Journal Article

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

Wesley D. Block, Dennis Merkle, Katheryn Meek and Susan P. Lees‐Miller

in Nucleic Acids Research

Volume 32, issue 6, pages 1967-1972
Published in print March 2004 | ISSN: 0305-1048
Published online March 2004 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkh508
Selective inhibition of the DNA‐dependent protein kinase (DNA‐PK) by the radiosensitizing agent caffeine

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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.

Journal Article.  4422 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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