Luppi Fabrizio, Aarbiou Jamil, van Wetering Sandra, Rahman Irfan, de Boer Willem I, Rabe Klaus F, Hiemstra Pieter S
Department of Pulmonology, Leiden University Medical Center, PO Box 9600, 2300RC, Leiden, The Netherlands.
Respir Res. 2005 Nov 25;6(1):140. doi: 10.1186/1465-9921-6-140.
Increased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation.
Cells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies.
At low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels.
These results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer.
吸烟者气道上皮细胞增殖增加的现象较为常见。为阐明导致这些上皮细胞变化的分子机制，我们研究了香烟烟雾冷凝物（CSC）对细胞增殖、伤口愈合以及丝裂原活化蛋白激酶（MAPK）激活的影响。我们还研究了细胞内谷胱甘肽/硫醇水平的调节是否能减弱CSC诱导的细胞增殖。
本研究使用支气管上皮细胞系NCI-H292细胞和原代支气管上皮细胞亚培养物。采用5-溴-2-脱氧尿苷（BrdU）掺入法评估CSC对上皮细胞增殖的影响。通过分析培养72小时期间3毫米圆形刮伤伤口的闭合情况来研究上皮伤口修复的调节。伤口闭合情况根据每隔24小时获取的数字图像计算得出。使用磷酸化特异性抗体通过蛋白质印迹法评估丝裂原活化蛋白激酶的激活情况。
低浓度的CSC可增加NCI-H292细胞的增殖，而高浓度则因细胞毒性而具有抑制作用。低浓度的CSC还可增加NCI-H292细胞和原代支气管上皮细胞（PBEC）的上皮伤口闭合，而高浓度时闭合受到抑制。在低促有丝分裂浓度下，CSC可导致参与细胞增殖的MAPK即细胞外信号调节激酶1/2（ERK1/2）持续激活。高浓度CSC对细胞增殖的抑制与促凋亡MAP激酶p38和应激活化蛋白激酶（JNK）的激活有关。使用N-乙酰-L-半胱氨酸、谷胱甘肽或丁硫氨酸亚砜胺（BSO）调节细胞内谷胱甘肽（GSH）/硫醇水平表明，CSC的刺激和抑制作用部分受细胞内GSH水平调节。
这些结果表明，CSC可能根据香烟烟雾的局部浓度和抗氧化状态增加细胞增殖和伤口闭合。这些发现与吸烟者上皮细胞增殖增加一致，并可能为肺癌的发展提供进一步的见解。
