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.
Highlights
Radiotherapy is an important pillar of cancer management
Using the propidium iodide DNA intercalator and flow cytometry technology we investigated the effect of 2 and 5 Gy irradiation to the cell cycle phases of U251 cells
The irradiation of U251 cells caused significant cell cycle arrest in G0/G1 phase at the expense of the decrease of S and G2/M cell cycle phases measured after 24 h or 72 h postirradiation (Figures 2A,B)
Summary
Radiotherapy is an important pillar of cancer management. The main aim of using ionizing radiation is to diminish cancer cells with high efficacy and selectivity. Molecular radiobiology has revealed radiation induced clustered DNA damage, i.e. the complex arrangement of two or more lesions (single- and doublestrand breaks) within one to two helical turns of DNA. This clustered DNA damage compromises the base excision repair pathway, resulting in an increased lifetime of the lesions [4, 5]. The activation of the complex DNA damage response (DDR) machinery starts by the signaling of DSBs, arresting the cell cycle, and triggering the DNA repair pathways [7,8,9,10,11]. Homologous recombination (HR), a slow, but precise process using the undamaged DNA sequence as a template to reproduce the lost or changed molecules, may repair more serious DSBs [12, 13]
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