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Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy
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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8966514/ https://www.ncbi.nlm.nih.gov/pubmed/35370480 http://dx.doi.org/10.3389/pore.2021.1609971 |
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author | Brunner, Szilvia Varga, Dániel Bozó, Renáta Polanek, Róbert Tőkés, Tünde Szabó, Emília Rita Molnár, Réka Gémes, Nikolett Szebeni, Gábor J. Puskás, László G. Erdélyi, Miklós Hideghéty, Katalin |
author_facet | Brunner, Szilvia Varga, Dániel Bozó, Renáta Polanek, Róbert Tőkés, Tünde Szabó, Emília Rita Molnár, Réka Gémes, Nikolett Szebeni, Gábor J. Puskás, László G. Erdélyi, Miklós Hideghéty, Katalin |
author_sort | Brunner, Szilvia |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8966514 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-89665142022-03-31 Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy Brunner, Szilvia Varga, Dániel Bozó, Renáta Polanek, Róbert Tőkés, Tünde Szabó, Emília Rita Molnár, Réka Gémes, Nikolett Szebeni, Gábor J. Puskás, László G. Erdélyi, Miklós Hideghéty, Katalin Pathol Oncol Res Pathology and Oncology Archive 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. Frontiers Media S.A. 2021-11-08 /pmc/articles/PMC8966514/ /pubmed/35370480 http://dx.doi.org/10.3389/pore.2021.1609971 Text en Copyright © 2021 Brunner, Varga, Bozó, Polanek, Tőkés, Szabó, Molnár, Gémes, Szebeni, Puskás, Erdélyi and Hideghéty. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pathology and Oncology Archive Brunner, Szilvia Varga, Dániel Bozó, Renáta Polanek, Róbert Tőkés, Tünde Szabó, Emília Rita Molnár, Réka Gémes, Nikolett Szebeni, Gábor J. Puskás, László G. Erdélyi, Miklós Hideghéty, Katalin Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title | Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title_full | Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title_fullStr | Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title_full_unstemmed | Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title_short | Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy |
title_sort | analysis of ionizing radiation induced dna damage by superresolution dstorm microscopy |
topic | Pathology and Oncology Archive |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8966514/ https://www.ncbi.nlm.nih.gov/pubmed/35370480 http://dx.doi.org/10.3389/pore.2021.1609971 |
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