title: Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.
abstract: Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.
authors.affiliation: Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Rybak
authors.affiliation: Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Hoang
authors.affiliation: Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Bujnowicz
authors.affiliation: Laboratory for Imaging of Tissue Structure and Function, Nencki Institute of Experimental Biology PAS, Warsaw, Poland.
authors.forename: Tytus
authors.initials: T
authors.lastname: Bernas
authors.affiliation: Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Berniak
authors.affiliation: Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Mirosław
authors.initials: M
authors.lastname: Zarębski
authors.affiliation: Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York, USA.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.affiliation: Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Dobrucki
mesh_terms: D000072283:A549 Cells; D002166:Camptothecin / Q000008:administration & dosage; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D005047:Etoposide / Q000008:administration & dosage; D006657:Histones / Q000737:chemistry*; D006801:Humans; D018613:Microscopy, Confocal; D008942:Mitoxantrone / Q000008:administration & dosage; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D000071857:Tumor Suppressor p53-Binding Protein 1 / Q000378:metabolism
chemical_list: C468783:H2AX protein, human; D006657:Histones; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D012694:Serine; D005047:Etoposide; D008942:Mitoxantrone; D002166:Camptothecin
keywords: DNA damage; DNA replication; camptothecin; topoisomerase inhibitors; transcription