Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines.
The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.
McKenna
Declan J
DJ
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, BT52 1SA.
Rajab
Nor F
NF
McKeown
Stephanie R
SR
McKerr
George
G
McKelvey-Martin
Valerie J
VJ
D020552:Comet Assay / Q000379:methods; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D005720:Gamma Rays; D016158:Genes, p53 / Q000528:radiation effects*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics*