Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia

Abstract

Flow cytometric analysis of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used to characterize the effects of X-rays on cell-cycle kinetics in the DNA-repair deficiency disease ataxia telangiectasia (AT). Cultured fibroblasts from homozygotes ( at/ at), heterozygotes ( at/+) and normal controls (+/+) were either: (1) irradiated, cultured, then pulsed with BrdU and harvested, or (2) pulsed with BrdU, irradiated, cultured and then harvested. Cells were then fixed and stained with both a fluoresceinated monoclonal antibody against BrdU to identify S-phase cells and with propidium diiodide to measure total DNA content. Irradiation of +/+ and at/+ cells induced a similar, transient G 2/M arrest detectable within 8 h, which subsequently delayed by 6–8 h the passage of cells into G 1 and depleted early S phase. In contrast, at/ at cells failed to arrest G 2/M phase and entered the next cell cycle without pausing to repair radiation-induced damage. X-Rays also blocked entry of +/+ G 1 cells into S phase, subsequently reducing the total S-phase population. This effect was not observed in at/ at cells. These cell-cycle responses to radiation may be of diagnostic use and ultimately may help explain the basic defect in AT.