Dipartimento di Biologia, Unità di Genetica, Mutagenesi e Epidemiologia Ambientale, University of Pisa, Derna 1, 56100 Pisa, Italy.
Mutagenesis. 2013 Jul;28(4):465-73. doi: 10.1093/mutage/get024. Epub 2013 May 21.
After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.
双链断裂诱导后，组蛋白 H2AX 丝氨酸 139 磷酸化形成的 γ-H2AX 焦点代表 DNA 损伤反应 (DDR) 的早期事件。随后，焦点迅速去磷酸化，作为随后募集效应蛋白的信号。因此，可以使用焦点的诱导和消失来监测暴露于遗传毒性应激的细胞群体中 DDR 机制的功能。在这里，我们研究了未刺激或体外培养的外周血淋巴细胞中 γ-H2AX 的时间过程，这些细胞用 UVB、博来霉素和丝裂霉素 C (MMC) 处理。一旦进行诱变暴露，就会在 0.5 到 5 小时的不同时间间隔从细胞中收获细胞。结果表明：(i) 在 20 小时刺激的外周血淋巴细胞中，UVB 照射导致核磷酸化广泛且剂量依赖性增加，γ-H2AX 焦点的消失与 UV 剂量呈比例，随着收获时间的增加而增加；(ii) 照射后培养 20 小时的 G0 细胞 UVB 暴露显示出低水平的 DNA 磷酸化，描绘了一个时间过程，其中最大效应在 0.5 小时达到，1 小时后开始去磷酸化；(iii) 用博来霉素硫酸盐处理未刺激的淋巴细胞会导致核磷酸化增加几倍，高于未处理的细胞，动力学与观察到的 UVB 暴露的 G1 细胞相似；(iv) 在刺激的细胞中，MMC 引起严重且剂量依赖性的 H2AX 高度磷酸化，以及与其他实验处理相比非常缓慢的去磷酸化动力学。这项研究证实了 γ-H2AX 焦点测定作为遗传毒性终点的可行性，并支持这样一种观点，即应该在人类群体的生物监测研究中引入这种类型的分析。这也可能成为筛查和诊断癌前状态或其他疾病的早期阶段的可行且有用的工具。
