dna damage and cellular stress responsesmcr.aacrjournals.org/content/molcanres/10/7/945.full.pdfdna...

DNA Damage and Cellular Stress Responses

AktPromotesPost-IrradiationSurvival ofHumanTumorCellsthrough Initiation, Progression, and Termination ofDNA-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

AbstractAkt phosphorylation has previously been described to be involved in mediating DNA damage repair through the

nonhomologous end-joining (NHEJ) repair pathway. Yet themechanism how Akt stimulates DNA-protein kinasecatalytic subunit (DNA-PKcs)-dependent DNAdouble-strand break (DNA-DSB) repair has not been described sofar. In the present study, we investigated themechanism bywhichAkt can interact withDNA-PKcs and promote itsfunction during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 inthe regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays apivotal 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 forprogression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation ofDNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damagesite. Thus, targeting of Akt results in radiosensitization ofDNA-PKcs andKu80 expressing, but not of cells deficientfor, either of these proteins. The data showed indicate for the first time that Akt through an immediate complexformation withDNA-PKcs can stimulate the accumulation ofDNA-PKcs at DNA-DSBs and promoteDNA-PKcsactivity for efficient NHEJ DNA-DSB repair. Mol Cancer Res; 10(7); 945–57. �2012 AACR.

IntroductionThe serine/threonine kinase Akt/PKB is expressed as 3

isoforms, Akt1/PKBa, Akt2/PKBb and Akt3/PKBg . Aktactivation is efficiently induced by ionizing radiation (IR) orby growth factors, such as EGF receptor (EGFR) ligands,through the activation of phosphoinositide 3-kinase (PI3K;ref. 1). Our results and accumulated reports from otherlaboratories indicate that radiosensitization by targetingPI3K or Akt1 (2–5) is a consequence of impaired DNA

double-strand break (DNA-DSB) repair and subsequentreproductive cell death (6).DNA-DSBs are the most lethal type of DNA lesions that

lead to cell death following IR exposure. Two processes areprimarily involved in DNA-DSB repair, nonhomologousend-joining (NHEJ) and homologous recombination (7),but NHEJ is the predominant process in higher eukaryotesandmammals. The catalytic subunit of theDNA-dependentprotein kinase complex (DNA-PKcs) is a key enzyme in theNHEJ process. Activation of DNA-PKcs requires the phos-phorylation of specific amino acid residues, amongwhich theT2609 cluster and S2056 have been identified as essential forefficient rejoining of DNA-DSBs during NHEJ (8). Like-wise, mutations in these phosphorylation sites result inenhanced cellular sensitivity to IR (9, 10). In this context,our previous work provided the first new insights into thepossible function of Akt1 in modulating post-irradiationsurvival. This occurs most likely via phosphorylation ofDNA-PKcs and consequently via the NHEJ repair pathway(2). We showed that an Akt antagonist inhibits radiation-induced phosphorylation of DNA-PKcs. This finding cor-relates with cellular radiosensitization after treatmentwith anAkt inhibitor due to suppression of DNA-DSB repair, asmeasured by g-H2AX foci. We also discovered that DNA-PKcs co-immunoprecipitates with either Akt1 or p-Akt (2).

Authors' Affiliations: 1Division of Radiobiology and Molecular Environ-mental Research, Department of Radiation Oncology, 2Department ofDermatology, Eberhard Karls University T€ubingen, T€ubingen, Germany;and 3Division of Molecular Radiation Biology, Department of RadiationOncology, University of Texas Southwestern Medical Center at Dallas,Dallas, Texas

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

D.J. Chen and H.P. Rodemann shared corresponding authorship.

Corresponding Author: H. Peter Rodemann, Eberhard Karls UniversityT€ubingen, Roentgenweg 11, T€ubingen 72076, Germany. Phone: 49-7071-298-5962; Fax: 49-7071-29-5900; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0592

�2012 American Association for Cancer Research.

MolecularCancer

Research

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A similar interaction was reported by Bozulic and colleagues(11). On the basis of these data, we conclude that Akt mightbe partially necessary for the repair of DNA-DSBs inirradiated cells via activation of DNA-PKcs.In the present study, using different approaches in cells

lacking or expressing DNA-PKcs or Ku80, we providedetailed information for the function of Akt, especiallyAkt1, in DNA-PKcs–dependent repair of DNA-DSBs.Our results indicate for the first time that Akt1 is not onlyinvolved in DNA-DSB repair but also directly promotingand regulating repair process through the 2 complemen-tary mechanisms. First, Akt, especially Akt1, facilitates IR-induced Ku/DNA-PKcs complex formation and accumu-lation of DNA-PKcs to DNA damage site; and second,Akt induces DNA-PKcs kinase activity and its autopho-sphorylation that is needed for release of DNA-PKcs fromdamage site.

Materials and MethodsReagents and antibodiesThe Akt pathway inhibitor (API) and antibodies against

Akt1, p-Akt (S472/3), p-H2AX (S139), DNA-PKcs, andphospho-DNA-PKcs (S2056) have been previouslydescribed (2, 3). The DNA-PKcs inhibitors NU7441 andNU7026were purchased fromTocris Bioscience and Sigma-Aldrich, respectively. Hygromycin B was purchased fromInvitrogen. G418 and puromycin were purchased fromBiochrom. Sepharose bead–conjugated IgG antibody waspurchased from Cell Signaling. Active Akt1 and calf thymusDNAcellulose were purchased fromSigma-Aldrich.Controlnontargeting siRNA (catalog no:D-001810-10) and AKT1-siRNA (catalog no:NM-0010144219) were purchased fromThermo Fisher Scientific.

Cell linesThe colon carcinoma cell lines HCT116 wild-type

(HCT116wt), HCT116 DNA-PKcs–deficient (HCT116-DNA-PKcs�/�), and HCT116 DNA-PKcs–deficient com-plemented withGFP-taggedDNA-PKcs (GFP-DNA-PKcs-HCT116) were kindly provided by Dr. Eric Hendrickson(Department of Biochemistry, Molecular Biology and Bio-physics, University of Minnesota Medical School, Minnea-polis, MN; ref. 12). HT1080 is a human fibrosarcoma cellline that stably expresses yellow fluorescent protein (YFP)-tagged DNA-PKcs (YFP-DNA-PKcs-HT1080). The ham-ster Xrs6 cell line lacking Ku80 (Xrs6 Ku80-def) and itsKu80 stably transfected counterpart (Xrs6þKu80) was alsoused. A549 cells are human non–small cell lung cancer cells.Except for the A549 cells, all cell lines were cultured inMinimum Essential Medium routinely supplemented with10% fetal calf serum and 1% penicillin/streptomycin. A549cells were cultured in Dulbecco's Modified Eagle's Media(DMEM). Cells were incubated in a humidified atmosphereof 93% air and 7% CO2 at 37�C. Stably transfected cellswere maintained in medium containing various antibiotics.GFP-DNA-PKcs-HCT116 cells were maintained in medi-um containing 1 mg/mL puromycin. YFP-DNA-PKcs-

HT1080 cells were maintained in medium containing250 mg/mL of G418. Xrs6 þ Ku80 cells were maintainedin medium containing 150 mg/mL hygromycin B.Rad51D1-deficient Chinese hamster ovary (CHO) cellsoriginally established in the laboratory of Dr. Larry H.Thompson (Lawrence Livermore National Laboratory,Livermore, CA) were received from the Laboratory of Dr.Eckhardt Dikomey (Radiobiology and Experimental Radio-oncology, University Medical Center Hamburg-Eppendorf,Hamburg, Germany).

Glutathione S-transferase pull-down assayGlutathione S-transferase (GST), GST-Akt1 full-length,

GST-Akt1 N-terminal fragment (1–150 a.a.), and GST-Akt1 C-terminal fragment (151–480 a.a.) were expressed inBL21 Escherichia coli bacteria by induction of cultures atoptical 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 Sepharosebeads (GE Healthcare Life Sciences), and the GST-taggedproteins were pulled down and washed with PBS. Then, thebeads were incubated with 1 mg of purified human DNA-PKcs in HEPES buffer [50 mmol/L HEPES, pH 7.5, 150mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10%glycerol, 1%TritonX-100, 1mmol/L dithiothreitol (DTT),0.5 mmol/L phenylmethylsulfonylfluoride (PMSF), 1 mg/mL Pepstatin, and 2 mg/mL leupeptin] supplemented with0.01% bovine serum albumin (BSA) and 40 ng/mL ethi-dium bromide (EtBr) for 2 hours at 4�C. Beads were washed3 times with HEPES buffer and then subjected to Westernblot analysis.

Clonogenic assay, immunoprecipitation, Westernblotting, g-H2AX foci formation assay, siRNAtransfection, fluorescence-activated cell-sorting analysis,immunostaining, and microscopyThese assays have been described previously (2, 13).

DNA-PKcs in vitro kinase assayA DNA-PKcs kinase assay was conducted using the

recombinant protein substrate GST-X4 as described earlier(14). Each phosphorylation reaction contained 25 mmol/LTris-HCl (pH 7.9), 25mmol/LMgCl2, 1mmol/LDTT, 25mmol/L KCl, 10% glycerol, [g-32P]ATP (6,000 Ci/mmol),the indicated concentration of DNA-PKcs, 24 nmol/L Kudimer, and the indicated concentration of inhibitors ordimethyl sulfoxide (DMSO) in a final volume of 10 mL.Reactions were incubated for 30 minutes at 30�C and thenterminated by the addition of SDS-PAGE sample buffer.Reaction products were analyzed using 8% SDS-PAGE anddetected by PhosphorImager analysis (AmershamPharmaciaBiotech). Thereafter, the gels were stained with 0.1% Coo-massie blue.The ability of Akt to activate DNA-PKcs was also inves-

tigated using kinase assay. Purified DNA-PKcs was incu-bated with active Akt and the reaction mixture describedabove in a final volume of 10 mL for 30 minutes on ice.Following electrophoresis and blotting, the phosphorylation

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Mol Cancer Res; 10(7) July 2012 Molecular Cancer Research946




of DNA-PKcs was analyzed using a phospho-specific S2056antibody.

DNA-binding assayA DNA-binding assay was conducted as described previ-

ously (15). Briefly, calf thymus DNA cellulose was sus-pended in 3 mL of binding buffer (15) and incubated withrotation at 4�C overnight. Cellulose suspension was thenwashed with fresh binding buffer and centrifuged. Thesupernatant was replaced cell lysate, suspended, incubatedwith rotation for 1 hour at 4�C, and centrifuged. The beadswere washed with binding buffer, and the amounts of DNA-PKcs and Akt1 retained on the resin were determined byWestern blotting.

Live-cell imaging and laser microirradiationThe indicated cells were treated withDMSOor 2.5mmol/

L API for 48 hours, trypsinized, and cultured on glass slides.After 24 hours, live-cell imaging combined with laser micro-irradiation was conducted (16). The fluorescence of livingcells was monitored with an Axiovert 200M microscope,MicroImaging Inc. A 365-nm pulsed nitrogen laser (Spec-tra-Physics) was directly coupled to the epifluorescence pathof the microscope. DNA-DSBs were generated in a definedarea of the nucleus by microirradiation with the 365-nmlaser. For quantitative analyses, we used standardized irra-diation conditions (80% laser output at 10Hz for 400ms) togenerate the same amount of DNA-DSBs in each experi-ment. Time lapse images were obtained, and the fluores-cence intensities of GFP/YFP-tagged DNA-PKcs before andafter microirradiation at the site of irradiation within the cellnucleus were determined using Axiovision Software, version4.5 (Carl Zeiss). All measurements were corrected for non-specific bleaching during monitoring.

Electrophoretic mobility shift assayA standard electrophoretic mobility shift assay (EMSA)

was conducted using purified Ku70/80 or nuclear proteinfractions. The effects of API on the DNA binding of theKu70/80 heterodimer were investigated using either 600nmol/L purified protein or 4 mg nuclear protein fraction.The effect of API on Ku/DNA complex formation innuclear fractions was investigated either by directly incu-bating protein samples with the inhibitor or in samplesisolated from cells pretreated with the inhibitor. Reactionswere incubated with [g-32P]-labeled 30-bp DNA oligo-nucleotides for 30 minutes on ice. DNA–protein com-plexes were separated from unbound oligonucleotides byelectrophoresis and detected using a PhosphorImager. Forsupershift analysis, the reactions were incubated with theKu80 antibody.

ResultsIR induces Akt1/DNA-PKcs complex formation in thenucleusPreviously, we have shown that Akt1 or phospho-Akt (p-

Akt; S472/3) and DNA-PKcs can be co-immunoprecipi-

tated after radiation exposure or stimulation of cells withEGF (2). These data suggest a regulatory interaction betweenAkt1 andDNA-PKcs. To analyze the nature of this potentialinteraction, we further investigated whether an interactionbetween p-Akt and the DNA-PK regulatory subunits Ku70/80 can also be observed by co-immunoprecipitation (co-IP).After radiation exposure or stimulation with EGF or insulin,enhanced co-IP of Ku70/80 can be observed (Fig. 1A and B).Importantly, in this experiment as calculated by the ratio ofKu80/Akt1, a marked increase by about 50% to 60% of theproportion of Ku80 in complex with Akt1 after irradiation aswell as EGF treatment was observed. After insulin treatmentan approximately 100% increase in the proportion of Ku80was apparent. In a control immunoprecipitation, a slightbinding of DNA-PKcs to the control antibody IgG isapparent which is not affected by radiation exposure (Fig.1C). Because Akt1/DNA-PK complex formation wasinduced by ligand stimulation (EGF or insulin) as well asby IR, we concluded that the presence of activated Akt is aprerequisite for Akt/DNA-PK complex formation. Next, weshowed that IR induces immediate Akt1/DNA-PKcs com-plex formation as well as Ku70/80 co-IP with Akt1 in thenucleus (Fig. 1D), but not in the cytoplasm. Co-IP of DNA-PKcs with Akt1 was observed immediately after irradiationand was enhanced in a time-dependentmanner (Fig. 1D andE). Interaction between Akt1 and DNA-PKcs might bemediated by direct or indirect binding of both proteins toDNA. To rule out this possibility, we conducted immuno-precipitation by p-Akt (S472/3) antibody in the nuclearfraction of A549 cells after mock irradiation or irradiationwith 4 Gy. Thereafter, immunoprecipitates from irradiatedsamples were not treated or treated for 30 minutes with 50mg/mL of EtBr, which is known to disrupt protein–DNAinteractions. The results presented in Fig. 1F (top) show thatDNAdid notmediate Akt1/DNA-PKcs complex formation.The IR-induced Akt1/DNA-PKcs complex formationshowed for A549 cells could also be observed in HCT116wild-type cells (Fig. 1G).Because we could show that Akt1 forms a complex with

DNA-PKcs, we analyzed whether binding of Ku70/80 toAkt1 isDNA-PKcs–dependent. As shown in Fig. 1G,Ku70/80 mainly co-IPs with Akt1 in HCT116-DNA-PKcs wild-type, but not in HCT116-DNA-PKcs�/� cells. These dataindicate that the Ku70/80 dimer may not directly interactwith Akt1, and its appearance in the complexwithAkt1mostlikely occurs through binding to DNA-PKcs. Ku-indepen-dent interaction of Akt with DNA-PKcs was further inves-tigated in Ku80-deficient (Xrs6) and Ku80-complementedcells (Xrs6 þ Ku80). The results shown in Fig. 1H indicatethat IR induces Akt1/DNA-PKcs complex formation inboth Ku80-deficient and -proficient cells. These data pro-vide evidence that Ku80 is not necessary for Akt1/DNA-PKcs complex formation.We further investigated whether a direct physical inter-

action between Akt1 and DNA-PKcs can be detected. AGST pull-down experiment in the presence of EtBr usingdifferent Akt1 constructs was carried out. As shown inFig. 1E, full-length Akt1 and the C-terminal fragment

Function of Akt in DNA-DSB Repair

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Toulany et al.





containing the kinase domain of Akt1 were able to pull-downDNA-PKcs, whereas theN-terminal fragment of Akt1was unable to pull-down DNA-PKcs (Fig. 1I).

Akt regulates the accumulation of DNA-PKcs at damagesitesNHEJ repair pathway is initiated by detection of DSBs

through binding of the Ku70/80 heterodimer to the ends ofthe DNA-DSBs, which results in the recruitment andbinding of DNA-PKcs to the Ku70/80 dimer (17–19).IR-induced Ku/DNA-PKcs complex formation occurred inHCT116 wild-type in a time-dependent manner after irra-diation (Fig. 2A). Because Akt1 forms a direct complex withDNA-PKcs through its kinase domain and is also present in acomplex with Ku70/80, we examined whether targeting ofAkt interferes with Ku/DNA-PKcs complex formation. Tothis aim the Akt inhibitor, API-59CJ-OH (API) was used(20). In A549 cells, API (2.5 mmol/L) markedly inhibitedIR-induced phosphorylation of Akt at S473 and T308 aswell as the phosphorylation of Akt substrate PRAS40 atT246 (Supplementary Fig. S1).Most importantly, however,API disturbed radiation-induced Ku/DNA-PKcs complexformation (Fig. 2A, bottom). In line with our previousreports (2), radiation-inducible phosphorylation of DNA-PKcs at T2609 was reduced to about 50% by API. Asradiation-induced ataxia telangiectasia mutated (ATM)phosphorylationwas not affected, we canmost likely excludeunspecific targeting of PI3K-like kinases by API. On thebasis of these results, we propose that Akt inhibition alsointerferes with DNA binding of DNA-PKcs. We conducteda DNA-PKcs pull-down assay with cells pretreated with orwithout the Akt inhibitor and showed that Akt inhibitionalso blocks DNA-PKcs binding to Ku and accumulation ofDNA-PKcs at damage sites (Fig. 2B, top). Importantly, inthis experiment, Akt inhibition did not affect the binding ofKu80 to DNA. Yet although the EGFR molecule does notcontain a DNA-binding domain (21), the observed bindingof EGFR toDNA cellulose, which, however, is not altered bythe experimental conditions tested, is most likely due to thehigh amount of EGFR loaded (see input) and contaminationof the pull-down assay. In a similar experiment, IR-inducedDNAbinding of Akt1 was blocked by the Akt inhibitor. Thelevels of total Akt1 in whole-cell lysates remained constant(Fig. 2B, bottom).

The function of Akt in the accumulation of DNA-PKcs atDNA-DSB sites was further investigated by live-cell imag-ing. GFP-DNA-PKcs–expressing HCT116 cells were trea-ted with API (2.5 mmol/L) and irradiated with a microbeamlaser. Immediately after irradiation, DNA-PKcs accumulat-ed at sites of DNA damage; however, this accumulation wasmarkedly reduced by API treatment (Fig. 2C). Likewise,downregulating Akt1 by siRNA (Western blotting data) inHT1080 cells markedly reduced the accumulation of YFP-DNA-PKcs at DNA-DSBs (Fig. 2D). In this context and tofurther prove the appearance of p-Akt at DNA damage sites,co-localization of p-Akt foci with g-H2AX foci was inves-tigated by confocal microscopy after laser and is shownin Fig. 2E.The data from the pull-down assays (Fig. 2B, top) indicate

that DNA binding of Ku80 was not affected by Akt inhi-bition. This aspect of Ku/DNA complex formation wasfurther investigated more specifically by EMSA using bothpurified proteins and nuclear fractions of whole-cell lysates.As a specificity control, we used theKu80 antibody at variousconcentrations to show the presence of a supershift of theKu/DNA complex using purified Ku or nuclear lysates ofA549 cells irradiated with a dose of 4 Gy (SupplementaryFig. S2A). Thereafter, the effect of 2.5 mmol/L API on Ku/DNA complex formation was analyzed using purified Ku70/80 dimers. In parallel, we examined the effect of API atconcentrations of 0, 2.5, 5, and 12.5 mmol/L on the Ku/DNA complex in the nuclear fraction of irradiated A549cells. As shown in Supplementary Fig. S2B, Ku binding toDNA was not affected by API at concentrations up to 12.5mmol/L. As a specificity control, the supershift induced by aKu80 antibody is shown in Supplementary Fig. S2B, lane 3.In an additional experiment, cells were pretreated with

API and mock-irradiated or irradiated with a dose of 4 Gy.Five minutes after irradiation, nuclear extracts were preparedand used for an EMSA. As shown in Supplementary Fig.S2C, API (2.5 mmol/L) did not affect Ku/DNA complexformation. As a specificity control, the Ku80 antibodyinduced a supershift (Supplementary Fig. S2C, lane 1).

Radiation-induced DNA-PKcs activity is partially Akt1-dependentOn the basis of the direct interaction between Akt1 and

DNA-PKcs, the potential of Akt1 to activateDNA-PKcswas

Figure 1. IR induces Akt1/DNA-PKcs complex formation in the nucleus. A, A549 cells were irradiatedwith 4Gy or treatedwith 100 ng/mL of EGF or insulin, andimmunoprecipitation (IP) of phospho-Akt (S473) was conducted. Whole-cell lysates from control and EGF-stimulated cells were used as inputs.After SDS-PAGE,membraneswere stainedwithPonceauSand thenWestern blotted for p-Akt (S473), Akt1, Ku70, andKu80.B, the intensities of p-Akt (S473),Akt1, Ku70, and Ku80 were analyzed by densitometry and normalized to the nonstimulated control (�), set to 1. Ins, insulin. C and D, A549 cells weremock-irradiated/irradiated. At the indicated timepoints post-IR, cytoplasmic andnuclear fractionswere isolated, and IPwas conducted byusing IgGantibody(C) or antibody against Akt1 (D). Co-IP of DNA-PKcs, Ku70, and Ku80 was verified. Total lysates from cytoplasmic and nuclear fractions, isolated5 minutes post-IR were used as inputs. Lamin A/C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected as purity controls. E, thedensitometric values represent the ratio of DNA-PKcs to Akt1 in nuclear fraction, normalized to nonirradiated control (�), set to 1. F, A549 cells weremock-irradiated/irradiated, and 10 minutes post-IR, cytoplasmic and nuclear fractions were isolated. IP was conducted using Akt1 antibody.Immunoprecipitationsweremock-treated or treatedwith 50mg/mLof EtBr for 30minutes and followedbywashingwithwashing buffer (2). As purity control fornuclear (Nuc.) and cytoplasmic (Cyt.) preparations, the levels of lamin A/C andGAPDHwere detected fromwhole lysates of both fractions (bottom). G and H,cells weremock-irradiated/irradiated. At indicated time (G) and 10minutes after IR (H), nuclear fractionswere isolated and IP of Akt1 was conducted. Co-IP ofDNA-PKcs and Ku70/80 was analyzed. Akt1 was detected as loading. In 100-mg protein samples from the same samples used for IP, the levels of DNA-PKcsand Ku80 were analyzed. I, GST pull-down experiment was conducted using different Akt1 constructs. Pull-down of DNA-PKcs was analyzed by Westernblotting. Fl, full length.






Figure2. Akt1 regulates the accumulation ofDNA-PKcs todamagesites. A, cellswere irradiated, and following IPofKu80, co-IP ofDNA-PKcswas verified. Thedensitometric values represent the ratio of DNA-PKcs to Ku80 normalized to the nonirradiated control (�), set to 1. Total lysates fromDNA-PKcs�/� cells wereused as inputs. Bottom,A549 cellswere pretreatedwith orwithout API (2.5mmol/L for 72 hours) before IR. Thereafter, IP ofDNA-PKcswas conducted, and co-IP of Ku80 with DNA-PKcs was analyzed. Hundredmicrograms of whole-cell lysates fromDMSO-treated nonirradiated cells was used as input. B, A549 cellswere treatedwith orwithout API (2.5 mmol/L) for 72 hours andweremock-irradiated/irradiatedwith 10Gy. At indicated times post-IR (top) or 5minutes post-IR(bottom), protein samples were prepared, and a DNA-binding assay was conducted. DNA-bound proteins were subjected to SDS-PAGE. After Ponceaustaining, levels of Ku80, DNA-PKcs, and Akt1 were analyzed. EGFR with a lack of DNA-binding domain was detected as negative control. Lysates ofnonirradiated, DMSO-treated cells were used as input. Levels of Akt1 in whole-cell lysates were used to indicate constant amounts of Akt under different

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analyzed. In an in vitro kinase assay, active Akt1 wasincubated with DNA-PKcs in the presence of ATP andkinase buffer. Active Akt1 clearly induced autophosphoryla-tion of DNA-PKcs at S2056 (Fig. 3A).Furthermore, the effect of API on DNA-PKcs kinase

activity was analyzed in a [g-32P]ATP-based in vitro kinaseassay. AGST-tagged recombinant XRCC4protein fragment(251–334 a.a., GST-X4) was used as a DNA-PKcs–specificsubstrate (14). A549 cells were either pretreated or untreatedwith API (2.5 mmol/L) and mock-irradiated or irradiatedwith a dose of 4Gy.Nuclear lysates were obtained from thesecells and were used in a kinase assay. As shown in Fig. 3B,API treatment reduced basal and radiation-induced DNA-PKcs activity in both the cell lines analyzed. As a positivecontrol, the DNA-PKcs inhibitor NU7026 completelyblocked phosphorylation of GST-X4 (Fig. 3B). SpecificDNA-PKcs–dependent phosphorylation of GST-X4 wasconfirmed by a lack of substrate phosphorylation underbasal conditions or post-irradiation in HCT116-DNA-PKcs�/� cells (Fig. 3B).Because Akt inhibition markedly affected DNA-PKcs

kinase activity in cell culture (Fig. 3B), we investigatedwhether API had any off-target effects on Ku-dependentDNA-PKcs kinase activity in the absence of Akt. As shownin Supplementary Fig. S3A, in the presence of Ku70/80

protein, GST-X4 was highly phosphorylated. API did notmarkedly affect the phosphorylation of the DNA-PKcssubstrate, but theDNA-PKcs inhibitorNU7441 completelyblocked GST-X4 phosphorylation (Supplementary Fig.S3A).To investigate whether Ku-independent and DNA-

PKcs–dependent phosphorylation of GST-X4 were affect-ed by API, DNA-PKcs kinase assays were conducted in theabsence of Ku heterodimer. Reactions were incubatedwith different concentrations of DNA-PKcs (0, 12.5,30, or 60 nmol/L). A comparison of the phosphorylationof GST-X4 in either the presence or absence of Ku70/80indicated that approximately 80% of DNA-PKcs activityis Ku70/80-dependent (Supplementary Fig. S3B, lane 1).However, even in the absence of Ku70/80, a minorproportion of phosphorylated GST-X4 was observed asa function of the DNA-PKcs concentration (Supplemen-tary Fig. S3B). On the basis of these results, we investi-gated the effect of API on GST-X4 phosphorylation byDNA-PKcs in the absence of Ku70/80. As shown inSupplementary Fig. S3C, incubation of the reaction mix-tures with API at concentrations of 2.5 to 25 mmol/L didnot affect GST-X4 phosphorylation. Again, in the pres-ence of the Ku dimer, strong phosphorylation of GST-X4was observed (Supplementary Fig. S3C, lane 1).

treatment conditions (bottom). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. C, GFP-DNA-PKcs-HCT116 cells weretreated with API (2.5 mmol/L) for 48 hours and plated onto glass slides. After 24 hours, cells were irradiated with amicrobeam laser. The fluorescence intensityofGFP-taggedDNA-PKcsat the irradiation site before and at 0 to 320 seconds after irradiationwasdeterminedand tested for statistical significant differences(P < 0.001; Student t test). Arrows point to the microirradiated site. D, YFP-DNA-PKcs-HT1080 cells were transfected with control (ctrl) or AKT1-siRNA. Fourdays after transfection, levels of Akt1 and actin were determined. In parallel, 3 days after transfection with AKT1-siRNA, cells were transferred to glass slidesand irradiated withmicrobeam laser 24 hours later. Intensity of YFP-DNA-PKcs accumulation at the irradiation site was quantified. Each data point in C and Drepresents the average of 15 independent measurements. Data points represent the mean fluorescence intensity values� SEM. Statistical significance (P <0.01) was analyzed by Student t test. E, GFP-DNA-PKcs–expressing HCT116 cells were laser-irradiated, incubated for 30minutes, fixed, and immunostainedwith p-Akt (S473) and g-H2AX. Ctrl, control.

Figure 3. Radiation-induced DNA-PKcs activity is partially Akt-dependent. A, purified human DNA-PKcs was incubated with or without active recombinanthuman Akt1 in the presence of ATP, and a kinase reaction was conducted. Levels of p-DNA-PKcs (S2056), DNA-PKcs, and Akt1 were detected by Westernblotting. B, cells were treatedwith/without API (2.5mmol/L) for 72 hours andmock-irradiated/irradiatedwith 4Gy. Fiveminutes post-IR, nuclear fractionswereisolated, and4mgof proteinwasused in theassay. Following thedetectionof phospho-GST-X4 (p-GST-X4), gelswere stainedwith0.1%Coomassieblue. TheDNA-PKcs inhibitor NU7026 (10 mmol/L, 1-hour pretreatment) was used as positive control.






The radiosensitizing effect of API depends on theexpression of DNA-PKBecause Akt1 directly interacts with DNA-PKcs and

regulates its activation following radiation exposure, patternof radiosensitization was investigated in DNA-PKcs andKu70/80-proficient and -deficient cells after Akt targeting.API increased radiation sensitivity in HCT116wt cells andXrs6 þ Ku80 cells, but not in NHEJ-deficient HCT116-DNA-PKcs�/� and Xrs6þKu80�/� (Ku80-def.). In CHORad51D1�/� homologous recombination cells (Rad51D1-def.), a similar radiosensitization by API as seen for NHEJrepair–proficient cells (HCT116, Xrs6þ KU80, A549, andHT1080) was observed (Fig. 4B and D). Moreover, asexpected, the high intrinsic radiosensitivity of DNA-PKcs-and Ku80-deficient cells was not affected after high-dose (1–4 Gy) or low-dose (0.2–1 Gy) irradiation in combinationwith API pretreatment (Fig. 4B and C). These controlexperiments support the assumption that targeting Aktresults in an impairment of NHEJ repair and consequentlyradiosensitization.Moreover, we investigated whether API treatment

affected cell proliferation and consequently a redistribu-tion of cells through the cell cycle. As shown by the slopeof the growth curve in Fig. 4E, API did not affect thedoubling time but prolonged the lag phase of cells beforeentering logarithmic growth. This led to a significantdifference in the number of cells at the indicated timepoints (Fig. 4E). Moreover, cell-cycle analysis indicatedthat API treatment alone did not affect the cell-cycledistribution significantly but resulted in combination withirradiation in a significant enhancement of the IR-medi-ated G1 arrest 24 hours after irradiation. The percentage ofsub-G1cells indicating the fraction of apoptotic cells wasnot increased after treatment with API alone or in com-bination with irradiation (control, 0.21% � 0.01%; API,0.17% � 0.03%; IR, 0.24% � 0.02%; API þ IR, 0.21%� 0.03%; data not shown). These data suggest thatinhibition of Akt activity by API (Supplementary Fig.S1) does not result in a potential IR-induced apoptosis.

Targeting of Akt1 impairs the repair of radiation-induced DNA-DSBs and prevents dissociation of DNA-PKcs from damage sitesBecause Akt inhibition markedly reduced DNA-PKcs

kinase activity in living cells (see Fig. 3B), we investigatedwhether Akt inhibition reduces autophosphorylation ofDNA-PKcs at S2056. This was accomplished using A549and HCT116wt cells. As shown in Fig. 5A, 30 minutes afterirradiation, both cells lines showed similar levels of totalDNA-PKcs; however, radiation-induced phosphorylation ofDNA-PKcs was much stronger in A549 cells than inHCT116wt cells. Because the reduction in S2056 phos-phorylation mediated by API was much stronger in irradi-ated A549 cells than in HCT116wt cells, this result couldexplain the stronger API-induced radiosensitization of A549cells thanHCT116wt cells (A549: SF3�API¼ 0.23, SF3þAPI¼ 0.09; HCT116wt: SF3 � API¼ 0.06, SF3þ API¼0.03). In addition, we showed that Akt1 knockdown by

siRNA also resulted in a marked reduction in radiation-induced DNA-PKcs autophosphorylation at S2056 in A549cells (Fig. 5B). Moreover, in A549 and HCT116 cells, thefrequency of residual DNA-DSBs was significantlyenhanced by API treatment (Fig. 5C).Autophosphorylation of DNA-PKcs at 2056 is an

essential step in the release of DNA-PKcs from damagesites after DSB repair (16, 22) and is reviewed by Dobbsand colleagues (23). Therefore, we investigated whetherAkt1 expression is required for dissociation of DNA-PKcsfrom damage sites. Cells were transfected with AKT1-siRNA and irradiated with a dose of 4 Gy. The cells weresubsequently stained with antibodies specific for p-S2056and g-H2AX foci 24 hours after irradiation. As shownin Fig. 5D, 24 hours after radiation exposure, phosphor-ylation of DNA-PKcs at S2056 appears as foci and co-localizes with g-H2AX foci. Interestingly, similar to theincrease in g-H2AX foci after Akt reduction, the frequen-cy of p-S2056 foci after downregulation of Akt1 wassignificantly enhanced (Fig. 5D and E). The magnifiedpicture of p-DNA-PKcs (S2056) resulting from ctrl-siRNA–transfected cells indicates that phosphorylatedDNA-PKcs appears in the focus as well as throughoutthe nucleus, whereas in AKT1-siRNA–transfected cells,the S2056 signal is mainly co-localized with g-H2AX focibut not throughout the nucleus. These data indicatethat targeting of Akt impairs DNA-DSB repair and thesubsequent release of DNA-PKcs from the damage sites(Fig. 5D).

DiscussionPreviously, it was described that phosphorylation of

DNA-PKcs mediated by IR is markedly antagonized byspecific inhibitors of EGFR, PI3K, and Akt such aserlotinib, LY294002, PI-103, and Akt inhibitor VIII(3, 6). Consequently, these inhibitors have been shownto impair DNA-DSB repair and to induce radiosensitiza-tion of a variety of tumor cells in vitro and in vivo(3, 6, 24). Increased cellular radiosensitivity followingPI3K/Akt targeting coincides with enhanced residualDNA-DSBs (2, 5, 6, 25, 26) most likely through inter-ference with DNA-PKcs phosphorylation during theNHEJ repair process (2). However, until now, the specificregulatory function of Akt in NHEJ repair mechanism hasnot been investigated. Yet this topic was specificallyaddressed in the present study. The results presentedherein indicate that Akt in general and specifically Akt1through 3 functions can regulate DNA-DSB repair. First,complex formation of Akt and DNA-PKcs stimulatesbinding of DNA-PKcs to DNA duplex ends marked byKu dimers. Second, Akt in the complex with DNA-PKcspromotes kinase activity of DNA-PKcs, which is necessaryfor executing an efficient DNA-DSB repair. Third, Akt-stimulated autophosphorylation of DNA-PKcs facilitatesthe release of DNA-PKcs from the damage site that isknown to be a necessary step for ligation and terminationof DNA-DSB repair (16, 23).

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Figure 4. Radiosensitizing effect of API depends onDNA-PKcs and Ku80 expression. A, whole-cell lysateswere applied for analysis of the expression levels ofDNA-PKcs and Ku80. Actin was used as a loading control. B–D, cells were treated with/without API (2.5 mmol/L) for 72 hours and plated to assess colonyformation. After 24 hours, cells were irradiated with the indicated doses of IR. Colonies that formed after 10 days were counted, and the clonogenicfraction of irradiated cells was normalized to the plating efficiency of nonirradiated controls. The data bars in B to D represent the mean survivingfraction (SF) � SEM from 6 parallel experiments in HCT116, Xrs6, and A549 cells and 3 parallel experiments in HT1080 cells. �, statistically significantradiosensitization in response to API (�, P < 0.05; ��, P < 0.01; ��, P < 0.001; Student t test). E, A549 cells were treated with API (2.5 mmol/L) for 72 hours andplated in 60-mm culture dishes. After the time periods, indicated cells were counted and graphed. Data points shown represent the mean � SEM of12 data points from 2 independent experiments. �, statistically significant antiproliferative effect of API (��, P < 0.01; ��, P < 0.001; Student t test).F, API-pretreated A549 cells were seeded in 6-cm dishes and after 24 hours were irradiated. After irradiation for 24 hours, cells were collected, andfluorescence-activated cell-sorting (FACS) analysis was conducted. Percentage of cells in different cell cycles were calculated and graphed. A significantIR-induced G1 arrest (��, P < 0.001) and enhancement of IR-induced G1 arrest by API (��, P < 0.001) was observed. Combination of API with radiationreduced the population of cells in S and G2/M significantly (��, P < 0.01). Ctrl, control.






In line with the report by Park and colleagues (27), in ourstudy, we showed that complex formation of Akt1 withDNA-PKcs requires the C-terminal domain of Akt1.Because of this direct interaction, exposure of cells to IRleads to an immediate Akt1/DNA-PKcs complex formation.Bozulic and colleagues (11) also reported a complex forma-tion of Akt1 and DNA-PKcs 30 minutes after radiationexposure. However, in that study, no further investigation ofthe regulatory function of this complex in DNA repair wasconducted. Convincing data have been published by Jeggoand colleagues (28) and Goodarzi and colleagues (29)

showing that the kinetics of DNA-DSB repair through theNHEJ mechanism is dominated by "fast" and "slow" com-ponents. Approximately 85% of DNA-DSBs induced by IRare repaired within the first 2 to 3 hours of post-irradiationvia the fast component, which has been shown to beindependent of ATM function (28). The remaining 15%of DNA-DSBs, which are mainly composed of complexlesions, are repaired in an ATM-dependent manner via theslow component (29, 30). Thus, in this context, the imme-diate post-irradiation formation of the nuclear Akt1/DNA-PKcs complex can be assumed to be primarily an important

Figure 5. Targeting Akt1 impairs radiation-induced DNA-DSB repair and prevents dissociation of DNA-PKcs from damage sites. A, HCT116wt and A549 cellswere pretreatedwith/without API (2.5mmol/L) for 72 hours and irradiatedwith 4Gy. At 10 and 30minutes post-IR, protein sampleswere prepared, and levels ofp-DNA-PKcs (S2056) and DNA-PKcs were analyzed by Western blot analysis. B, A549 cells were transfected with control (ctrl)- or AKT1-siRNA andirradiated with 4 Gy, 4 days later. Expression levels of Akt1, p-DNA-PKcs (S2056), and DNA-PKcs were analyzed by Western blotting. Actin was used as aloading control. C, A549 andHCT116wt cells were treated with/without API (2.5 mmol/L) for 72 hours and irradiated with 2 or 4 Gy. Twenty-four hours post-IR,the cells were stained with an antibody-specific for p-H2AX (S139). Using a fluorescence microscope, the number of g-H2AX foci was counted in 160to 260 cells per treatment condition. �, statistically significant enhancement of residual g-H2AX foci following API treatment (��, P < 0.001; Student t test).D1–4, A549 cells were transfected with control (ctrl)-siRNA or AKT1-specific siRNA. Three days after transfection, the cells were irradiated with 4 Gyand were stained with antibodies specific for p-H2AX (S139) and p-DNA-PKcs (S2056) 24 hours post-IR. DNA was stained with YO-PRO (green). E, thenumber of S2056 foci in 100 to 135 nuclei was counted and graphed. �, statistically significant enhancement of residual S2056 foci following AKT1-siRNAtransfection (��, P < 0.001; Student t test).

Toulany et al.





step in the execution of DNA-DSB repair via the fastcomponent. This assumption is further substantiated byour immunostaining results showing that activated Akt, thatis, p-Akt (S473), co-localizes with g-H2AX foci in micro-beam laser–irradiated cells. This observation is in agreementwith our previous report (31) and the report by Fraser andcolleagues (32) reporting co-localization of p-Akt (S473)with g-H2AX foci after exposure to IR. However, it isunclear whether the IR-induced S473 signal is solely orig-inated from p-Akt1 or from the phosphorylated isoformsAkt2 and Akt3. Because the commercially available p-Akt(S473) antibodies are used detect the phosphorylation ofAkt2 at S474 as well as the phosphorylation of Akt3 at S472,the involvement of Akt2 andAkt3 in the regulation ofDNA-DSB repair cannot be ruled out at present and needs to befurther investigated.The first step in the initiation of the DNA-DSB repair via

NHEJ pathway requires the binding of the Ku70/80 hetero-dimer to the 30 and 50 ends of the DNA break-site whichmarks the DNA-DSB for binding of DNA-PKcs and itsfurther activity (33). As shown, binding of DNA-PKcs toKu70/80 can be significantly inhibited by pretreating cellswith the Akt inhibitor API (see Fig. 2A, bottom). Thus, it

can be assumed that Akt promotes the kinase activity ofDNA-PKcs, which seems to be prerequisite for binding ofDNA-PKcs to Ku70/80 (34). On the basis of the observa-tion that inhibition of Akt interferes with the binding ofDNA-PKcs to Ku70/80, we addressed the question whetherAkt targeting interferes with the recruitment of DNA-PKcsto the damage site. Results from live-cell imaging indicatethat Akt targeting prevents accumulation of DNA-PKcs atthe site of DNA damage. Yet the repression of DNA-PKcsaccumulation is much more prominent in cells treated withAPI than in cells transfected with AKT1-siRNA. Thisdifferential effect can most likely be explained by the factthat API inhibits all 3 isoforms of Akt (20), whereas thesiRNA approach depletes specifically Akt1. Thus, these datacannot exclude the involvement of Akt2 and/or Akt3 in theregulation of NHEJ. Nevertheless, because AKT1-siRNAresults in an approximately 50% reduction of DNA-PKcsaccumulation, it can also be argued that the Akt1 is themajorisoform of Akt, which is directly involved in the efficientactivation step of DNA-PK–dependent DSB repair.As reported byViniegra and colleagues (35), full activation

of Akt in response to IR is mediated through ATM. Regard-ing the role of ATM in Akt phosphorylation, Fraser and

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Figure 6. Proposed interaction of Akt and DNA-PKcs during NHEJ repair process. On the basis of the data shown, the scheme illustrates the potentialinteraction of Akt with DNA-PKcs with respect to the described classical NHEJ (18, 23). Step 1, after induction of DNA-DSBs by IR, DNAbreak-sites are detected and marked by Ku70/80 heterodimer. Step 2, for recruitment of DNA-PKcs and binding to Ku70/80, direct interaction and bindingof Akt to DNA-PKcs is needed. Step 3, DNA-PKcs undergoes trans/autophosphorylation, resulting in the release of DNA-PKcs from the repaireddamage site. Step 4, various additional factors, such as XLF, XRCC4, DNA-ligase IV, and polynucleotide kinase/phosphatase process the final steps ofNHEJ repair.






colleagues (32) showed that Akt phosphorylation dependson MRE11-ATM-RNF168 signaling in direct response toDNA-DSB–induced IR (32). Transphosphorylation ofDNA-PKcs at T2609 seems not only to be dependent onATM as reported by Chen and colleagues (36) but also onAkt as previously shown by Choi and colleagues (6) and ourlaboratory (2). Although the involvement of Akt in ATM-dependent slow component of DNA-DSB repair needs to befurther investigated, it can be assumed that Akt acts as a linkbetween ATM and DNA-PKcs. This conclusion is furthersupported by the results shown in Supplementary Fig. S1,which indicates that Akt inhibition without affecting ATMphosphorylation inhibits DNA-PKcs phosphorylation.DNA-PKcs kinase activity and its autophosphorylation,

partially activated by Akt, are necessary steps for the pro-gression and the termination of DNA-DSB repair. Espe-cially, autophosphorylation at DNA-PKcs at S2056 (36) isnecessary for efficient DNA-DSB repair through NHEJmechanism (10, 36–39) and for subsequent release ofDNA-PKcs from the damage site (16, 22, 23). Akt-depen-dent DNA-PKcs activation/autophosphorylation impliesthat Akt1 functions as a mediator of the dissociation ofDNA-PKcs from the DNA-DSB which is a prerequisite stepfor ligation and subsequent termination of DNA-DSBrepair. This conclusion is supported by a significantlyenhanced amount of DNA-PKcs foci co-localizing withunrepaired DNA-DSBs after Akt1 knockdown. Yet theAKT1-siRNA–mediated increase in the number of residualS2056 foci 24 hours after irradiation seems to be in conflictwith Akt targeting–mediated inhibition ofDNA-PKcs phos-phorylation at S2056 for up to 30 minutes after IR observedby Western blotting (see Fig. 5A and B). This potentialconflict can be explained by a decreased repair efficiency inAKT-targeted cells, which results 24 hours after radiation inan increased number of S2056 foci because of nonreleasedDNA-PKcs. In contrast, the noncompromised DNA repairefficacy in control cells results in a decreased number ofDNA-PKcs foci, reflecting the termination of DSB repairand released DNA-PKcs from the damage site. With respectto the stimulation of DNA-PKcs by Akt, it needs to befurther investigated whether phosphorylation of DNA-PKcsis taking place before or after recruitment of Akt/DNA-PKcscomplex to DNA damage site. Furthermore, it needs to beclarified whether Akt is modified upon recruitment of theAkt/DNA-PKcs complex to DNA. On the basis of the datapresented herein and in reflection of the current model ofhow DNA-PKcs is acting at the DNA damage site describedbyWeterings andChen (18) andDobbs and colleagues (23),we suggest an expanded model by implementing the func-tion of Akt in the regulation of NHEJ repair (Fig. 6). Afterinduction of DNA-DSBs by IR, DNA-DSB sites are

detected and bound by Ku70/80 heterodimers, whichrepresents the initial step of NHEJ repair (Fig. 6, step 1).For recruitment of DNA-PKcs and binding to Ku70/80,the direct interaction and complex formation of Akt toDNA-PKcs seem to warrant full activity of DNA-PKcs(Fig. 6, step 2). After processing of DNA break ends byvarious proteins, such as XLF, XRCC4, DNA-ligase IVand polynucleotide kinase/phosphatase (Fig. 6, step 3),efficient autophosphorylation of DNA-PKcs at S2056occurs under co-control of Akt. As a consequence of thisAkt-stimulated autophosphorylation, DNA-PKcs will bereleased from the repaired damage site (Fig. 6, step 4).Thus, in this model, Akt is stimulating the efficientaccumulation of DNA-PKcs at the damage site, initiationof the repair process, as well as autophosphorylation ofDNA-PKcs, which is prerequisite for the release of DNA-PKcs after repair has been executed.In conclusion, the present study provides the first mech-

anistic evidence for a stimulatory role of Akt in DNA-PKcs–dependent NHEJ repair pathway in human tumor cells afterradiation exposure. On the basis of the results shown,specific experiments can be designed to answer open ques-tions of specific details of Akt/DNA-PKcs interaction duringDNA-DSB repair in irradiated tumor cells.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.

Authors' ContributionsConception and design: M. Toulany, K.R. Fattah, D.J. Chen, H.P. RodemannDevelopment of methodology:M. Toulany, K.-J. Lee, B. Fehrenbacher, B.P. Chen,D.J. ChenAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.):M. Toulany, K.-J. Lee, K.R. Fattah, Y.-F. Lin, M. Schaller, B.P. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis):M. Toulany, K.-J. Lee, K.R. Fattah, B. Fehrenbacher, M. Schaller,B.P. Chen, D.J. Chen, H.P. RodemannWriting, review, and/or revision of the manuscript: M. Toulany, K.R. Fattah, M.Schaller, B.P. Chen, D.J. Chen, H.P. RodemannAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. Toulany, B. Fehrenbacher, H.P. RodemannStudy supervision: M. Toulany, H.P. Rodemann

AcknowledgmentsThe authors thank Dr. Rainer Kehlbach and Dr. Stephan M. Huber for

fluorescence-activated cell-sorting (FACS) analysis and Tim-Andre Schickfluß andShih-Ya Wang for technical assistance.

Grant SupportThe study was supported by grants from the Deutsche Forschungsgemeinschaft

[Ro527/5-1 (DFG-PAK190); SFB-773-TP B02] awarded to H.P. Rodemann; 03-MTO-80000903 awarded to M. Toulany/H.P. Rodemann; and SFB 773-TP Z2awarded to M. Schaller.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received December 12, 2011; revised April 19, 2012; accepted May 4, 2012;published OnlineFirst May 17, 2012.

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2012;10:945-957. Published OnlineFirst May 17, 2012.Mol Cancer Res Mahmoud Toulany, Kyung-Jong Lee, Kazi R. Fattah, et al. Dependent DNA Double-Strand Break Repair

−through Initiation, Progression, and Termination of DNA-PKcs Akt Promotes Post-Irradiation Survival of Human Tumor Cells

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