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Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis

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    SYSNO ASEP0024908
    Document TypeJ - Journal Article
    R&D Document TypeJournal Article
    Subsidiary JOstatní články
    TitleEvaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis
    TitleHodnocení jaderného DNA difúzního testu k detekci apoptózy a apoptózy
    Author(s) Gichner, Tomáš (UEB-Q)
    Mukherjee, A. (IN)
    Wagner, E. D. (US)
    Plewa, M. J. (US)
    Source TitleMutation Research - Genetic Toxicology and Environmental Mutagenesis. - : Elsevier - ISSN 1383-5718
    Roč. 586, č. 1 (2005), s. 38-46
    Number of pages9 s.
    Languageeng - English
    CountryNL - Netherlands
    KeywordsDNase-I ; Ethyl methanesulphonate ; Hydrogen peroxide
    Subject RIVEB - Genetics ; Molecular Biology
    R&D ProjectsGA521/05/0500 GA ČR - Czech Science Foundation (CSF)
    CEZAV0Z50380511 - UEB-Q (2005-2011)
    AnnotationWe applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apo-ptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA da-mage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) fol-lowed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bro-mide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treat-ments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants. (c) 2005 Elsevier B.V. All rights reserved.
    WorkplaceInstitute of Experimental Botany
    ContactDavid Klier, knihovna@ueb.cas.cz, Tel.: 220 390 469
    Year of Publishing2006
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