Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

Figure 1

(a) Radiation-induced apoptosis measured by means of Annexin-V/PI and TUNEL methodologies. Cytometry plots were used for gating cells stained using Annexin-V (An) and propidium iodide (PI) before and after irradiation. In all plots, the lower left quadrant corresponds to the viable, nonapoptotic cell population (An−/PI−). The lower right quadrant corresponds to the cell population An+PI–, which is undergoing early apoptosis (EA) and is shown in green. The upper right quadrant corresponds to the cell population An+PI+, which is undergoing late apoptosis (LA) and is shown in red. Frequencies of EA and LA are shown in each graph at 0, 24, 48, and 72 hours after irradiation in normal and AT cells and they correspond to the mean of 3 different experiments with two replicas each. A minimal number of 10000 cells were analyzed in each experiment. The asterisks indicate statistical differences in the apoptotic levels between AT and normal cells when comparing the EA fraction, the LA fraction, and the sum of Annexin-V-positive cells (EA + LA). In all cases, test was applied and the values were <0.005. Frequencies of TUNEL-positive cells for each cell type at 0, 24, 48, and 72 hours pIR are shown over each cytometry plot. The asterisks indicate statistical differences between AT and normal cells ( test; values < 0.007). The values for TUNEL were obtained after scoring 1000 cells for each time point and each cell line using an epifluorescence microscope. (b) Scoring of TUNEL-positive cells. On the left, a general view under the microscope (40x) showing irradiated cells in which a combination of TUNEL staining (green) and γH2AX immunofluorescence (red) has been applied. DNA is stained with DAPI (blue). TUNEL-positive cells (white arrowheads) depict intense TUNEL staining and they show the morphological features of apoptotic cells (right panel): smaller nuclei with highly condensed chromatin—intensely stained with blue—undergoing variable levels of nuclear fragmentation. Also, TUNEL-positive cells could depict a pan-nuclear γH2AX staining but never had γH2AX foci. (c) Western blot detection of apoptotic markers. Normal and AT cells were irradiated with 5 Gy of γ-rays and expression of p53, its activated form phospho-p53 (Ser15), and other p53 targets such as p21, Bax, and the cleaved fraction of caspase 3 were analyzed at 0, 24, 48, and 72 hours after irradiation. Proteins were detected in two different experiments and GADPH was used as the housekeeping gene.

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