| Background and objective Esophageal Carcinoma is a familiar malignant tumor, itsincidence of disease and mortality are rather high. When cisplatin-based combination chemotherapy is used to treat Esophageal Carcinoma, acquired resistance often happened, which will affect the therapy. A more comprehensive understanding of cisplatin resistance is significant to enhance the action of chemotherapy and increase the viability of patient. Cisplatin is a cytotoxicity agent and its target is DNA once it enters a cell. Cisplatin forms intrastrand crosslinks, interstrand crosslinks and monofunctional adducts, which results in the inhibition of DNA replication, RNA transcription, and arrest at the G2 phase of the cell cycle and/or programmed cell death. These years, the research of cisplatin resistance has been focused on increased repair of adducts and increased adduct tolerance. DNA polymerase B is a house-keeping gene, its base sequence is highly conservative from yeast to mammalian. The major role of DNA polymerase B was thought to be limited in its involvement in short patch research indicated that polymerase B might take part in a wild spectrum of DNA metabolism reaction, including long patch base excision repair, DNA replication, recombination and transleisional DNA synthesis. High level Pol B have been detected in tumor tissue from colon, breast and prostate adenocarcinomas. Enhanced Pol B mRNA have been detected in some cisplatin resistance tumor cell lines such as ovarian and colon carcinoma and leucocythemia. Those probablycaused by that excessive Pol B would disturb or replace the natural function of other polymerases in cell. Furthermore, Pol B is an error-prone enzyme and its fidelity of repair is low, so the outstretched function would induce inheritance instability and drug resistance.To determine whether or not the enhanced expression of DNA polymerase B combination is related with cisplatin resistance, we established a Cisplatin-Induced Human Esophageal Carcinoma cisplatin resistant cell line(ECa109/cDDP), then the protein levels of DNA polymerase B in ECa109cell and ECa109/cDDP cell were studied.MethodsECa109 cells were maintained in DMEM medium supplemented with 10% FCS. Using the corresponding dose calculated from clinical chemotherapy ECa109/cDDP was established from Esophageal Carcinoma cell line ECa109 , with ECa109 exposed intermittently and repeatedly to high - level concentration of cisplatin at 10 u g/ml for 24 hours each time , drug sensitivity was detected by MTT assay , the change of cellular configuration was detected by inverse microscope , cellular cycle distributing of two type cells was detected by flow cytometry ( FCM ) and DNA polymerase B expression of two types of cells were detected by western blotting . The half inhibit concentration(IC5o) of Chinese hamster ovary(CHO) cells which transinfect 58bp deleted pol B wild pol B and empty plasmid by cisplatin were detected by MTT.Result(1) The resistance index of ECa109/cDDP to cisplain was 1.57. The proliferation of ECa109/cDDP cells is quicker than ECa109 cells (P<0.01) .Under reverse microscope, ECa109 and ECa109/cDDP cells have epithelioial mono layer array, the size of cells is different and the shape is polygonal. The bordline of ECa109 cells are clear, while the karyon is big, cycoidal and elliptic. The bordline of ECa109/cDDP cells is not clear, while the karyon is big, anomalous, and the bulk slightly expanded. Parts of cells are distorted and gigantic cells emerge. Cellular cycle assay display the cell number of S-phase increased and G0/G1 phase decreased (P<0.01) .(2) DNA polymerase B expression level of ECa109/cDDP cells is higher thanECa109/cDDP (P<0.01) .The IC50 of CHO cells which transinfect 58bp deleted type Pol B plasmid is higher than CHO cells which transinfect wild type Pol B plasmid and empty plasmid (P<0.01) . The IC50 of cells which transinfect empty plasmid is higher than wild type Pol B plasmid (P<0.01) .Conclusion(1) ECa109/cDDP cells indicate that resistant phenotype an... |
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