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

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

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 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

OBJECTIVETo construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair.

METHODSHuman lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed.

RESULTSpEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK.

CONCLUSIONThe hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.

Antigens, Nuclear ; analysis ; Cell Division ; DNA Damage ; DNA Helicases ; DNA Repair ; DNA-Binding Proteins ; analysis ; deficiency ; Humans ; Ku Autoantigen ; RNA, Antisense ; Transfection

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

Animals ; Antigens, Nuclear ; genetics ; Apoptosis ; DNA Repair ; DNA-Activated Protein Kinase ; DNA-Binding Proteins ; genetics ; Humans ; Ku Autoantigen ; Neoplasms ; genetics ; pathology ; Nuclear Proteins ; Protein-Serine-Threonine Kinases ; genetics ; pharmacology

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

BACKGROUNDRecent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination.

METHODSTCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique.

RESULTSRAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation.

CONCLUSIONSRAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

Antigens, Nuclear ; genetics ; Base Sequence ; Complementarity Determining Regions ; DNA Breaks ; DNA-Binding Proteins ; genetics ; Genes, RAG-1 ; Genes, T-Cell Receptor ; Humans ; Jurkat Cells ; Ku Autoantigen ; Leukemia, T-Cell ; genetics ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Recombination, Genetic

OBJECTIVETo characterize DNA-PKcs and Ku70 expressions in hepato- and cholangio-neoplastic tissues and the association with the degree of malignancy and invasiveness of the tumors.

METHODSThe expression of DNA-PKcs and Ku70 was examined in 47 cases of hepato- or cholangio-neoplasm by immunohistochemistry.

RESULTSKu70 was expressed in all of the neoplastic tissues examined and with a little variation in levels. The highest expression was observed in adenocarcinomas and adenomas. There was no statistically significant association between Ku70 expression level and the degree of their malignancy extent or invasiveness. In contrast to Ku 70, a wide variation in expression levels of DNA-Pkcs was observed among different types of neoplastic tissues. The highest ratio of positive expressing cells was detected in hepatocellular carcinomas (92.1%), which was significantly higher than that in cholangioadeno carcinomas (65.3%) and biliary cystadenocarcinomas (51.9%). Low or no expression level was detected in papillary adenoma cases. DNA-PKcs expression of invasive adenomas and adeno-carcinomas (61.2%) was significantly higher than that of non-invasive adenomas and adeno-carcinomas (30.4%). There was no expression observed in the normal tissues adjacent to the tumors.

CONCLUSIONDNA-PKcs is expressed in hepato- and cholangio-neoplasms and its variable level of expression is associated with the types of the tumor and their degree of malignancy and invasiveness. DNA-PKcs could be recognized as a new biomarker for liver neoplasm.

Adenocarcinoma ; enzymology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, Nuclear ; biosynthesis ; genetics ; Bile Duct Neoplasms ; enzymology ; Bile Ducts, Intrahepatic ; enzymology ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; enzymology ; DNA-Activated Protein Kinase ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Female ; Humans ; Ku Autoantigen ; Liver Neoplasms ; enzymology ; Male ; Middle Aged