Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Abstract

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research.

Keywords: DNA-DSB; confocal microscopy; dSTORM; ionizing radiation; superresolution; γ-H2AX.

FIGURE 1

(A) A selected area from a confocal image slice (red: Alexa Flour 647-γH2AX, blue: DAPI-DNA). (B) Segmented γH2AX clusters in the area shown in Figure (A). (C) Segmented nuclei in the same area. (D) The whole volumetric image of the area shown in Figure (A). (E) Segmented nuclei (blue), a filtered out object (yellow–protruding) and γH2AX clusters (red dots). Scalebar: 5 µm.

FIGURE 2

Irradiation induces cell cycle arrest in G0/G1 phase and apoptosis of U251 cells. (A) Representative dot plots of cell cycle analysis after 24 h (upper part). Irradiation caused significant G0/G1 cell cycle arrest, decrease both in S and G/2M phase (lower part) after 24 h. (B) Representative dot plots of cell cycle analysis after 72 h (upper part). Irradiation caused significant G0/G1 cell cycle arrest, decrease both in S and G/2M phase (lower part) after 72 h. (C) Representative dot plots of Sub-G1 analysis after 72 h (upper part). The internucleosomal DNA fragmentation increased upon 2 and 5 Gy irradiation after 72 h *p <0.05, **p <0.01, ***p <0.001.

FIGURE 3

Irradiation induces the increase of pγ-H2AX+ cells in U251 cells. (A) Representative dot plots of pγ-H2AX+ immunofluorescent staining detected by flow cytometry (upper part) and the percentage of U251 cells with the increase of pγ-H2AX signal (lower part) following irradiation. (B) Representative dot plots of the distribution of pγ-H2AX+ U251 cells within the G0/G1, S, and G2/M cell cycle phases, separately (left panel). Representative cell cycle distribution of untreated cells (right panel). Irradiation caused significant increase of pγ-H2AX+ U251 cells both within G0/G1, S, and G2M phases after 30 min (right lower graph). *p <0.05, **p <0.01, ***p <0.001.

FIGURE 4

(A) Confocal images represent repair foci in U251 cell nuclei. 30 min after irradiation, the median number of γ-H2AX foci per cell increased significantly to a 15 times higher level than in the unirradiated cells. At 24 h, the foci number decreased in the 2 Gy group, but remained four times higher than the control, and in the 5 Gy group, a higher portion of DSBs remained unrepaired. (B) Detection and characterization of γ-H2AX clusters by dSTORM in U251 cell nuclei. The dSTORM images show the substructures of the repair foci. After irradiation with 2 Gy or 5 Gy, the cluster number and area reach their maxima at 30 min, and decrease at24 h and 72 h in a dose dependent manner. Scalebar: 6 µm.

FIGURE 5

(A) Graphic representation of the confocal image analysis. Mean (black line) and median (red line) density (foci per µm²) of γ-H2AX foci/cell irradiated at different dose levels (control (blue), 2 Gy (yellow), 5 Gy (red) and fixed at the defined time points after treatment (0.5, 24, and 72 h). The median density of γ-H2AX foci was five times higher than the control 30 min after 2 Gy and lower, and only four times higher after 5 Gy. At 24 h, the density of γ-H2AX foci was still elevated, and at 72 h it decreased to the control value or below at both dose levels. (B) Evaluation of dSTORM images. The figure shows the cluster density (the number of DNA DSB repair foci per µm²) in the control and in the irradiated (2 Gy,5 Gy) cells 0.5, 24, and 72 h after treatment (mean: black line, median:red line). The cluster density (cluster number/cell nucleus area) increased considerably 0.5 h after x-ray irradiation, in a clear correlation with the dose level. At 24 h, the measured density remained three times higher after 2 Gy and six times higher after 5 Gy than in the control. A small amount of residual foci could be detected in both dose groups at 72 h.

FIGURE 6

Violin plots represent the foci volume and cluster area of cell nuclei with confocal (A) and dSTORM (B) analysis. The median (red line) and the mean (black line) values of the γ-H2AX foci volume (A) and cluster area (B) in the control and the treated samples at 0.5, 24, and 72 h after irradiation. The foci volume (A) and cluster area (B) are smaller at 30 min after irradiation with 5 Gy, and larger after irradiation with 2 Gy. At 24 h after irradiation, the foci volume (A) and cluster area (B) increased compared to the values measured at 0.5 h min after irradiation.

FIGURE 7

The distribution of the epitope number/clusters. Black lines: mean values; red lines: median values. There was a significant increase in the mean of epitopes/clusters over the control both after 2 and 5 Gy. At 24 h, it decreased to the control level and stayed there at 72 h in the 2 Gy group. However, in the 5 Gy irradiated group we could observe a very slow decrease by 72 h, when a considerable number of epitopes could still be detected.