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Mutation Research 683 (2010) 115–122

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

fficient repair of DNA double-strand breaks in malignant cells with structuralnstability

ue Cheng, Zhenhua Zhang, Bridget Keenan, Anna V. Roschke, Kenneth Nakahara, Peter D. Aplan ∗

enetics Branch, National Cancer Institute, Navy 8, Room 5101, 8901 Wisconsin Ave., Bethesda, MD 20889-5105, USA

r t i c l e i n f o

rticle history:eceived 30 March 2009eceived in revised form 22 October 2009ccepted 30 October 2009vailable online 10 November 2009

eywords:NA double-strand break (DSB)hromosomal rearrangement

-SceIvarian cancer

a b s t r a c t

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of grosschromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigatedthe repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introducedinto OVCAR-8 cells between a constitutive promoter (EF1�) and the Herpes simplex virus thymidine kinase(TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a singleDSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1� promoterfrom the TK gene, or deletion of either the EF1� promoter or the TK gene. All mutations that we identifiedwere interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNADSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitialdeletions using the aforementioned negative selection system, we developed a positive selection system

to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycinphosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs wereproduced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked thehygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portionof the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cellline, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without

.

the introduction of a GCR

. Introduction

Mammalian cells have developed effective systems to respondo and repair DNA double-strand breaks (DSBs). Unfaithful repairf these breaks can lead to cell death or gross chromosomal rear-angements (GCR), including deletions, amplifications, inversions,nd translocations [1–4]. GCRs, in turn, can lead to amplification ofroto-oncogenes, or generation of novel, chimeric fusion oncopro-eins [5–7].

Given that these oncogenic GCRs are important causes ofalignant transformation, it is therefore important to under-

tand the mechanisms that lead to these GCRs and generationf oncogenic fusion genes. Two basic DNA repair pathways,

omologous recombination (HR) and non-homologous end join-

ng (NHEJ) have been identified in human cells [8–11]. Since mostncogenic GCRs are thought to be mediated via improper NHEJ-ediated repair of two DNA DSBs, the study of improper NHEJ

Abbreviations: DSB, double-strand break; GCRs, gross chromosomal rearrange-ents; NHEJ, non-homologous end joining.∗ Corresponding author. Tel.: +1 301 435 5005; fax: +1 301 496 0047.

E-mail address: aplanp@mail.nih.gov (P.D. Aplan).

027-5107/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.mrfmmm.2009.10.016

Published by Elsevier B.V.

repair should yield insights into the mechanisms that lead toGCRs.

There are limited techniques available that can be used to pro-duce a specific DNA DSB in living cells for these studies. Using asystem to assay DNA DSB repair by HR in embryonic stem (ES) cells,investigators have previously demonstrated that two induced DNADSBs were required to produce a GCR [12]. We wondered whetherthose results might be specific for ES cells, or DNA DSB repair viaHR, and hypothesized that in non-embryonic, hematopoietic cellsa single induced break might recombine with a spontaneous DNADSB, since it has been estimated that approximately 50 DNA DSBsoccur per cell cycle [13]. We used the yeast I-SceI endonuclease todevelop a loss of function reporter system in which a DNA DSB wasintroduced in a predetermined region. In this system, the 18-bp I-SceI recognition site was inserted between a constitutive promoter(EF1�) and the Herpes simplex virus thymidine kinase (TK) gene,which confers sensitivity to gancyclovir (GCV). The ensuing chro-mosomal changes flanking the breakage site can then be studied

after transfection of an I-SceI expression vector into these cells.Using hematopoietic U937 cells, we found that approximately 50%of the cells transfected by an I-SceI expression vector showed clearevidence of I-SceI cleavage, suggesting that I-SceI mediated cleavageof genomic DNA was reasonably efficient. The most common muta-

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ion that occurred in the cells that survived GCV selection was annterstitial deletion. More complex rearrangements, such as inser-ions or duplications, were also noted. At the breakpoint junction,

any clones showed hallmarks of NHEJ, such as direct or invertedepeats or micro-homology [14]. However, we did not detect anylear evidence of GCRs in over 100 independent clones analyzed.

In order to determine whether the above findings were uniqueo hematopoietic U937 cells, or were more generalizable to otherell types, we extended this system to an epithelial cell line. In anffort to produce GCRs, we chose the OVCAR-8 ovarian cancer celline for these studies, since this cell line displayed a high degree ofngoing structural chromosomal instability [15].

. Materials and methods

.1. Cell line, DNA transfection and drug treatment

OVCAR-8 cells were obtained from the NCI-60 anticancer drug discovery panel15,16]. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovineerum, 100 U/ml penicillin and 100 �g/ml streptomycin. Cells were maintained at7 ◦C in a humidified atmosphere of 5% CO2.

Transfections of linearized vector pEF1�TK, containing I-SceI recognitionequences, were performed using electroporation as previously described [14].ransfections of the I-SceI expression vector, pCBASce, were performed usingipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s recom-ended protocol. OVCAR-8 cells were at approximately 75–90% confluence before

he transfection. The next day cells were split into ten 100 mm tissue culture dishesrom one 75 cm2 flask. On the third day following the transfection, 500 �g/ml G418Invitrogen, Carlsbad, CA), 40 �M GCV (InvivoGen, San Diego, CA) or 200 �g/mlygromycin B (Invitrogen, Carlsbad, CA) was added, depending on the selectionsed. All resistant clones were isolated and expanded for further analysis.

For etoposide treatment, the A15 cells were transfected with I-SceI expressionectors, cultured for 48 h, and incubated with etoposide at 100 �M for 1 h, 10 �Mor 4 h or 5 �M for 4 h. For bleomycin treatment, A15 cells were transfected with I-ceI expression vectors, cultured for 48 h, and subsequently treated with bleomycint 5 �g/ml or 20 �g/ml for 30 min. Following treatment with either etoposide orleomycin, the cells were washed with PBS and expanded for 3 days. The cells werehen split into eight 100 mm tissue culture dishes and incubated for 24 h beforeddition of GCV (40 �M).

.2. Vector construction

Both pEF1�TK (containing I-SceI recognition site) and I-SceI expression vectorsPCBASce) were previous described by Varga and Aplan [14] and Richardson et al.17]. A vector (named pTBGHygro) containing the hygromycin phosphotransferaseDNA (HygroR; confers resistance to hygromycin) fused in frame to human �-globinxon 3, preceded by �-globin intron 2, exon 2, and an I-SceI recognition sequenceas generated as follows. A BamHI/EcoRI fragment containing the I-SceI recogni-

ion sequence, �-globin exon 2, intron 2 and exon 3 was generated by PCR andigated into the BamHI and EcoRI sites of pcDNA3 (Invitrogen). This plasmid washen digested with BamHI and BglII and religated to delete the CMV promoter fromhe pcDNA3 backbone. Finally, this plasmid was digested with EcoRV and NotI, andPCR-generated hygromycin phosphotransferase cDNA was ligated in frame into-globin exon 3 to generate the pTBGHygro vector.

.3. Conventional and inverse PCR

All PCR amplifications, unless otherwise indicated, were performed using PCRuperMix High Fidelity enzyme and buffers (Invitrogen, Carlsbad, CA). DNA extrac-ion, digestion and ligation were as previously reported [14]. To verify DNA quality,e routinely amplified the SVCT locus for all clones. For inverse PCR, DNA wasigested with either HindIII or MboI, and re-ligated with T4 Ligase (Promega, Madi-on, WI). Long PCR fragments for either conventional or inverse PCR were amplifiedsing the TaKaRa LA Taq kit (TaKaRa Bio, Otsu, Shiga, Japan), following the manu-acturer’s recommended protocol. Details of primer sequences and locations in theector are listed in Supplementary Table 1.

.4. Fluorescence in situ hybridization and chromosome painting

Chromosome preparations were made from OVCAR-8 cell line and clones OV-1 and A-15 by conventional methods. Slides were pretreated and denatured asescribed elsewhere [18]. The pEF1aTK and pTBGHygro plasmids were labeled by

ick translation with digoxigenin-11-dUTP (Roche, Indianapolis, IN), precipitated inthanol with 50× excess of human Cot-1 DNA (Roche, Indianapolis, IN), and resus-ended to a final concentration of 50 ng/�l in Hybrizol solution (Qbiogene, Montréal,anada). Whole chromosome painting probes for chromosomes 2 and 9 (labeled inreen) were obtained from Applied Spectral Imaging (Vista, CA). After denaturing at0 ◦C for 10 min and preannealing at 37 ◦C for 60 min, 10 �l of probe mixtures were

rch 683 (2010) 115–122

applied under 22 mm × 22 mm coverslips. Slides were incubated in a moist cham-ber overnight at 37 ◦C. After detection with anti-digoxigenin antibodies labeled withrhodamine (Roche, Indianapolis, IN) they were mounted in antifade solution (Vec-tor Laboratories, Burlingham, CA) containing DAPI. Leica microscope equipped withDAPI, FITC and rhodamine filters (Chroma Technology, Rockingham, VT) and Sen-sys CCD camera (Photometrics, Tucson, AZ) connected with Q-FISH software (LeicaMicrosystems Imaging Solutions, Cambrige, UK) were used for image acquisitions.

2.5. Southern blot analysis

Duplicate samples of 10 �g of genomic DNA were isolated from the indicatedclones, digested with HindIII, and size fractionated using a 0.8% agarose gel. TheDNA fragments were transferred to nitrocellulose membranes and hybridized to32P-labeled probes as previously described [14].

2.6. Nucleotide sequence analysis

PCR products were cloned into the pGEM-T easy vector (Promega, Madison, WI)and transformed into DH-5� cells. Plasmid DNA was extracted (Qiagen, Valencia, CA)and sequenced (NAPCORE, The Children’s Hospital of Philadelphia or Retrogen, SanDiego, CA). Nucleotide sequences were compared to the human genome assemblybased on NCBI Human Genome Build 34.

3. Results

3.1. Creation of a reporter cell line

In an attempt to produce GCRs, we generated a cell line whichcould serve as a reporter for unfaithful repair of a DNA DSB.A plasmid that expresses the TK gene under the control of theEF1� promoter, with the 18-bp recognition sequence for the I-SceIrestriction enzyme placed between the EF1� promoter and TK gene(Fig. 1A and Supplementary Fig. 1), was linearized and introducedinto OVCAR-8 cells. Stable transfectants were selected with G418,and several clones that had integrated a single copy of the con-struct were identified by Southern blot hybridization. These cloneswere then treated with 40 �M GCV to verify expression of Hsv-tk and sensitivity to GCV. One clone, named A15, had integrateda single copy of the EF1aTK vector and was shown to be sensitiveto GCV. We used inverse PCR to clone the integration sites, anddetermined that the construct was integrated on chromosome 2,between nucleotides 24378945 and 24378974 (Fig. 1B). To confirmthis integration event, we synthesized a probe (named CHR2; nuc24378079–24378704) immediately downstream of the HindIII siteshown in Fig. 1B, and within an intron of the C2orf84 gene. Thisprobe was hybridized to a Southern blot of genomic DNA from theparental OVCAR-8 cell line and the A15 clone. As shown in Fig. 1C, anovel, rearranged fragment generated by the insertion is seen in theA15 subclone, as well as the endogenous band from the remainingintact allele. To further characterize the insertion, we co-hybridizedmetaphase chromosomes to a chromosome 2 paint and the EF1aTKvector. Comparison of the parental OVCAR-8 cell line with the A15cell line demonstrates that both the parental and daughter celllines had one morphologically normal copy of chromosome 2, and3 additional chromosomes with a portion of chromosome 2. Fig. 1Ddemonstrates that the vector is integrated on the telomeric portionof a derivative chromosome 2.

3.2. Generation and analysis of GCVR clones

The A15 clone was transfected with the I-SceI expression vec-tor pCBASce [12], and incubated without GCV selection for 3 days.GCV resistant (GCVR) clones were then selected by the addition ofGCV to a final concentration of 40 �M. After three weeks selection

in GCV, no GCVR clones were obtained from the control (vectoralone) transfectants. However, 5–10 GCVR clones/10 cm dish weredetected. We isolated 59 GCVR clones and attempted to expandthem. Thirty-six clones were successfully expanded and subjectedto DNA analysis; the remainder either did not expand or became

Y. Cheng et al. / Mutation Research 683 (2010) 115–122 117

Fig. 1. Integration of pEF1�TK construct into chromosome 2 in A15 cells. (A) Integration of the pEF1�TK construct in OVCAR-8-derived clones by Southern blot analysis. DNAswere digested with HindIII and hybridized with probe Neo. A15 cells, with a single integration site, were used for further analysis. (B) The integration sites on chromosometwo (based on NCBI build 34) are indicated. Gray squares indicate the CHR2, EF1�, and Neo probes used for Southern blot analyses. I-SceI (I) and HindIII (H) sites are indicated.( ridizen or A15E

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C) Genomic DNA from parental (OV8) or A15 cells, digested with HindIII, and hybovel fragment containing the EF1aTK vector is indicated with an arrow. OVCAR-8F1aTK plasmid (red). Note the red sister chromatid signals on the magnified inset.

ontaminated and were discarded. Following PCR and sequencenalysis, 2 clones were determined to be duplicates (C23 and C35).our clones (C1, C27, C34, and C36) had evidence of I-SceI cleavage,s evidenced by inability to amplify across the I-SceI site, and lossf exogenous EF1� sequences on Southern blot, but were unableo be characterized further. Finally, 31 independent clones werenalyzed by Southern blot and sequencing analyses.

The GCVR clones were screened with PCR primers that flankedhe I-SceI cleavage site (primers IPCREF2 and INVALU5R in Fig. 2And and Supplementary Table 1). Based on our previous obser-ations in U937 cells [14], we anticipated that small interstitialeletions and insertions would be detected by this assay, as shown

n Fig. 2C. The parental cell line A15 showed a PCR product of thexpected size (1204 bp), whereas clones C30, C33, C35, C40, C44,nd C53 all generated variable sized, smaller PCR products, sug-esting an interstitial deletion. Additional clones (for example, C34,43, C47, C54 of Fig. 2C) did not produce a PCR product, suggestinghat either one or both primer binding sites had been deleted, orhat the primer binding sites had been separated by a large distance

for instance, by a large insertion or GCR). Direct sequencing of PCRroducts showed that 7 clones (C4, C9, C23, C30, C33, C44 and C53)ad small (less than 1 kb) interstitial deletions (Table 1).

We used Southern blot analysis to further evaluate cloneshat did not generate PCR products with primers IPCREF2 and

d to the CHR2 probe. The endogenous fragment is indicated with an asterisk, thecells (panels D and E respectively) hybridized to chromosome 2 paint (green) and

INVALU5R. Since the EF1aTk vector does not contain any HindIIIsites, EF1� and Neo probes hybridize to the same HindIII fragmentin the parental A15 clone (see Fig. 1B for location of probes). Fur-thermore, if the A15 clone is cleaved at the I-SceI site, and undergoesan interstitial deletion during DNA DSB repair, the resulting daugh-ter clone will also contain EF1� and G418 sequences on the sameHindIII fragment (which is smaller than the HindIII fragment in theparental A15 clone). As demonstrated in Fig. 2D, clones C17, C18,C23, C24, C28, and C29 had variably sized HindIII fragments, dif-ferent from that of the parental A15 clone, which hybridized to theNeo probe. In each case, the same novel (i.e., smaller than the frag-ment from the parental A15 clone) HindIII fragments hybridizedto the EF1� probe, suggesting that these clones had undergone aninterstitial deletion.

If the clone had undergone a GCR, the size of the HindIII frag-ments that hybridized to the EF1� and Neo probes would dependon the location of endogenous HindIII sites. Therefore, if the clonehad undergone a GCR, we would expect the EF1� and Neo probesto hybridize to HindIII fragments that were not the size of the wild-

type fragment, and not of the same size. This situation is in contrastto the interstitial deletions, in which the non-wild-type fragmentsthat hybridized to the EF1� and Neo probes were the same size.

As shown in Fig. 2D, 5 clones (C16, C19, C20, C21 and C26) lostthe EF1� region, but had variable, non-wild-type sized fragments

118 Y. Cheng et al. / Mutation Research 683 (2010) 115–122

Fig. 2. Analyses of DNA DSB repair events in GCV resistant clones. (A) Location of PCR primers used for forward PCR. (B) Location of primers used for inverse PCR. MboI andHindIII restriction sites are indicated by M and H respectively. (C) Example of PCR amplification across I-SceI site. Parental A15 cells generated a 1.2 kb DNA fragment, clonesC30, C33, C35, C40, C44, and C53 generated variable sized fragments, and clones C34, C43, C47, and C54 generated no PCR products. Bottom panel shows PCR amplificationo derive( C23, Ct tion o

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f the SVCT locus, used as a DNA quality control. (D) Southern blot analysis of A15-top) and EF1� (bottom). (E) Endogenous allele of EF1�. Note that clones C17, C18,he Neo and EF1� probes. C16, C19, C20, C21 and C26 clones have lost the EF1� por

hat hybridized to the Neo probe, suggesting that these clonesad either a larger interstitial deletion (which had deleted EF1�equences), or had a GCR that had deleted EF1� sequences. UsingindIII or MboI digested DNA samples, we performed inverse PCR

o clone the rearrangement from 12 clones (Fig. 2B and Table 1).ll of these clones analyzed by inverse PCR represented larger

nterstitial deletions. In sum, all 31 clones analyzed, including2 that were analyzed by Southern blot alone and 19 that wereequenced, represented interstitial deletions (Table 1). 13 of the 19equenced clones were simple interstitial deletions, and 6 clonesepresented more complex rearrangements, due to DNA insertion

nd inversion events. Two clones had insertions that were derivedrom the I-SceI expression vector, and two other clones had smallnsertions of undetermined origin. Clone C19 had an inverted dupli-ation at the I-SceI site, and clone C26 had a 357 bp insertion thatatched sequences, from a gene encoding a hypothetical protein

d clones. Genomic DNAs were digested with HindIII and hybridized to probes Neo24, C28 and C29 have non-wild-type sized fragments, which are detected by bothf the vector.

(LOC730045) located on chromosome 18. Thirteen clones showedmicro-homology (1–8 bp) at the junction sites (Table 1).

3.3. Characterization of clones induced by I-SceI and genotoxicagents

Since we were unable to produce GCRs by induction of a sin-gle DNA DSB, we reasoned that we might be able to produce GCRsby generating two or more simultaneous DNA DSBs. Therefore, weused etoposide or bleomycin to generate random DNA DSBs, andI-SceI to produce a specific DNA DSB. Following transfection of the

A15 cell line with an I-SceI expression vector, cells were treatedwith 100 �M etoposide for 1 h or 10 �M etopside for 4 h, and thenallowed to recover for 4 days before GCV selection. A total of 7 GCVR

clones were identified; 1 clone (V1) from the 100 �M/1 h treatment,the other 6 clones from the 10 �M/4 h treatment. The A15 clone was

Y. Cheng et al. / Mutation Research 683 (2010) 115–122 119

Table 1Summary of DNA break/repair events induced by I-SceI endonuclease, bleomycin and etoposide.

Clonea Result Bp deletedfrom EF1� side

Bp deletedfrom TK side

Total deletionb Micro-homology 5′ Micro-homology 3′c Comments

C2 Deletion ∼1200C3 Deletion ∼500C4 Deletion 62 235 315 TAC6 Deletion ∼1400C9 Deletion 7 151 176 CGGGC11 Deletion 3060 3075 Insertion: TAGAATAC13 Deletion ∼1800C15 Deletion 274 539 831C16 Deletion 702 575 1295 AC17 Deletion ∼1200C18 Deletion ∼1600C19 Deletion 391 583 992 AG CC Inversion: 218 bp of I-SceI + TKC20 Deletion 434 1535 1987C21 Deletion 556 884 1458 ACTC22 Deletion 12 2982 3012 GGC23/35 Deletion 568 584 Insertion: 135 bp from vectorC24 Deletion 23 367 408 GTCGA GCCC Insertion: 86 bp from vectorC26 Deletion 1028 305 1351 AAA CA Insertion: 357 bp from chro. 18C28 Deletion ∼1000C29 Deletion 10 854 882 CC30 Deletion 5 843 866 Insertion: AGGAC31 Deletion 626 892 1536C33 Deletion 8 660 686 GGGCGCGCC39 Deletion 2388 878 3284 GC40 Deletion ∼1500C43 Deletion ∼1800C44 Deletion 208 213 439 GAACCCC47 Deletion ∼1600C53 Deletion 20 662 700 CC54 Deletion ∼1500C59 Deletion ∼800

B1 Deletion 100 892 1010 GB3 Deletion 7 2893 2918 CGB4 Deletion 12 460 490 CGGB6 Deletion 2294 972 3284B8 Deletion ∼2100B9 Deletion ∼2200B10 Deletion 25 136 179 Insertion: 22bpB13 Deletion 426 859 1303 TAAB17 Deletion ∼500B20 Deletion 822 1701 2541 AAB21 Deletion 1962 751 2731 Insertion: CB23 Deletion 5 1475 1498 Insertion: 379 bp from vectorB24 Deletion 180 180 CGT I-SceI is intactB26 Deletion 529 830 1377 Insertion: GCB27 Deletion ∼1600

V1 Deletion ∼500V3 Deletion 1515 1668 3201V4 Deletion 10 714 742 CGGGCV5 Deletion 1276 1294 G C Insertion: 21 bp from vectorV7 Deletion 7 546 571 CCCCV12 Deletion 1098 395 1511V17 Deletion 515 1905 2438

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Approximate DNA sizes detected by Southern blot analyses.a C clones: I-SceI only; B clones: I-SceI + bleomycin; V clones: I-SceI + etoposide.b Includes 18 bp of I-SceI sequence.c Two micro-homology sites caused by two religations (both 5′ and 3′) were only

reated with bleomycin in a similar fashion, for 30 min with eitheror 20 �g/ml bleomycin. A total of 15 GCVR clones were identi-

ed, four from the 5 �g/ml treatment, and 11 from the 20 �g/mlreatment.

Compared to clones generated by expression of I-SceI only,ells treated with both I-SceI and etoposide or bleomycin had aower yield of GCVR clones, several times less than that of the I-

ceI only group. However, analysis of the GCVR clones from allhree groups demonstrated only interstitial deletions; no GCRsere identified (Table 1). The size of the deletions was simi-

ar in all three groups, with the maximum deletion identifiedeing 3.2 kb (Table 2). All three groups showed similar frequen-

ted in some clones.

cies of micro-homology and insertions at the breakpoint junctions(Table 1).

3.4. Use of a positive selection system to produce GCR

Given that the negative selection (i.e., detection of clones miss-ing a gene product) approach described above produced only

clones with interstitial deletions leading to loss of TK expression,we devised a positive selection approach. This positive selectionapproach would detect the presence of a gene product, as opposedto the absence of a gene product, and would not detect clones witha simple interstitial deletion. A vector (pTBGHygro) containing the

120 Y. Cheng et al. / Mutation Research 683 (2010) 115–122

Table 2Comparison of DNA break and recombination events occurred in A15 cells.

Groups I-SceI I-SceI + bleomycin I-SceI + etoposide

Characterized clones 31 15 7

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Table 3Hygromycin resistant clones.

Clone # Rearrangement I-SceI fusion site Micro-homology

8 Vector fusion 123 None10 Vector fusion 116 A14 Vector fusion 135 None15 Vector fusion 113 A17 Vector fusion 115 A20 Vector fusion 124 None21 Vector fusion 116 None26 Vector fusion 124 ATT28 Vector fusion 125 None1A Vector fusion 115 None3A Vector fusion 130 None7A Vector fusion 98 None8A Vector fusion 111 CCTA

FIDpCps

Deletion ranges (bp) 176–3284 179–3284 500–3201Median deletions (bp) 1200 1600 1294

ygromycin phosphotransferase cDNA (HygroR; confers resistanceo hygromycin) fused in frame to human �-globin exon 3, precededy �-globin intron 2, exon 2, and an I-SceI recognition sequenceas generated (Fig. 3 and Supplementary Fig. 2). The vector also

ontains a G418R expression cassette to allow for selection of cellshat have integrated the vector. We transfected this vector intoVCAR-8 cells, and identified a clone (named OVCAR-8-11) thatad integrated a single copy of the vector. As anticipated, sincehe HygroR cDNA lacks a promoter, these cells were hygromycinensitive. As described above, we used inverse PCR to determinehat the insertion site for the OVCAR-8-11 clone was chromosome, nucleotide 65632493, 20 kb telomeric of AK094938. We usedouthern blot analysis with a PCR-generated probe (CHR9 nuc.5631975–65632438, see Fig. 3) to verify the insertion site (Fig. 3B);ultiple cross-hybridizing bands are seen since this integration site

s within a low copy number repeat region. We further charac-erized the insertion by chromosome painting and FISH. Neitherhe parental OVCAR-8 nor the daughter OVCAR-8-11 clone had an

ntact chromosome 9; instead, there were 3 chromosomes in bothell lines that contained chromosome 9 material (Fig. 3B and C).he pTBGHygro vector hybridized to a subcentromeric region ofhromosome 9 (Fig. 3C).

ig. 3. I-SceI induced cleavage of the pTGHygro cassette leads to DNA capture events. (A-SceI recognition site (I) was inserted upstream of the �-globin exons 2 and 3. HindIII (HNA from OVCAR-8, A15, or OVCAR-8-11 clones, hybridized to the CHR9 probe. The endTGHygro vector is indicated with an arrow. Several endogenous fragments, including onHR9 probe is within a low copy number repeat region. OVCAR-8 or OVCAR-8-11 cells (palasmid (red). Note the red signals on the magnified inset. (E) Subclones generated by Iequences derived from the I-SceI expression vector, and subsequent religation between

9A Vector fusion 112 None

The OVCAR-8-11 subclone was transfected with an I-SceIexpression vector, and selected with hygromycin, in the hopes ofrecovering rare clones that had undergone a GCR, and juxtaposedgene regulatory sequences from a distant genomic region to theHygroR gene, thus allowing expression of the HygroR gene, leadingto hygromycin resistance. No HygroR clones were recovered fromcontrol experiments with an empty vector (pBluescript II) transfec-tion, and we recovered few clones following transfection with theI-SceI expression vector. All 14 of the clones analyzed were vector

capture events, in which a portion of SV40 regulatory sequencesderived from the I-SceI expression vector became juxtaposed to �-globin exon 2 (Fig. 3 and Table 3), leading to hygromycin resistance.

) Integration of pTGHygro construct into chromosome 9 in OVCAR-8-11 cells. The) site and CHR9 probe are indicated. (B) Southern blot of HindIII digested genomicogenous fragment is indicated with an asterisk, the novel fragment containing thee that is more intense than the novel OVCAR-8-11 fragment, are seen because the

nels C and D respectively) hybridized to chromosome 9 paint (green) and pTGHygro-SceI expression vector resulted from cleavage at the I-SceI site, insertion of SV40the chromosomal end and �-globin sequences from the pTGHygro construct.

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. Discussion

Aberrant repair of DNA DSBs is likely to play a role in generatinghe GCRs that are hallmarks of human malignancies. We previouslyeported that induction of a single DNA DSB in a hematopoietic celline did not generate GCRs, but instead only small interstitial dele-ions [14]. However, given that solid tumors typically display moreCR, and genetic instability than do hematopoietic malignancies

6,7,19,20], we wished to determine if GCRs could be induced by aingle DNA DSB in an epithelial cell line. For these experiments, wesed the OVCAR-8 cell line, which has previously been reported toave a high degree of ongoing chromosomal instability [15].

We induced DNA DSBs in OVCAR-8 cells by first introducing theecognition sequence for the I-SceI restriction enzyme, followed byransfection with an I-SceI expression vector. Clones that had beenleaved by I-SceI were analyzed using a combination of PCR assaysnd Southern blot hybridizations. The simplest form of repair forDNA DSB induced by I-SceI would be a perfect religation event

hat recreates an I-SceI recognition sequence which would remainusceptible to I-SceI cleavage. Although our experiment was notesigned to detect perfect repair of I-SceI cleavage, other studiesave utilized a substrate that includes sequences flanked by I-SceIites [21]. In those studies, cleavage by I-SceI and religation at the-SceI sites could be detected by loss of intervening sequence. Thenvestigators noted that 33–65% of the clones recovered had beenepaired by a precise religation event [21]. If the cleaved recog-ition sequence is repaired by an imprecise mechanism, such ason-homologous end joining, clones can be isolated due to loss ofK function.

We characterized 31 clones that sustained mutations followinghe repair of a single I-SceI-induced DNA DSB in OVCAR-8 cells. Sim-lar to previous reports [10,14,22–28], the most common DNA DSBepair leading to loss of TK expression was an interstitial deletion,ften accompanied by additional mutations, such as insertions andnversions. Many of these clones showed hallmarks of NHEJ, such as

icro-homology at the site of DNA DSB repair in these cells. Severallones displayed more complex DNA DSB repair events. One cloneontained 357 bp of sequence derived from LOC730045, located onhromosome 18q, and 2 clones captured I-SceI expression vectorequences similar to previous findings [14,23]. However, none ofhe clones analyzed showed evidence of a GCR. It remains possi-le that cleavage at the I-SceI site induced a GCR at a site distantrom the I-SceI site; a distant GCR would not have been identified byhe Southern blot or PCR assays, which focused on aberrant repairvents at the I-SceI site.

Prior studies have demonstrated that balanced chromosomalranslocations are extraordinarily uncommon events when a singleNA DSB is induced by I-SceI in murine embryonic stem cells [12].owever, if two simultaneous DNA DSB breaks are introduced, the

requency of balanced translocations increases more than 2000-old [12]. A special case exists at telomeric DNA, where integrationf a vector containing an I-SceI recognition site near a telomerereatly increases the frequency of clone that has undergone eitherlarge (∼300 bp or greater) deletion or GCR [26].

To determine if simultaneous production of a specific DNA DSBby expression of I-SceI) and a non-specific DNA DSB would lead toroduction of a GCR, we treated cells with etoposide or bleomycin.e characterized 22 clones that had been transfected with an I-SceI

xpression vector and subsequently treated with either etoposider bleomycin. Similar to the I-SceI-induced cells, none of theselones showed evidence of a GCR. However, it remains possible

hat the timing which was employed for this experiment (transfec-ion of the I-SceI plasmid 48 h prior to treatment with etoposide orleomycin) was sub-optimal.

A potential disadvantage of the above approach is that it utilizesnegative selection system. Results obtained by us [14] and others

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[23,26] with this type of approach (a negative selection based on TKand GCV selection) have yielded primarily interstitial deletions of5 kb or less. Therefore, we developed a complementary vector thatallowed for positive selection. However, as described above, all ofthe clones HygroR clones identified were vector capture events, inwhich a portion of SV40 regulatory sequences derived from the I-SceI expression vector became juxtaposed to �-globin exon 2 orintron 2. These vector capture events at I-SceI cleavage sites havebeen described previously [14,23], and are presumably due to theability of large amounts of excess plasmid DNA in the nucleus toserve as “patches” at the DNA DSB site.

In summary, we have established two complementary systemsto generate a specific, single DNA DSB in an epithelial cancer cellline, which may be useful for elaborating DNA DSB repair mech-anisms. Despite examination of over 65 mutant clones, PCR andSouthern blot analysis revealed no evidence for a GCR in any of theclones analyzed. Instead, we detected principally small deletionsor insertions, with features suggesting repair by NHEJ. These find-ings indicate that GCRs are likely to be a rare consequence of DNADSB repair, even in a cell line such as OVCAR-8, which is prone tostructural instability. It is possible that use of a cell line that is defi-cient in proteins required for conventional NHEJ will be needed toproduce GCRs following a single induced DNA DSB.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank Tamas Varga, Ilan R. Kirsch and Michael Kuehl forsuggestions and helpful advice, and Maria Jasin for the gift of thepCBASce expression vector. This work was supported by the Intra-mural Research Program of the NIH, NCI.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mrfmmm.2009.10.016.

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