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Cance8748

Published OnlineFirst October 12, 2010; DOI: 10.1158/0008-5472.CAN-10-0944

Canceresearch

apeutics, Targets, and Chemical Biology

or Cell Kill by c-MYC Depletion: Role of MYC-Regulated

R

es that Control DNA Double-Strand Break Repair

R. Luoto1, Alice X. Meng1, Amanda R. Wasylishen1,2, Helen Zhao1, Carla L. Coackley1,
Z. Penn1,2, and Robert G. Bristow1,2,3
ractMY

MYC apotentIn thisin respwith sDNA-Pincreation ousingrecomresulteMYC-d

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C regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis.lso controls the expression of DNA double-strand break (DSB) repair genes and therefore may be aial target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability.report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survivalonse to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associateseveral DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2,Kcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated withsed RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induc-f MYC in G0-G1 and S-G2-M cells resulted in upregulation of Rad51 gene expression. MYC knockdownsmall interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologousbination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYCd in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However,ependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycinonizing radiation, two agents selectively toxic to DSB repair–deficient cells. Our results suggest that
C or i
anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensi-tization or chemosensitization. Cancer Res; 70(21); 8748–59. ©2010 AACR.

ical musingantagoof lunarreststudyapeuttumorsuch(siRN(PMOarrestPre

with t

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C is a basic helix-loop-helix leucine zipper transcrip-actor that dimerizes with its binding partner MAXssociates with gene promoters containing the E-boxCACGTG or CACATG to induce gene transcription

YC controls a broad spectrum of functions includingration and cell cycle, differentiation, sensitization totic stimuli, and genetic instability (1). These functionsregulated in most human cancers by a variety ofnisms including gene amplification, insertional muta-or chromosomal translocation of the myc gene. Thisl role in oncogenesis makes MYC a promising targetnd-alone molecular cancer therapies in cells undergo-

ddiction (2). Recent data from Soucek andave shown that MYC inhibition in a preclin-

genesPKcs(12–14gous r(NHEJsisterDNA bparaloas weparticmycinhomoacrossof KU

ns: 1Campbell Family Cancer Research Institute,edical Biophysics, and 3Department of Radiationity of Toronto, Radiation Medicine Program, Ontarioincess Margaret Hospital, Toronto, Ontario, Canada

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

thor: Robert G. Bristow, Radiation Medicine Program,t Hospital, 5th Floor, Room 5-808, 610 UniversityOntario, Canada M5G 2M9. Phone: 416-946-2936;; E-mail: [email protected].

5472.CAN-10-0944

ssociation for Cancer Research.

21) November 1, 2010

Research. on May 23, 2021cancerres.aacrjournals.org aded from

ouse model of RAS-induced lung adenocarcinomaa reversible, systemic expression of a MYC mutant thatnizes MYC activity triggered apoptosis and regressiong tumors. MYC inhibition exerted profound growthin normal tissues, which were well tolerated (3). Thissuggested that targeting MYC could maintain the ther-ic ratio of cancer treatment by preferential killing ofcells relative to normal cells. Indeed, anti-MYC agentsas antisense oligonucleotides, small interfering RNAA), or phosphorodiamidate morpholino oligomers) have been developed to induce tumor cell growth, differentiation, or apoptosis (4–7).vious studies have indicated the association of MYChe promoters of DNA double-strand break (DSB) repairNbs1, Ku70, Rad51, BRCA2, Rad50, Rad54L, and DNA-(8–11) and the expression of mismatch repair genes). DSBs are repaired by two major pathways: homolo-ecombination (HR) and nonhomologous end joining). HR predominates during S and G2 phases, as it useschromatid in a template-guided manner to repair thereak (15). HR relies on the function of RAD51 and itsgs RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3,ll as BRCA1 and BRCA2 (15). HR-deficient cells areularly sensitive to the DNA cross-linking agent mito-C (MMC). In contrast, NHEJ does not require strandlogy and is considered error prone, and it predominates
all phases of the cell cycle. NHEJ requires the action70, KU80, DNA-PKcs, DNA ligase IV, and XRCC4.
. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

NHEJ-(IR; resensitmitotirepairsensitidamaghave cdard tand deGive

tentiahibitioand sethat acells).promodownindepeknocknot fu

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Repos6 mon(16). DcarcinCollecas preMCF7H1299PinthuCanadthe OToronrial Sltively,MCF1(2003;ToronpreviofactordownrticatedThe Cdren (with tlack aticatedtive geto beInd

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deficient cells are in turn sensitive to ionizing radiationf. 15). Inhibition of MYC-regulated DNA repair couldize tumor cells by inducing genetic instability andc catastrophe. Additionally, if MYC regulation of DSBgene expression altered chemosensitivity and radio-vity, using combinations of MYC inhibitors with DNA-ing agents could be beneficial. However, few studiesombined molecular targeting of MYC with now stan-reatments such as radiochemotherapy or chemotherapytermined long-term clonogenic cell survival.n that MYC promotes genetic instability and has a po-l role in DNA repair, we determined whether MYC in-n leads to decreased DSB repair protein expressionnsitization of tumor cells to IR or MMC (two agentsre selectively toxic to NHEJ- or HR-deficient cancerWe show that MYC occupies most DSB repair geneters and regulates RAD51 expression. MYC knock-alone results in loss of long-term clonogenic survivalndent of inducing apoptosis. However, partial MYC
down, which allowed for colony formation assays, did Chr
essentmodif(pH 8itor coTris-HinhibifragmnopreX-100167 mthe Mbit IgGproteialbum(QiagePCR p(26), C(chrom

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rials and Methods

ulture, generation of inducible MYC cell lines,iRNA tranfectionsmal human diploid fibroblasts GMO5757 (Coriell Cellitory, 2008) up to 13 passages were used withinths of purchase, and cultured as previously describedU145 and PC-3 prostate cancer cells and RKO colonoma cells were obtained from American Type Culturetion (2001, 2001, and 2007, respectively) and culturedviously described (17, 18). 22RV1 prostate cancer cells,breast cancer cells, Rat-1/myc cells, and DR-GFPlung cancer cells were kind gifts from Drs. Yonis (2004; Juravinski Cancer Center, Hamilton, Ontario,a), Fei-Fei Liu (2009) and Linda Penn (2001; both atntario Cancer Institute/Princess Margaret Hospital,to, Ontario, Canada), and Simon Powell (2004; Memo-oan-Kettering Cancer Center, New York, NY), respec-and were cultured as described previously (17–22).0A cells were a kind gift from Dr. Senthil MuthuswamyOntario Cancer Institute/Princess Margaret Hospital,to, Ontario, Canada) and were cultured as describedusly either in full growth medium (23) or in growthwithdrawal (0.05% horse serum, 10 μg/mL insulin) toegulate endogenous MYC. Each cell line was reauthen-by short tandem repeat (STR) analyses performed by

entre for Applied Genomics, The Hospital for Sick Chil-Toronto, Ontario, Canada; June to September 2010),he exception of RKO, Rat-1/myc, and GMO5757, whichvailable STR profiles. The latter cell lines were authen-by multiple gene expression analyses and compara-nomic hybridization. All cells were tested and foundMycoplasma-free during experiments.
ucible pF vector control and pF-MYC cell lines wereted using the 4-hydroxytamoxifen (4-OHT)–inducible
mentthe co

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iral expression system as described previously (24).presence of 4-OHT, the GEV16 transcription factorlled by the Ub promoter (GEV16-Gal4 DNA-bindingin, estrogen receptor ligand-binding domain, andtransactivation domain) translocates to the nucleus,the 5xUAS, and activates transcription of the gene ofst (24). Briefly, lentiviruses were first generated incells. Cells were then infected with viral supernatantspresence of 0.8 μg/mL polybrene. Forty-eight hoursnfection, stable cell pools were selected in 200 μg/mLycin and 1 μg/mL puromycin.

s were transfected with Validated Stealth Select RNAiMYC duplexes 1 or 2 at a concentration of 10 nmol/Li-MEM, or with RAD51 duplexes 1 and 2 at 0.25 nmol/L,th Stealth RNAi Negative Universal Control usingectamine 2000 (all from Invitrogen) as previouslybed (17).

atin immunoprecipitationomatin immunoprecipitations (ChIP) were performedially as previously described (25) with the followingications. Nuclei were isolated in 5 mmol/L Tris-HCl.0), 85 mmol/L KCl, 0.5% NP40, and 1× protease inhib-cktail (Roche). Pelleted nuclei were lysed in 50 mmol/LCl (pH 8.0), 10 mmol/L EDTA, 1% SDS, and 1× proteasetor cocktail. Chromatin was sheared to 0.2- to 1.5-kbents using a Bioruptor sonicator (Diagenode). Immu-cipitations were performed in 0.01% SDS, 1.1% Triton, 1.2 mmol/L EDTA, 16.7 mmol/L Tris-HCl (pH 8.0),mol/L NaCl, and 1× protease inhibitor cocktail usingYC N-262 antibody (Santa Cruz Biotechnology) or rab-control (Jackson ImmunoResearch Laboratories) withn A/G beads (Pierce) blocked in 1% bovine serumin. DNA was purified using a PCR Purification kitn). Immunoprecipitated DNA was amplified usingrimers (see Supplementary Table S1) Rad51 (9), BRCA1AD (carbamoyl-phosphate synthetase 2), and Chr22osome 22; ref. 9).

rn blottingls were lysed directly in boiling 2× SDS buffer [2%78 mmol/L Tris-HCl (pH 6.8), 4.5% glycerol]. Proteinsion was detected and quantified using primary anti-s MYC (9E10), RAD51 (H-92), KU70 (A-9), and ATMSanta Cruz Biotechnology), cyclin D1 (Ab-3; Onco-RB (retinoblastoma protein; Pharmingen), α-tubulinCalbiochem), and β-actin (Sigma) and IRDye second-tibodies with Odyssey IR Imaging System (LI-CORences).

se transcription and real-time quantitative PCRwas extracted using the RNeasy kit (Qiagen). cDNA

ynthesized using the High Capacity cDNA Archivepplied Biosystems). Real-time quantitative PCR wasmed in triplicates using the Taqman Gene Expressionon a StepOnePlus real-time quantitative PCR instru-
(Applied Biosystems). Analysis was performed usingmparative Ct value method.
Cancer Res; 70(21) November 1, 2010 8749



Cell ccell soFor

5-bromfor 1(BD Bwere aQuest(27). Gstainin4-OHTdetach10 μg/supple37°C.the M

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ter 48MMCThe Dticals)mediufixed a15 daya lighas precells (MMCtransfcytomdescri

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Prethat Mfore dgeneanaly10 kbgene tweregene pin viv22RV1their rfocusestart smajorRad51refs. 9targetsignalnot asMYC tKu70,refs. 1genesref. 9)the prasynchNex

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ycle analysis and fluorescence-activatedrtingcell cycle analysis, cells were treated with 10 μmol/Lo-2′-deoxyuridine (BrdUrd; Sigma) in culture medium

hour. FITC-conjugated anti-BrdUrd antibody-labelediosciences) and propidium iodide (PI)–stained cellsnalyzed with a FACSCalibur flow cytometer and Cell-Pro software (BD Biosciences) as previously describedMO5757 cell cycle profiles were obtained using PIg. For cell sorting, MYC expression was induced withfor 16 hours in pF-MYC MCF10A cells. Cells wereed by trypsin (Invitrogen) digestion and stained withmL Hoechst 33342 in medium lacking phenol red andmented with 10% fetal bovine serum for 90 minutes atCells were sorted into G0-G1 and S-G2-M pools usingoFlo XDP cell sorter (Beckman Coulter).

genic survival and apoptosis assaysA-transfected DU145 or H1299 cells were trypsinized af-hours and subsequently treated either with 0.5 μg/mLfor 1 hour or with 2 Gy IR. Cells were plated in triplicates.NA-PKcs inhibitor Ku-0057788 (KuDOS Pharmaceu-was used at 1 μmol/L for 1 hour before 2 Gy IR, andm was changed 16 hours later. For all assays, cells werend stained in 1% methylene blue in 50% ethanol 10 tos later. Surviving clones with >50 cells were scored undert microscope, and resulting survival was calculatedviously described (27, 28). For apoptosis assays, H1299or Rat-1/myc controls) were treated with 0.5 μg/mLfor 1 hour or with 2 or 10 Gy IR at 48 hours after siRNAection and assessed for sub-G1 populations by flowetry or nuclear apoptotic bodies, as previouslybed (21).

saywas assessed using a direct repeat green fluorescentn (DR-GFP) assay essentially as previously describedhese H1299 cells possess an integrated DR-GFP con-, whose expression is prevented by an insert with therestriction site in the reading frame, whereby transfec-f I-SceI endonuclease creates a DSB, which whened by error-free HR leads to GFP-expressing cells). Briefly, H1299 cells were transiently transfected withthe negative control phCMV-1 I-SceI, the functionaluclease pCMV3xnlsI-SceI, or pGFP (as control forection efficiency) together with control, MYC, orsiRNA. Cells were trypsinized 72 hours after transfec-

nd assessed for GFP expression with FACSCalibur floweter and CellQuest Pro software. The percentage-positive cells in 50,000 to 100,000 events was normal-o the negative control and corrected for transfectionncy. There were no significant differences in transfec-fficiencies between treatments.

tical analysisistical analysis was performed using a one-way ANOVA
ukey's multiple comparison test or a nonlinear curveg the Prism 5 software (GraphPad), with P < 0.05 con-
starveafter 8

r Res; 70(21) November 1, 2010


d significant. Data derived from multiple independentments are shown as mean ± SE.

lts

vious ChIP studies completed by our group showedYC can bind the Rad51 gene promoter (9). We there-etermined whether MYC also binds to other DSB repairpromoters involved in HR or NHEJ (30–32). In silicoses were performed on sequences spanning fromupstream to 3 kb downstream of selected DSB repairranscription start sites (33). This revealed that therecandidate MYC-binding sites in other DSB repairromoter regions (Fig. 1A). To show these interactionso using ChIP, we used the prostate cancer cell lines, PC-3, and DU145, given our previous publication onelative HR and NHEJ repair gene expression (34). Wed on candidate sequences nearest to the transcriptionites (Fig. 1A). MYC was found to associate with theity of the tested HR gene promoters, including Rad51,B, Rad51C, XRCC2, Rad50, BRCA1, and BRCA2 (Fig. 1A;–11). Two primer pairs flanking the two most proximalsequences of XRCC3 showed weak or no enhancedover the negative controls, indicating that MYC wassociated with this promoter region (Fig. 1A). Candidatearget sites in NHEJ gene promoters DNA-PKcs, XRCC4,and DNA ligase IV were all occupied by MYC (Fig. 1A;0, 11). An E-box motif, not associated with MYC targeton Chr22, was used as a negative control (Fig. 1A;. We conclude that MYC can endogenously occupyomoters of the majority of the DSB repair genes inronously growing cancer cell lines.t, we compared the protein expression levels of MYCo MYC-occupied targets (RAD51 and KU70) in a panellines with varying histopathology. Endogenous MYCn expression was associated with increased RAD51U70 protein levels (Fig. 1B). To assess the correlationC and DSB repair protein levels in nontransformedwe serum starved normal human fibroblasts757 and followed MYC and DSB repair protein expres-ollowing serum add back and subsequent cell cycleession. As expected, MYC protein was negligible incells, but rapidly increased following addition of serumC; Supplementary Fig. S1). RAD51 was induced on cellprogression following cyclin D1 expression (Fig. 1C;mentary Fig. S1; ref. 35). These results are consistentrevious observations that MYC and RAD51 proteinincrease with an increase in S-phase fraction (35, 36).assess whether MYC is sufficient to activate the expres-f DSB repair genes in nontransformed human cells, weated a stable GEV16-inducible pF-MYC MCF10A cellee Materials and Methods; ref. 24). MCF10A cells werein medium containing reduced growth factors to

regulate the high levels of endogenous MYC and toiate the biological effects of endogenous and 4-OHT–d exogenous MYC. MYC induction in growth factor–
d cells was sufficient to induce Rad51 gene expressionhours (Fig. 2A), whereas RAD51 protein expression
Cancer Research



was upresultelittle eylationof MYafter iincrea

and CpF-MYtein aand Sand R

Figuregenes.sequeninvolveChIP ascontrol(right).gene ptranscrtarget s(arrowsChr22 winputs;RAD51was asMCF7,a loadinGMO57releaseCells wLeft, prand cyctubulinobtainemean oAsynch, asynchronous.


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regulated after 16 hours (Fig. 2B). MYC induction alsod in a modest upregulation of Rad51C, whereas it hadffect on NHEJ gene expression (Fig. 2A). RB phosphor-status and cell cycle analyses showed that induction

C was associated with S-phase entry only after 16 hours
nduction (Fig. 2C). In contrast, Rad51 gene expressionsed at 8 hours before S-phase progression (Fig. 2A
increaCyclin

acrjournals.org


). To further eliminate cell cycle bias, we flow sortedC cells into G0-G1 and S-G2-M pools followed by pro-nd RNA analyses. MYC was upregulated in both G0-G1

-G2-M phases after 16 hours of induction (Fig. 2D),AD51 could be induced in G0-G1 cells and was already

1. MYC association with DSB repairA, candidate MYC target consensusces within the promoters of genesd in HR and NHEJ were validated insays using a MYC antibody, rabbit IgG, or no-antibody control in 22RV1 cellsA schematic presentation of DSB repairromoters spanning 2 kb from theiption start site shows candidate MYCequences and ChIP primer pairs). An E-box not targeted by MYC onas used as a negative control. Inp,IP, immunoprecipitation. B, MYC,, and KU70 basal protein expressionsessed in 22RV1, PC-3, DU145, H1299,and RKO cell lines. Tubulin is shown asg control. C, normal human fibroblasts57 were serum starved for 48 h andd into cell cycle by serum stimulation.ere harvested 2, 4, 8, 16, and 24 h later.otein expression of MYC, RAD51,lin D1 is presented as normalized toexpression; right, cell cycle profilesd by PI staining are presented asf four independent experiments.

sed in S-G2-M cells, independent of MYC (Fig. 2D).A2 mRNA controls were selectively induced in S-G2-M




Figurefactor wrepair gand KUand ducontrolpools, fis prese

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2. MYC induces Rad51 expression in nontransformed cells. A to C, inducible pF vector control or pF-MYC MCF10A cells were cultured in growthithdrawal for 24 h. Cells were treated with ethanol as a vehicle control or induced with 100 nmol/L 4-OHT for 8 to 16 h. A, Myc and DSBene expression at 8 and 16 h after induction is presented relative to B2M and normalized to vehicle-treated pF control cells. B, MYC, RAD51,70 protein expression under conditions in A. Tubulin is shown as a loading control. C, hyperphosphorylated RB protein (see top band after induction)al-parameter (BrdUrd-PI) flow cytometry indicates S-phase entry at 16 h after MYC induction. ATM is used as a high–molecular weight loading. D, pF-MYC cells were vehicle treated or induced by 100 nmol/L 4-OHT for 16 h before fluorescence-activated cell sorting into G0-G1 and S-G2-Mollowed by protein and RNA analysis. Tubulin is shown as a loading control for Western blotting. Myc, Rad51, and cyclin A2 gene expression
nted relative to B2M and normalized to the vehicle control at G0-G1. Columns, mean of two to three independent experiments; bars, SE.
r Res; 70(21) November 1, 2010 Cancer Research

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(Fig. 2can renontraadditiexpresTo

was fMYC,promoMYC.low antectabMYC-C

resultgene ptarget(Fig. 3Nex

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Figurein maligwere tror MYCharvestlater. ORad51investigor rabbused ascontrolMYC onegativthe ChIfor MYCblottingloadingcells weMYC (1(0.25 nm48 h lato 72 hanalyseobtaine(BrdUrdpresentfour indBars, S


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D; ref. 37). Collectively, these data indicated that MYCgulate Rad51 gene and protein expression in G0-G1

nsiting cells and during G1 to S cell cycle progression;onal factors in addition to MYC may control Rad51sion in S phase.determine if MYC occupancy of the Rad51 promoterunctionally important in cells with downregulatedwe characterized the level of MYC and DSB repair geneter binding and protein expression following siRNA toWe used DU145 and H1299 cell lines, which expressd high endogenous MYC, respectively, and have de-
le endogenous levels of RAD51 protein (see Fig. 1B).hIP specificity was confirmed wit
contr

E.

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ed in reduced MYC occupation on Rad51 and Ku70romoters (Fig. 3A). CAD was used as a positive MYCcontrol, whereas Chr22 was used as a negative controlA; ref. 9).t, we determined the effect of MYC knockdown on DSBprotein expression. We used an optimal siRNA con-tion such that MYC protein levels were reduced toatest extent and the control siRNA had the least toxic-ta not shown). MYC siRNA (10 nmol/L) resulted in 60%reduction of MYC expression and in reduced RAD51

n levels in DU145 and H1299 cells (Figs. 3B, 5, and 7). In
ast, KU70 protein levels were changed minimally
h MYC siRNA, which by similar treatment (Fig. 3B), consistent with the lack of

3. MYC regulates RAD51nant cells. A, H1299 cellsansfected with control (Ctrl)siRNA (10 nmol/L) and

ed for ChIP assays 48 hccupation of MYC onand Ku70 promoters wasated using a MYC antibodyit IgG control. CAD wasa positive MYC target

. An E-box not targeted byn Chr22 was used as ae control. Right, fractions ofP cell lysates were analyzedexpression by Western

. Actin is shown as acontrol. DU145 and H1299re transfected with control,0 nmol/L), or RAD51ol/L) siRNA and harvested

ter for protein (B) and 24later for cell cycle (C)s. Cell cycle profilesd using dual-parameter-PI) flow cytometry areed as a mean of two toependent experiments.




MYC-dsamedetermincreaand inHowevdownmentain RADand thKU70,

Givperformodifcell suvival oby condecreaH1299RAD5

FiguresiRNAassesswas de 1

PI stain ; bars,

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ependent Ku70 expression (Fig. 2A). Next, using theconditions of MYC siRNA, cell cycle distribution wasined. MYC knockdown resulted in a nonsignificantse in G1 fraction in DU145 cells at 24 and 48 hours,an inhibition of S-phase progression in H1299 (Fig. 3C).er, we observed reduced proliferation of MYC knock-DU145 cells in growth assays at later time points (Supple-ry Fig. S2). We conclude that MYC-dependent reduction51 expression is not solely a consequence of a G1 arrest

termined 72 h after DNA damage using Hoechst 33342 staining; right, subing. Columns, mean of 2 to 14 independent experiments for each cell line

at MYC knockdown directly inhibits RAD51, but notprotein expression.

DU145and B



en the effect of MYC on RAD51 expression, we nextmed colony-forming assays to investigate whetherying MYC or RAD51 would affect long-term clonogenicrvival based on inhibited RAD51 expression. The sur-f DU145 or H1299 cells was not significantly affectedtrol siRNA transfection (data not shown). We observedsed cell survival by 65% in DU145 cells and by 77% incells following MYC siRNA (Fig. 4A and B). In contrast,1 knockdown resulted in 55% reduced survival in

ll populations were assessed 72 h after DNA damage usingSE. *, P < 0.05.

4. MYC is necessary for cell survival. DU145 (A) and H1299 (B) cells were transfected with control, MYC (10 nmol/L), or RAD51 (0.25 nmol/L)and plated for colony-forming assays 48 h after treatment. MYC siRNA reduced both surviving cell colony number and size. C, apoptosis wased in H1299 cells transfected as in B. Forty-eight hours after transfection, cells were treated with MMC and 2 or 10 Gy IR. Left, apoptotic morphology

-G ce

, but did not affect survival in H1299 cells (Fig. 4A). The combination of MYC and RAD51 knockdown

Cancer Research



resulte(Fig. 4vivingin botgrowtcells wgrowthas pand giwe detin thisbinedmeasuor subwith tFig. SMYC cas prelines (MM

lapsinicallyassayssee w

specifsion inwas ntent wdowncell kin signobservin theAdd

directFig. 6H1299and evestedbinatiby 85%and CtreatmHR min MYFina

Figuredoes nDU145were trMYC (1(0.25 nCells wMMC asurvivamean sindepeneach ceright, mproteinblots anin para*, P < 0


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d in a trend toward additive cell kill in both cell linesA and B). In addition to reducing the number of sur-cells, MYC siRNA led to smaller surviving colony sizesh cell lines (Fig. 4A and B). This suggests inhibitedh rate and/or postmitotic cell death of the daughterithin a colony, and is consistent with DU145 reducedh rate (Supplementary Fig. S2). MYC downregulationreviously been shown to promote apoptosis (3, 12),ven that siRNA to MYC was more toxic in H1299 cells,ermined whether apoptosis was the mode of cell deathcell system (Fig. 4C). MYC knockdown alone or com-with DSB-inducing agents did not induce apoptosis asred by nuclear morphology (Hoechst 33342 staining)-G1 peak analysis (flow cytometry; Fig. 4C) comparedhe positive control, Rat-1/myc cells (Supplementary3). This indicates that cell death following siRNA toan involve modes of cell death other than apoptosis,viously documented in irradiated prostate cancer cell21).C causes intrastrand DNA cross-links that lead to col-g replication forks and generation of DSBs that are typ-repaired by HR (38). We performed colony-forming
using a combination of MYC siRNA and MMC to
hether MYC knockdown cells were sensitive to HR-kill foNHEJ

.05.

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ic agent. An average of 70% knockdown of MYC expres-DU145 cells led to 40% reduction in RAD51, but this

ot sufficient to enhance MMC toxicity (Fig. 5A). Consis-ith this observation, an average of 80% MYC knock-in MMC-treated H1299 cells did not result in furtherill (Fig. 5B). In contrast, RAD51 knockdown resultedificant MMC sensitivity in both cell lines (Fig. 5). Theseations suggest that RAD51, but not MYC, plays a rolecellular MMC sensitivity of tumor cell clonogens.itionally, we performed DR-GFP assays (17) thatly measure HR function (see Materials and Methods;A) to ascertain MYC involvement in functional HR.cells were cotransfected with the I-SceI endonuclease

ither control, MYC, or RAD51 siRNA. Cells were har-72 hours after transfection to allow sufficient recom-

on to occur. Whereas RAD51 knockdown reduced HR, MYC knockdown decreased HR by only 14% (Fig. 6B). This was independent of cell cycle following siRNAent (Fig. 3C). The mild effect of MYC inhibition onay therefore explain the lack of MMC-induced cell killC knockdown cells.lly, we investigated the role of MYC knockdown in cell
llowing DSBs induced by IR. Cells deficient in HR orwill be sensitized to IR treatment (15). Neither
5. MYC downregulationot sensitize cells to MMC.(A) and H1299 (B) cellsansfected with control,0 nmol/L), or RAD51mol/L) siRNA for 48 h.ere treated with 0.5 μg/mLnd plated for clonogenicl assays. Columns,urvival of two to sevendent experiments forll line; bars, SE. A and B,ean MYC and RAD51expression on Westernd quantifications collected

llel to clonogenic assays.




ure. A,orteonuularultsflowressco

5 nlyzesfewn.led.prea poependent experiments; bars, SE. *, P < 0.05.

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6. The effect of MYC downregulation onH1299 cells that possess a DR-GFPr construct express GFP after I-SceIclease cleavage and repair by functionalHR. This restores the reading frame andin GFP expression, which is detectedcytometry. B, I-SceI endonucleaseion vector was transfected togetherntrol, MYC (10 nmol/L), or RAD51mol/L) siRNA. Recombination wasd by flow cytometry 72 h afterction. Representative dot blots areCells that have undergone HR areC, MYC knockdown led to minorssion of HR. RAD51 siRNA was usedsitive control. Columns, mean of three

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Association for Cancer


DU145relevaThis isame(Suppsitizatwith tTheseto cell

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nor H1299 cells exhibited sensitization to a clinicallynt dose of 2 Gy following MYC knockdown (Fig. 7).s in contrast to the profound radiosensitization of thecells through inhibition of the NHEJ protein, DNA-PKcslementary Fig. S4). RAD51 knockdown led to radiosen-ion in H1299, but not in DU145, cells (Fig. 7), consistenthe variable IR sensitivity of HR-deficient cells (15).results indicate that MYC knockdown does not leadular radiosensitization.

ssion

ious ChIP array studies have shown the association ofto several DSB repair promoters (9–11), and we nowthe novel findings that MYC directly transactivatesand associates with the Ku70 promoter with little ef-n KU70 expression. The MYC-dependent differentialtion of HR versus NHEJ proteins is unknown, bute a result of differential translational control and pro-alf-life (17). We also observed that knockdown of MYCto inhibition of RAD51, a central factor in HR. Yet, thisegulation was not sufficient to sensitize cells to DNA-ing agents such as MMC and IR, which preferentially
-deficient cells. MYC has been estimated to regulate15% of human genes (10, 11, 33). Global MYC ChIP
can diactiva

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data combined with gene expression data revealed thatmajority of MYC-associated targets did not changesion following ectopic MYC expression in B cells (33).also been speculated that MYC amplification in tumoray bind low-affinity E boxes that would be out of itslogic target range in normal cells (10, 33, 39). Together,data show the importance of comparing gene and pro-xpression data with functional assays to ascertain theology associated with MYC promoter binding.results show that under conditions that allow suffi-cell proliferation and clonogenic potential, 60% toeduction of MYC levels alone caused substantial cell. Targeting MYC by overexpression, antisense, siRNA,tors, or a dominant-interfering MYC mutant can in-poptosis and growth delay, resulting in tumor regres-2, 3, 40, 41). However, whereas apoptosis does notarily affect long-term cell survival (21), fewer studieshown the effect of MYC inhibition in cellular radiosen-ion or chemosensitization in colony-forming survival. As such, the 65% to 77% cell kill we observed follow-YC siRNA was not explained by apoptotic cell death, asiRNA alone or its combination with MMC or IR did note apoptosis. Additionally, MYC-addicted tumor cells

7. MYC knockdownot sensitize cells to IR.(A) and H1299 (B) cellsansfected with control,0 nmol/L), or RAD51ol/L) siRNA. Forty-eight

fter transfection, cellsated for clonogenicl assays and subjectedIR. Columns, meanl of two to four independentents for each cell line;E. Right, mean MYC andprotein expression andcations completed into clonogenic assays.

fferentiate, senesce, or cease to proliferate on MYC in-tion (42), and highlight the importance of assessing the




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y of possible therapeutic applications using long-clonogenic potential. There are inconsistent conclu-from studies comparing cellular sensitivity withdepletion or overexpression, and the outcome mayl type dependent. Studies have reported NHEJ repair,atch repair, and disparate chemotherapy-inducedsis or proliferation in the context of both increasedecreased MYC expression in differing tumor cell lines–46). We conclude that attempts to combine MYC-effects in DSB repair gene expression with chemo-

y or radiotherapy may not lead to sensitization ofclonogens.ucing RAD51 expression by 80-90% resulted in MMCsensitivity in H1299 cells and in MMC sensitivity in

5 cells. RAD51 is overexpressed in several cancers,creased levels of RAD51 correlate with increased erro-recombination and resistance to DNA-damaging(47). Given that HR-deficient cells are sensitive toies such as MMC, cisplatin, and IR, targeting RAD51ereby inhibiting HR is a potential target for combinedies. For example, imatinib leads to decreased RAD51sion. Combining imatinib with IR resulted in decreasedenic survival in vitro, as well as in tumor growth delay3 prostate cancer xenografts (28). Hence, directly tar-the key players in HR may provide a potent tool for
cell radiosensitization, given that indirect HR targetingh MYC did not lead to enhanced sensitivity.
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nscriptional regulatory role for c-Myc in Burkitt's lymphoma cells.c Natl Acad Sci U S A 2003;100:8164–9.cci B, D'Agnano I, Amendola D, et al. Myc down-regulation

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onclusion, our work suggests that although MYC occu-he majority of DSB repair promoters and controls1 expression, MYC downregulation by siRNA is not suf-to induce cellular radiosensitivity and MMC sensitivity.ver, MYC depletion can lead to cell death directly. Smallules and PMO as single agents that are not limited byection-based methods should be further assessed, asstrategy may directly target cell proliferation, tumorssion, and genetic instability.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Gaetano Zafarana for helpful discussions.

Support

y Fox Foundation Program grant 15004, CCSRI Operating grants 1715498, and Canadian Foundation for Innovation grant to the STTARRion Facility. This research was funded in part by the Ontario Ministryh and Long Term Care. The views expressed do not necessarily reflectf the Ontario Ministry of Health and Long Term Care.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/16/2010; revised 07/12/2010; accepted 08/08/2010; publishedirst 10/12/2010.

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2010;70:8748-8759. Published OnlineFirst October 12, 2010.Cancer Res Kaisa R. Luoto, Alice X. Meng, Amanda R. Wasylishen, et al. Genes that Control DNA Double-Strand Break RepairTumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated

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