OBJECTIVE: To investigate the influence of Ku80 inhibition on the chemotherapeutic sensitivity of the T-acute lymphoblastic leukemia(T-ALL) cell line Jurkat, and to explore the potential mechanism. METHODS: The transcription and expression level of Ku80 in 6 hematological malignant cell lines were detected by RT-qPCR and Western blot, respectively. The expression of Ku80 in Jurkat cells was detected by Western blot after transfection with the recombinant shKu80 lentiviral vector. The proliferation capacity of Jurkat cells was explored by CCK-8 after Ku80 inhibition. The colony formation ability, apoptosis, and γH2AX(a protein marker of DNA damage) expression in Jurkat cells were investigated after Ku80 silencing and co-treated with etoposide(VP16) for 4 hours through soft agar assay, flow cytometry and Western blot, respectively. RESULTS: The mRNA level and protein expression of Ku80 were both highest in Jurkat among 6 hematological malignant cell lines. Ku80 expression was successfully down regulated in Jurkat cells after relative plasmid transfected. The proliferative ability of cells was significantly decreased after Ku80 inhibition(P < 0.05). The colony formation capacity of Jurkat cells was obviously reduced and the cells apoptosis and γH2AX expression were increased after Ku80 inhibition, with or without VP16 incubation. CONCLUSION Targeted silencing of Ku80 could enhance the sensitivity of VP16 in Jurkat cells, which might be associated with the elevated level of DNA damage accumulation.
Humans ; Ku Autoantigen/metabolism* ; Jurkat Cells ; Apoptosis/drug effects* ; Cell Proliferation ; Etoposide/pharmacology* ; DNA Damage ; Cell Line, Tumor ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
CRISPR-Cas Systems ; DNA Ligase ATP ; genetics ; Gene Expression Regulation ; genetics ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ku Autoantigen ; genetics

OBJECTIVETo investigate the clinical value of serum anti-Ku86 in early detection of hepatocellular carcinoma (HCC).

METHODSExpression levels of Ku86 protein in HCC and adjacent normal liver tissues were detected by Western blotting. Serum anti-Ku86 level in 83 patients with early HCC and 124 patients with liver cirrhosis were detected by enzyme-linked immunosorbent assay (ELISA). Chemiluminescence was used to measure the serum level of α-fetoprotein (AFP).

RESULTSExpression of Ku86 protein in HCC was increased when compared with the adjacent normal liver tissues (0.21 ± 0.05 vs. 0.08 ± 0.02, P < 0.01). Serum anti-Ku86 level was significantly elevated in HCC patients compared with that in liver cirrhosis patients (0.47 ± 0.22 vs. 0.22 ± 0.06 Abs at 450 nm, P < 0.01), but there was no significant difference between HBV infection and HCV infection in HCC patients (0.51 ± 0.19 vs. 0.47 ± 0.24, P = 0.267). Of note, serum anti-Ku86 level was significantly decreased after surgical resection of the tumors in the 30 HCC cases tested (P < 0.01). The results of ROC analysis indicated a better performance of anti-Ku86 (0.857) than AFP (0.739) for early detection of HCC. In 83 HCC patients, the positive rate of anti-Ku86 was 61.4% (51/83), significantly higher than that of the AFP positive rate (27.7%, 23/83). The anti-Ku86 level was positive in 37 of 60 HCC cases with negative AFP. Combination assay of AFP and anti-Ku86 could detect 60 of 83 HCC cases (72.3%, 60/83). There was no significant correlation of anti-Ku86 and AFP (r = 0.156, P = 0.161).

CONCLUSIONSSerum anti-Ku86 level is significantly elevated and is not related to HBV and HCV infection in HCC patients. Serum anti-Ku86 antibody may be a potential biomarker for early detection of HCC, and can be used in combination with AFP in clinics.

Adult ; Aged ; Antigens, Nuclear ; immunology ; Autoantibodies ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; DNA-Binding Proteins ; immunology ; Early Detection of Cancer ; Female ; Hepatitis B ; blood ; Hepatitis C ; blood ; Humans ; Ku Autoantigen ; Liver Cirrhosis ; blood ; Liver Neoplasms ; blood ; diagnosis ; virology ; Male ; Middle Aged ; ROC Curve ; alpha-Fetoproteins ; metabolism

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism

OBJECTIVEThe objective of this study is to investigate the effect of epidermal growth factor receptor (EGFR) monoclonal antibody MAb225 on repair of DNA double strand break (DNA-DSB) after radiation in tongue squamous cell carcinoma cell.

METHODSThe single cell gel electrophoresis (SCGE) was performed to estimate the repair of DNA-DSB induced by radiation in human tongue carcinoma cells Tca8113 treated with or without MAb225. Expression of Ku70 and Ku80 were detected by semiquantitative reverse transcription polymerase chain reaction and Western bolt.

RESULTSComet tail moment of MAb225 treated cell was significantly higher than untreated cell (P < 0.05). The expression of Ku70 and Ku80 were inhibited by MAb225.

CONCLUSIONMAb225 can inhibit repair of DNA-DSB induced by down-regulated expression of Ku70 and Ku80.

Antibodies, Monoclonal ; Antigens, Nuclear ; DNA Repair ; DNA-Binding Proteins ; Humans ; Ku Autoantigen ; Receptor, Epidermal Growth Factor ; Tongue Neoplasms

OBJECTIVETo study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF).

METHODSKu80 siRNA expression vectors were transfected into HELF by lipofectamine. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression level of Ku80, p53 and p21 proteins or the phosphorylation levels of p53-ser15 after cells were exposed to silica.

RESULTSThe expression levels of Ku80 protein increased in concentration-dependent and time-dependent manners after cells were exposed to silica. The proportion of G1 phases in H-NC cells (controls) decreased from 89.28% +/- 2.19% to 68.93% +/- 3.79% after exposure to silica, and the proportion of G1 phases in HELF cells (H-Ku80) decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% after exposure to silica (P<0.05). The expression levels of Ku80, p53 proteins or p21 proteins or phosphorylation level of p53-ser15 were obviously suppressed in H-Ku80, as compared with H-NC.

CONCLUSIONKu80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.

Antigens, Nuclear ; metabolism ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA-Binding Proteins ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Ku Autoantigen ; Lung ; cytology ; metabolism ; Phosphorylation ; Quartz ; toxicity ; Signal Transduction ; Tumor Suppressor Protein p53 ; metabolism

Animals ; Antigens, Nuclear ; DNA ; genetics ; metabolism ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA-Binding Proteins ; Humans ; Ku Autoantigen

OBJECTIVETo study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF).

METHODSControl HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h.

RESULTSAfter treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P < 0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P < 0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%.

CONCLUSIONSilica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.

Antigens, Nuclear ; metabolism ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Comet Assay ; DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; DNA-Binding Proteins ; metabolism ; Fibroblasts ; enzymology ; Histones ; metabolism ; Humans ; Ku Autoantigen ; Lung ; cytology ; Phosphatidylinositol 3-Kinase ; metabolism ; Silicon Dioxide ; toxicity

INTRODUCTIONMultiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.

MATERIALS AND METHODSHere, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.

RESULTSAnti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.

CONCLUSIONSThese studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.

Antibodies, Monoclonal ; isolation & purification ; Antibody Affinity ; Cell Line ; DNA Helicases ; immunology ; Humans ; Immunoglobulin Idiotypes ; immunology ; isolation & purification ; Immunoglobulin Variable Region ; isolation & purification ; Ku Autoantigen ; Multiple Myeloma ; immunology ; Peptide Library ; Recombinant Proteins

OBJECTIVETo extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.

METHODSConstruct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.

RESULTSThe shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.

CONCLUSIONThe vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.

Antigens, Nuclear ; genetics ; DNA-Binding Proteins ; genetics ; Gene Knockdown Techniques ; Hep G2 Cells ; Humans ; Ku Autoantigen ; Plasmids ; RNA ; genetics ; RNA, Small Interfering