DNA-PK, The Target For Enhancing Radio-and Chemo-Sensitivity Of Cancer Cells

PartⅠThe expressions of DSB repair protein in benign and malignant tissues of breast and cervix[Objective] To study the expressions of DSB repair proteins (including Ku80, DNA-PKcs and ATM) in breast carcinoma, breast fibroma, cervical carcinoma and cervical intra-epithelial neoplasia (CIN), and to explore their roles in neoplasia and tumor progression of breast and cervical carcinoma.[Methods] Immunohistochemistry was applied to detect the expressions of Ku80, DNA-PKcs, and ATM in 55 cases of breast carcinoma, 14 cases of breast fibroma, 41 cases of cervical carcinoma and 15 cases of CIN.[Results] The positive percentages of Ku80, DNA-PKcs and ATM were 74.55%,54.55% and 52.73% respectively in breast carcinoma, and were 92.86%,92.86% and 71.43% respectively in breast fibroma; The expression of three proteins in fibroma were all higher than that in carcinoma, but the differences of Ku80 and ATM were no significant (P>0.05), only the expression difference of DNA-PKcs was significant (X2=6.98,P=0.01). The expressions of DNA-PKcs and ATM protein were not associated with the pathology, tumor diameter, clinical stage and the lymph node metastasis of breast carcinoma patients (P>0.05), while Ku80 expression associated with tumor diameter and clinical stage of breast carcinoma patients (P<0.01). In breast carcinoma cases, Ku80 was positively correlated with DNA-PKcs (r=0.36, P=0.01) and ATM (r=0.36, P=0.02). The positive percentages of Ku80, DNA-PKcs and ATM were 70.73%, 68.29% and 19.51% respectively in cervical carcinoma, and were 80.00%, 73.33% and 33.33% respectively in CIN; the expression of three proteins in CIN were all higher than that in cervical carcinoma, but the differences were no significant (P>0.05). The expression of these proteins were not associated with the age of patients, pathology, differentiation, and clinical stage (P>0.05).in all 56 cases, Ku80 was positively correlated with DNA-PKcs (r=0.58, P=0.00) and ATM (r=0.27, P=0.04).[Conclusions] DNA-PKcs and Ku80 are more possible to be the target of cancer gene-therapy; DNA-PKcs might play the major role in the neoplasia and tumor progression of breast carcinoma; Ku80 had close relationship with DNA-PKcs and ATM in breast and cervical carcinoma.PartⅡResponse of cervical carcinoma and normal interstitial cell lines after X-ray irradiation[Objective] Study the response of cervical carcinoma cell lines, normal vascular endothelial cell line and fibroblast cell line which simulate as cervical carcinoma tissues after a serial of doses of X-ray irradiation.[Methods] Human cervical adencarcinoma cell line HeLa, human cervical squamous carcinoma cell lines SiHa, C33A, Caski, human umbilical vein endothelial cell line ECV304 and mouse fibroblast cell line NIH/3T3 were irradiated with 6 MV X-ray at the absorb doses of 6Gy and 10Gy. After irradiated for 24h and 48h, cells were collected and analyzed by flow cytometry for apoptosis and cell cycle.[Results] The apoptosis rate of C33A was highest, while the SiHa cell line was most resistant for X-ray; The apoptosis rates of ECV304 after irradiation with 10 Gy (13.04%±1.08%) was higher than 6Gy (6.51%±0.61%), P<0.05, while ones of the other cell lines were similar (P>0.05); Every cell lines were blocked at G2/M phase after irradiation, the cell percentages of G2/M phase increased with the doses of irradiation and got to maximum at 24-48h after irradiation.[Conclusions] High doses of irradiation could improve the apoptosis of tumor vascular endothelial cell and increase the cell accumulation of G2/M phase, which provide the theory evidences for high dose of irradiation at cervical carcinoma radiotherapy, boosting dose of irradiation, selecting the specific drugs aim directly at G2/M phase, and specifically inhibiting the genes of G2/M check point for gene therapy.PartⅢCorrelativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells[Objective] To explore the correlation between the expressions of DSB (DNA double-strand break) repair protein (including Ku80, DNA-PKcs and ATM) and radiosensitivity parameters in human tumor cell lines, and to study which protein could prognosticate the radiosensitivity of tumor cell.[Methods] SF2 (survival fraction at 2Gy),αandβvalues of eight tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation array,apoptosis rates at 48h after 10Gy of 6MV X-ray irradiation were analyzed by flow cytometry analysis, and Western Blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein, the correlativity between protein expression and SF2,α,βvalue or apoptosis rate was analyzed by Pearson linear correlation analysis.[Results]The expression of same protein in different cell lines and the expression of three proteins in the same cell line were various; there was a positive correlativity between the expression of DNA-PKcs and SF2 (r=0.72, P=0.04), but no correlativity was found between Ku80 and SF2 or ATM and SF2 (P>0.05). Bothαand apoptosis rate had no correlativity with the expression of DSB repair protein (P>0.05).[Conclusions] The expression of DNA-PKcs protein may be able to prognosticate the radiosensitivity of tumor cells, and could be an ideal target to enhance the radiosensitivity.PartⅣTo study the effect on radio- and chemo- sensitivity of HeLa cells after Ku80 inhibition[Objective] The HeLa cell model of Ku80 expression suppressed by siRNA was constructed and utilized to study the role of Ku80 in enhancing the radio- and chemo- sensitivity[Methods] Ku80-siRNA expression plasmids were constructed and HeLa cells were transfected with these plasmids by lipofectamine. Western blot was applied to measure the expression of Ku80;The proliferation of HeLa cells were determined by clone formation assay, MTT assay and tumor subcutaneously formation studies on nude mice. After irradiation with 6MV X-ray,cells were collected and analyzed by flow cytometry for apoptosis and cell cycle at 24h, 48h and 72h; The radiobiology parameters of three cell lines were acquired by clone formation array. The inhibition rate of cells treated with Topotecan, Etoposide or DDP was detected by MTT assay.[Results] Two cell clones stable transfected were got, and the inhibition rates of Ku80 protein expression of positive clone got to 96.40%; The clone formation efficiency of HeLa/Ku80-siRNA was 0.46±0.05, lower than the control cells (t=5.11, P<0.01). The proliferation rate of HeLa/Ku80-siRNA were slower than control cells 48h and 72h after transfection (P<0.05). And after transplanted for 25 days, the mean tumor volume of HeLa/Ku80-siRNA was only (18.92±3.60) mm3, while the mean tumor volume of control cells got to (194.88±30.61) mm3, the difference was significant (t=12.69, P<0.01), tumor inhibition rate got to 90.06%. The apoptosis rates of HeLa cells Ku80 inhibited were higher than control cells at 48h and 72h after X-ray radiation (P<0.05), but the differences of cell cycle change after irradiation were not significant (P>0.05); HeLa cells silenced of Ku80 had lower SF2 and D0 than control cells, and their SER (sensitization enhancement ratio) based on D10 got to 1.365. HeLa/Ku80-siRNA was more sensitivity to Topotecan and Etoposide than control cells (P<0.05).[Conclusion] The HeLa cell models Ku80 expression suppressed were successfully established; the inhibition of Ku80 by siRNA could enhance the radio- and chemo- sensitivity of HeLa, and suppress the cell proliferation.PartⅤStudy of Enhanced Radiosensitivity in Cervical Carcinoma Cell Line HeLa by Inhibition of DNA-PKcs[Objective] This study tried to probe potential role of DNA-PKcs in enhancing the radiosensitivity of cervical carcinoma.[Methods] The DNA-PKcs-targeted shRNA (small hairpin interfering RNA) and a competive DNA-PKcs inhibitor LY294002, were applied to inhibit the DNA-PKcs expression or activity in HeLa cells. Then the SF2,αvalues and apoptosis rates were analyzed by clonogenic survival assay and flow cytometry analysis respectively. [Results] The DNA-PKcs-targeted shRNA could enhance the radiosensitivity of HeLa, and its SF2 was 0.37, lower than the control cells clearly (0.53). The apoptosis rates of HeLa cells only administrated by 50μmol/L LY294002 management for 1 hour did not increase significantly (P>0.05), but LY294002 sensitized HeLa cells to 6 MV X-ray, after HeLa cells were administrated LY294002 and irradiation at 48h and 72h after 6Gy irradiation, the apoptosis rates were higher than those of cells only accepted irradiation (48h: t=3.25, P=0.03; 72h: t=3.01, P=0.04).[Conclusion] Inhibition of DNA-PKcs expression or activities may sensitize HeLa cells to X-ray. As the results, DNA-PKcs may be an ideal target for enhancing radiosensitivity of cervical carcinoma.PartⅥThe effect on radiobiology of HeLa after co-inhibition of DNA-PKcs and Ku80[Objective] To study the radiosensibility and cell cycle distribution of HeLa inhibited of one or several DNA double-strand break (DSB) repair proteins after being treated with small interfering RNA (siRNA) or LY294002.[Methods] Ku80 silenced cell HeLa/Ku80-siRNA and control cell HeLa/Neg-siRNA were applied, and transfected with DNA-PKcs-targeted siRNA or pretreated with 50μM LY294002, a chemically specific phosphatidylinositol (PI) 3-kinase (DNA-PKcs and ATM are both members of PI 3-kinase gene family) inhibitor, after irradiated with 6MV X-ray, cells were detected by clonogenic survival assay for radiosensibility and analyzed by flow cytometry for cell cycle distribution and apoptosis. [Results] HeLa/Ku80-siRNA transfected with DNA-PKcs-siRNA was more sensitive to radiation than HeLa/Ku80-siRNA, SF2 were 0.08±0.01 and 0.20±0.05 respectively, the SF2 of HeLa/Ku80-siRNA pretreated with LY294002 even got to 0.03±0.01, while the SF2 of control cell line HeLa/Neg-siRNA was 0.51±0.07; G2/M accumulation was observed at all cell lines after 6Gy X-ray exposure, the percentage of DNA-PKcs-siRNA transfected HeLa/Neg-siRNA and LY294002 treated cells in G2/M phase even didn't get to highest at 72h post-irradiation, while other cell lines had a shorter G2/M delay, the G2/M accumulation got to vertex at 48h post-irradiation.[Conclusions] After 95% Ku80 was inhibited, HeLa cells could have other proteins, for example DNA-PKcs or ATM to compensate the DSB repair function, co-inhibition of these proteins resulted in markedly sensitive to X-ray; Ku80, DNA-PKcs and ATM may play different role in cell cycle accumulation after irradiation.