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Published online 1 April 2004

Nucleic Acids Research, 2004, Vol. 32, No. 6 1967-1972
© 2004 Oxford University Press

Selective inhibition of the DNA-dependent protein kinase (DNA-PK) by the radiosensitizing agent caffeine

Wesley D. Block, Dennis Merkle1, Katheryn Meek2 and Susan P. Lees-Miller*

Department of Biological Sciences, Cancer Biology Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary AB, T2N 4N1, Canada, 1 Department of Chemistry, University of Calgary, 2500 University Drive, N.W. Calgary, AB T2N 1N4, Canada and 2 College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA

*To whom correspondence should be addressed. Tel: +1 403 220 7628; Fax: +1 403 210 3899; Email: leesmill{at}ucalgary.ca

Received March 4, 2004; Accepted March 5, 2004

Caffeine inhibits cell cycle checkpoints, sensitizes cells to ionizing radiation-induced cell killing and inhibits the protein kinase activity of two cell cycle checkpoint regulators, Ataxia-Telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). In contrast, caffeine has been reported to have little effect on the protein kinase activity of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks. Previously, we reported that DNA-PK phosphorylates Thr21 of the 32 kDa subunit of replication protein A (RPA32) in response to camptothecin. In this report we demonstrate that the camptothecin-induced phosphorylation of RPA32 on Thr21 is inhibited by 2 mM caffeine. In addition, we show that caffeine inhibits immunoprecipitated and purified DNA-PK, as well as DNA-PK in cell extracts, with an IC50 of 0.2–0.6 mM. Caffeine inhibited DNA-PK activity through a mixed non-competitive mechanism with respect to ATP. In contrast, 10-fold higher concentrations of caffeine were required to inhibit DNA-PK autophosphorylation in vitro and caffeine failed to inhibit DNA-PKcs dependent double-strand break repair in vivo. These data suggest that while DNA-PK does not appear to be the target of caffeine-induced radiosensitization, caffeine cannot be used to differentiate between ATM, ATR and DNA- PK-dependent substrate phosphorylation in vivo.


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