Arsenite-induced alterations in Ku70-deficient cells: a model to study genotoxic effects.
As one of three subunits of DNA-dependent protein kinase (DNA-PK), Ku70 protein plays an important role in repair of DNA double-strand breaks (DNA DSB). To further understand the functions of Ku70 protein and the mechanisms underlying arsenite-induced genotoxic effects, the effects of Ku70 deficiency were examined. The Ku70-deficient cell line HLFK and null vector cell line HLFC were established after recombinant plasmid of Ku70 gene antisense RNA and null pEGFP-C1 vector were transferred into human embryo lung fibroblasts (HLF) cells. Experiments were undertaken to detect DNA DSB damage by neutral single-cell gel electrophoresis assay (SCGE), chromosomal alterations by micronucleus test, and cell cycle progression by flow cytometry in HLFC and HLFK cells treated with control, 1, 2.5, 5, or 10 microM sodium arsenite for 2, 4, or 24 h, respectively. Western blot analysis results showed that Ku70 protein content in HLFK cells decreased to 38% of those in HLFC cells. The median lethal concentrations (LC50) of sodium arsenite to HLFC and HLFK cells for 24 h were 27.38 microM and 21.80 microM, respectively. Results of neutral SCGE assay showed that there were concentration-dependent increases in tail length of DNA DSB, in percent of cells with DNA DSB tails, and in severity of DNA DSB damage in HLFK and HLFC cells. The increases in these indices in HLFK cells were significantly higher than those found in HLFC cells exposed to similar amounts of metal. The ability of DNA DSB to repair in HLFK cells was less than that seen in HLFC cells. Sodium arsenite produced concentration-dependent elevation in micronuclei and abnormal nuclei formation. The Ku70-deficiency enhanced the susceptibility to chromosomal alterations induced by sodium arsenite. Low concentrations of sodium arsenite induced cell arrest at G1; however, at high concentrations of metal this G1 arrest effect disappeared. These results suggested that Ku70 protein plays an important role in repair of DNA DSB damage and for maintainance of genome stability.
Liu
Qi-Zhan
QZ
Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.
Jiang
Gao-Feng
GF
He
Yun
Y
Wang
Xin-Ru
XR
Zhou
Jian-Wei
JW
Zhuang
Zhi-Xiong
ZX
D018053:Arsenites / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D020552:Comet Assay; D004249:DNA Damage; D004265:DNA Helicases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004785:Environmental Pollutants / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D000072200:Ku Autoantigen; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D015162:Micronucleus Tests; D016296:Mutagenesis / Q000187:drug effects*; D009153:Mutagens / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D017670:Sodium Compounds / Q000633:toxicity*; D014162:Transfection
D018053:Arsenites; D004785:Environmental Pollutants; D009153:Mutagens; D012333:RNA, Messenger; D017670:Sodium Compounds; C017947:sodium arsenite; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen