Selective inhibition of the DNA-dependent protein kinase (DNA-PK) by the radiosensitizing agent caffeine.
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.
Block
Wesley D
WD
Department of Biological Sciences, Cancer Biology Research Group, University of Calgary, 3330 Hospital Drive N.W., Calgary AB, T2N 4N1, Canada.
Merkle
Dennis
D
Meek
Katheryn
K
Lees-Miller
Susan P
SP
D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology; D002457:Cell Extracts; D002460:Cell Line; D006224:Cricetinae; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D051716:Replication Protein A
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D011838:Radiation-Sensitizing Agents; D051716:Replication Protein A; D002110:Caffeine; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D002166:Camptothecin