title: Replication stress induces micronuclei comprising of aggregated DNA double-strand breaks.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Key Laboratory of Experimental Teratology, Ministry of Education, and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Xu
authors.forename: Zhaoliang
authors.initials: Z
authors.lastname: Sun
authors.forename: Zhaojian
authors.initials: Z
authors.lastname: Liu
authors.forename: Haiyang
authors.initials: H
authors.lastname: Guo
authors.forename: Qiao
authors.initials: Q
authors.lastname: Liu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Jiang
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Zou
authors.forename: Yaoqin
authors.initials: Y
authors.lastname: Gong
authors.forename: Jay A
authors.initials: JA
authors.lastname: Tischfield
authors.forename: Changshun
authors.initials: C
authors.lastname: Shao
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D017239:Paclitaxel / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D013312:Stress, Physiological / Q000187:drug effects; D013936:Thymidine / Q000494:pharmacology
chemical_list: D006657:Histones; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D016590:Aphidicolin; D017239:Paclitaxel; D013936:Thymidine; D006918:Hydroxyurea