title: Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair.
abstract: 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.
authors.affiliation: Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Eberhard-Karls University Tuebingen, Roentgenweg 11, 72076 Tuebingen, Germany.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Toulany
authors.forename: Rainer
authors.initials: R
authors.lastname: Kehlbach
authors.forename: Urszula
authors.initials: U
authors.lastname: Florczak
authors.forename: Ali
authors.initials: A
authors.lastname: Sak
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
authors.forename: Jianyong
authors.initials: J
authors.lastname: Chen
authors.forename: Markus
authors.initials: M
authors.lastname: Lobrich
authors.forename: H Peter
authors.initials: HP
authors.lastname: Rodemann
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D016283:Proto-Oncogene Proteins p21(ras); D011836:Radiation Tolerance / Q000187:drug effects / Q000528:radiation effects; D011839:Radiation, Ionizing; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins