DeLoughery Zachary, Luczak Michal W, Ortega-Atienza Sara, Zhitkovich Anatoly
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
Toxicol Sci. 2015 Jan;143(1):54-63. doi: 10.1093/toxsci/kfu207. Epub 2014 Oct 6.
Hexavalent chromium is a human respiratory carcinogen that undergoes intracellular activation in vivo primarily via reduction with ascorbate. Replication of Cr-adducted DNA triggers mismatch repair that generates toxic DNA double-strand breaks (DSBs) as secondary lesions. Here, we examined the intranuclear distribution of chromate-induced breaks and a central DSB signaling branch targeting histone H2AX. Using ascorbate-restored cells (H460 human lung epithelial cells, normal human lung and normal mouse embryonic fibroblasts (MEFs)), we found that Cr(VI) produced a typical DSB-associated spectrum of H2AX modifications, including its Ser139-phosphorylated (known as γH2AX) and mono- and diubiquitinated forms. However, whereas canonical DSB signaling relies on ATM, the formation of γH2AX and its ubiquitinated products by Cr(VI) was dependent on ATR kinase. Based on the established mode of ATR activation, this suggests that Cr-induced DSB are not blunt-ended and likely contain single-stranded tails. Confocal imaging with markers of active and inactive chromatin revealed a selective formation of Cr-induced DSB in euchromatin of mouse and human cells. In contrast to DSB, Cr-DNA adducts were produced in both types of chromatin. The euchromatin targeting of Cr-induced DSB makes these lesions particularly dangerous by increasing the probability of deleting active tumor suppressors and producing oncogenic translocations. Accumulation of transcription-inhibiting ubiquitinated forms of γH2AX in euchromatin is expected to contribute to the ability of Cr(VI) to suppress upregulation of inducible genes.
六价铬是一种人类呼吸道致癌物，在体内主要通过与抗坏血酸还原而发生细胞内活化。铬加合物DNA的复制引发错配修复，产生有毒的DNA双链断裂（DSB）作为继发性损伤。在此，我们研究了铬酸盐诱导的断裂的核内分布以及靶向组蛋白H2AX的中心DSB信号分支。使用抗坏血酸恢复的细胞（H460人肺上皮细胞、正常人肺和正常小鼠胚胎成纤维细胞（MEF）），我们发现Cr(VI)产生了典型的与DSB相关的H2AX修饰谱，包括其Ser139磷酸化（称为γH2AX）以及单泛素化和双泛素化形式。然而，虽然经典的DSB信号传导依赖于ATM，但Cr(VI)诱导γH2AX及其泛素化产物的形成却依赖于ATR激酶。基于已确立的ATR激活模式，这表明Cr诱导的DSB不是平头的，可能含有单链尾巴。用活性和非活性染色质标记物进行的共聚焦成像显示，Cr诱导的DSB在小鼠和人类细胞的常染色质中选择性形成。与DSB不同，Cr-DNA加合物在两种染色质中均有产生。Cr诱导的DSB对常染色质的靶向作用通过增加删除活性肿瘤抑制因子和产生致癌易位的可能性，使这些损伤特别危险。预计常染色质中γH2AX的转录抑制性泛素化形式的积累将有助于Cr(VI)抑制诱导基因上调的能力。
