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

I. Management of invasive transitional human bladder carcinoma. The tw o main treatment options for invasive transitional cell bladder carcin oma are radiotherapy or primary cystectomy with urinary diversion or b ladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the abs ence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastic spread before cystectom y 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 consum ing and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell line (HT1 376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 GY). We hav e compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) a ssay. A very good inverse correlation was obtained between cell surviv al (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict th e radio-responsiveness of individual cell lines. The clinical usefulne ss of the assay for predicting response to radiotherapy in bladder can cer patients is currently being investigated. II. Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repa ired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably d ependent 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 i s relatively simple and allows individual cells to be examined. Here w e present a protocol for combination of the Comet assay with fluoresce nt 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 und erway to facilitate future application of the technique to study selec tive DNA damage and repair in defined sequences in single mammalian ce lls.