[Role of DNA-dependent protein kinase catalytic subunit in silica-induced DNA double-strand break repair in human embryo lung fibroblasts].
OBJECTIVE
To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF).


METHODS
Two stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay.


RESULTS
After treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P < 0.05).


CONCLUSIONS
Silica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.
Zhang
Feng-mei
FM
National Institute of Occupation Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Liu
Bing-ci
BC
Liu
Hai-feng
HF
Jia
Xiao-wei
XW
Ye
Meng
M
D002460:Cell Line; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D012822:Silicon Dioxide / Q000494:pharmacology*; D014162:Transfection
C468783:H2AX protein, human; D006657:Histones; D012822:Silicon Dioxide; D051747:DNA-Activated Protein Kinase