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Nucleic Acids Research, 2002, Vol. 30, No. 2 e1
© 2002 Oxford University Press

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells

S. J. Collis1,2, V. K. Sangar1,5, A. Tighe1,2, S. A. Roberts3, N. W. Clarke4,5, J. H. Hendry1 and G. P. Margison2,*

1CRC Experimental Radiation Oncology Group, 2Carcinogenesis Group and 3Biostatistics Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK, 4Department of Urology, Christie Hospital NHS Trust, Manchester M20 4BX, UK and 5Salford Royal Hospitals NHS Trust, Salford M6 8HD, UK

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.

* To whom correspondence should be addressed. Tel: +44 161 446 3183; Fax: +44 161 446 3109; Email: gmargison{at}picr.man.ac.uk Present addresses:S. J. Collis, Johns Hopkins Oncology Center, Cancer Research Building, Department of Radiation Biology, Room 132, 1650 Orleans Street, Baltimore, MD 21218, USAA. Tighe, The School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK


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C. P. Diggle, J. Bentley, and A. E. Kiltie
Development of a rapid, small-scale DNA repair assay for use on clinical samples
Nucleic Acids Res., August 1, 2003; 31(15): e83 - e83.
[Abstract] [Full Text] [PDF]


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