Demonstration of DNA damage/repair in individual cells using in situ end labelling: association of p53 with sites of DNA damage.
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-stimulated 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.
Coates
P J
PJ
Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, U.K.
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Ansari
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Hall
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D000818:Animals; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D004848:Epithelium / Q000378:metabolism / Q000528:radiation effects; D005047:Etoposide / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D051379:Mice; D051381:Rats; D012867:Skin / Q000528:radiation effects; D013194:Staining and Labeling / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014466:Ultraviolet Rays
D016159:Tumor Suppressor Protein p53; D005047:Etoposide