title: Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry".
abstract: Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .
authors: Department of Pathology, New York Medical College, Valhalla, NY 10595, USA.
authors: Hong
authors: H
authors: Zhao
authors: Jurek
authors: J
authors: Dobrucki
authors: Paulina
authors: P
authors: Rybak
authors: Frank
authors: F
authors: Traganos
authors: H
authors: H
authors: Dorota Halicka
authors: Zbigniew
authors: Z
authors: Darzynkiewicz
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D057930:Click Chemistry / Q000379:methods*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000009:adverse effects; D047569:Laser Scanning Cytometry / Q000379:methods*; D018613:Microscopy, Confocal; D018384:Oxidative Stress; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; D006861:Hydrogen Peroxide; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine