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Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells

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

McKelvey-Martin, Valerie, Ho, ETS, McKeown, Stephanie, Johnston, SR, McCarthy, PJ, Rajab, NF and Downes, Stephen (1998) Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. MUTAGENESIS, 13 (1). pp. 1-8. [Journal article]

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Abstract

I. Management of invasive transitional human bladder carcinoma. The two main treatment options for invasive transitional cell bladder carcinoma are radiotherapy or primary cystectomy with urinary diversion or bladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the absence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastic spread before cystectomy is performed. The SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, is regarded as a good measure of radiosensitivity. However, the assay is time consuming and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell line (HT1376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 GY). We have compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) assay. A very good inverse correlation was obtained between cell survival (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict the radio-responsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. II. Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repaired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably dependent on DNA position and transcription. This has been established by biochemical techniques which are labour intensive and require large numbers of cells. The Comet assay for overall DNA damage and repair is relatively simple and allows individual cells to be examined. Here we present a protocol for combination of the Comet assay with fluorescent in situ hybridization (FISH) using a p53 sequences with DNA comets. Chromosome-specific probes can also be used. Optimization of the FISH/Comet protocol to include automation of the analysis is currently underway to facilitate future application of the technique to study selective DNA damage and repair in defined sequences in single mammalian cells.

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 > Pharmaceutical Science and Practice
Biomedical Sciences Research Institute > Molecular Medicine > Nano Systems Biology
ID Code:3406
Deposited By:Professor Stephen Downes
Deposited On:15 Dec 2009 16:06
Last Modified:04 Dec 2012 12:30

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