Toulany Mahmoud, Kehlbach Rainer, Florczak Urszula, Sak Ali, Wang Shaomeng, Chen Jianyong, Lobrich Markus, Rodemann H Peter
Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Eberhard-Karls University Tuebingen, Roentgenweg 11, 72076 Tuebingen, Germany.
Mol Cancer Ther. 2008 Jul;7(7):1772-81. doi: 10.1158/1535-7163.MCT-07-2200.
We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59 CJ-OH (API; 1-5 micromol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G(1) cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the gamma-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.
我们已经报道过，表皮生长因子受体/磷脂酰肌醇3-激酶/AKT信号通路是调节人类肿瘤细胞辐射敏感性和DNA双链断裂（DNA-dsb）修复的重要途径。在本研究中，我们调查了AKT1对受辐照的非小细胞肺癌细胞系A549和H460中DNA依赖性蛋白激酶催化亚基（DNA-PKcs）活性及DNA-dsb修复的影响。用特异性AKT通路抑制剂API-59 CJ-OH（API；1-5微摩尔/升）处理细胞，使两个细胞系的克隆形成存活率降低了40%至85%，并显著增强了它们的辐射敏感性。荧光激活细胞分选分析（亚G1期细胞）和聚（ADP-核糖）聚合酶裂解显示，API处理或用AKT1小干扰RNA（siRNA）转染可诱导H460细胞凋亡，但不诱导A549细胞凋亡。然而，在抗凋亡的A549细胞或凋亡敏感的H460细胞中，API和/或AKT1-siRNA在辐照后均未增强聚（ADP-核糖）聚合酶裂解和凋亡。用API预处理细胞或用AKT1-siRNA转染可强烈抑制辐射诱导的DNA-PKcs在T2609和S2056位点的磷酸化，以及通过γ-H2AX焦点分析测定的DNA-dsb修复。免疫共沉淀实验显示活化的AKT和DNA-PKcs形成复合物，支持AKT在受辐照细胞中DNA-PKcs激活中起重要调节作用的假设。因此，靶向AKT最有可能通过特异性抑制DNA-PKcs依赖性DNA-dsb修复，而不是通过增强辐射诱导的凋亡来增强肺癌细胞系A549和H460的辐射敏感性。
