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

Valerie J. McKelvey-Martin, Edwin T S Ho, Stephanie R. McKeown, S. Robin Johnston, Patsy J. McCarthy, Nor Fadilah Rajab, C. Stephen Downes

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

I. 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 metastatic 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 ~ 70% of human tumours. In this paper three bladder transitional cell carcinoma cell lines (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 radioresponsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. II. 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 gene probe which allows specific observation of p53 sequences within 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.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalMutagenesis
Volume13
Issue number1
DOIs
Publication statusPublished - Jan 1998
Externally publishedYes

Fingerprint

Comet Assay
DNA sequences
Electrophoresis
Fluorescence In Situ Hybridization
Assays
Urinary Bladder
Gels
Carcinoma
Cells
DNA Repair
DNA Damage
Radiotherapy
Transitional Cell Carcinoma
Cystectomy
Radiation Tolerance
Cell Line
DNA
Tumors
Repair
Neoplasms

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

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. / McKelvey-Martin, Valerie J.; Ho, Edwin T S; McKeown, Stephanie R.; Johnston, S. Robin; McCarthy, Patsy J.; Rajab, Nor Fadilah; Downes, C. Stephen.

In: Mutagenesis, Vol. 13, No. 1, 01.1998, p. 1-8.

Research output: Contribution to journalArticle

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abstract = "I. 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 metastatic 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 ~ 70{\%} of human tumours. In this paper three bladder transitional cell carcinoma cell lines (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 radioresponsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. II. 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 gene probe which allows specific observation of p53 sequences within 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.",
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