title: Demonstration of DNA damage/repair in individual cells using in situ end labelling: association of p53 with sites of DNA damage.
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-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.
authors.affiliation: Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, U.K.
authors.forename: P J
authors.initials: PJ
authors.lastname: Coates
authors.forename: V
authors.initials: V
authors.lastname: Save
authors.forename: B
authors.initials: B
authors.lastname: Ansari
authors.forename: P A
authors.initials: PA
authors.lastname: Hall
mesh_terms: 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
chemical_list: D016159:Tumor Suppressor Protein p53; D005047:Etoposide