Carcinogenesis, Vol. 20, No. 2, 199-203,
February 1999
© 1999 Oxford University Press
Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage
Cancer Research Unit, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 µM) or NU1025 (300 µM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 ± 0.6 and 1.7 ± 0.2, respectively. When used in combination, a DEF10 of 7.8 ± 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 ± 0.04 and 1.20 ± 0.05, respectively, compared with 1.01 ± 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 ± 0.05 with NU1025 compared with IR alone, 1.02 ± 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 µM WM and 300 µM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential.
Abbreviations: DEF10, dose enhancement factor at 10% survival; DMSO, dimethyl sulfoxide; DNA-PK, DNA-dependent protein kinase; DSB, double-strand break; IR, ionizing radiation; PARP, poly(ADP-ribose) polymerase; RR, relative retention; SSB, single-strand break; WM, wortmannin.
1 Present address: Department of Ophthalmology, Manchester Royal Eye Hospital, Oxford Road, Manchester M13 9WH, UK
2 To whom correspondence should be addressed Email: b.w.durkacz{at}newcastle.ac.uk
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