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Cancers 2019, 11(3), 355; https://doi.org/10.3390/cancers11030355

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

1
Biotechnologie et Signalisation Cellulaire, UMR 7242, CNRS/Université de Strasbourg, Boulevard S. Brant, 67412 Illlkirch, France
2
Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, France
3
Centre National de la Recherche Scientifique, UMR 7104, 67404 Illkirch, France
4
Institut National de la Santé et de la Recherche Médicale, U1258, 67404 Illkirch, France
5
Université de Strasbourg, 67404 Illkirch, France
6
Laboratoire de Bioimagerie et Pathologies, UMR 7213, CNRS/Université de Strasbourg, Route du Rhin, 67401 Illkirch, France
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Received: 9 January 2019 / Revised: 1 March 2019 / Accepted: 8 March 2019 / Published: 13 March 2019
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Abstract

Phosphorylated histone H2AX (γ-H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of γ-H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear γ-H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear γ-H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear γ-H2AX pattern. Delivery of labeled anti-γ-H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear γ-H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of γ-H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy. View Full-Text
Keywords: histone variant; H2AX phosphorylation; γ-H2AX; pan-nuclear pattern; monoclonal antibody; cancer cells; cell death; replication stress; chemotherapy; H2AFX gene; knock-out histone variant; H2AX phosphorylation; γ-H2AX; pan-nuclear pattern; monoclonal antibody; cancer cells; cell death; replication stress; chemotherapy; H2AFX gene; knock-out
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Moeglin, E.; Desplancq, D.; Conic, S.; Oulad-Abdelghani, M.; Stoessel, A.; Chiper, M.; Vigneron, M.; Didier, P.; Tora, L.; Weiss, E. Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress. Cancers 2019, 11, 355.

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