
Objective There is a basic radiotherapy principle that cancer cells have different features possessing radiotherapy in contract to that of normal tissue cells. CDC25A expresses at a high level in various cancer cells tissues, thus the quantity of CDC25A may serve as the determine factors for curative effects of radiotherapy. To study the anti-tumor effect and mechanisms of CDC25A in radiation environment, we build high expression CDC25A lung cancer cells H1299and low expression lung cancer cells H1299, to investigate the effects of the different expressing CDC25A on cell growth, cycle, and radiosensitivity in H1299cells, and to explore its mechanism.MethodIn the first part of this study, we built up a human lung carcinoma H1299pEGFP-N1-CDC25A-GFP cell line by liposome mediated, which could stably express high level of CDC25A protein, a low level CDC25A expressing group by pGPU-GFP-shRNA liposome transfection, a control low CDC25A expressing group by pGPU-GFP, and a control group; CDC25A protein secretion and CDC25A mRNA expression were detected by Western blot and RT-PCR, and were compared with transfection efficiency. MTT assay was applied for cell proliferation detection, while Flow Cytometry (FCM) for cell cycle analysis.In the second part of this study, Immunofluorescence technique was used to investigate whether there was CDC25A in H1299cells under the fluorescence microscope and the confocal microscopy.16hours after4Gy X ray exposure, the cell percentages of apoptosis and each cell cycle phase were measured with flow cytometry among three cell line, that is pEGFP-N1-CDC25A-GFP cell line, pGPU-GFP-shRNA cell line, a control group. Cell growth inhibition was assessed by MTT-microculture tetrazolium assay after4Gy and12Gy X ray exposure at1d,4d,7d. Cloning experiment to compare the radiation sensitivity and survivability of cells based on the quality of CDC25A.In the third part of this study, the expression of Ku70in three cell lines was detected with WB. Confocal fluorescence immunoassay was detected the different expressing of yH2AX in three cell lines before and after radiotherapy. The above would explain the radiosensitivity effect of CDC25A and its mechanismResult In the first part of this study, we transferred the plasmids successfully; RT-PCR and WB showed high level expressing CDC25A group, low level expressing CDC25A group, moreover, there is no different among these groups either mRNA level or protein level. Our studies also demonstrated that after transfected with plasmid12h,24h,72h,there was no significant difference of the cell cycle distribution. MTT results showed cell proliferation between high level expressing and low level expressing CDC25A group are significantly different (P<0.05), The apoptosis rates of the other three groups were lower, no significant difference between the groups (P>0.05).In the second part of this study, the positive clones were selected by G418, highly or lowly expressing clones were more selected by flow cytometry, and get stable highly expressing CHO cell lines. G2/M cells lied in most high expressing CDC25A cell lines that were observed16h after irradiation(41.4±1.685%) while G0/G1cells lied fewest(37.5±1.579%).However,G2/M cells lied in few low expressing CDC25A cell lines that were observed (23.2±1.584%,P<0.01) while most G0/G1cells lied in low expressing CDC25A (P<0.01). After4Gy and12Gy X ray irradiation, the growth curve and adhesive rate showed that low expression cell line grew fastest and high expressing CDC25A grew slowest(P<0.01). The effect with12Gy group is more significant than the4Gy group. Colony-forming unit assays had the similar results (P<0.01).Above all, overexpression of CDC25A in non small cell lung cancer may play an important role in the development of radiosensitivity.In the third part of this study, The expression of Ku70in high expressing CDC25A group is the highest and the expression of γH2AX is the lowest without radiotherapy. The result is contrary to the low expressing CDC25A group. There is no different in other three groups. After4Gy X ray expoused,the expressing of Ku70in low expressing CDC25A group was increasing to top while it was no change in high expressing CDC25A group. The immunfluresene showed that the expressions ofγH2AX were higher in high expressing CDC25A group than those in low expressing experimental groups at1h after2Gy X ray expouse.ConclusionCDC25A can significantly accelerate the process of cell cycle and promote the proliferation of lung cancer cell line. This may be related to positive regulation the expressing of Ku70could increase capability of DNA damage repair and DNA double-strand break repair after ionizing radiation.CDC25A can regulate G2/M and G1/S transition in cell cycle. Up-regulate the expressing of CDC25A could not arrested cells in G0/G1and G2/M phase, or induce apoptosis. But down-regulate the expressing of CDC25A could apparently arrested cells in G0/G1, block the cell cycle process in order to leave more time for DNA damage repair.Over expression of CDC25A can inhibit lung cancer cell proliferate and enhanced the radiation-sensitivity. Low expression of CDC25a group had the opposite effect. Our study also slowed that the expressions of yH2AX were higher in high expressing CDC25A group than those in low expressing experimental groups at lh after2Gy X ray expose, it may be explained that over expressing CDC25a can cause the abolition of blocking function of DNA damage checkpoint, making cells to undertake mitosis in DNA damage repair status.Cdc25a can be a target genes that make tumor cells more sensitive to radiation therapy, or to predict whether the cancer is sensitive to radiation.