DEMONSTRATION OF DNA-DAMAGE REPAIR IN INDIVIDUAL CELLS USING IN-SITU END LABELING - 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-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.