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Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

Biomedical Sciences Research Institute Computer Science Research Institute Environmental Sciences Research Institute Nanotechnology & Advanced Materials Research Institute

McKenna, DJ, Gallus, M, McKeown, Stephanie, Downes, Stephen and McKelvey-Martin, Valerie (2003) Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells. DNA REPAIR, 2 (8). pp. 879-890. [Journal article]

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DOI: 10.1016/S1568-7864(03)00086-7

Abstract

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 mug/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 mug/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reductio in in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents. (C) 2003 Elsevier Science B.V. All rights reserved.

Item Type:Journal article
Faculties and Schools:Faculty of Life and Health Sciences
Faculty of Life and Health Sciences > School of Biomedical Sciences
Faculty of Life and Health Sciences > School of Health Sciences
Faculty of Life and Health Sciences > School of Pharmacy and Pharmaceutical Science
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Molecular Medicine
Biomedical Sciences Research Institute > Molecular Medicine > Nano Systems Biology
ID Code:3389
Deposited By:Professor Stephen Downes
Deposited On:15 Dec 2009 20:37
Last Modified:04 Dec 2012 12:24

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