Use of Comet-FISH in the study of DNA damage and repair: review

Abstract

The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest, including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA regions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene regions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the measured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.