DNA damage response induced by tobacco smoke in normal human bronchial epithelial and A549 pulmonary adenocarcinoma cells assessed by laser scanning cytometry

Abstract

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.

Fig. 1. Effect of exposure of A549 cells to CS on expression of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P

The cells were exposed for 15 min to CS from 2R4F cigarette then transferred to a CO₂ incubator and cultured for 1 h. The induction of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P was detected immunocytochemically and measured by LSC, in conjunction with cellular DNA content, as described in Materials and Methods. Mock-treated cells served as a control. The DNA content frequency histogram of the CS-treated cells from the representative culture is shown in the right panel; control cells had nearly identical histograms (not shown).

Fig. 2. Expression of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P in NHBE cells mock-treated (Ctrl) or exposed to CS

The cells were treated with CS for 15 min then cultured for 1 h prior to fixation. As in Fig. 1 the DNA content frequency histogram of a representative culture is shown (right panel)

Fig. 3. Effect of length of cells exposure to CS on the increase (Δ) in expression of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P

A549 cells (top panels) and NHBE cells (bottom panels) were exposed to CS as described in Materials and Methods for periods of 5 – 20 min and then were transferred to culture for 1 h before being harvested and fixed. The mean values of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P immunofluorescence were measured for populations of cells in G₁, S and G₂M phases of the cell cycle by gating analysis based on differences in DNA content. The data are expressed as the n-fold increase of these mean values (Δ) over the respective values of the mock-treated cells.

Fig. 4. Effect of duration of cell culturing after their exposure to CS on the expression of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P

A549 cells (top panels) or NHBE cells (bottom panels) were treated with CS for 20 min, then transferred into culture and grown for 0.25 h, 0.5 h, 1 h, 2 h, and 4 h before the cultures were terminated. The mean values of ATM-Ser1981^P, Chk2-Thr68^P, γH2AX and p53-Ser15^P immunofluorescence were estimated for populations of G₁, S and G₂M-phase cells. The data are expressed as the n-fold increase of these mean values (Δ) over the respective values of the mock-treated cells.