title: 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.
abstract: ABSTRACT I: Management of invasive transitional cell 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 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 approximately 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 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. ABSTRACT 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 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.
authors.affiliation: Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, UK. v.mckelvey@ulst.ac.uk
authors.forename: V J
authors.initials: VJ
authors.lastname: McKelvey-Martin
authors.forename: E T
authors.initials: ET
authors.lastname: Ho
authors.forename: S R
authors.initials: SR
authors.lastname: McKeown
authors.forename: S R
authors.initials: SR
authors.lastname: Johnston
authors.forename: P J
authors.initials: PJ
authors.lastname: McCarthy
authors.forename: N F
authors.initials: NF
authors.lastname: Rajab
authors.forename: C S
authors.initials: CS
authors.lastname: Downes
mesh_terms: D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004587:Electrophoresis, Agar Gel / Q000379:methods / Q000639:trends*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009361:Neoplasm Invasiveness; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D014965:X-Rays