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
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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