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Objective:To investigate the correlation of GNG4 with DNA damage repair and chemosensitivity of ovarian cancer cisplatin-resistant A2780/DDP cells.Methods:A2780/DDP cells were divided into 500 ng/ml cisplatin group (cDDP group), short hairpin RNA (shRNA)-GNG4 silencing GNG4 expression group (shRNA group), 500 ng/ml cisplatin and shRNA-GNG4 intervention group (shRNA+cDDP group), and non cisplatin and shRNA-GNG4 intervention group (blank control group). Western blot was used to detect the expressions of GNG4 and γH2AX proteins in each group; DNA damage in each group was detected by single cell gel electrophoresis. The focus formation of γH2AX gene at the injury site was detected by immunofluorescence. The ability of cell clone formation was detected by plate clone formation experiment.Results:Compared with the other three groups, the expression level of GNG4 protein in shRNA+cDDP group was the lowest, the expression level of γH2AX protein was the highest, and the differences were statistically significant (all P < 0.01). Single cell gel electrophoresis assay showed that the comet tail DNA% in blank control group, cDDP group, shRNA group and shRNA+cDDP group were (7.7±2.5)%, (12.3±3.6)%, (20.1±2.1)%, (38.6±2.8)%, respectively, and Olive trailing distance were 5.12±1.89, 8.23±2.97, 14.99±3.65, 22.43±3.17, respectively, the comet tail DNA% and Olive tail distance in shRNA+cDDP group were higher than those in the other three groups, and the differences were statistically significant (all P < 0.05). Immunofluorescence assay showed that the focus numbers of γH2AX in each cell of blank control group, cDDP group, shRNA group and shRNA+cDDP group were 4.2±0.7, 5.1±0.5, 26.8±3.3, 71.3±6.2, respectively, the shRNA+cDDP group was higher than the other three groups, and the differences were statistically significant (all P < 0.05). The clone formation rates of blank control group, cDDP group, shRNA group and shRNA+cDDP group were (78.27±5.01)%, (45.67±3.29)%, (26.20±5.76)%, (1.56±0.21)%, respectively, the shRNA+cDDP group was lower than the other three groups, and the differences were statistically significant (all P < 0.001). Conclusions:Down-regulation of GNG4 expression can increase the cisplatin sensitivity of ovarian cancer A2780/DDP cells, which may be achieved by inhibiting the DNA damage repair function induced by cisplatin.
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In the face of DNA damage caused by various factors, cells have a set of response and repair mechanisms. Cell cycle arrest plays an important role in the DNA damage repair, which provides enough time for repairing damaged DNA. Research on cell cycle regulation focuses on cyclin-dependent protein kinases (CDKs) and cell cycle checkpoints. In the process of DNA damage repair, phosphatidylinositol-3-kinase like kinases (PIKKs) which are recruited to the DNA damage sites can activate cell cycle checkpoint-related proteins to halt cell cycle. In the common DNA damage repair pathways, such as base excision repair (BER), nucleotide excision repair (NER) , mismatch repair (MMR) , and DNA double-strand break repair, the recruitment of repair-related proteins also plays a role in the cell cycle regulation. In this paper, the relationship between the main forms of DNA damage repair and cell cycle arrest and relevant research progress were reviewed.
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BACKGROUND/AIMS: DNA double strand breaks (DSB) is one of the critical types of DNA damage. If unrepaired, DSB is accumulated in the nucleus of cells, the cells become apoptotic or transform to tumor by way of genomic instability. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. There was no report that Helicobacter pylori (H. pylori), the gastric carcinogen, induce DNA DSB in gastric epithelium in vivo. The aim of this study was to investigate whether H. pylori induce DSB in the gastric epithelial cells of chronic gastritis. METHODS: Immunohistochemical stains were performed for the DSB markers, phospho-53BP1 and gammaH2AX, in the gastric epithelium derived from 44 peptic ulcer disease patients before and after H. pylori eradication. DNA fragmentation assay was performed in the cell line to investigate the DNA damage by H. pylori infection. RESULTS: The mean expression score of gammaH2AX was significantly higher in the H. pylori infected gastric epithelium as compared to the H. pylori eradicated gastric epithelium (8.8+/-5.5 vs. 6.2+/-5.3 respectively; p=0.008). The expression score of phospho-53BP1 between before and after eradication of H. pylori was not statistically different, but tended to be higher in H. pylori infection. DNA fragmentation was developed significantly more in the cell lines after infection with H. pylori. CONCLUSIONS: DSB of DNA damage was typical feature of H. pylori infection in the gastric epithelium.