Key Laboratory of Experimental Teratology, Ministry of Education, and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.
PLoS One. 2011 Apr 15;6(4):e18618. doi: 10.1371/journal.pone.0018618.
Micronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing intrinsic genomic instability and in normal cells exposed to genotoxic agents. DNA double-strand breaks are marked by phosphorylation of H2AX at serine 139 (γ-H2AX). One subclass of MN contains massive and uniform γ-H2AX signals. This study tested whether this subclass of MN can be induced by replication stress.
We observed that a large proportion of MN, from 20% to nearly 50%, showed uniform staining by antibodies against γ-H2AX, a marker of DNA double-strand breaks (DSBs). Such micronuclei were designated as MN-γ-H2AX (+). We showed that such MN can be induced by chemicals that are known to cause DNA replication stress and S phase arrest. Hydroxyurea, aphidicolin and thymidine could all significantly induce MN-γ-H2AX (+), which were formed during S phase and appeared to be derived from aggregation of DSBs. MN-γ-H2AX (-), MN that were devoid of uniform γ-H2AX signals, were induced to a lesser extent in terms of fold change. Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-γ-H2AX (-). The frequency of MN-γ-H2AX (+), but not that of MN-γ-H2AX (-), was also significantly increased in cells that experience S phase prolongation due to depletion of cell cycle regulator CUL4B. Depletion of replication protein A1 (RPA1) by RNA interference resulted in an elevation of both MN-γ-H2AX (+) and MN-γ-H2AX (-).
CONCLUSIONS/SIGNIFICANCE: A subclass of MN, MN-γ-H2AX (+), can be preferentially induced by replication stress. Classification of MN according to their γ-H2AX status may provide a more refined evaluation of intrinsic genomic instabilities and the various environmental genotoxicants.
哺乳动物细胞中的微核（MN）可用作基因组不稳定性和遗传毒性暴露的可靠生物标志物。内在基因组不稳定性的细胞和暴露于遗传毒性剂的正常细胞中通常会观察到 MN 的升高。DNA 双链断裂通过丝氨酸 139 处的 H2AX 磷酸化（γ-H2AX）来标记。MN 的一个亚类包含大量且均匀的 γ-H2AX 信号。本研究测试了这个 MN 亚类是否可以由复制应激诱导。
我们观察到，大量 MN（从 20%到近 50%）表现出针对 γ-H2AX 的抗体的均匀染色，γ-H2AX 是 DNA 双链断裂（DSBs）的标志物。这种微核被指定为 MN-γ-H2AX（+）。我们表明，这种 MN 可以由已知引起 DNA 复制应激和 S 期阻滞的化学物质诱导。羟基脲、阿非迪可林和胸苷均可显著诱导 MN-γ-H2AX（+），其在 S 期形成，似乎来自 DSBs 的聚集。在倍数变化方面，缺乏均匀 γ-H2AX 信号的 MN-γ-H2AX（-）则较少诱导。微管解聚抑制剂紫杉醇仅诱导 MN-γ-H2AX（-）。由于细胞周期调节剂 CUL4B 的消耗导致 S 期延长，MN-γ-H2AX（+）的频率显著增加，但 MN-γ-H2AX（-）的频率没有显著增加。通过 RNA 干扰耗尽复制蛋白 A1（RPA1）导致 MN-γ-H2AX（+）和 MN-γ-H2AX（-）的升高。
结论/意义：MN 的一个亚类，MN-γ-H2AX（+），可以优先由复制应激诱导。根据其 γ-H2AX 状态对 MN 进行分类，可能为内在基因组不稳定性和各种环境遗传毒性剂提供更精细的评估。
