Radiation dose determines the method for quantification of DNA double strand breaks

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more a...

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Bibliographic Details
Published in:Anais da Academia Brasileira de Ciências 2016-03, Vol.88 (1), p.127-136
Main Authors: Bulat, Tanja, Keta, Otilija, Korićanac, Lela, Žakula, Jelena, Petrović, Ivan, Ristić-Fira, Aleksandra, Todorović, Danijela
Format: Article
Language:English
Subjects:
Blotting, Western
Cell Line, Tumor - radiation effects
DNA Breaks, Double-Stranded - radiation effects
Dose-Response Relationship, Radiation
Histones - radiation effects
Humans
Melanoma - radiotherapy
microscopia de imunofluorescência
Microscopy, Fluorescence
microscópio AxioImagerA1
MULTIDISCIPLINARY SCIENCES
Phosphorylation
Radiometry - methods
software ApoTome
Western blot
γH2AX
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Summary:Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.
ISSN:0001-3765
1678-2690
1678-2690
DOI:10.1590/0001-3765201620140553