Demonstration of DNA damage/repair in individual cells using in situ end labelling: Association of p53 with sites of DNA damage

Authors

  • Dr. Philip J. Coates,

    Corresponding author
    1. Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, U.K.
    • Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, U.K.
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  • Vicki Save,

    1. Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, U.K.
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  • Bijan Ansari,

    1. Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, U.K.
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  • Peter A. Hall

    1. Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, U.K.
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Abstract

We describe the development and application of in situ end labelling (ISEL) to identity sites of damaged DNA in the nuclei of individual cells. In cell culture, exposure to a variety of genotoxic agents induced a dose and time-dependent increase in nuclear labelling. In addition, examination of histological sections of human skin exposed to solar-simulated UV light showed ISEL in both keratinocytes and superficial dermal cells, with the same spatial and temporal distribution as that of a marker of DNA repair, PCNA (proliferating cell nuclear antigen). Using co-localization techniques and confocal microscopy, we found increased levels of p53 in many ISEL-positive cells in vitro, with a similar distribution of labelling in the nucleus. This observation provides further evidence for a direct role of p53 in the recognition of damaged DNA. Thus, ISEL should prove a convenient method for demonstrating genotoxic insult in individual cells and in histological material, and may have value in toxicological screening. This high-resolution microscopy technique can also be used to compare the spatial distribution of various proteins implicated in the response to DNA damage with the sites of the lesion.

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