Use of the Comet-FISH Assay to Compare DNA Damage and Repair in p53 and hTERT Genes following Ionizing Radiation

Abstract

The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.

The Circadian Gene Per1 Plays an Important Role in Radiation-Induced Apoptosis and DNA Damage in Glioma

“…Simultaneously, they also established that Per1 directly acts on the p53 node, as checkpoint components upstream of p53 remained active in response to DNA damage. Quantitative transcriptional analyses of p53 target genes demonstrated that unbound p53 was absolutely required for the activation of the DNA-damage response (Dakup et al, 2016;McKenna et al, 2012).…”

Down-Regulation of PER2 Increases Apoptosis of Gliomas after X-Ray Irradiation

“…Moreover, C-MYC is also controlled by the circadian clock [30]; together with the two proteins described above, it can induce the apoptosis of U343 cells.…”

Development of a comet-FISH assay for the detection of DNA damage in hemocytes of Crassostrea gigas

“…This study demonstrates the utility of the comet-FISH to analyse the fragmentation of NORs on hemocytes of Crassostrea gigas and reveals its potential use to analyse damaged induced by genotoxicant exposure in specific genes. However, despite of its efficacy in the assignment of DNA damage to genes and specific chromosomal regions (Mladinic et al, 2012;Reiter et al, 2012;Volpato et al, 2008, Hovhannisyan et al, 2005 and in the analysis of cellular DNA repair capacity (Guo et al, 2013;McKenna et al, 2012), this technique has not been widely employed in the field of ecotoxicology. This can be explained by the fact that its application, optimization and interpretation of the results require experience in gene mapping by FISH.…”