title: DNA double-strand breaks by Cr(VI) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: Zachary
authors.initials: Z
authors.lastname: DeLoughery
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: Michal W
authors.initials: MW
authors.lastname: Luczak
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: Sara
authors.initials: S
authors.lastname: Ortega-Atienza
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912 anatoly_zhitkovich@brown.edu.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Zhitkovich
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002840:Chromates / Q000633:toxicity*; D002857:Chromium / Q000633:toxicity*; D053903:DNA Breaks, Double-Stranded; D004305:Dose-Response Relationship, Drug; D022041:Euchromatin / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D010084:Oxidation-Reduction; D010766:Phosphorylation; D017680:Potassium Compounds / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects; D054875:Ubiquitination
chemical_list: D001205:Ascorbic Acid; D064007:Ataxia Telangiectasia Mutated Proteins; D002840:Chromates; D002857:Chromium; D022041:Euchromatin; D006657:Histones; D017680:Potassium Compounds; C468783:H2AX protein, human; C074702:chromium hexavalent ion; C027373:potassium chromate(VI); C576897:Atr protein, mouse; C576597:ATR protein, human; C475961:gamma-H2AX protein, mouse
keywords: ATR; cancer; chromate; euchromatin; hexavalent chromium; γ-H2AX