An optimized method for detecting gamma-H2AX in blood cells reveals a significant interindividual variation in the gamma-H2AX response among humans.
Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, gammaH2AX) 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.
Ismail
Ismail Hassan
IH
Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, SE-413 45 Göteborg, Sweden.
Wadhra
Tabasum Imran
TI
Hammarsten
Ola
O
D000617:Aminoglycosides / Q000633:toxicity; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D053281:Enediynes / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006657:Histones / Q000032:analysis*; D006801:Humans; D007962:Leukocytes / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D011839:Radiation, Ionizing
D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D053281:Enediynes; D005456:Fluorescent Dyes; C468783:H2AX protein, human; D006657:Histones; C055955:calicheamicin gamma(1)I; D016650:Fluorescein-5-isothiocyanate