Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.
Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.
Collis
S J
SJ
CRC Experimental Radiation Oncology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
Sangar
V K
VK
Tighe
A
A
Roberts
S A
SA
Clarke
N W
NW
Hendry
J H
JH
Margison
G P
GP
D002470:Cell Survival; D004249:DNA Damage / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004260:DNA Repair / Q000235:genetics*; D005434:Flow Cytometry; D005453:Fluorescence; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics*; D011836:Radiation Tolerance; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics*