Published OnlineFirst May 17, 2012; DOI: 10.1158/1541-7786.MCR-11-0592 Molecular Cancer DNA Damage and Cellular Stress Responses Research Akt Promotes Post-Irradiation Survival of Human Tumor Cells through Initiation, Progression, and Termination of DNA-PKcs–Dependent DNA Double-Strand Break Repair Mahmoud Toulany1, Kyung-Jong Lee3, Kazi R. Fattah3, Yu-Fen Lin3, Brigit Fehrenbacher2, Martin Schaller2, Benjamin P. Chen3, David J. Chen3, and H. Peter Rodemann1 Abstract Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site. Thus, targeting of Akt results in radiosensitization of DNA-PKcs and Ku80 expressing, but not of cells deficient for, either of these proteins. The data showed indicate for the first time that Akt through an immediate complex formation with DNA-PKcs can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity for efficient NHEJ DNA-DSB repair. Mol Cancer Res; 10(7); 945–57. Ó2012 AACR. Introduction double-strand break (DNA-DSB) repair and subsequent The serine/threonine kinase Akt/PKB is expressed as 3 reproductive cell death (6). isoforms, Akt1/PKBa, Akt2/PKBb and Akt3/PKBg. Akt DNA-DSBs are the most lethal type of DNA lesions that activation is efficiently induced by ionizing radiation (IR) or lead to cell death following IR exposure. Two processes are by growth factors, such as EGF receptor (EGFR) ligands, primarily involved in DNA-DSB repair, nonhomologous through the activation of phosphoinositide 3-kinase (PI3K; end-joining (NHEJ) and homologous recombination (7), ref. 1). Our results and accumulated reports from other but NHEJ is the predominant process in higher eukaryotes laboratories indicate that radiosensitization by targeting and mammals. The catalytic subunit of the DNA-dependent PI3K or Akt1 (2–5) is a consequence of impaired DNA protein kinase complex (DNA-PKcs) is a key enzyme in the NHEJ process. Activation of DNA-PKcs requires the phos- phorylation of specific amino acid residues, among which the fi Authors' Affiliations: 1Division of Radiobiology and Molecular Environ- T2609 cluster and S2056 have been identi ed as essential for mental Research, Department of Radiation Oncology, 2Department of efficient rejoining of DNA-DSBs during NHEJ (8). Like- Dermatology, Eberhard Karls University Tubingen,€ Tubingen,€ Germany; and 3Division of Molecular Radiation Biology, Department of Radiation wise, mutations in these phosphorylation sites result in Oncology, University of Texas Southwestern Medical Center at Dallas, enhanced cellular sensitivity to IR (9, 10). In this context, Dallas, Texas our previous work provided the first new insights into the Note: Supplementary data for this article are available at Molecular Cancer possible function of Akt1 in modulating post-irradiation Research Online (http://mcr.aacrjournals.org/). survival. This occurs most likely via phosphorylation of D.J. Chen and H.P. Rodemann shared corresponding authorship. DNA-PKcs and consequently via the NHEJ repair pathway (2). We showed that an Akt antagonist inhibits radiation- Corresponding Author: H. Peter Rodemann, Eberhard Karls University fi Tubingen,€ Roentgenweg 11, Tubingen€ 72076, Germany. Phone: 49-7071- induced phosphorylation of DNA-PKcs. This nding cor- 298-5962; Fax: 49-7071-29-5900; E-mail: hans-peter.rodemann@uni- relates with cellular radiosensitization after treatment with an tuebingen.de Akt inhibitor due to suppression of DNA-DSB repair, as doi: 10.1158/1541-7786.MCR-11-0592 measured by g-H2AX foci. We also discovered that DNA- Ó2012 American Association for Cancer Research. PKcs co-immunoprecipitates with either Akt1 or p-Akt (2). www.aacrjournals.org 945 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst May 17, 2012; DOI: 10.1158/1541-7786.MCR-11-0592 Toulany et al. A similar interaction was reported by Bozulic and colleagues HT1080 cells were maintained in medium containing (11). On the basis of these data, we conclude that Akt might 250 mg/mL of G418. Xrs6 þ Ku80 cells were maintained be partially necessary for the repair of DNA-DSBs in in medium containing 150 mg/mL hygromycin B. irradiated cells via activation of DNA-PKcs. Rad51D1-deficient Chinese hamster ovary (CHO) cells In the present study, using different approaches in cells originally established in the laboratory of Dr. Larry H. lackingorexpressingDNA-PKcsorKu80,weprovide Thompson (Lawrence Livermore National Laboratory, detailed information for the function of Akt, especially Livermore, CA) were received from the Laboratory of Dr. Akt1, in DNA-PKcs–dependent repair of DNA-DSBs. Eckhardt Dikomey (Radiobiology and Experimental Radio- Ourresultsindicateforthefirst time that Akt1 is not only oncology, University Medical Center Hamburg-Eppendorf, involved in DNA-DSB repair but also directly promoting Hamburg, Germany). and regulating repair process through the 2 complemen- tary mechanisms. First, Akt, especially Akt1, facilitates IR- Glutathione S-transferase pull-down assay induced Ku/DNA-PKcs complex formation and accumu- Glutathione S-transferase (GST), GST-Akt1 full-length, lation of DNA-PKcs to DNA damage site; and second, GST-Akt1 N-terminal fragment (1–150 a.a.), and GST- Akt induces DNA-PKcs kinase activity and its autopho- Akt1 C-terminal fragment (151–480 a.a.) were expressed in sphorylation that is needed for release of DNA-PKcs from BL21 Escherichia coli bacteria by induction of cultures at damage site. optical density (OD)600 of 0.6 with 0.2 mmol/L isopropyl- b-D-thiogalactopyranoside (IPTG) at 37 C for 4 hours. Bacterial lysates were applied to Glutathione Sepharose Materials and Methods beads (GE Healthcare Life Sciences), and the GST-tagged Reagents and antibodies proteins were pulled down and washed with PBS. Then, the The Akt pathway inhibitor (API) and antibodies against beads were incubated with 1 mg of purified human DNA- Akt1, p-Akt (S472/3), p-H2AX (S139), DNA-PKcs, and PKcs in HEPES buffer [50 mmol/L HEPES, pH 7.5, 150 phospho-DNA-PKcs (S2056) have been previously mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% described (2, 3). The DNA-PKcs inhibitors NU7441 and glycerol, 1% Triton X-100, 1 mmol/L dithiothreitol (DTT), NU7026 were purchased from Tocris Bioscience and Sigma- 0.5 mmol/L phenylmethylsulfonylfluoride (PMSF), 1 mg/ Aldrich, respectively. Hygromycin B was purchased from mL Pepstatin, and 2 mg/mL leupeptin] supplemented with Invitrogen. G418 and puromycin were purchased from 0.01% bovine serum albumin (BSA) and 40 ng/mL ethi- Biochrom. Sepharose bead–conjugated IgG antibody was dium bromide (EtBr) for 2 hours at 4C. Beads were washed purchased from Cell Signaling. Active Akt1 and calf thymus 3 times with HEPES buffer and then subjected to Western DNA cellulose were purchased from Sigma-Aldrich. Control blot analysis. nontargeting siRNA (catalog no: D-001810-10) and AKT1- siRNA (catalog no: NM-0010144219) were purchased from Clonogenic assay, immunoprecipitation, Western Thermo Fisher Scientific. blotting, g-H2AX foci formation assay, siRNA transfection, fluorescence-activated cell-sorting analysis, Cell lines immunostaining, and microscopy The colon carcinoma cell lines HCT116 wild-type These assays have been described previously (2, 13). (HCT116wt), HCT116 DNA-PKcs–deficient (HCT116- À À DNA-PKcs / ), and HCT116 DNA-PKcs–deficient com- DNA-PKcs in vitro kinase assay plemented with GFP-tagged DNA-PKcs (GFP-DNA-PKcs- A DNA-PKcs kinase assay was conducted using the HCT116) were kindly provided by Dr. Eric Hendrickson recombinant protein substrate GST-X4 as described earlier (Department of Biochemistry, Molecular Biology and Bio- (14). Each phosphorylation reaction contained 25 mmol/L physics, University of Minnesota Medical School, Minnea- Tris-HCl (pH 7.9), 25 mmol/L MgCl2, 1 mmol/L DTT, 25 polis, MN; ref. 12). HT1080 is a human fibrosarcoma cell mmol/L KCl, 10% glycerol, [g-32P]ATP (6,000 Ci/mmol), line that stably expresses yellow fluorescent protein (YFP)- the indicated concentration of DNA-PKcs, 24 nmol/L Ku tagged DNA-PKcs (YFP-DNA-PKcs-HT1080). The ham- dimer, and the indicated concentration of inhibitors or ster Xrs6 cell line lacking Ku80 (Xrs6 Ku80-def) and its dimethyl sulfoxide (DMSO) in a final volume of 10 mL. Ku80 stably transfected counterpart (Xrs6 þ Ku80) was also Reactions were incubated for 30 minutes at 30C and then used. A549 cells are human non–small cell lung cancer cells. terminated by the addition of SDS-PAGE sample buffer. Except for the A549 cells, all cell lines were cultured in Reaction products were analyzed using 8% SDS-PAGE and Minimum Essential Medium routinely supplemented with detected by PhosphorImager analysis (Amersham Pharmacia 10% fetal calf serum and 1% penicillin/streptomycin. A549 Biotech). Thereafter, the gels were stained with 0.1% Coo- cells were cultured in Dulbecco's Modified Eagle's Media massie blue.