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

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

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