New York Medical College, Valhalla, New York 10595, USA.
Cytometry A. 2009 Oct;75(10):840-7. doi: 10.1002/cyto.a.20778.
Cigarette smoke (CS) is a major cause of lung cancer and a contributor to the development of a wide range of other malignancies. There is an acute need to develop a methodology that can rapidly assess the potential carcinogenic properties of the genotoxic agents present in CS. We recently reported that exposure of normal human bronchial epithelial cells (NHBEs) or A549 pulmonary carcinoma cells to CS induces the activation of ATM through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 (gammaH2AX) most likely in response to the formation of potentially carcinogenic DNA double-strand breaks (DSBs). To obtain a more complete view of the DNA damage response (DDR) we explored the correlation between ATM activation, H2AX phosphorylation, activation of Chk2 through its phosphorylation on Thr68, and phosphorylation of p53 on Ser15 in NHBE and A549 cell exposed to CS. Multiparameter analysis by laser scanning cytometry made it possible to relate these DDR events, detected immunocytochemically, with cell cycle phase. The CS-dose-dependent induction and increase in the extent of phosphorylation of ATM, Chk2, H2AX, and p53 were seen in both cell types. ATM and Chk2 were phosphorylated approximately 1 h prior to phosphorylation of H2AX and p53. The dephosphorylation of ATM, Chk2, and H2AX was seen after 2 h following CS exposure. The dose-dependency and kinetics of DDR were essentially similar in both cell types, which provide justification for the use of A549 cells in the assessment of genotoxicity of CS in lieu of normal bronchial epithelial cells. The observation that DDR was more pronounced in S-phase cells is consistent with the mechanism of induction of DSBs occurring as a result of collision of replication forks with primary lesions such as DNA adducts that can be caused by CS-generated oxidants. The cytometric assessment of CS-induced DDR provides a means to estimate the genotoxicity of CS and to explore the mechanisms of the response as a function of cell cycle phase and cell type.
香烟烟雾（CS）是肺癌的主要原因，也是广泛存在的其他恶性肿瘤的发展因素。目前迫切需要开发一种方法，能够快速评估 CS 中存在的遗传毒性物质的潜在致癌特性。我们最近报道，正常人类支气管上皮细胞（NHBE）或 A549 肺癌细胞暴露于 CS 会通过其丝氨酸 1981 的磷酸化和组蛋白 H2AX 丝氨酸 139 的磷酸化（γH2AX）激活 ATM，这很可能是为了应对潜在致癌性 DNA 双链断裂（DSB）的形成。为了更全面地了解 DNA 损伤反应（DDR），我们探索了 ATM 激活、H2AX 磷酸化、Chk2 通过其丝氨酸 68 的磷酸化激活以及 CS 暴露的 NHBE 和 A549 细胞中 p53 丝氨酸 15 的磷酸化之间的相关性。激光扫描细胞术的多参数分析使我们能够将这些 DDR 事件与免疫细胞化学检测到的细胞周期阶段相关联。在两种细胞类型中，都观察到 CS 剂量依赖性诱导和增加 ATM、Chk2、H2AX 和 p53 的磷酸化程度。ATM 和 Chk2 的磷酸化发生在 H2AX 和 p53 的磷酸化之前约 1 小时。CS 暴露后 2 小时观察到 ATM、Chk2 和 H2AX 的去磷酸化。两种细胞类型的 DDR 剂量依赖性和动力学基本相似，这为使用 A549 细胞评估 CS 的遗传毒性提供了依据，而不是正常的支气管上皮细胞。DDR 在 S 期细胞中更为明显的观察结果与 DSB 的诱导机制一致，即复制叉与原发性损伤（如 CS 产生的氧化剂引起的 DNA 加合物）碰撞会导致 DSB 的发生。CS 诱导的 DDR 的细胞计量评估提供了一种估计 CS 遗传毒性的方法，并探索了作为细胞周期阶段和细胞类型函数的反应机制。
