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Mutation Research 769 (2014) 1–10

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

j ourna l h om epage: www.elsev ier .com/ l ocate /molmutComm uni t y ad dress : www.elsev ier .com/ locate /mutres

uppression of DNA-dependent protein kinase sensitize cells toadiation without affecting DSB repair

nn-Sofie Gustafsson ∗, Andris Abramenkovs, Bo Stenerlöwection of Biomedical Radiation Sciences, Department of Radiology, Oncology and Radiation Science, Rudbeck Laboratory, Uppsala University,ag Hammarskjölds Väg 20, SE-751 85 Uppsala, Sweden

r t i c l e i n f o

rticle history:eceived 9 April 2014eceived in revised form 4 June 2014ccepted 16 June 2014vailable online 22 June 2014

eywords:NA repairNA-PKcs

onizing radiationNA-PK deficiencyU7441

a b s t r a c t

Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in theDNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit ofDNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining(NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNAagainst DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellularresponse to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, leadto extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels ofDNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis.Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or �-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lackingDNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severelycompromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functionscan sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-

localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was onlyseen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survivalafter radiation exposure which is uncoupled from its essential function in DSB repair. This could haveimplications for the development of therapeutic strategies aiming to radiosensitize tumors by affectingthe DNA-PKcs function.

Publis

© 2014 The Authors.

. Introduction

Ionizing radiation and endogenous processes, e.g. free radicalsormed in normal cellular metabolism or collapse of replicationorks during replication of DNA, may result in complex dam-ge to the DNA. Among the different types of DNA damage,NA double-strand break (DSB) is the most unfavorable typef DNA lesion, and unrepaired or misrepaired DSB can lead toene mutations, chromosomal aberrations, permanent cell cyclerrest, apoptosis, mitotic cell death and cancer development1–3].

In DSB repair two pathways have been implicated to processhe break, non-homologous end joining (NHEJ) and homologous

ecombination (HR) [4]. Some of the key proteins involved inHEJ are DNA-dependent protein kinase catalytic subunit (DNA-Kcs), Ku70 and Ku80 that together form the DNA-dependent

∗ Corresponding author. Tel.: +46 184713887; fax: +46 184713432.E-mail address: ann-sofie.gustafsson@bms.uu.se (A.-S. Gustafsson).

ttp://dx.doi.org/10.1016/j.mrfmmm.2014.06.004027-5107/© 2014 The Authors. Published by Elsevier B.V. This is an open access article un

hed by Elsevier B.V. This is an open access article under the CC BY-NC-SAlicense (http://creativecommons.org/licenses/by-nc-sa/3.0/).

protein kinase (DNA-PK) complex [5]. DNA-PKcs is a member of thephosphatidylinositol 3 (PI-3)-like kinase family that includes ATM(ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related)[6]. Besides functioning in NHEJ, DNA-PKcs is also required forV(D)J recombination of immune-globulin genes and T-cell recep-tor genes, and telomere length maintenance [7]. NHEJ is believedto play a more important role then HR in mammalian cells, and ispresent at all time during the cell cycle, HR is limited to late S, G2and M phase [8–10].

Proteins involved in the repair and signaling of DSB play a vitalrole in cell survival and healthy human life [11–13]. Many tumorstypes characterized by genomic instability are caused by mutationsin DSB-responsive genes. The expression of DNA-PKcs in tumorbiopsies can be both up- and down-regulated in specific tumortypes [14]. Several cancer types with decreased levels of both DNA-PKcs and Ku70/80 have been found [15–17] which could indicate

that partial deficiency of proteins involved in DSB repair may con-tribute to increased risk of developing cancer [14,18]. In contrast,elevated levels of DNA-PKcs have been found in various tumor typesand linked to poor survival [19].

der the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Besides its direct role in repair, it is becoming evident that DNA-Kcs may have other functions that promote cell survival. Arlandert al. (2008) suggested that DNA-PKcs contributes to the G2 check-oints and cell cycle regulation [20]. Recently it was also shownhat DNA-PKcs is involved in stabilization of the mitotic spindleormation in a way that reduced levels of DNA-PKcs may directhe cell toward mitotic catastrophe [21–23]. Further data indicateshat DNA-PKcs is a central player in G2 progression and low lev-ls of DNA-PKcs promotes a fast progression from S phase to G2,ut interferes with progression through mitosis and leads to cellccumulation in G2 [22]. Human cells deficient in DNA-PK show aevere sensitivity to radiation and reduced capacity to repair DSBs11,24,25] and it is possible that this inability to progress throughhe cell-cycle is affected by the presence of unrepaired DNA [26].

In the present investigation, we studied DNA repair in cells witheduced levels of DNA-PKcs. Human cells transfected with siRNAargeting DNA-PKcs, resulting in 5–20% of the original DNA-PKcsontent, exhibited similar extreme radiosensitivity as cells com-letely lacking DNA-PKcs. Surprisingly, the low DNA-PKcs-levelsad no significant effect on DSB repair capacity, but low levelsf DNA-PKcs affected the cell cycle, resulting in siRNA treatedell accumulation in G2 after irradiation and inability to proceedhrough mitosis. These data support recent findings suggesting thatNA-PKcs is an important regulator of the cell-cycle while we here

how that this function seems to be uncoupled from the cellularapacity to repair DSBs.

. Materials and methods

.1. Cell lines and culture conditions

Human epithelial cancer cell line A431 (ATCC, Manassas, VA,SA) was grown in Ham’s F-10. Human oral squamous carci-oma cell line H314 (ATCC) was grown in DMEM/Ham’s F-12.uman colorectal carcinoma cell line HCT116 (ATCC) was grown incCoy’s. Normal human skin fibroblast GM5758 cell line (Humanenetic Mutant Cell Repository, Camden, NJ, USA) was grown inEM, supplemented with 2× concentration of MEM vitamins,

ssential and non-essential amino acids. The human glioma cellines M059J deficient in DNA-PKcs (ATCC) and M059K (ATCC)

ith wild-type DNA-PKcs were cultured in DMEM/Ham’s F12 sup-lemented with non-essential amino acids. Culture medium forll the cells was supplemented with 10% FCS (Sigma, St. Louis,O), 2 mM l-glutamine, 100 IU/ml penicillin and 10 �g/ml strep-

omycin (Biochrom Kg, Berlin Germany). All cells were cultured in humidified incubator with 5% CO2 at 37 ◦C and trypsinized withrypsin–EDTA (0.25% trypsin, 0.02% EDTA, Biochrom Kg).

.2. Irradiation and inhibitor treatment

Cells were irradiated with 137Cs �-ray photons (Gammacell 40xactor, MDS Nordion, Kanata, Canada) at a dose rate of ∼1 Gy/min.U7441 was obtained from Selleckchem (Houston, TX). NU7441as dissolved in dimethyl sulfoxide. For treatment, cells were pre-

reated in 37 ◦C for 1 h with 2–10 �M NU7441 before irradiation.lonogenic survival assay NU7441 was removed 24 h after irradi-tion with fresh medium. Dimethyl sulfoxide was used in mockreated cells.

.3. Antibodies

Primary mouse-monoclonal antibodies used were, DNA-PKcs

ab1832, Abcam), p-DNA-PKcs (Thr2609) (ab18356, Abcam), �-ctin (Sigma) and �-H2AX (Millipore). Primary rabbit-polyclonalntibody used were 53BP1 (Bethyl Lab, Montgomery, TX), p-DNA-Kcs (Ser2056) (ab18192, Abcam), p-DNA-PKcs (Thr2609) (Santa

Research 769 (2014) 1–10

Cruz), p-AKT (Ser437) (Cell Signaling) and p-Histone H3 (Millipore).Nucleus was stained with DAPI (Invitrogen). Secondary antibod-ies Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 donkeyanti-rabbit (Invitrogen).

2.4. siRNA transfection

For the siRNA transfection of DNA-PKcs a SMART pool providedby Dharmacon (Thermo Scientific) was used with four siRNA spe-cific for PRKDC (catalogue no. L-005030-00-0020) and a SMART poolas non-specific control (mock) (catalogue no. D-001810-10-20).Transfections were performed with DharmaFECT 1 TransfectionReagent (catalogue no T-2001-01) according to the instructionsof the supplier. The concentration of siRNA was 100 nM and wasadded to the cells together with DharmaFECT I. Cells were seededone day prior transfection, and cells were analyzed three days aftertransfection.

2.5. SDS-PAGE and Western blot analysis

Cells were lysed in ice cold lysis buffer containing 1% Tween-20,20 mM Tris (pH 8.0) 137 mM NaCl, 10% glycerol, 2 nM EDTA, 1 mMactivated sodium orthovanadate (Sigma) and protease inhibitorcocktail (P8340, Sigma). Cell extracts were loaded and elec-trophoresed on NuPAGE Novex 3–8% Tris–acetate gels (Invitrogen).Wet transfer to nitrocellulose membranes was done overnight andthen the membranes were blocked for 1 h in 5% BSA, PBS and thenincubated with the primary antibody overnight at 4 ◦C. Secondaryantibodies (626520 and 656120) (Invitrogen) labeled with HorseRadish Peroxidase were incubated for 1 h at room temperature.Immunoreactive bands were visualized in a CCD camera (SuperCCDHR, Fujifilm, Japan) after treatment with electrochemiluminescentsolution (Immobilon). The intensity of the bands was analyzedusing Image J software (NIH, US).

2.6. Cell survival

Cells were counted and an appropriate number was seeded intoT-25 cell flask in triplicate for each dose. Cells were irradiated with1–4 Gy, 4 h after seeding. After 1–2 weeks cells were fixed in 95%EtOH and stained with Mayer’s hematoxylin and colonies with >50cells were scored.

2.7. Mitotic analysis

Three days after transfection with siRNA cells were irradiatedwith 5 Gy, and let to repair for 24 h in fresh medium before fixationand immunostaining with p-Histone H3 (p-H3) antibody. At themicroscope p-H3 positive cells were scored, at least >250 cells perdata point.

For nocodazole treatment, cells were treated with 1 �l/mg noco-dazole (Sigma), 3 days after transfection with siRNA, and incubatedfor 6 h. After 6 h, nocodazole was removed and cells were irradiatedwith 2 Gy and let to repair for 18 h in fresh medium before fixationand immunostaining with p-H3 antibody. At the microscope p-H3positive cells were scored, at least >400 cells per data point.

2.8. Immunofluorescence staining and quantification of foci

Cells were grown on microscope slides for three days after trans-fection and were irradiated with 1–5 Gy. After repair of DNA forvarious times at 37 ◦C, the cells were fixed in methanol for 20 min

at −20 ◦C followed by permeabilization in ice cold acetone for 10 s.Blocking was performed in 10% FCS-PBS for 1 h at room temperatureand then incubation overnight at 4 ◦C with primary antibodies. Sec-ondary antibodies were incubated for 1 h at 37 ◦C (dilution 1:400).

A.-S. Gustafsson et al. / Mutation Research 769 (2014) 1–10 3

Fig. 1. Reduction of DNA-PKcs leads to extreme radiosensitivity. (A) WB: expression of DNA-PKcs after siDNA-PKcs transfection for 3 days in A431, HCT116, H314, GM5758cells and in DNA-PKcs deficient M059J cells. Quantification of band intensity with Image J, fraction of residual DNA-PKcs is shown for each cell line, ±SD from three experiments.(B) Representative images of A431 cell mock-transfected and siDNA-PKcs transfected for 3 days, fixed and immunostained with antibodies against DNA-PKcs. Nuclei werevisualized with DAPI staining. (C) Clongenic survival in three cell lines, A431, HCT116 and H314, transfected with siRNA against DNA-PKcs (siDNA-PKcs) for 3 days or treatedwith non-specific siRNA (mock). In comparison, survival for M059J cells (lacking DNA-PKcs) and M059K (wt DNA-PKcs) are shown. Colonies with >50 cells were scored 10–15d three

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ays after irradiation. SF = surviving fraction. Data represent mean ± SD of at least

ean surviving fraction at 2 Gy.

lides were captured with Zeiss LSM 510 Meta confocal micro-cope. Images were processed and foci were counted using Image

software (NIH, US).

.9. DSB Rejoining and pulsed-field gel electrophoresis

Cells were labeled with 2 kBq/ml [methyl-14C] thymidinePerkin Elmer), together with siRNA transfection, three days prioro irradiation. Cells were irradiated with 40 Gy and embedded in0 �l agarose plugs (0.6% w/v, InCert, Cambrex). Cell plugs were

ysed using a two-step cold lysis protocol in ESP lysis buffer (2% N-auroylsarcosine (Sigma), 1 mg/ml Proteinase K (Roche), all dilutedn 0.5 M EDTA (Na3) at pH 8.0 [27]. After >20 h the cell plugs were

oved to HS-buffer and incubated overnight at 4 ◦C (HS: High Salt;.85 M NaCl, 0.15 M KCl, 5 mM MgCl2, 2 mM EDTA, 4 mM Tris, 0.5%riton X-100, pH 7.5, Triton X-100 is added just before use). Plugsere washed twice in 0.1 M EDTA and once in 0.5× TBE at 4 ◦Crior to electrophoresis. The plugs were then loaded into wells

n a chilled (4 ◦C) agarose gel (0.8% SeaKem Gold, Lonza). The gelas placed into a PFGE unit (Gene Navigator, Amersham Pharmaciaiotech, Uppsala, Sweden) with 120◦ between the fields. Followinglectrophoresis in 0.5× TBE at 10 ◦C, the gels were sliced at the posi-ion of the 5.7 Mbp chromosome from Schizosaccharomyces pombe

BioRad), and 14C in the gel segments were measured by liquid scin-illation. The fraction of radioactivity corresponding to DNA of sizeess than 5.7 Mbp was divided by the total radioactivity in the lane,iving the Fraction Activity Released (FAR) which is a measure of

independent experiments for each cell line. (D) Radiation sensitivity, summary of

DNA double-strand breaks. Data was normalized, set to 100% att = 0.

3. Results

3.1. Reduction of DNA-PKcs leads to extreme radiosensitivity

The reduction of DNA-PKcs was confirmed on Western blot per-formed in parallel with each experiment (Fig. 1A). Protein levelswere markedly decreased two days following initial transfectionand were stable for at least four days (Supplementary Fig. S1A).The transfection efficiency varied slightly between experimentsand cell lines, but there was a large 80–95% reduction in DNA-PKcslevels in all of the tested human cell lines, A431 epithelial cancercells, H314 oral squamous carcinoma cells, HCT116 colorectal car-cinoma cells and GM5758 normal fibroblasts. The depletion wasalso evident on images with immunofluorescent staining againstDNA-PKcs (Fig. 1B). ATM levels were also analyzed for potentialloss as a consequence of DNA-PKcs knock-down, however ATM lev-els were not altered (Supplementary Fig. S1B). The M059J gliomacell line which completely lacks DNA-PKcs is sensitive to ionizingradiation and is shown for comparison. To test whether an 80–95%depletion of DNA-PKcs influence the survival and proliferation of

cells, a clonogenic survival assay was performed on three differ-ent cell lines, A431, H314 and HCT116 (Fig. 1C). siRNA mediateddepletion of DNA-PKcs markedly reduced the surviving fraction inall cell lines and at a dose of 2 Gy there was a 3–10 fold reduction

4 A.-S. Gustafsson et al. / Mutation Research 769 (2014) 1–10

Fig. 2. Cells with low levels of DNA-PKcs are delayed in the mitotic progression. (A) Accumulation of mitotic cells in asynchronous A431 cells with low levels of DNA-PKcs.Cells were irradiated with 5 Gy and scored for p-H3-positive cells 24 h after irradiation. Data represent mean ± SD of two independent experiments. At least 250 cells/timepoint were scored per experiment. (B) Suppression of DNA-PKcs leads to accumulation of mitotic cells following irradiation and treatment with nocodazole. Cells weresynchronized for 6 h with nocodazole and irradiated, and after 18 h incubation in medium without nocodazole, cells were fixed and p-H3-positive cells were scored. Ctrcells were unirradiated. Data represent mean ± SD of two independent experiments. At least 400 cells/time point were scored per experiment. (C) Phosphorylated DNA-PKcs(Thr2609) is dislocated from chromatin in mitotic cells and located at the midbody (white arrows). Thr2609 (green) is merged with p-H3-positive (red). In comparison, totalD ined w

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NA-PKcs (green) in interphase and mitotic cells are shown (right). Nuclei were sta

n the survival (Fig. 1C and D). In comparison, M059J cells, whichompletely lacks DNA-PKcs, are shown. Thus, partial reduction inNA-PKcs may lead to similar extreme radiosensitivity as seen inells completely lacking DNA-PKcs.

.2. Cells with low levels of DNA-PKcs are delayed in the mitoticrogression

Recent data suggest that DNA-PKcs, beyond its role in DNAepair, may have other cellular functions and could be an impor-

ant regulator of the cell-cycle [21–23]. To test the hypothesis thatow DNA-PKcs levels could lead to aberrant mitosis, asynchronous431 cells were scored for phosphorylated Histone H3 (p-H3) 24 hfter irradiation. p-H3 is related to several phases of mitosis and

ith DAPI (blue).

used as a marker herein for positive G2/M cells. Cells treated withsiRNA against DNA-PKcs exhibited two-fold increase of p-H3 posi-tive cells when compared to mock treated cells (Fig. 2A). Further, insiRNA treated cells a substantial number of the mitotic cells werein late mitosis (metaphase, anaphase or telophase), which was instrong contrast to mock treated cells (data not shown). To enrichthe fraction of cells in mitosis, cells were treated with nocodazole,an agent used to arrest and synchronize cells in G2/M-phase. Cellswere incubated for 6 h with nocodazole, irradiated and releasedfor 18 h by removing the drug before fixation and scoring of p-H3-

positive cells. After irradiation with 2 Gy there was a 2-fold increasein the fraction of p-H3-positive cells when treated with siRNAagainst DNA-PKcs compared to mock treated cells (Fig. 2B). Thus,these results suggest that low amounts of DNA-PKcs could lead to

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ccumulation of cells in G2/M phase after irradiation. The resultsndicate that irradiated cells with low levels of DNA-PKcs enter

itosis as cells with normal protein levels, but are then delayedn the mitotic progression. This further strengthens the hypothesishat DNA-PKcs has a role in regulation of the mitotic progres-ion. Indeed, in the mitotic A431 cells DNA-PKcs was dislocatedrom chromatin and mitosis-specific DNA-PKcs phosphorylationThr2609) appeared to concentrate at two sites on opposite sidef the nucleus implying a different role than in DSB repair (Fig. 2C),lthough there was no evident difference in the protein dis-ribution between siDNA-PKcs or mock treated cells (data nothown).

.3. Cells with low residual levels of DNA-PKcs have apparentlyormal DSB repair

Since cells with defective DNA-PK have a reduced repair capac-ty, it is reasonable to assume that the extreme radiosensitivity andhe mitotic failure, seen in DNA-PKcs depleted cells above, can bexplained by inability to repair DSB. To test this hypothesis, theormation and disappearance of 53BP1 (p53 binding protein1) focind �-H2AX was analyzed in single cells after irradiation. GM5758nd A431 cells were transfected with siRNA against DNA-PKcs orock treated and after 3 days cells were irradiated and incubated

or repair. 53BP1 foci were rapidly formed in both cell lines afterrradiation and the initial number of 53BP1 foci was similar inells transfected with siRNA against DNA-PKcs and mock treatedFig. 3A). However, somewhat surprising, the reduced amount ofNA-PKcs did not significantly affect the kinetic properties of either3BP1 or �-H2AX in either cell line (Fig. 3B and D) and 1–24 h after

rradiation there were similar numbers of foci in cells transfectedith siRNA against DNA-PKcs and mock treated cells. Similarly,

here was no difference between siDNA-PKcs and mock treatedells when the residual number of 53BP1 foci was scored 24 h afterrradiation with different doses (Fig. 3C). These results show thatiDNA-PKcs does not affect the formation and disappearance ofoth the DSB marker 53BP1 and �-H2AX after irradiation.

To further investigate if low DNA-PKcs levels affect the capacityo repair radiation-induced DSBs, DSB rejoining was analyzed usingulsed-field gel electrophoresis (PFGE). Despite the large reduction

n the levels of DNA-PKcs, there was no effect on DSB rejoining inny of the four tested cell lines (Fig. 3E), not even for radiation dosesp to 200 Gy, which induces at least 5000–6000 DSB per cell (Sup-lementary Fig. S2). In contrast, M059J cells, completely lackingNA-PKcs, showed only marginal rejoining up to 24 h post irra-iation (Fig. 3E). In summary, these data suggest that despite lowesidual levels of DNA-PKcs, i.e. 5–20% of the original protein levels,ells have apparently normal DSB repair.

.4. Inhibition of the DNA-PKcs kinase activity sensitize cells toadiation and reduce DSB repair

Because siDNA-PKcs treatment markedly reduced cell survivalfter irradiation, despite an efficient DNA repair, we wanted to com-are siDNA-PKcs treated cells with cells treated with the specificNA-PKcs inhibitor NU7441. Cell survival was markedly reduced

n A431 cells treated with NU7441 (Fig. 4A), and the dose-responseas similar to that in M059J cells, lacking DNA-PKcs (Fig. 1C). Weext investigated whether treatment with NU7441 affected the for-ation and disappearance 53BP1 in A431 and GM5758 cells. The

nitial number of 53BP1 foci after 1 h was similar in untreated cellsnd cells treated with NU7441, however, after 4–24 h of repair,

he residual numbers of 53BP1 foci were higher in the NU7441reated cells (Fig. 4B). Further, in GM5758 cells DSB rejoining waslmost completely inhibited by NU7441 (Fig. 4C). Evidently DSBepair is differentially affected whether the activity of DNA-PKcs

Research 769 (2014) 1–10 5

is inhibited, or if there are low amounts of DNA-PKcs present.Our data show that inhibition of DNA-PKcs activity reduce bothDSB repair and cell survival after exposure to ionizing radiation(Fig. 4). In contrast, lowering of DNA-PKcs levels via siRNA treat-ment markedly reduced cell survival without any clear effects onrepair (Figs. 1 and 3). Thus, the present data implicate that there aredifferent mechanisms by which loss of DNA-PKcs function sensitizecells to ionizing radiation and suggest that DNA-PKcs has a criticalrole in maintenance of survival after radiation exposure, besides itsimportant function in DSB repair.

A number of studies suggest a close interaction between AKT andDNA-PKcs [28,29]. Treatment with NU7441 or siDNA-PKcs abol-ished the activity of AKT (Ser473) after irradiation (SupplementaryFig. S3A), supporting the role of DNA-PKcs in activation of AKT andthat low amounts of DNA-PKcs, via down regulation of AKT, maypromote apoptosis. To test this, cells were irradiated and scored forapoptosis by measuring cleaved PARP. Compared to unirradiatedcells there was a clear increase in PARP cleavage 72 h after irradi-ation with 8 Gy, but there was no difference between siDNA-PKcsand mock treated cells (Supplementary Fig. S3B).

3.5. Phosphorylation of DNA-PKcs at Thr2609 and Ser2056

ATM and DNA-PKcs are the major kinases activated followingradiation. DNA-PKcs is rapidly activated by both phosphorylationand autophosphorylation upon irradiation and several phosphor-ylation sites have been identified so far. ATM is essential forphosphorylation of Thr2609 [30] and DNA-PKcs is responsiblefor Ser2056 phosphorylation [31]. Western blot analysis was per-formed on A431 cells to test if radiation-induced phosphorylationof Thr2609 and Ser2056 are affected by a depletion of DNA-PKcs.The overall phosphorylation of both Thr2609 and Ser2056 wasalmost completely abolished in cells targeted with siRNA againstDNA-PKcs (Fig. 5A), most likely due to decreased levels of DNA-PKcs. However, cells inhibited with the DNA-PKcs inhibitor NU7441showed no difference in DNA-PKcs phosphorylation.

Despite the reduced amount of phosphorylated DNA-PKcsin Western blot, formation and disappearance of Thr2609 andSer2056 foci after irradiation were similar in mock- and siRNA-treated cells, and these foci co-localized with the DSB markers53BP1 and �-H2AX, indicating that the phosphorylation of DNA-PKcs at DSB sites were not affected by the amount of DNA-PKcs(Fig. 5B and C). Further, no apparent difference could be seen in fociintensity between siDNA-PKcs and mock treated cells. These resultssuggest that the proteins left in the cells are gathering around DSBand are fully activated at these sites. Cells treated with the DNA-PKcs inhibitor NU7441 exhibited an apparently normal formationof Thr2609 and Ser2056 foci at early time-points after irradiation.However, 4–24 h post-irradiation, the numbers of Thr2609 andSer2056 foci showed only a small decrease (Fig. 5C), which coin-cided with the 53BP1 analysis and is in line with the hypothesis thatATM activation promotes DNA-PKcs binding to DNA but inhibitionby NU7441 prevents DNA-PKcs to participate in DSB repair.

Interestingly, foci of DNA-PKcs were clearly visible 60 min afterirradiation of cells treated with target siRNA (Fig. 6). The DNA-PKcsfoci overlapped with the DSB marker 53BP1 and these foci werenot seen in cells with normal levels of DNA-PKcs (mock-treated).This could be explained by the high levels of DNA-PKcs in normallypresent in human cells but when the protein level is decreased by80–95% the allocation around DSB of active proteins can be seen.Notably, the DNA-PKcs foci became visible 60 min after irradiation

and after 4–24 h they became more distinct, whereas cells analyzedat shorter repair times just displayed a weaker signal of DNA-PKcsover the whole nucleus. Thus, low levels of DNA-PKcs may cause adelay in the redistribution to the DSB sites.

6 A.-S. Gustafsson et al. / Mutation Research 769 (2014) 1–10

Fig. 3. Cells with low residual levels of DNA-PKcs have apparently normal DSB repair. (A) representative images of A431 and GM5758 cells mock treated or siDNA-PKcstransfected, irradiated with 2 Gy or 1 Gy, respectively, and allowed to recover for the indicated times. 0 Gy was used as control. Cells were fixed and immunostained with53BP1 (red) and nuclei were stained with DAPI (blue). (B) Kinetics of 53BP1 foci in cells treated as in (A). At least 100 cells/time points were scored for foci. Data representmean ± SD of three independent experiments. (C) Dose response by average number of 53BP1 foci per cell. Cells were either mock transfected or siDNA-PKcs transfected andthen irradiated at indicated doses and allowed to recover for 24 h before fixation and immunostaining. Counting and statistics as in (B). (D) Time response of �-H2AX foci inc , HCTC K andt .

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ells treated as in (A). Counting and statistics as in (B). (E) Rejoining of DSBs in A431ells were allowed to recover for the indicated times prior to PFGE analysis. M059

= 0 h and each data point represents mean ± SD of three independent experiments

. Discussion

It is well known that decreased capacity to repair DNA damage

y radiation or other DNA damaging agents is a cancer risk factornd DNA-PK has been implied to be down regulated in many differ-nt tumor types. The expression seems to be tissue dependent andt has been demonstrated that tissue from uterine, breast and lung

116 and H314 cells mock-transfected or siDNA-PKcs transfected before irradiation. M059J data are shown as comparison. Data were normalized to the DSB level at

cancer have a tendency of low DNA-PK levels whereas head andneck cancer show normal levels [15,16,32]. Human cells maintaina high reservoir of proteins involved in NHEJ of DSB repair and high

level of proteins allows the cell to protect the genome by the imme-diate presence of repair proteins at the breaks, minimizing the timefor assembly and activation at the break site. There are nearly half amillion Ku molecules and around one hundred thousand DNA-PKcs

A.-S. Gustafsson et al. / Mutation Research 769 (2014) 1–10 7

Fig. 4. Inhibition of the DNA-PKcs kinase activity sensitize cells to radiation and reduce DSB repair. (A) Clonogenic survival in A431 cells treated with the DNA-PKcs inhibitorNU7441 (2 �M). Cells were preincubated with NU7441 1 h before irradiation at indicated doses. NU7441 was removed 24 h after irradiation and colonies with >50 cells werescored after 10 days. (B) 53BP1 foci formation and removal in A431 and GM5758 cells. Cells were incubated with 10 �M NU7441 1 h before irradiation at indicated doses andallowed to recover at indicated times before fixation and immunostaining. For time response cells were irradiated with 2 Gy, and for dose response foci were counted after2 D of t5 and aD epen

mistcastomesctDaantcPatilDtdamIictwuaAt

4 h. At least 100 cells/time points were scored for foci and data represent mean ± S �M NU7441. Cells were incubated with NU7441 1 h prior to irradiation with 40 Gyata was normalized (t = 0 h) and each data point represents mean ± SD of three ind

olecules per cell [33]. The data presented here provide furtherndications of the importance of DNA-PKcs for promoting cellularurvival. Irrespective of the strong radiosensitizing effect, we showhat with levels of DNA-PKcs below 20% compared to normal cells,ells retain their ability to rejoin DNA fragments and remove 53BP1nd �-H2AX foci with normal speed. To our knowledge, the presenttudy is the first to show that knock-down of critical DSB repair pro-eins leads to extreme radiosensitivity without any apparent effectn the repair. Thus, the high levels of DNA-PKcs in normal cellsay indicate important roles in other cellular functions and that an

xcess of molecules need to be present in the stress response uponevere DNA damage. Our observations show that after irradiation,ells with limited levels of DNA-PKcs have a two-fold increase inhe fraction of cells arresting in G2/M, indicating that low levels ofNA-PKcs may stall mitosis. This was both shown in synchronizednd asynchronous cells (Fig. 2). These observations are in line with

study by Shang et al., showing that DNA-PKcs contributes to theormal spindle formation and centrosome stability as well as inac-ivation of DNA-PKcs could cause multipolar spindle and mitoticatastrophe after DNA damage [21]. We here also show that DNA-Kcs is dislocated from the chromatin in the mitotic nucleus and

DSB-independent Thr2609 phosphorylation was concentrated atwo distinct sites on each side of the chromatin (Fig. 2C), indicat-ng a role of DNA-PK at centrosomes in the mitotic cell. This is inine with recent data showing co-localization of phosphorylatedNA-PKcs (Thr2609) and Plk1 in mitotic cells [21–23,34]. Deple-

ion of this mitotic phosphorylation of DNA-PKcs could result inelay and dysfunction in the mitotic transition. This could lead to

loss or gain of a whole chromosome, which is the most com-on chromosomal instability associated with human cancers [23].

t would be rational to assume that the presence of unrepaired DSBn DNA-PKcs deficient/suppressed cells should cause delay in cell-ycle progression and mitotic failure. Importantly, our data suggesthat DNA DSB repair capacity is not the limiting factor in cellsith low DNA-PKcs levels. This information could be crucial for the

nderstanding of the complexity of DNA damage stress response,nd specifically, the multifunctional roles of DNA-PK. ATM andTR have been firmly established as central players in the activa-

ion of the check point pathways that relay on the DNA damage

hree independent experiments. (C) Rejoining of DSBs in GM5758 cells treated withllowed to recover for the indicated times before preparation and analysis by PFGE.

dent experiments.

signaling to downstream effectors through phosphorylation[35,36] and perhaps DNA-PK plays a part in this role as well.Inhibition of PI-3 kinases increase mitotic arrest and mitotic celldeath [37] and ATM, ATR and DNA-PK have all been confirmedto localize to centrosomes during mitosis [34]. This suggests thatthese PI3-like kinases complement each other on centrosomes andthe following downstream phosphorylations are necessary for cellcycle progression and synthesis of microtubules [34]. We cannotexclude the possibility of alternative pathways (e.g. homologousrecombination) may rejoin the DSBs, however this does not explainthe arrest of cells in G2/M phase.

Cells with total deficiency of DNA-PKcs, like the M059J cell line,display a hypersensitivity to ionizing radiation that reflects a crucialrole of this enzyme in maintaining repair capacity [24,25,38,39] andchromosome stability [40]. Phosphorylation of DNA-PKcs is criticalfor the binding and release from the DNA end and inhibition ofDNA-PKcs will result in blocked DNA ends, which inhibit furtherprocessing and efficient ligation. The majority of phosphorylationsites are located at the ABCDE-and PQR cluster and mutations ateither of these sites impairs DNA repair [30,35,39,41–44]. There-fore, targeting and inhibiting DNA-PK activity is an attractive cancertherapy strategy and the most successful approach has been withthe use of small molecules targeting the ATP binding site of thekinase domain [45]. A promising drug is the NU7441, based onthe LY294002 backbone, which shows a strong inhibition of DNA-PKcs and DNA repair in cells [46]. Inhibition by NU7441 does notresult in the same radiosensitivity as cells totally lacking DNA-PKcs[47], however we observed an almost complete inhibition of DNArepair in cells treated with NU7441, which was in strong contrast tocells with decreased levels of DNA-PKcs. Even though the inhibitorshold promising results for improving cancer therapy, the moleculesknown so far are limited by their poor pharmacokinetics [45,48].Further, our data implicates that suppression of DNA-PKcs could bea preferable therapeutic strategy; whereas inhibition of the kinaseactivity by NU7441 blocks the DSB repair in both dividing and non-

dividing cells (Fig. 4), while suppression of DNA-PKcs by siRNAmainly affects the dividing cells.

The high sensitivity to ionizing radiation in cells with low DNA-PKcs levels could be due to dysregulation of downstream molecules.

8 A.-S. Gustafsson et al. / Mutation Research 769 (2014) 1–10

Fig. 5. Phosphorylation of DNA-PKcs at Thr2609 and Ser2056. (A) WB: relative levels of DNA-PKcs, p-Thr2609 (DNA-PKcs), p-Ser2056 (DNA-PKcs) in A431 cells, mock-transfected, siDNA-PKcs transfected or treated with 10 �M NU7441 1 h before irradiation with 2 Gy. Cells were allowed to repair for 1 h before preparation for WB. Equalloading was confirmed by �-actin immunoblot. (B) Representative images of A431 cells, mock-transfected, siDNA-PKcs transfected or treated with 10 �M NU7441 1 h priorto irradiation with 2 Gy. Cells were allowed to repair for 1 h or 24 h before fixation and immunostaining with either p-Thr2609 (DNA-PKcs) (green) and co-stained with theDSB marker 53BP1 (red), or p-Ser2056 (DNA-PKcs) (red) and co-stained with the DSB marker �-H2AX (green). Co-localization between the different antibodies is depictedin the merged images as yellow, nuclei were stained with DAPI (blue). (C) Average number of p-Thr2609 (DNA-PKcs) and p-Ser2056 (DNA-PKcs) foci in A431 and GM5758c owedr nt wer

Icictnbr

t5fc

ells mock-transfected, siDNA-PKcs transfected or treated with 10 �M NU7441 follepresent mean ± SD of three independent experiments. At least 100 cells/time poi

ndeed, AKT phosphorylation (Ser473) decreased in siRNA treatedells after irradiation. However, increased apoptosis was observedn both, DNA-PKcs silenced cells and mock treated cells whenleaved PARP pathway was studied. (Supplementary Fig. S3). Fur-her, we could not detect any signs of accelerated senescence (dataot shown), which was recently reported when DNA-PKcs waslocked with the PI3-inhibitor NVP-BEZ235 in combination withadiation [49].

In our study we show that in cells with low levels of DNA-PKcshere was clear visualization of DNA-PKcs foci that overlapped with

3BP1, which was not seen in mock treated cells. However, theormation of DNA-PKcs foci was relatively slow and this could indi-ate a delay in the re-localization of DNA-PKcs to the DSB sites

by irradiation at indicated doses and allowed to recover the indicated times. Datae scored per data point.

in siRNA-treated cells. We could not identify a difference in thenumbers of Thr2609 and Ser2056 foci between cells with low DNA-PKcs levels and cells with normal DNA-PKcs content, however inNU7441 treated cells the majority of foci retained even after 24 hrepair. This implies that the proteins left are active and repairingDSB as in normal cells, but inhibition of DNA-PKcs kinase activityleaves the cell unable to rejoin DNA ends. Our observations alsoshow a re-localization of p-DNA-PKcs during mitosis, indicating aphosphorylation event that may be critical for mitosis.

In summary, our data show that DNA-PKcs is necessary for reg-

ulation of mitotic progression after irradiation, and this processseems to be independent of its role in DSB repair. This increase ofradiation sensitivity, compared to non-dividing cells, could be an

A.-S. Gustafsson et al. / Mutation

Fig. 6. Low levels of DNA-PKcs display foci formation. Representative images ofA431 cells mock-transfected or siDNA-PKcs transfected. Cells were irradiated with2 Gy, fixed and immunostained after 15 min or 4 h with DNA-PKcs (green) and theDSB marker 53BP1 (red). The two images were merged and co-localization of DNA-P

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Kcs and 53BP1 are depicted as yellow, nuclei were stained with DAPI (blue).

dvantage in radiotherapy of tumors. Identification of additionalunctions of DNA-PKcs, beyond its strong role in DSB repair, couldive important knowledge about regulation of genomic stabilitynd this complexity would be important to keep in mind for theevelopment of new therapeutic approaches targeting the DNAamage signaling and repair pathways.

onflict of interest statement

The authors declare that there are no conflicts of interest.

cknowledgements

Microscopic imaging was performed with equipment main-ained by the Science for Life Lab BioVis Platform, Uppsala. Weincerely thank K. Karlsson for help with data for the M059 cells.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.mrfmmm.014.06.004.

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