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.
Highlights
The comet-fluorescent in situ hybridisation (FISH) assay is a method that allows DNA damage and repair to be detected in specific gene regions relative to the overall genome [1]
Our results demonstrate that the p53 gene region is more rapidly repaired than the hTERT gene region in the bladder cancer cell lines RT4 and RT112 (Figure 3a & 3b), which expands on our previous observations that the p53 gene region is repaired more rapidly than the overall genome following DNA damage [21,22]
This preferential repair of the p53 gene region compared to both the overall genome and the hTERT gene region was observed in normal fibroblast cells (Figure 5a and 6a), indicating that repair of strand breaks within, or in the vicinity of, the p53 probe region is carried out quickly in a significant number of analysed cells, whereas breaks in or near the hTERT gene region were not
Summary
The comet-FISH assay is a method that allows DNA damage and repair to be detected in specific gene regions relative to the overall genome [1]. The ability to obtain this type of information would be useful because this assay could benefit many areas of clinical investigation by providing valuable information about the intrinsic DNA characteristics of individual cells and their responses to various external factors, such as radiation, chemicals and drugs. This information would prove relevant in the diagnosis, prognosis and treatment of cancer by allowing analysis of tumour cells, since the repair of important gene regions is integral in determining individual patient response to therapy [3]. The ability to predict the radiosensitivity of individual tumours would represent a major step forward in radiation biology, since there is still no definitive way of predicting whether an individual patient will respond to radiotherapy or not
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