Block Wesley D, Merkle Dennis, Meek Katheryn, Lees-Miller Susan P
Department of Biological Sciences, Cancer Biology Research Group, University of Calgary, 3330 Hospital Drive N.W., Calgary AB, T2N 4N1, Canada.
Nucleic Acids Res. 2004 Apr 1;32(6):1967-72. doi: 10.1093/nar/gkh508. Print 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.
咖啡因可抑制细胞周期检查点，使细胞对电离辐射诱导的细胞杀伤更加敏感，并抑制两种细胞周期检查点调节因子——共济失调毛细血管扩张症突变基因（ATM）和ATM及Rad3相关蛋白（ATR）的蛋白激酶活性。相比之下，据报道咖啡因对DNA依赖性蛋白激酶（DNA-PK）的蛋白激酶活性影响较小，而DNA-PK对于DNA双链断裂的修复至关重要。此前，我们报道过DNA-PK会在喜树碱作用下使复制蛋白A（RPA32）32 kDa亚基的苏氨酸21位点发生磷酸化。在本报告中，我们证明2 mM咖啡因可抑制喜树碱诱导的RPA32苏氨酸21位点的磷酸化。此外，我们还表明咖啡因可抑制免疫沉淀并纯化的DNA-PK以及细胞提取物中的DNA-PK，其半数抑制浓度（IC50）为0.2 - 0.6 mM。咖啡因通过对ATP的混合非竞争性机制抑制DNA-PK活性。相比之下，在体外抑制DNA-PK自身磷酸化需要浓度高10倍的咖啡因，并且咖啡因在体内无法抑制DNA-PK催化亚基（DNA-PKcs）依赖性双链断裂修复。这些数据表明，虽然DNA-PK似乎不是咖啡因诱导放射增敏作用的靶点，但咖啡因无法用于在体内区分ATM、ATR和DNA-PK依赖性底物磷酸化。
