[DNA damage and apoptosis of human airway epithelial cell lines caused by cigarette smoke extract].
OBJECTIVE
Cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types. This study was to investigate DNA damage and apoptosis in normal human bronchial epithelial cell line NHBE and lung carcinoma cell line SPC-A1 caused by cigarette smoke extract.


METHODS
NHBE and SPC-A1 cells were incubated with different concentrations of cigarette smoke extract. Cell viability was evaluated by MTT assay. Fluorescence-labeled anti-histone gamma-H2AX polyclonal antibody was used to detect DNA double-strand breaks (DSBS) in chromatin. DNA damage was analyzed by flow cytometry. The expression of gamma-H2AX was detected by Western blot. Cigarette smoke extract-induced cell apoptosis was detected by sub G1 peak method and annexin V-FITC/propidium iodide (PI) staining assay. Cell morphology of DNA damage and apoptosis was observed by confocal laser microscopy.


RESULTS
MTT assay results showed that cigarette smoke extract decreased the viability of NHBE and SPC-A1 cells in time-and concentration-dependent manners. Cigarette smoke extract induced DSBS in NHBE cells and SPC-A1 cells, and led to H2AX phosphorylation (denoted gamma-H2AX) in time-and concentration-dependent manners. The maximal value of gamma-H2AX was seen about 4 h after the treatment, and the value decreased subsequently, but did not reduce to a normal level. Cell apoptosis appeared about 12 h after DNA damage. Cigarette smoke extract also initiated the accumulation of gamma-H2AX in these cells, and cell apoptosis morphology was observed 4 h later.


CONCLUSIONS
Cigarette smoke extract can induce DSBS and apoptosis in NHBE and SPC-A1 cells in time-and concentration-dependent manners.
Fu
Qiang
Q
Molecular Medical Center, Cancer Research Institute, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Cheng
Jing
J
Han
Zhong-Bo
ZB
Li
Xiao-Lan
XL
Chen
Xiao-Yan
XY
Zhang
Peng
P
Xiao
Hui
H
Tao
De-Ding
DD
Hu
Jun-Bo
JB
Gong
Jian-Ping
JP
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects*; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D012906:Smoke / Q000009:adverse effects* / Q000032:analysis; D013997:Time Factors; D014026:Nicotiana
C468783:H2AX protein, human; D006657:Histones; D012906:Smoke