9.Role of p53 in silica-induced cell cycle alternation and DNA double-strand break repair in human embryo lung fibroblasts.
Feng-Mei ZHANG ; Bing-Ci LIU ; Hai-Feng LIU ; Xiao-Wei JIA ; Meng YE
Chinese Journal of Industrial Hygiene and Occupational Diseases 2010;28(4):246-249
OBJECTIVETo study the role of p53 in silica-induced cell cycle alternation and DNA double strand breaks repair in human embryo lung fibroblasts (HELF).
METHODSNeutral comet assay was applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair competence was calculated. The expression levels and phosphorylation of protein in HELF were determined by Western blot. Cell cycle changes were identified by flow cytometry in HELF.
RESULTSAfter treatment with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h), the expression levels and phosphorylation of p53 increased in a time-dependent manner, reaching maximum at 12 h and then decreasing at 24 h. After treatment with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h, the expression levels and phosphorylation of p53 increased in concentration-dependent manner. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased (DRC = 87.68%), compared with the negative control cell induced by silica (DRC = 57.19%). Silica increased the percentage of S phase (31.8 +/- 1.1)% compared with the controls (24.3 +/- 3.8)% (P < 0.05). When p53 expression was inhibited, the number of S phase cells was significantly increased, (41.4 +/- 0.6)% compared with the controls (25.4 +/- 1.9)% (P < 0.05).
CONCLUSIONThe silica dramatically increases the expression levels and phosphorylation of p53. The increased expression of p53 mediates silica-induced cell cycle change and inhibits silica-induced DNA double strand breaks repair.
Cell Cycle ; Cell Line ; Comet Assay ; DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung ; cytology ; Silicon Dioxide ; toxicity ; Tumor Suppressor Protein p53 ; metabolism
10.Roles of phosphatidylinositol 3 kinase in silica-induced DNA double strand breaks damage repair in human embryo lung fibroblasts.
Hai-Feng LIU ; Feng-Mei ZHANG ; Bing-Ci LIU ; Xiao-Wei JIA ; Meng YE
Chinese Journal of Industrial Hygiene and Occupational Diseases 2010;28(4):241-245
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