DNA-PKcs functions in cellular hyper-radiosensitivity / 中华放射医学与防护杂志

Objective@#To explore the functions of DNA-PKcs in cellular low dose hyper-radiosensitivity.@*Methods@#Colony-formation assay was used to detect the survival fractions of M059K and M059J cell lines treated by X-ray irradiation. Micronucleus assay and γ-H2AX foci assay were used to measure the radiation-induced DNA damage. Western blot was used to detect the relative expression levels of phospho-Chk1, total Chk1, phospho-Chk2 and total Chk2 of M059K and M059J cells.@*Results@#The hyper-radiosensitivity was observed in M059K cells irradiated with X-ray of doses lower than 1 Gy. DNA damage levels did not show HRS/IRR in the cell lines we used. pChk1/Chk1 in M059K cells was significantly increased during 20 min to 60 min after 0.2 Gy X-ray irradiation (t=14.157, 13.661, 14.177, 11.317, 14.512, P<0.05); pChk2/Chk2 in M059K cells was markedly increased during 20 min to 50 min after 0.2 Gy X-ray irradiation (t=13.182, 13.868, 14.155, 14.477, P<0.05).@*Conclusions@#M059K cells show the phenomenon of low dose hyper-radiosensitivity, which may be related to activation of proteins in G2/M phase checkpoints regulated by DNA-PKcs.

Screening and verifying the proteins interacting with phosphorylation cluster of DNA-PKcs by yeast two-hybrid assay / 中华放射医学与防护杂志

Objective To screen and verify the proteins interacting with phosphorylation cluster of DNA dependent protein kinase catalytic subunit((DNA-PKcs) by yeast two-hybrid assay.Methods To know the proteins interacting with DNA-PKcs phosphorylation cluster,yeast two-hybrid assay was applied to screen the cDNA library of human hepatic tissue with a previously constructed plasmid pGBKT7-DPC.The positive clones were further identified by PCR,rotary validation and sequence analysis.Then the eukaryotic expression vectors of the bait protein and screened positive clone proteins were constructed and transfected into human embryonic kidney 293T cells to detect whether the proteins could been expressed correctly.At last,the bait protein and screened positive clone proteins were co-transfected into 293T cells and protein interaction was detected with Co-Immunoprecipitation (Co-IP) assay.Results After two rounds of screening using the yeast two-hybrid assay,12 candidate clones were obtained.Then 7 clones with different insert fragments were identified by PCR,and 3 positive proteins interacted with DNA-PKcs phosphorylation cluster were further verified by rotary validation.Sequencing analysis demonstrated that these 3 proteins were MBNL1,SIK2 and YY1AP1,respectively.Accordingly,the eukaryotic expression vectors of bait protein and 3 positive clone proteins were constructed successfully and expressed correctly in 293T ceils.Finally,the Co-IP assay confirmed that these 3 positive clone proteins could interact with DNA-PKcs phosphorylation cluster.Conclusions Proteins interacting with DNA-PKcs phosphorylation cluster are successfully screened and identified.

The role of DNA-PKcs in autophagy induced by ionizing radiation / 军事医学

Objective To investigate the effect of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on autophagy induction by ionizing radiation ( IR ) .Methods The cell model of knocking-down DNA-PKcs expression was constructed by transfecting HeLa cells with a pSicoR-based lentivirus vector expressing DNA-PKcs specific shRNA .Cellular growth activity and radiosensitivity were detected by cell countingkit ( CCK)-8 assay.The expression of autophagy related proteins was detected by Western blotting hybridization .Autophagy was also detected by monitoring the autophagic marker green fluorescere protein ( GFP )-light chain 3 ( LC3 ) puncta per cell under an immunofluorescent microscope .Results A cellular model of knocking-down DNA-PKcs expression was successfully generated by transfecting the specific shRNA against DNA-PKcs.Depression of DNA-PKcs significantly decreased the growth activity of HeLa cells and increased the cellular sensitivity to ionizing radiation .Both the expression changes of P 62 and LC3 proteins and immunofluorescent GFP-LC3 puncta observation indicated that knocking-down DNA-PKcs prompted the induction of autophagy by ionizing radiation . Moreover, inactivation of DNA-PKcs led to a decreased phosphorylation of mammalian target of sirolimus ( Rapamycin, RAPA) ( mTOR) at S2481 site.Conclusion Depression of DNA-PKcs expression prompts the induction of autophagy by IR and cellular radiosensitivity .mTOR signaling may be involved in the regulation of autophagy processing by DNA-PKcs.

Radiosensitization of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells / 中华放射医学与防护杂志

Objective To evaluate the radiosensitization and mechanism of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells.Methods The flow cytometry was used to determine the sub-population of cancer stem cells with the markers CD133/ CD44.The cells were divided into four groups:control group,20 μmol/L NU7926 group,2 Gy irradiation group,and 20 μmol/L NU7026 combined with 2 Gy irradiation group.Cell proliferation and survival were evaluated by colony-formation experiment.Flow cytometry was used to analyze the cell cycle distribution and apoptosis induction.γ-H2AX foci were detected with immune-fluorescence staining analysis by a laser confocal microscopy for investiating the DNA double-strand break repair.Results The flow cytometric data of CD133 +/CD44 + positive cells indicated that the sub-population of cancer stem cell (CSC) took the ratio of (88.14 ± 0.47)％ of the cultured HCT116 cells.The colony-formation efficiency of HCT116 cells was (84.75 ± 1.35) ％ in serum-free mediums in vitro culture.Compared to 2 Gy irradiation alone group,the NU7026 combined with 2 Gy irradiation group had a lower cell colony-forming ratio (t =7.21,P ＜0.01) and a lower survival ratio (t =7.22,P ＜ 0.01).The proportion of CSCs sub-population increased at 48 h post-2 Gy irradiation,suggesting that HCT116 CSCs was more resistant to ionizing radiation.Importantly,NU7026 largely decreased the proportion of CSCs sub-population in 2 Gy-irradiated cells.The difference of CSC proportion between 2 Gy irradiation alone group and 2 Gy combined with NU7026 treatment group was statistically significant (t =9.55,P ＜ 0.01).In addition,the group of NU7026 combined with 2 Gy irradiation had a higher ratio of G2/M arrest 24 h post-irradiation (t =7.67,P ＜ 0.01) and an increased induction of cell early apoptosis (t =8.24,P ＜ 0.05).48 h post irradiation as compared to 2 Gy irradiation alone group.NU7026 treatment significantly inhibited the cellular capacity of repairing DNA double-strand breaks induced by γ-ray irradiation.The γ-H2AX foci of the combined treatments group were much higher than that of 2 Gy irradiation alone group at 2,4,8,24 h postirradiation (t=19.58,11.95,7.01,9.45,P＜0.01).Conclusions DNA-dependent protein kinase catalytic subunit inhibitor NU7026 can significantly sensitize the cancer stem cells of colorectal carcinoma HCT116 cells to γ-ray irradiation.Multiple mechanisms are involved in the radiosensitization effect of NU7026,including DNA repair inhibition,elongation of G2/M arrest,and increase of radiation-induced apoptosis.

The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism

OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein expression levels were detected using western blotting, and the correlation between protein levels was analyzed. RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene combined with radiation was significantly greater (p<0.01) than that elicited by either radiation or elemene alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a combination of elemene and radiation was greater than that of cells treated with either elemene or radiation alone (p<0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene alone (p<0.01), while p53 protein expression was increased (p<0.01). A negative correlation was observed between DNA-PKcs and p53 expression (r=−0.569, p=0.040), while a positive correlation was found between DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012). CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular mechanism of action may be related to the downregulation of DNA-PKcs gene expression. .

Effect of elemene on radiosensitivity of A549 cells and its possible molecular mechanism / 中华放射医学与防护杂志

Objective To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism.Methods The effect of radiosensitivity was detected by colony forming assay.The protein expressions of DNA-PKcs,Bcl-2 and P53 were detected with Western blot.The correlation between the protein expression of DNA-PKcs and Bcl-2,DNA-PKcs and P53 was analyzed.Results Elemene had radiosensitizing effect on A549 cells,with the SERDo and SERDq 1.54 ± 0.20 and 1.43±0.15,respectively for 10 μg/ml elemene,and 1.63 ±0.32 and 1.75 ±0.19,respectively for 20 μg/ml elemene.Compared with irradiation group,the expression of DNA-PKcs was reduced significantly in 10,20 μg/ml elemene combined with radiation group ( t =7.52,8.33,P ＜ 0.05 ),so was for Bcl-2(t =10.74,11.33,P ＜0.05).The expression of P53 protein increased significantly (t =-9.25,7.66,P ＜0.05).There was a remarkable negative correlation between the expression of DNA-PKcs and P53(r =-0.569,P ＜0.05),and a remarkable positive correlation between DNA-PKcs and Bcl-2 (r =0.755,P ＜0.05 ).Conclusions Elemene has radiosensitizing effect on A549 cells,which might be related to down-regulation of DNA-PKcs gene expression,up-regulation of P53 and down-regulation of Bcl-2.

Deficiency of DNA double-strand break repair and enhanced radiosensitivity in Tip60 silenced cells / 中华放射医学与防护杂志

Objective To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism.Methods siRNA and anacardic acid (AA,an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity,respectively.Radiosensitivity was analyzed by colony-forming ability assay.γ-H2AX foci were detected to analyze the DNA double-strand break (DSB).Immunoprecipitation was used to determine the interaction of proteins.Results siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1,2 Gy after γ-ray irradiation,but had no significant effect at 4 Gy post-irradiation ( t =3.364,3.979,P ＜ 0.05 ).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA doublestrand break repair at 1,4 and 8 h after irradiation( t =3.875,3.183 and 3.175,respectively,P ＜ 0.05 ).The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation.Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation.Conclusions Tip60plays a role in the cellular response to ionizing radiation-induced DNA damage through,at least in part,interacting with DNA-PKcs and regulating its phosphorylation.

Up-regulation of DNA-PKcs and Its Mechanism in Human Glioma / 中国肿瘤临床

Objective: To detect the gene expression of Ku70, Ku80, ERCC4, lig4 and DNA-PKcs in non-homologous end joining pathway in human pdmary glioma tissues and normal brain tissues and to explore the underlying mechanism. Methods: The expression levels of Ku70, Ku80, ERCC4, lig4 and DNA-PKcsin in 36 glioma samples and 12 normal brain tissue samples were measured by SYBR Green real-time quantitative PCR. Methylation of DNA-PKcs was detected by methylation-specific PCR (MSP). Results: There was no significant difference in Ku70, Ku80, ERCC4 and lig4 expression between human primary glioma and normal brain tissues (P<0.05), while DNA-PKcs was significantly up-regulated (P= 0.002). The expression of DNA-PKcs was significantly higher in grade Ⅲ and Ⅳ glioma than that in grade Ⅱ glioma and normal brain tissues (P<0.05). Moreover, glioma tissues showed weaker methylation than normal brain tissues. Conclusion: The up-regulation of DNA-PKcs may be associated with pathogenesis of glioma. Demethylation of DNA-PKcs promoter is an important reason for its up-regulated expression in glioma.

DNA-PKcs expression in hepatoma and normal mouse liver tissues of various developmental stages and its influence on cell proliferation / 第二军医大学学报

Objective: To observe the expression of DNA dependent protein kinase catalytic subunit (DNA-PKcs) in the hepatoma tissues and mouse liver tissues of different developmental stages, so as to understand the role of DNA-PKcs in cell proliferation and tumorigenesis. Methods: Immunohistochemistry and Western blotting assay were used to observe the protein expression of DNA-PKcs in liver tissues and hepatoma tissues. The siRNA technique was used to silence the expression of DNA-PKcs in the HepG2 cells; cell proliferation assay and tumor transplantation test in nude mice were performed to evaluate the changes of the proliferation ability and tumorigenesis. Western blotting assay was also conducted to examine the expression of proliferation related proteins p-GSK3β and c-myc. Results: DNA-PKcs expression decreased in the liver tissues with the decrease of cell proliferation ability during the development of mice, and the expression level of DNA-PKcs was weak in liver tissues of adult mouse; but the DNA-PKcs protein level in the hepatoma tissues was significantly elevated (P<0.01). Cell growth curve showed that the proliferation of HepG2 cells was significantly decreased after suppression of DNA-PKcs with siRNA(P< 0.01), accompanied by a suppressed tumorigenesis ability. The expression of signal pathway related protein p-GSK3β and c-myc was inhibited after DNA-PKcs silencing in HepG2 cells(P<0.01). Conclusion: DNA-PKcs expression level is closely related to the proliferation ability of liver cells. Overexpression of DNA-PKcs may participate in the development and progression of hepatoma through mediating cell proliferation via the Wnt/GSK/c-myc related signal pathway.

Radiosensitization and relative mechanisms of vanillin derivative BVAN08 on human glioma U-251 cells / 中华放射医学与防护杂志

Objective To provide more convincing evidences and experimental data for exploring vanillin derivative BVAN08,6-bromine-5-hydroxy-4-methoxy-benzaldehyde,as a new anticancer drug,and to investigate the effect on the growth,radiosensitization of human glioma cell line U-251 and the relative mechanism.Methods The effect of BVAN08 on cell proliferation of U-251 and radiosensitivity to 60Co γ-rays (irradiation dose rate 2.3 Gy/min) were analyzed with MTT and colony-forming ability assay.Change in cellular morphology was observed by using light microscope.Change in cell cycle and apoptosis was detected with flow cytometry.The autophagy was observed by using TEM (irradiation dose rate is transmission electron microscope).DNA-PKcs protein level was detected through Western blot analysis.Results BVAN08 exhibited a dose- and time-dependent inhibition on the proliferation of U-251 cells during the concentration range of 10-100 mol/L (t = 1.83-3.07,P ＜ 0.05).IC50 at 48 h and 72 h after administration with BVAN08 were 55.3 and 52.7 mol/L,respectively.Obvious G2/M arrest was induced in U-251 cells after 4 h administration with BVAN08,and reached peak at 12 h.The G2/M population reached 63.3% in U-251 cells after 12 h administration of 60 μmol/L BVAN08 and kept increasing with the time,while both apoptosis and autophagic cell death were induced.The most effective radiosensitization time for BVAN08 treatment was 12 h before irradiation.The enhancement ratio of radiosensitivity was 3.14 for 20 μmol/L of BVAN08 12 h before 2 Gy irradiation.Conclusions BVAN08 can nduce apoptosis as well as autophygic cell death of U-251 cells,and sensitize U-251 cells.The mechanism of its radiosensitizing effect might be associated with the induction of G2/M arrest and inhibition of DNA-PKcs expression.BVAN08 seemed to be a romising radiosensitizing anticancer drug.

Correlation of expression DNA double-strand break repair protein with radiosensitivity of tumor cells / 肿瘤

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 of human tumor cell lines, and to reveal the value of the three proteins for the prognosis of the radiosensitivity of tumor cells. Methods: Eight tumor cell lines were selected including four human cervical carcinoma cell lines (HeLa, SiHa, C33A, and Caski), three human breast carcinoma cell lines (MCF-7, MDA-MB-231, MDA-MB453), and one human lung carcinoma cell line (A549). The expressions of Ku80, DNA-PKcs and ATM protein were measured by Western blotting. The apoptotic ratio of tumor cells was analyzed by flow cytometry after 48 h X-ray irradiation at 10 Gy of 6 MV. SF2 value (survival fraction at 2 Gy) and α and β values were obtained by clone formation assay. The correlation of protein expression with SF2, α/β value or apoptotic ratio was analyzed by Pearson linear correlation analysis. Results: The expression of same protein in different cell lines and the expression of the three proteins in the same cell line had significant difference. There was a positive correlation between the expression of DNA-PKcs and SF2 (r=0.723, P=0.043 0.05). The expression of the three proteins had no correlation with either apoptotic ratio or α/ β value (P>0.05). Conclusions: Tumor cells with higher expression of DNA-PKcs protein will have higher radioresistance. The expression level of DNA-PKcs protein in tumor cells may be an indicator for predicting the radiosensitivity of tumor cells.

The Significance of Ku70 Expression in Patients with Nasopharyngeal Carcinoma / 대한방사선종양학회지

PURPOSE: The objective of this study was to determine whether the expressions of the two components of DNA-dependent protein kinase, Ku70 and DNA-PKcs, influence the response to radiotherapy (RT) and outcome of treatment of non-disseminated nasopharyngeal carcinoma (NPC) in patients who received definitive RT. MATERIASL AND METHODS: Sixty-six patients with NPC who were treated with radiotherapy alone or with concurrent chemotherapy between June 1995 and December 2001 were divided into groups based on the levels of immunoreactivity for Ku70 and DNA-PKcs in pretreatment biopsy specimens. The over-expression of Ku70 or DNA-PKcs groups included patients whose biopsy specimens showed at least 50% immunopositive tumor cells; patients in which less than 50% of the tumor cells in the biopsy tissues were immunopositive were placed in the low Ku70 and DNA-PKcs groups. The immunoreactivities for Ku70 and DNA-PKcs were retrospectively compared with the sensitivity of the tumor to radiation and the patterns of therapy failure. Univariate analyses were performed to determine the prognostic factors that influenced locoregional control of NPC. RESULTS: The five-year locoregional control rate was significantly higher in the low Ku70 group (Ku(-)) (85%) than in the high Ku70 group (Ku(+)) (42%) (p=0.0042). However, there were no differences in the metastases-free survival rates between the two groups (Ku70 (+), 82%; Ku70 (-), 78%; p=0.8672). Univariate analysis indicated that the over-expression of Ku70 surpassed other well-known predictive clinocopathologic parameters as an independent prognostic factor for locoregional control. Eighteen of 22 patients who had locoregional recurrences of the tumor displayed an over-expression of Ku70. No significant association was found between the level of DNA-PKcs expression and the clinical outcome. CONCLUSION: Our data suggest that the level of Ku70 expression can be used as a molecular marker to predict the response to RT and the locoregional control after RT and concurrent chemotherapy in patients with non-disseminated NPC.

Loss of DNA-dependent Protein Kinase Catalytic Subunit (DNA- PKcs) Expression in Gastric Cancers / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: DNA-PKcs is one of the DNA repair genes. It was recently found that hyperplasia and dysplasia of the intestinal mucosa and the production of aberrant crypt foci were developed in DNA-PKcs-null mice, and this suggests a suppressive role for DNA-PKcs in tumorigenesis. MATERIALS AND METHODS: To investigate the possible relationship between the clinico-pathologic characteristics and the survival of gastric cancer patients, the expression status of DNA-PKcs was determined in 279 consecutive gastric cancers. Immunohistochemical analysis was performed to evaluate the expression levels of DNA-PKcs protein by using the tissue array method. RESULTS: Out of 279 consecutive gastric cancers, 63 cases (22.6%) showed the loss of DNA-PKcs expression. The loss of DNA-PKcs expression was significantly associated with advanced cancer (p <0.001), lymphatic invasion (p=0.001), lymph node metastasis (p=0.009), and advanced pTNM stage (p=0.009). Univariate survival analysis revealed that patients with the loss of DNA-PKcs expression had significantly poorer survival than those patients with intact DNA-PKcs expression (p=0.004). Moreover, the loss of DNA-PKcs expression was identified to correlate with a lower survival in the subgroup of stage I gastric cancer patients (p=0.037). CONCLUSION: The loss of DNA-PKcs expression was found in 23% of human gastric cancers and this was identified to significantly correlate with poor patient survival, especially for stage I gastric cancer patients.