An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans

• Published in: Nucleic acids research Vol. 35; no. 5; p. e36
• Main Authors: Ismail, Ismail Hassan, Wadhra, Tabasum Imran, Hammarsten, Ola
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.03.2007; Oxford Publishing Limited (England)
• Subjects: Aminoglycosides - toxicity; Animals; Antibiotics, Antineoplastic - toxicity; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Cells, Cultured; cleavage; comet assay; DNA Breaks, Double-Stranded; dna-damage; double-strand breaks; Enediynes - toxicity; Flow Cytometry - methods; Fluorescein-5-isothiocyanate - analysis; Fluorescent Dyes - analysis; Histones - analysis; Humans; in-vivo; Leukocytes - chemistry; Leukocytes - drug effects; Leukocytes - radiation effects; mdc1; Methods Online; Mice; phosphorylated histone h2ax; Radiation, Ionizing; radiosensitivity; repair; sensitivity
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkl1169

Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, γH2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. Here we describe a rapid and simple flow-cytometry-based method, optimized to measure gamma-H2AX in non-fixed peripheral blood cells. No DSB induced signal was observed in H2AX−/− cells indicating that our FACS method specifically recognized gamma-H2AX accumulation. The gamma-H2AX assay was capable of detecting DNA damage at levels 100-fold below the detection limit of the alkaline comet assay. The gamma-H2AX signal was quantitative with a linear increase of the gamma-H2AX signal over two orders of magnitude. We found that all nucleated blood cell types examined, including the short-lived neutrophils induce gamma-H2AX in response to DSBs. Interindividual difference in the gamma-H2AX signal in response to ionizing radiation and the DSB-inducing drug calicheamicin was almost 2-fold in blood cells from patients, indicating that the amount of gamma-H2AX produced in response to a given dose of radiation varies significantly in the human population. This simple method could be used to monitor response to radiation or DNA-damaging drugs.