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

• Main Authors: Eric Moeglin, Dominique Desplancq, Sascha Conic, Mustapha Oulad-Abdelghani, Audrey Stoessel, Manuela Chiper, Marc Vigneron, Pascal Didier, Laszlo Tora, Etienne Weiss
• Format: Article
• Language: English
• Published: MDPI AG 2019-03-01
• Series: Cancers
• Subjects: histone variant; H2AX phosphorylation; γ-H2AX; pan-nuclear pattern; monoclonal antibody; cancer cells; cell death; replication stress; chemotherapy; H2AFX gene; knock-out
• Online Access: http://www.mdpi.com/2072-6694/11/3/355

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.