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DNA-PK, the Target for Enhancing Radio-and Chemo-Sensitivity of Cancer Cells

Author: ZhuangLiang
Tutor: YuShiYing
School: Huazhong University of Science and Technology
Course: Oncology
Keywords: Ku80 DNA-PKcs ATM Breast Cancer Adenofibroma Cervical Cancer Cervical intraepithelial neoplasia Radiotherapy Apoptosis G2 / M arrest Tumor cell lines Radiosensitivity RNA interference Stable transfection Human cervical carcinoma cell line HeLa Cell Cycle Cells Chemosensitivity Cisplatin Topotecan Etoposide LY294002 Radiotherapy Cervical Cancer Small interfering RNA Cell cycle arrest
CLC: R730.5
Type: PhD thesis
Year: 2007
Downloads: 275
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The first part of the DNA double-strand break repair proteins in benign and malignant lesions of breast and cervical tissues [Objective] To study DNA double-strand break repair proteins (Ku80, DNA-PKcs and ATM) in breast cancer and breast and cervical fibroid tissue cancer and cervical intraepithelial neoplasia (CIN) expression, and explore three kinds of proteins in tumor development role and relationship. [Methods] Immunohistochemical SP method detected 55 cases of breast cancer and 14 cases of breast fibroma organizations, and 41 cases of cervical cancer and 15 cases of CIN tissues Ku80, DNA-PKcs and ATM protein expression. [Results] Ku80, DNA-PKcs and ATM protein in breast cancer patients with positive rates were 74.55%, 54.55% and 52.73%, in patients with breast fibroma positive rates were 92.86%, 92.86% and 71.43%; DNA-PKcs protein in breast cancer tissues was significantly lower breast fibroid tumor (X2 = 6.98, P = 0.01), while the expression of Ku80 and ATM, although the expression in breast cancer than breast fibroid tissue also, but the difference was not statistically significant (P gt; 0.05). ATM and DNA-PKcs protein expression in different pathological types, tumor size, clinical stage and lymph node metastasis of breast cancer patients showed no significant difference (P gt; 0.05); however Ku80 expression and tumor size and the patient's clinical staging (P lt; 0.01). Spearman rank correlation analysis, in 55 cases of breast cancer patients, Ku80 and DNA-PKcs expression was positively correlated (r = 0.36, P = 0.01); Ku80 and ATM expression also positively correlated (r = 0.33, P = 0.02). Ku80, DNA-PKcs and ATM protein in cervical cancer patients, the positive rate was 70.73%, 68.29% and 19.51% in CIN patients positive rates were 80.00%, 73.33% and 33.33%; three kinds of protein in cervical cancer tissues were lower than CIN tissues, but the difference was not statistically significant (P gt; 0.05). Three kinds of protein expression in different age, pathological type, differentiation and clinical stage cervical cancer patients showed no significant difference (P gt; 0.05). Spearman rank correlation analysis, in 56 patients, Ku80 and DNA-PKcs expression was positively correlated (r = 0.58, P = 0.00); Ku80 and ATM expression also positively correlated (r = 0.27, P = 0.04). [Conclusion] DNA-PKcs and Ku80 may become a target for sensitizing tumor therapy; DNA-PKcs protein in breast cancer development may play an important role; Ku80 and DNA-PKcs and ATM's close relationship exists between both. The second part of cervical cancer cell lines and normal stromal cells after irradiation by X-ray radiation effects [Objective] To take advantage of cervical cancer cell lines and normal vascular epithelial cell lines, normal fibroblasts in vitro cancer and stromal vascular substance organization of the subject after different doses of radiation response. [Methods] cervical adenocarcinoma cell line HeLa, cervical carcinoma cell line SiHa, C33A, Caski, and human umbilical vein endothelial cell line ECV304, mouse fibroblast cell line NIH/3T3 cells, respectively 6MV X-ray (300cGy / min) exposure 6Gy and 10Gy, 24h and 48h after the cells were collected propidium iodide staining, flow cytometry apoptosis and cell cycle changes. [Results] suffer the highest rates of apoptosis after irradiation, C33A, and SiHa the most resistant to the remaining cell lines in between; various cancer cell lines and NIH/3T3 48h after 10Gy irradiation produced similar 6Gy apoptotic rate (P gt; 0.05), and 48h after 10Gy irradiation produced ECV304 apoptosis rate (13.04% ± 1.08%) than 6Gy (6.51% ± 0.61%) is much higher (P lt; 0.05); various cell lines by irradiation, were showed significant G2 / M phase arrest and G2 / M arrest cell percentage increased with dose; being irradiated generally 24h ~ 48h G2 / M phase arrest highest percentage of cells. [Conclusion] High doses of radiation increase the apoptosis rate of tumor blood vessels, cancer cells after irradiation G2 / M phase arrest in a dose-dependent, high-dose radiation for the treatment of cervical cancer, the additional radiation dose to continue, select cycle specific chemotherapy drugs, as well as for the G2 / M checkpoint genes provide a basis for targeting blocked. The third part of the DNA double-strand break repair protein expression and tumor cell radiosensitivity [Objective] To detect tumor cell lines of DNA double-strand breaks (DNA double-strand break, DSB) repair proteins (Ku80, DNA-PKcs and ATM) expression levels and radiosensitivity parameters to explore three proteins indicates that the value of the radiosensitivity of tumor cells. [Method] 4 human cervical carcinoma cell line HeLa, SiHa, C33A and Caski, 3 株 human breast cancer cell line MCF-7, MDA-MB-231 and MDA-MB-453, and a human lung cancer cell line A549, Western blot analysis 8 cells Ku80, DNA-PKcs and ATM protein levels, flow cytometry 10Gy 6MV X-ray irradiation after 48h apoptosis rate, colony formation assay SF2 (Surviving fraction after 2Gy) values, and α , β values, Pearson linear correlation analysis of protein expression and apoptosis after irradiation, SF2 value and the α value correlation. [Results] three kinds of proteins in cells with one and the same protein expression in different cell lines were significantly different; DNA-PKcs expression level of positive correlation exists between SF2 (r = 0.72, P = 0.04 lt ; 0.05); Ku80 and ATM expression and SF2 values ??were not significantly correlated (P gt; 0.05); 3 proteins and apoptosis rate and α values ??were not correlated (P gt; 0.05). Conclusion The higher expression of DNA-PKcs cells more resistant to radiation, and its expression levels may be indicators of radiosensitivity of tumor cells; DNA-PKcs and Ku80 may be able to become a tumor radiosensitization ideal targets. Part IV siRNA inhibited Ku80 expression of HeLa cells after chemotherapy sensitivity [Objective] To establish the use of small interfering RNA (small interfering RNA, siRNA) inhibition of Ku80 expression in HeLa cell model, in order to explore the Ku80 increase in chemotherapy sensitive aspects of the potential applications. [Method] build targeted inhibition of Ku80 siRNA expression plasmid transfected HeLa cells stably expressing the transformation screened clones, Western blot detection of Ku80 expression; colony formation assay, MTT method and form tumors in nude mice clonogenic assay detects rate, and cell proliferation in vitro and in vivo situation; 6MV X-ray irradiation 6Gy later, flow cytometry apoptosis and cell cycle, cell colony formation assay SF2, D0 equivalents; MTT test cell by different concentrations ADS Topotecan, etoposide and cisplatin role in cell proliferation rate after the change. [Results] constructed plasmid transfected into HeLa cells stably transfected with access to two clones, Western blot analysis showed that the positive clones Ku80 protein inhibition rate reached 96.4%, named HeLa/Ku80-siRNA; HeLa / Neg-siRNA cell colony formation rate 0.62 ± 0.02, while HeLa/Ku80-siRNA cell colony formation was 0.46 ± 0.05, significantly lower than the control cells (t = 5.11, P lt; 0.01); MTT shows 48h and 72h, cell culture, HeLa/Ku80-siRNA cell proliferation was significantly lower than the control cells (P lt; 0.05); tumor growth in nude mice experiments show planted 25 days, HeLa/Ku80-siRNA average volume of tumor cell seeding (18.92 ± 3.60) mm3, significantly lower than the control cell seeding tumor volume (194.88 ± 30.61) mm3, (t = 12.69, P lt; 0.01), the inhibition rate reached 90.06%. HeLa/Ku80-siRNA by X-ray irradiation in vitro after 48h and 72h apoptosis higher than control cells (P lt; 0.05), but the three changes in the cell cycle was not statistically significant (P gt; 0.05); positive clones cell lines D0 and SF2 significantly lower in D10 sensitizing dose ratio of 1.365. Ku80 inhibited cells to etoposide, topotecan and increased sensitivity (P lt; 0.05). Conclusion Ku80-siRNA can inhibit the expression of Ku80 in vivo inhibition of proliferation of HeLa cells, HeLa cells and promote the X lines and topoisomerase inhibitor sensitivity. Fifth partially inhibited DNA-PKcs expression promoting the radiosensitivity of HeLa cells [Objective] To investigate the DSB repair protein DNA-PKcs become cervical cancer radiosensitization target possibilities. [Method] targeted inhibition of DNA-PKcs small hairpin interfering RNA (small hairpin interfering RNA, shRNA) expression plasmid and small molecule inhibitors LY294002, HeLa cells were inhibited DNA-PKcs protein expression and activity, the colony formation assay and HeLa cells by flow cytometry 6MV X-ray irradiation after SF2, α values ??and the apoptosis rate changes. [Results] targeted inhibition of DNA-PKcs shRNA can promote the radiosensitivity of HeLa cells, which SF2 is 0.37, which was significantly lower than the control HeLa cells 0.53; separate acceptance 50μmol / L LY294002 for 1h unused HeLa cells apoptosis was significantly increased (P gt; 0.05), but the first by LY294002 treatment and then irradiated 6Gy HeLa cells at 48h and 72h apoptosis rates than irradiation alone 6Gy apoptosis in HeLa cells was significantly increased (48h points: t = 3.25 , P = 0.03; 72h points: t = 3.01, P = 0.04). [Conclusion] inhibition of DNA-PKcs expression or activity can promote the radiosensitivity of HeLa cells; prompt DNA-PKcs may be able to become cervical cancer radiosensitization ideal targets. Part VI synergistic inhibition Ku80 and DNA-PKcs in HeLa cells to ionizing radiation biological function [Objective] siRNA and LY29400 inhibited single or multiple DNA double-strand break repair protein radiosensitivity of HeLa cells and cell cycle changes. [Methods] Ku80 inhibited cell HeLa/Ku80-siRNA and control cells HeLa / Neg-siRNA were transfected be targeted inhibition inhibition of DNA-PKcs siRNA or PI-3-K inhibitor LY294002 treatment by 6MV X-ray irradiation After clonogenic assay to detect cell radiosensitivity changes, flow cytometry cell cycle and apoptosis rate changes. [Results] The transfected DNA-PKcs-siRNA after HeLa/Ku80-siRNA radiosensitivity was significantly higher than HeLa/Ku80-siRNA, SF2 were 0.08 ± 0.01 and 0.20 ± 0.05, LY294002 role of SF2 after HeLa/Ku80-siRNA reached 0.03 ± 0.01, as the control cells, HeLa / Neg-siRNA its SF2 value of 0.51 ± 0.07; each cell have appeared after irradiation 6Gy G2 / M phase arrest, transfected DNA-PKcs-siRNA in HeLa / Neg-siRNA cells and LY294002 role HeLa/Ku80-siRNA, HeLa / Neg-siRNA cell G2 / M phase arrest occurs gradually slow, 72h after irradiation has not yet reached its peak, while the other cell G2 / M phase arrest were peaked at 48h after irradiation . [Conclusion] in the inhibition of Ku80 has reached 95%, based on, DSB repair by other DSB repair proteins such as DNA-PKcs and ATM compensatory, synergistic inhibition of these proteins can be significantly increased radiosensitivity of HeLa cells; Ku80, DNA- PKcs and ATM in cell G2 / M phase arrest process played a different role.

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