The Study Of Relationship Between The Expression Of DNA-PK And Proliferation Of Cancer Cells And Enffection In The Sensitivity To DNA Damage Agents

Objective The nonhomologous ending-joining (NHE J) is the most important pathway of the DNA repair in mammalian cells. DNA-PK play an importent role in this pathway and is a nuclear erine/threonine protein kinase that comprises a catalytic subunit (DNAPKcs), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. To study the function of NHEJ in the genesis of tumor and chemoresistance, and to explore the potential of DNA-PK as the target to reversal of chemoresistance and enhancing the sensitivity of cell to chemotherapeutic agents , we examined the expression of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and regulatory subject(Ku70) in ovarian carcinoma tissue and some tumor cell lines and investigated connection between the dwonregulation of DNA-PKcs or Ku70 and tumor cell proliferation and the sensitivity of cell to some chemotherapeutic agents.Methods The expressions of Ku70 and DNA-PKcs were determined by immunohistochemical S-P method in normal ovarian tissues, benign tumors , primary and recurrent ovarian carcinoma, ovarian cancer cell line A2780 and HeLa cells. A 63bp of DNA template targeting Ku70 or DNA-PKcs coding siRNA was synthesized to reconstruct pSIRENKu70shRNA and pSIRENDNA-PKcs shRNA. The inserted sequences were verified by DNA sequencing. The recombinant plasmid was cloned into A2780 and HeLa cell lines by lipid-mediated transfection and code small interfering RNA to make target genes silence. The cells were divided into four groups: control group ,iRNA group, chemotherapy group (Cisplatin , etoposide or topotecan) and transfected + chemotherapy group. The expression of DNA-PKcs and Ku70 in the levels of mRNA and protein was detected by RT-PCR and Western-blot. Survival rates and IC50 of cells were measured with MTT methods, Cell cycle and apoptosis were analyzed by flow cytometry (FCM).Results The expression of DNA-PKcs and Ku70 is different in normal ovarian tissues, benign tumors and ovarian carcinoma , the expression in ovarian carcinoma is higher than that in benign tumors, the expression in benign tumors is higher than that in normal ovarian tissues, and The expression of DNA-PKcs and Ku70 was significantly increased with the increase of pathological grades and the decrease of differentiation degree, the expression of DNA-PKcs were significantly correlated with the expression of Ku70, the expression of DNA-PKcs and Ku70 has not different in different histopathologic type; The intense expression of DNA- PKcs in recurrent cancer tissues was significantly higher than that in primary ovarian cancer , 29 of 36 cases with primary and their own recurrent ovarian cancers had enhanced expression of DNA- PKcs, The change of DNA -PKcs expression was not related with pathologic type, pathologic grade and clinical stage of the cancer. The expression of Ku70 in primary ovarian cancers is not relevant to that in recurrent ovarian cancers.The recombinant plasmids pSIRENKu70shRNA and pSIRENDNA-PKcs shRNA were constructed successfully and testified by DNA sequencing. The expression of DNA-PKcs in the levels of mRNA and protein was significantly suppressed when A2780 and HeLa cells were transfected by pSIRENDNA-PKcs shRNA . The growth of A2780 cells and HeLa cells, was inhibited significantly after transfected by pSIRENDNA-PKcs shRNA . The S phase cells increased meanwhile the G1 phase cells decreased . We used Cisplatin, etoposide or topotecan to treat the cells at 48h after transfected . IC50 of cells was distinctly decreased than untransfected cells. Conclusion DNA — PK should play an important role in the genesis and development of ovarian carcinoma. DNA repair function of NHEJ enhanced in ovarian carcinoma treated with chemotherapy, this may be an important mechanism of Cisplatin resistance in ovarian carcinoma . A 63bp of DNA template with loop structure targeting DNA-PKcs or Ku70 genes was synthesized and inserted into the plasmid, the recombinant plasmid could express stably and code small interfering RNA in cells to make target genes silence. Inhibiting the expression of DNA-PK could suppress the growth of tumor cells and enhance the sensitivity of cells to chemotherapeutic agents Cisplatin , etoposide and topotecan. DNA-PK may be one of the causes of drug resistanc, Inhibiting the expression of DNA-PK to decrease the function of DNA repair could enhance the sensitivity of cell to chemotherapeutic agents, our date strongly support the potential of DNA-PK as the target to reversal of drug resistance.