BCL2 Suppresses PARP1 Function and Nonapoptotic Cell Deathcancerres.aacrjournals.org/content/canres/72/16/4193.full.pdf · Therapeutics, Targets, and Chemical Biology BCL2 Suppresses

Therapeutics, Targets, and Chemical Biology

BCL2 Suppresses PARP1 Function and NonapoptoticCell Death

Chaitali Dutta, Tovah Day, Nadja Kopp, Diederik van Bodegom, Matthew S. Davids, Jeremy Ryan, Liat Bird,Naveen Kommajosyula, Oliver Weigert, Akinori Yoda, Hua Fung, Jennifer R. Brown, Geoffrey I. Shapiro,Anthony Letai, and David M. Weinstock

AbstractBCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer

mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chroniclymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment.Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, whichsuppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphomacells. The BH3mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected byresistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On thebasis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would killPARP inhibitor–sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor–sensitivebreast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together,our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity andsuppresses PARP1-dependent repair. Targeted disruption of the BCL2–PARP1 interaction therefore mayrepresent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis. Cancer Res;72(16); 4193–203. �2012 AACR.

IntroductionMany tumors harbor genetic, epigenetic, or posttranscrip-

tional alterations that result in overexpression of the antia-poptotic protein BCL2 (1–3). In these tumors, BCL2 inhibitsapoptosis from a number of stresses, including DNA damage,microtubule perturbation, and oncogene activation (4), bybinding the BH3 (Bcl-2 homology-3) domain of proapoptoticfactors. Small molecules that occupy the BH3-binding pocketof BCL2, such as ABT-737 (5), are under development for thetreatment of malignancies dependent on BCL2 (6, 7). Theseagents function by disrupting the interaction between BCL2and proapoptotic BH3 domain–containing proteins, such asBIM. Upon displacement, certain proapoptotic proteins caninitiate oligomerization of the apoptosis effector proteins BAXand BAK.

Cells can overcome dependence on BCL2 in 3 ways (8). First,they can upregulate other antiapoptotic factors through cell-autonomous or non–cell-autonomous processes. For example,long-term selection of BCL2-overexpressing diffuse large B-celllymphoma (DLBCL) cells in the presence of ABT-737 promotesresistance through upregulation of the antiapoptotic BCL2family members MCL1 and/or BFL1 (9). Second, cells candownregulate proapoptotic signaling through changes ineither gene expression or posttranslational modifications.Third, cells can lose BAX and/or BAK and thereby becomeincapable of orchestrating intrinsic apoptosis. Each of thesemechanisms can confer broad resistance to apoptosis inducedby various stimuli, includingDNA-damaging agents andmicro-tubule binders (10, 11).

Essentially, all cases of follicular lymphoma and many casesof DLBCL, chronic lymphocytic leukemia (CLL), and othersubsets of non–Hodgkin lymphoma depend on BCL2 forsurvival (8). In the upfront setting, these diseases are broadlysensitive to an array of antineoplastic agents. However, relapseafter treatment with one or more therapeutic regimens iscommonly associated with resistance to multiple classes ofchemotherapy as a consequence of epigenetic, genetic, and/orposttranslational modifications in the intrinsic pathway ofapoptosis. Even in cells lacking autonomous resistance toapoptosis, bone marrow and lymphoid node stroma provideattachment sites and surface bound growth factors as survivalsignals that reduce the apoptotic response to a broad range ofchemotherapies (12, 13).

Authors' Affiliation: Departments of Medical Oncology and PediatricOncology, Dana-Farber Cancer Institute, HarvardMedical School, Boston,Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: David M. Weinstock, Dana-Farber Cancer Insti-tute, 450 Brookline Avenue, Dana 510B, Boston, MA 02115. Phone: 617-632-4245; Fax: 617-632-5167; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-4204

�2012 American Association for Cancer Research.

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Published OnlineFirst June 11, 2012; DOI: 10.1158/0008-5472.CAN-11-4204

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Novel approaches are clearly needed for inducing death intumor cells with resistance to apoptosis. Here, we identify anovel mechanism for target nonapoptotic cell death that isdependent on PARP1 and suppressed by BCL2.

Materials and MethodsThese studies were approved by the Dana-Farber Cancer

Institute (DFCI; Boston, MA) Institutional Review Board. All Pvalues were calculated by 2-sided t test using GraphPadsoftware. Additional details are provided in the SupplementaryMethods.

Cell culture and reagentsHT, Toledo, and OCI-LY8 were provided by Margaret Shipp.

1OCI-Ly1-7R, OCI-Ly1-10R, 1863, and 3256 were previouslydescribed (14, 15). Cell lines were not authenticated beforethese studies.N-Methyl-N 0-nitro-N-nitrosoguanidine (MNNG),etoposide, and hydrogen peroxidewere purchased fromSigma.BCL2-DKO cells were generated from a pool of DKO cellstransfected with pcDNA3.1-BCL2 and selected in 1 mg/mLG418 (Gold Biotechnology). ABT-737 and ABT-888 were kindlysupplied by Abbott Laboratories.

Cell death assayCells were treated with 500 mmol/L MNNG for 15 minutes,

washed with media, and cultured for 16 hours with dimethylsulfoxide (DMSO), 100 nmol/L ABT-737, 100 nmol/L ABT-888,or combination. Cells were stained with propidium iodide (PI;1 mg/mL; Roche) and Annexin V-fluorescein isothiocyanate(FITC; Upstate), and flow cytometry was conducted on aFACSCanto II (BD BioSciences) at the DFCI Division of Hema-tologic Neoplasia FACS Core Facility.

Determination of NAD and ATP concentrationsMouse embryonic fibroblasts (MEF) were treated 30 min-

utes with 500 mmol/LMNNG in the presence of ABT-737, ABT-888, DMSO, or combinations. MTT (Sigma) and CellTiter-GloLuminescent Cell Viability Assay (Promega) were used todetermine cellular NAD and ATP, respectively, using SoftMaxPro5.

ELISAIncreasing concentrations of purified glutathione S-

transferase (GST)-BCL2 were added to fractionated lysatesfrom HT cells. Similarly, increasing concentrations of ABT-737were added to fractionated lysates from OCI-Ly1-10R. Theprotein concentration of each fraction was measured usingthe bicinchoninic acid method (Thermo-Fisher). PARP1 enzy-matic activity on immobilized histones was measured usingan ELISA (Trevigen).

Comet assayOCI-LY1-10R cells were treated with 50 mmol/L MNNG for

15minutes. Cells were collected beforeMNNG treatment (pre),immediately after washing out MNNG (post-MNNG), or afterwashing followed by 3-hour treatment with DMSO, ABT-888(100 nmol/L), or ABT-737 (100 nmol/L). Cells were subjected tothe alkaline comet assay (Trevigen).

ImmunofluorescenceCells were fixed in 4% paraformaldehyde for 30 minutes at

room temperature and permeabilized for 10 minutes in 5%bovine serum albumin (BSA)/PBS containing 0.25% Triton X-100. Cells werewashedwith PBS containing 0.02%Triton X-100and incubated with antibodies diluted in 2.5% BSA/PBS con-taining 0.1% Triton X-100 overnight. Images were captured ona Zeiss Axio Observer.A1 fluorescence microscope. Confocalimages were captured on a Zeiss 710 laser scanning confocalmicroscope and analyzed using ImageJ software.

Primary CLL cell cultureThe day before the experiment, 5� 104 human stromal cells

per well were seeded onto 48-well plates. CLL cells werecollected from peripheral blood, isolated by Ficoll separation,and viably frozen in liquid nitrogen. On the day of the exper-iment, the CLL cells were thawed and added to the stromalculture at 20:1 ratio. For comparison, CLL cells were alsocultured in suspension at 3 � 106 cells/mL. For assessmentof stroma-derived resistance, 50 mmol/L MNNG, 100 nmol/LABT-737, and/or 200 nmol/L ABT-888 were added 24 hourslater. At the indicated time points, CLL cells were collected bygently pipetting off the CLL cells. After appropriate treatments,cells were washed once with PBS and resuspended in PBScontaining 1% FBS and FcR blocking reagent. Cells werestained with pacific blue-CD19 and APC-CXCR4 antibody todifferentiate B cells from stromal cells. Cells were resuspendedin 50 mL of Annexin-binding buffer and stained with AnnexinV-FITC and PI. Cell numbers were quantified after the additionof 2,500 Fluoresbrite-counting beads (Polyscience) per sample.Mitochondrial sensitivity was determined by BH3 profiling, asdescribed (11).

BCL2 transduction colony assayCellswere plated in 6-well plates (1,000 cells perwell) the day

before the retroviral infection. The cells were transduced and24 hours later and were selected in 1 mg/mL G418 (GoldBiotechnology). After 24 hours, cells were washed and incu-bated in the presence or absence of ABT-737. Cells werefixed inmethanol for 1 hour and stained with Giemsa.

ResultsOverexpressed BCL2 localizes in the nucleus of lymphoidtumor cells

Multiple previous studies have identified BCL2 within thenucleus of tumor cell lines and fibroblasts with high levels ofBCL2 expression (16–19). We first examined BCL2 localizationin the DLBCL cell lines OCI-LY1, OCI-LY8, and Toledo. All3 lines harbor translocation t(14;18), which results in expres-sion of BCL2 under IGH transcriptional control. Immunoblot-ting of nuclear and cytoplasmic fractions showed BCL2 withinthe nucleus of all 3 cell lines in the presence or absence ofMNNG, ionizing radiation (IR), or ABT-737 (Fig. 1A). Immu-nofluorescence of OCI-LY8 cells confirmed the nuclear local-ization of BCL2 (Supplementary Fig. S1). BCL2 also localized tothe nucleus in 2 murine B lineage leukemia lines that over-express BCL2 and MYC (15).

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Cancer Res; 72(16) August 15, 2012 Cancer Research4194




To determinewhether ectopically expressed BCL2 can local-ize to the nucleus, we transfected hemagglutinin (HA)-taggedBCL2 into 293T cells. Immunoblotting with anti-HA antibodyon cellular fractions revealed that BCL2 was present in thenucleoplasm before and after irradiation (Fig. 1B). Irradiationalso promoted the recruitment of BCL2 to chromatin (Fig. 1B).Even with ectopic expression, the nuclear and cytoplasmiclevels of BCL2 remained lower than those observed in OCI-LY1and OCI-LY8 cells (Supplementary Fig. S2).

BCL2 and PARP1 interact in DLBCL cellsLocalization of BCL2 to irradiated chromatin suggested that

BCL2 interacts with one or more factors involved in the DNAdamage response. To identify proteins in the chromatin frac-tion that interact with BCL2, we isolated the chromatin frac-tions from OCI-LY8 cells after irradiation and conductedimmunoprecipitation with an anti-BCL2 antibody (Fig. 1C).Mass spectrometry of a 113-kDa band present only in theirradiated chromatin fraction (Fig. 1C) identified 18 distinctpeptides from PARP1 with greater than 99% confidence (Sup-plementary Table S1).

PARP1 plays a role in several nuclear processes, includingbase excision repair, transcription regulation, DNA methyla-tion, and chromatin modeling (20). PARP1 responds to DNAdamage by using NADþ to transfer PAR to acceptor proteins,including histones and PARP1 itself.

The BCL2–PARP1 interaction is disrupted by ABT-737To determine whether the BCL2–PARP1 interaction

involves the BH3-binding groove of BCL2, we exposed OCI-LY8 cells to irradiation followed by a 30-minute treatment withDMSO, 100 nmol/L ABT-737 or 100 nmol/L of an inactive ABT-737 enantiomer (5). ABT-737 displaced approximately 65% ofPARP1 from BCL2 whereas the enantiomer had no effect (Fig.1D). ABT-737 had little or no effect compared with its inactiveenantiomer on the interaction between BCL2 and the nonho-mologous end-joining protein KU70 (Fig. 1D), which does notbind within the BH3-binding groove (18).

PARP1 undergoes auto-PARylation in response to DNAdamage. This creates a negatively charged scaffold that canmediate nonspecific protein interactions. Thus, the BCL2–PARP1 interaction could involve PAR, rather than PARP1 itself.

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Figure 1. Nuclear BCL2 interacts with PARP1. A, nuclear (N) and cytoplasmic (C) fractions from Em-MYC/MMTV-BCL2 B-cell acute lymphoblastic leukemias(1863 and 3256; ref. 15) were immunoblotted for BCL2, tubulin (cytoplasmic marker), and histone H3 (nuclear marker). DLBCL lines were analyzed byimmunoblotting 1 hour after irradiation or treatmentwithMNNG in the presenceor absenceof ABT-737.B,HEK293T cellswere transfectedwith an expressionplasmid for HA-BCL2, and fractionated lysateswere collected after 24 hours. C, OCI-LY8 cells were exposed to IR. After 30minutes, whole-cell lysates (WCL)or subcellular fractions were isolated and subjected to immunoprecipitation (IP) with anti-BCL2 antibody followed by separation on SDS-PAGE gel andCoomassie staining. Mass spectrometry of the approximately 113-kDa band (top arrow) revealed multiple peptides from PARP1. Mass spectrometryof the lower 2 bands indicatedby arrows identifiedmultiple histones. D,OCI-LY8 cells were irradiated and then treatedwith vehicle (DMSO), ABT-737 (737), anABT-737 enantiomer (enant), or ABT-888 (888). Subcellular fractions were immunoprecipitated with anti-BCL2 antibody and immunoblotted for BCL2,PARP1, or KU70. Loading and fractionation were confirmed by immunoblotting (Input). E, immunoblotting against PAR in the presence or absenceof 100 mmol/L MNNG, 100 nmol/L ABT-888, the combination, or 100 nmol/L ABT-737.

BCL2 Inhibits PARP1-Mediated Cell Death

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Treatment with the PARP1 inhibitor ABT-888 (21) completelyblocked MNNG-induced PARylation (Fig. 1E) but had no effecton the BCL2–PARP1 interaction (Fig. 1D), indicating that theinteraction with BCL2 is independent of PAR.

BCL2 interacts directly with PARP1 in vitroTo clarify whether PARP1 interacts with BCL2 directly or via

intermediary proteins, we conducted co-immunoprecipitationon a mixture of purified PARP1 and GST-tagged BCL2. In theabsence of DNA and NADþ, glutathione (GSH) beads coatedwith GST-BCL2 recovered PARP1 (Fig. 2A). In contrast, noPARP1was recoveredwhenGSHbeadswere coatedwith eitherGST-BCL-xL or GST-BCL-w (Fig. 2A), 2 BCL2 family membersthat are also bound by ABT-737 (5).

We conducted a fluorescence polarization assay, as previ-ously described (22), to confirm the interaction between full-length PARP1 and BCL2. As expected, full-length ABT-737

avidly interacted with BCL2 (Ki, 24.9 nmol/L) whereas thenegative control NOXA failed to interact with BCL2 (Fig. 2B).Consistent with the findings by co-immunoprecipitation, full-length PARP1 interacted with BCL2 (Ki, 77.0 nmol/L; Fig. 2B).The addition of ABT-737 displaced purified PARP1 from GST-BCL2 in a dose-dependent fashion (Fig. 2C), confirming theability of ABT-737 to directly disrupt the BCL2-PARP1interaction.

The PARP1 BRCT domain is required for interactionwith BCL2

Full-length PARP1 includes an N-terminal DNA-bindingdomain, a C-terminal catalytic domain, and central PADR,BRCT, and WGR domains (Fig. 2D). To map the region thatinteracts with BCL2, we generated His-tagged C-terminaldeletion mutants of PARP1 (Fig. 2D). Immunoblot analysiswith antibody to PARP1 confirmed all protein products

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Figure 2. ABT-737 disrupts the BCL2–PARP1 interaction. A, purified PARP1 was incubated with GST-tagged BCL2, GST-BCL-xL, or GST-BCL-w. Mixtureswere subjected to immunoprecipitation (IP) with anti-GST antibody followed by immunoblotting (IB) for PARP1. Ponceau staining was conducted onimmunoprecipitates to confirm equal recovery. B, fluorescence polarization assay of GST-BCL2mixedwith increasing concentrations of ABT-737, NOXA, orPARP1, as previously described (22). Error bars indicate SEM. C, GST-BCL2 and PARP1 were incubated along with increasing concentrations of ABT-737.Immunoprecipitation and immunoblotting were conducted as in A. D, schematic representation of full-length PARP1, deletion constructs, and brct domainconstructs. F1 and F2 are zinc finger 1 and zinc finger 2 domains, respectively. N indicates the nuclear localization signal. E, bacterially expressed PARP1proteins were separated on SDS-PAGE gel and immunoblotted with anti-PARP1 antibody. Arrows indicate the expected sizes. F, His-PARP1constructs were incubatedwith purifiedGST-BCL2 followed by immunoprecipitation with anti-His antibody and immunoblotting against BCL2. PurifiedGST-BCL2 was used as a control for BCL2 immunoblotting. Incubation of the His-DBRCT and GST-BCL2 was conducted in the presence or absence of100 nmol/L ABT-737. G, GST-BCL2 was incubated with brct-only or brctDBH3 constructs followed by immunoprecipitation and immunoblotting with anantibody specific for the PARP1 BRCT domain. Ponceau staining was conducted to confirm equal loading. WB, Western blotting.

Dutta et al.





(Fig.2E). We mixed equimolar amounts of each His-PARP1protein with BCL2 and conducted co-immunoprecipitation.Western blot analysis of BCL2 in eluted His-tagged proteincomplex revealed that a C-terminal deletion mutant lackingthe BRCT domain did not interact with BCL2 (Fig. 2F).To confirm the involvement of the BRCT domain, we

generated an internal BRCT domain deletionmutant (DBRCT),which also hadmarkedly reduced binding of BCL2 (Fig. 2F). Todetermine whether the PARP1 BRCT domain alone can bindBCL2, we generated a 15-kDa fragment that contains thePARP1 BRCT domain (Fig. 2D). After mixing with GSH beadscoated with GST-BCL2, immunoblotting with an antibodyspecific for the PARP1 BRCT domain showed that the BRCTdomain alone is sufficient to bind BCL2 (Fig. 2G). The PARP1BRCT domain contains a single BH3 motif (LXXXXD) begin-ning at codon 401. Mutation of both L410 and D406 to alaninesmarkedly reduced binding of BCL2 (Fig. 2G).

BCL2 blocks PARP1 enzymatic activity in DLBCL cellsTo determine whether the BCL2–PARP1 interaction affects

PARP1 function, we measured in vitro PARylation of immobi-lized histones within cell lysates from the DLBCL cell line HT,which lacks significant BCL2 expression (11). Addition ofpurified BCL2 reduced PARP1 activity in the nucleoplasm andchromatin fractions of HT cells (Fig. 3A). In contrast, addition

of MCL1, BCL-xL, or BCL-w had no effect on PARP1 activity(Fig. 3B). These results are similar to a previous report in HL60and U937 cells, in which ectopic overexpression of BCL2suppressed both basal PARP1 activity and the PARP1 responseto a topoisomerase II inhibitor (23).

To determine whether ABT-737 can increase PARP1 activityby displacing BCL2 from PARP1, we measured the effect ofABT-737 on PARP1 activity in fractionated lysates from OCI-LY8 cells. ABT-737 potentiated PARP1 enzymatic activity in adose-dependent manner in both nucleoplasm and chromatinfractions (Fig. 3A). Together, these findings indicate that theBCL2–PARP1 interaction blocks PARP1 activity, whereas ABT-737 can displace PARP1 from BCL2 and restore that activity.

Next, we asked whether inhibition of PARP1 function byBCL2 suppresses PARP1-dependent DNA repair. Assaying theeffects of BCL2 inhibition on DNA repair is complicated by thedependence of most BCL2-overexpressing cells on BCL2 forsurvival. We used OCI-LY1 and SU-DHL4 clones previouslyselected for resistance to ABT-737 by long-term culture in thepresence of the drug (14). These clones maintain BCL2 expres-sion but have also upregulated the antiapoptotic BCL2 familymembers MCL1 and/or BFL1 (14).

OCI-LY1-10R cells were treated for 15 minutes with MNNG,which is known to activate PARP1-dependent DNA repair(24). After washing away MNNG, the cells were allowed torepair the damage for 3 hours in the presence of vehicle, ABT-888, or ABT-737. We conducted the alkaline comet assay,which quantifies both single- and double-stranded DNAbreaks. Cells treated with ABT-737 had significantly smallercomet tails, indicating more extensive resolution of MNNG-induced DNA damage than in cells treated with MNNG andvehicle (Fig. 3C). The addition of ABT-888 did not furthersuppress repair, suggesting that most of the PARP1-mediatedrepair in these cells is inactive (Fig. 3B). Of note, neither ABT-737 nor MNNG affect the nuclear localization of BCL2(Fig. 1A).

BCL2 blocks PARP1-dependent, nonapoptotic deathin DLBCL cells

PARP1 enzymatic activity is required for a form of non-apoptotic cell death previously termed "parthanatos" (25–27).This pathway can be induced by ischemia or treatment withthe alkylating agent MNNG, both of which result in DNAdamage that requires PARP1-dependent repair. This form ofcell death does not involve the intrinsic apoptosis pathway, aswild-type and Bax�/�Bak�/� MEFs have similar sensitivity toMNNG-induced nonapoptotic death (24, 28).

We hypothesized that inhibition of PARP1 enzymatic activ-ity by BCL2 would suppress this form of nonapoptotic celldeath. Supporting this hypothesis, previous studies havereported that BCL2 can inhibit nitric oxide–induced, nona-poptotic cell death of PC12 and HeLa cells (29) as well asnonapoptotic death of neural cells induced by GSH depletion(30). We assayed the survival of ABT-737–resistant clonestreated with MNNG, ABT-737, ABT-888, or combinations (Fig.4A). As expected, very little cell deathwas observedwith single-agent ABT-737 (Fig. 4A). However, ABT-737 significantlyincreased death induced by MNNG in ABT-737–resistant

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Figure 3. BCL2 suppresses PARP1 enzymatic function. A, purified GST-BCL2 was added to fractionated lysates from HT cells or ABT-737 wasadded to fractionated lysates from OCI-LY8 cells. PARP1 activity onimmobilized histoneswasmeasuredbyELISA. Error bars represent SEM.B, GST-MCL1, GST-BCL-xL, and GST-BCL-w were added tonucleoplasm extracts from HT cells and PARP1 activity was assayed, asabove. C, OCI-LY1-10R cells treated with MNNG for 15 minutes werewashed and exposed to vehicle (DMSO), ABT-888, or ABT-737. Cellswere collected before MNNG treatment (pre), immediately after washingMNNG (post-MNNG), or 3 hours after wash. Olive moments werequantified for 500 cells in each category. Error bars indicate SEM.Pictures are �100 magnification.






OCI-LY1 and SU-DHL4 clones as well as Toledo cells (Fig. 4Aand B; P < 0.05 compared with MNNG plus vehicle in all lines).Importantly, MNNG þ ABT-737–induced death was partiallyreversed by ABT-888, supporting a specific role for PARP1 (Fig.4A and B).

Transmission electron microscopy of OCI-LY1-10R and SU-DHL4-2R cells treated with MNNG þ ABT-737 showed ultra-structural features of nonapoptotic cell death, including loss ofmembrane integrity with preservation of nuclear architecture(Fig. 4C). These findings are most consistent with necrotic celldeath. Double-membrane structures characteristic of autop-hagy were not present. In contrast, treatment with ABT-737plus the topoisomerase II poison etoposide resulted in featurescharacteristic of apoptosis, including chromatin condensationand preservation of membrane integrity (Fig. 4C). Thus, ABT-737 can promote PARP1-dependent nonapoptotic death inDLBCL cells.

We treated Bax�/�Bak�/�MEFs (referred to as "DKO cells")with MNNG and confirmed that nonapoptotic cell death isindependent of BAX and BAK (Fig. 5A and B). DKO cells thatstably overexpress BCL2 ("BCL2-DKO cells") were less sensitiveto MNNG (P < 0.05 compared with DKO cells; Fig. 5A),consistent with the inhibition of PARP1-dependent cell deathby BCL2. In both lines, MNNG-induced death was completelyblocked by ABT-888 (Fig. 5A). Thus, BCL2 inhibition canpromote at least 2 forms of cell death. The first is BAX/BAK-dependent intrinsic apoptosis, whereas the secondinvolves PARP1-dependent nonapoptotic death that is inde-pendent of BAX and BAK.

PARP1-mediated cell death induced by MNNG is charac-terized by reductions in cellular NADþ and ATP (24, 28). Weconfirmed that both NADþ and ATP concentrations aresignificantly reduced in DKO cells after 30-minute treatmentwith MNNG (Fig. 5C). In DKO cells, reductions in NADþ and

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Figure 4. BCL2 suppressesMNNG-induced death inDLBCL cells. A, cells were treatedwith indicated agents for 20 hours and analyzed by flowcytometry afterstaining with PI and FITC-conjugated Annexin V. Percent alive is the fraction of Annexin V/PI-negative cells relative to the same cell line treated withDMSOandnoMNNG. Error bars indicateSD. �,P<0.05. B, example of flowcytometric plots ofOCI-LY1-10Rcells treatedwith agents for 20 hours and stainedwith PI and Annexin V-FITC. C, transmission electron micrographs of OCI-LY1-10R cells fixed 9 hours after treatment with DMSO, 500 mmol/L MNNG,100 nmol/L ABT-737, 50 mmol/L etoposide, or combinations.

Dutta et al.





ATP after MNNG treatment were attenuated by the PARP1inhibitor ABT-888 but were unaffected by ABT-737 (Fig. 5C).MNNG treatment led to a lesser reduction of ATP levels inBCL2-DKO cells, consistent with the attenuation of PARP1activity by BCL2 (Fig. 5C). Unlike DKO cells, ABT-737 reducedthe levels of NADþ and ATP inMNNG-treated BCL2-DKO cells(P < 0.05 for both NADþ and ATP), and these effects weresignificantly reversed by ABT-888 (P < 0.05 for both NADþ andATP; Fig. 5C).

ABT-737 promotes nonapoptotic death in primaryCLL cells independent of stromal protectionWe hypothesized that nonapoptotic cell death induced by

MNNGþ ABT-737 could be used to overcome stroma-inducedresistance to apoptosis. CLL cells are known to be dependenton BCL2 and sensitive to treatment with ABT-737 (31). Cocul-ture of primary CLL cells with murine or human marrowstromal cells (HMSC) reduces apoptosis of CLL cells upontreatment with antineoplastic agents or ABT-737 throughupregulation of antiapoptotic BCL2 family members (12, 13).We cultured primary CLL cells from 14 patients (Supple-

mentary Table S2) with or without HMSCs for 48 hours (Fig. 6).As expected, cells cultured in the presence of human HMSCsdownregulated CXCR4 (Fig. 6A), the CLL cell surface homingreceptor for SDF-1/CXCR12 (12, 32). ABT-737 potently inducedmitochondrial depolarization in primary CLL cells cultured in

the absence of HMSCs (Fig. 6B), whereas cells cocultured withHMSCs had significant resistance to 100 nmol/L ABT-737 (P <0.01; Fig. 6C and D). In contrast, coculture with HMSCs did notblock the additive killing from the combination of 50 mmol/LMNNG plus 100 nmol/L ABT-737 but killing was reduced byABT-888 (P < 0.05; Fig. 6C and D), showing partial dependenceon PARP1. Of note, 100 nmol/L ABT-737 plus 50mmol/LMNNGresulted in only 20% loss of viability among the HMSC cells(data not shown).

BCL2 expression induces death in PARP inhibitor–sensitive cells

A logical extension of our findings that BCL2 overexpressionphenocopies PARP1 inhibition is that cells sensitive to ther-apeutic PARP inhibitors could be killed by overexpression ofBCL2. As a proof-of-principle, we retrovirally transduced BCL2into the BRCA1-mutated breast cancer cell line MDA-MB-436(33). Strikingly, ectopic expression of BCL2 completely blockedcolony formation, which was partially reversed by co-admin-istration of ABT-737 (Fig. 7A).

To expand this finding, we screened approximately 30 lungcancer cell lines for sensitivity to the PARP1 inhibitorAG014699. The lung cancer cell lines H520 (GI50, 30 nmol/L)and HCC827 (GI50, 65 nmol/L) were highly sensitive toAG014699, compared with insensitive lines such as H1299(GI50, >1 mmol/L; Fig. 7B; Supplementary Fig. S3). Similar to

Figure 5. ABT-737 promotesMNNG-induced cell death in BCL2-overexpressing cells that isindependent of BAX/BAK.A, Bax�/�Bak�/� MEFs (DKO) andBCL2-transgenic Bax�/�Bak�/�

MEFs (BCL2-DKO) were treated withindicated agents for 20 hours andanalyzed by flow cytometry afterstainingwith PI and FITC-conjugatedAnnexin V. Percent alive is thefraction of Annexin V/PI-negativecells relative to the same cell linetreated with DMSO and no MNNG.Error bars indicate SD. �, P < 0.05.B, transmission electronmicrographs at �890 and �2,900magnifications fixed 9 hours aftertreatment with the indicated agents.C, concentrations of NADþ and ATPwere determined in lysates isolatedafter 9-hour treatment with MNNGand the indicated agents. All valuesare relative to the same cell linetreated with vehicle and no MNNG.

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MDA-MB-436 cells, ectopic expression of BCL2 in the PARPinhibitor–sensitive cell lines reduced colony formation by 90%to 100% but did not affect colony formation in H1290 cells (Fig.7C). The effect from BCL2 was reversed by ABT-737 at ther-apeutic concentrations (Fig. 7C).

DiscussionBCL2 suppresses the intrinsic pathway of apoptosis by

binding proapoptotic BCL2 family members (8). We haveidentified 2 additional pathways that can modulate survivalthrough BCL2. First, inhibition of PARP1 activity by BCL2delays the repair of DNA damage as well as nonapoptotic celldeath. The pharmacologic BH3 mimetic ABT-737, which wasdesigned to induce BAX/BAK-dependent apoptosis in cellsdependent on BCL2 (5), can also promote PARP1-dependentnonapoptotic death by dissociating PARP1 from BCL2 andthereby restoring PARP1 enzymatic activity.

The second new pathway of survival modulation involvingBCL2 results from the ability of BCL2 overexpression tophenocopy PARP deficiency. More specifically, BCL2 overex-pression in PARP inhibitor–sensitive cells can promote celldeath, and this death can be reversed by interrupting theinteraction between BCL2 and PARP1. The "pro-death" effect

from BCL2 and the "pro-survival" effect from ABT-737 arespecific to cells that lack the ability to repair damage thatresults from PARP deficiency. In this model (Fig. 7D), basallevels of damage are repaired through PARP1 without drasti-cally affecting cellular energy stores. PARP1 deficiency, eitherthrough interaction with BCL2 or pharmacologic inhibition,results in the conversion of single-strand lesions to double-strand breaks, which can be repaired by homology-directedrepair (HDR; refs. 34, 35). Extensive DNA damage resultingfrom exogenous clastogens can drive both apoptosis andPARP1-mediated nonapoptotic cell death, which are bothsuppressed by BCL2.

The extent of damage necessary to induce PARP1-mediatednonapoptotic death within any particular cell is likely todepend on several factors, including energymetabolism, repairefficiency, threshold for undergoing apoptosis (36), and thedownstreameffectors of PARP1-dependent death. One testableprediction based on our findings is that tumors with BCL2overexpression must maintain some extent of HDR, as con-current deficiencies in HDR and PARP1 result in syntheticlethality (34, 35). Challenging our model, overexpression ofBCL2 or BCL-xL can suppress HDR in rodent (37) and human(38, 39) cells. In addition, BCL2 inhibits the formation of

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Figure 6. ABT-737 promotes nonapoptotic death in primary CLL cells independent of stroma. A, CLL cells were cultured with or without primary HMSCs andsurface CXCR4 expression was quantified by flow cytometry. APC, allophycocyanin. B, mitochondria from 6CLL samples was exposed to ABT-737, and therelative depolarization was quantified on the basis of cytochrome c release, as previously described (31). Cyanide-4-trifluoromethoxyphenyl-hydrazone(FCCP) was used as a positive control. C, CLL cells from patient D1001 were cultured in the presence or absence of HMSCs for 24 hours and then treatedwith DMSO, 100 nmol/L ABT-737, 50 mmol/LMNNG, 200 nmol/L ABT-888, or combinations, as shown. The cells were stained with FITC-conjugated AnnexinV, PI, and pacific blue–conjugated CD19 antibodies. CD19-positive cells were gated, and cells negative for both PI and Annexin V were considered alive.D, percent of cells alive under various treatment conditions for all 16 CLL samples. Bars indicate mean and 95% confidence interval. �, P < 0.05.

Dutta et al.





radiation-induced BRCA1 foci in human lymphoma cells (39),further suggesting that BCL2 may negatively modulate HDR.However, another study in human lymphoblastoid cells notedthe opposite effect, with a 3-fold increase in HDR upon BCL-xLoverexpression (40).A series of studies have reported additional effects of BCL2 in

various cell types onotherDNApathways, including nucleotideexcision repair, base excision repair, mismatch repair, andhomologous recombination (16–19, 37, 38, 41–43). Thedescribed effects from BCL2 include both direct binding anddecreased transcription of DNA repair factors. The extent towhich these effects are present within lymphoid tumor cellsand their contribution to sustaining DNA damage after treat-ment with clastogens have not been addressed.Several aspects of the BCL2-mediated suppression of PARP1

activity remain unclear. First, the precise function of the PARP1BRCT domain is poorly understood. In chicken DT40 cells, thePARP1 BRCT domain appears to be involved in mutagenicrepair at immunoglobulin loci, possibly through an interactionwith nonhomologous end-joining factors (44, 45). Aberrantsomatic hypermutation at oncogene loci is a common feature

of lymphomas with BCL2 overexpression (46). Thus, it isintriguing to speculate that BCL2-mediated suppression ofPARP1 function could promote these events by inhibitingmorefidelitous repair.

It also remains unclear whether targeting nonapoptoticdeath would offer an adequate therapeutic index, as somepopulations of nonneoplastic cellsmay be similarly sensitive toPARP1-mediated death. Zong and colleagues identified greatersensitivity to MNNG-induced death in proliferating versusvegetative cells, despite equivalent amounts of DNA damageand PARP activity (47). The greater sensitivity among prolif-erating cells resulted from dependence on glycolysis for thegeneration of ATP (i.e., the Warburg effect).

In conclusion, we have identified a novel interactionbetween BCL2 and PARP1 that blocks PARP1 enzymaticactivity and suppresses PARP1-dependent repair. Targetingof the BCL2–PARP1 interaction with BH3 mimetics is a poten-tial approach for killing BCL2-expressing tumors that areresistant to apoptosis, either through upregulation of otherantiapoptotic BCL2 family members or from defects in theintrinsic pathway (11, 13).

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Figure 7. Model for PARP1 involvement in DNA repair and cell death. A, MDA-MB-436 breast cancer cells were transduced with BCL2 or control virus andselected in neomycin in the presence or absence of increasing concentrations of ABT-737. The experiment was carried out in triplicate. B, sensitivity of lungcancer cell lines to the PARP1 inhibitor AG014699. A replicate experiment is shown in Supplementary Fig. S3. C, lung cancer cells were transducedand selected, as in A. Representative data of 3 independent experiments are shown. D, basal DNA damage from endogenous processes such as replicationrequires PARP1 for repair. In the absence of functional PARP1, single-strand lesions can become double-strand breaks that require HDR for resolution.In cells with impaired HDR, the accumulation of DNA double-strand breaks induces apoptosis. Similarly, extensive DNA damage from irradiation ortreatment with alkylating agents can induce apoptosis, which is suppressed by BCL2. The same DNA damage can initiate repair and nonapoptotic cell deathdependent on PARP1. The latter pathway is also suppressed by BCL2.






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

Authors' ContributionsConception and design: C. Dutta, N. Kommajosyula, A. Yoda, A. Letai, D.M.WeinstockDevelopment of methodology: C. Dutta, N. Kommajosyula, A. Yoda, H. Fung,D.M. WeinstockAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Dutta, T. Day, N. Kopp, M.S. Davids, J. Ryan, L. Bird,N. Kommajosyula, O. Weigert, J.R. Brown, G.I. ShapiroAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Dutta, N. Kopp, D. van Bodegom, M.S. Davids,J. Ryan, N. Kommajosyula, G.I. Shapiro, A. Letai, D.M. WeinstockWriting, review, and/or revision of the manuscript: C. Dutta, T. Day, M.S.Davids, J. Ryan, J.R. Brown, G.I. Shapiro, D.M. WeinstockAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N. Kopp, D. van Bodegom, J. Ryan,L. Bird, O. Weigert, A. Yoda, J.R. BrownStudy supervision: D.M. Weinstock

AcknowledgmentsThe authors thankMargaret Shipp and Bjoern Chapuy for providing reagents

and helpful discussion and Maria Ericsson, Simon Dillon, and John Daley forassistance with electron microscopy, mass spectrometry, and flow cytometry,respectively.

Grant SupportThisworkwas supported by aBurroughs-Wellcome FundCareer Award in the

Biomedical Sciences (6150801), the Claudia Adams Barr Program in CancerResearch, and the John Stellato Fund. T. Day is supported by an American CancerSociety Fellowship. N. Kommajosyula and G.I. Shapiro are supported by the DF/HCC SPORE in Lung Cancer P50 CA90578.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 27, 2011; revised May 15, 2012; accepted May 31, 2012;published OnlineFirst June 11, 2012.

References1. Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, et al.

miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc NatlAcad Sci U S A 2005;102:13944–9.

2. Tsujimoto Y, Gorham J, Cossman J, Jaffe E, Croce CM. The t(14;18)chromosome translocations involved in B-cell neoplasms result frommistakes in VDJ joining. Science 1985;229:1390–3.

3. Gao M, Lola CM, Wang M, Miller KD, Sledge GW, Hutchins GD, et al.Synthesis of carbon-11-labeled tricyclic necroptosis inhibitors as newpotential PET agents for imaging of tumor necrosis factor alpha (TNF-alpha). Appl Radiat Isot 2010;68:1950–8.

4. Chipuk JE, Moldoveanu T, Llambi F, ParsonsMJ, Green DR. The BCL-2 family reunion. Mol Cell 2010;37:299–310.

5. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ,Belli BA, et al. An inhibitor ofBcl-2 family proteins induces regressionofsolid tumours. Nature 2005;435:677–81.

6. Zeitlin BD, Zeitlin IJ, Nor JE. Expanding circle of inhibition: small-molecule inhibitors of Bcl-2 as anticancer cell and antiangiogenicagents. J Clin Oncol 2008;26:4180–8.

7. WilsonWH,O'ConnorOA,CzuczmanMS, LacasceAS,Gerecitano JF,Leonard JP, et al. Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study ofsafety, pharmacokinetics, pharmacodynamics, and antitumour activ-ity. Lancet Oncol 2010;11:1149–59.

8. Letai AG. Diagnosing and exploiting cancer's addiction to blocks inapoptosis. Nat Rev Cancer 2008;8:121–32.

9. Krumschnabel G, Ebner HL, Hess MW, Villunger A. Apoptosis andnecroptosis are induced in rainbow trout cell lines exposed to cad-mium. Aquat Toxicol 2010;99:73–85.

10. DelGaizoMooreV, Schlis KD, SallanSE, ArmstrongSA, Letai A.BCL-2dependence andABT-737 sensitivity in acute lymphoblastic leukemia.Blood 2008;111:2300–9.

11. Deng J, Carlson N, Takeyama K, Dal Cin P, Shipp M, Letai A. BH3profiling identifies three distinct classes of apoptotic blocks to predictresponse to ABT-737 and conventional chemotherapeutic agents.Cancer Cell 2007;12:171–85.

12. Kurtova AV, Balakrishnan K, Chen R, DingW, Schnabl S, Quiroga MP,et al. Diversemarrow stromal cells protect CLL cells from spontaneousand drug-induced apoptosis: development of a reliable and reproduc-ible system to assess stromal cell adhesion-mediated drug resistance.Blood 2009;114:4441–50.

13. Vogler M, Butterworth M, Majid A, Walewska RJ, Sun XM, Dyer MJ,et al. Concurrent up-regulation of BCL-XL and BCL2A1 inducesapproximately 1000-fold resistance to ABT-737 in chronic lympho-cytic leukemia. Blood 2009;113:4403–13.

14. YeciesD,CarlsonNE, Deng J, Letai A. Acquired resistance to ABT-737in lymphoma cells that up-regulate MCL-1 and BFL-1. Blood 2010;115:3304–13.

15. Brunelle JK, Ryan J, Yecies D, Opferman JT, Letai A. MCL-1-depen-dent leukemia cells are more sensitive to chemotherapy than BCL-2-dependent counterparts. J Cell Biol 2009;187:429–42.

16. Hou Y, Gao F, Wang Q, Zhao J, Flagg T, Zhang Y, et al. Bcl2 impedesDNA mismatch repair by directly regulating the hMSH2-hMSH6 het-erodimeric complex. J Biol Chem 2007;282:9279–87.

17. Jin Z,MayWS,Gao F, Flagg T, Deng X. Bcl2 suppresses DNA repair byenhancing c-Myc transcriptional activity. J Biol Chem 2006;281:14446–56.

18. Wang Q, Gao F, May WS, Zhang Y, Flagg T, Deng X. Bcl2 negativelyregulates DNA double-strand-break repair through a nonhomologousend-joining pathway. Mol Cell 2008;29:488–98.

19. Zhao J, Gao F, Zhang Y, Wei K, Liu Y, Deng X. Bcl2 inhibits abasic siterepair by down-regulating APE1 endonuclease activity. J Biol Chem2008;283:9925–32.

20. Krishnakumar R, Kraus WL. The PARP side of the nucleus: molecularactions, physiological outcomes, and clinical targets. Mol Cell 2010;39:8–24.

21. Penning TD, Zhu GD, Gandhi VB, Gong J, Liu X, Shi Y, et al. Discoveryof the Poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2-methyl-pyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-888) for thetreatment of cancer. J Med Chem 2009;52:514–23.

22. Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, KorsmeyerSJ. Distinct BH3 domains either sensitize or activate mitochondrialapoptosis, serving as prototype cancer therapeutics. Cancer Cell2002;2:183–92.

23. Kuo ML, Shen SC, Yang CH, Chuang SE, Cheng AL, Huang TS. Bcl-2prevents topoisomerase II inhibitor GL331-induced apoptosis ismedi-ated by down-regulation of poly(ADP-ribose)polymerase activity.Oncogene 1998;17:2225–34.

24. Challa S, Chan FK. Going up in flames: necrotic cell injury andinflammatory diseases. Cell Mol Life Sci 2010;67:3241–53.

25. Andrabi SA, Dawson TM, Dawson VL.Mitochondrial and nuclear crosstalk in cell death: parthanatos. Ann N Y Acad Sci 2008;1147:233–41.

26. David KK, Andrabi SA, Dawson TM, Dawson VL. Parthanatos, amessenger of death. Front Biosci 2009;14:1116–28.

27. Wang Y, Dawson VL, Dawson TM. Poly(ADP-ribose) signals to mito-chondrial AIF: a key event in parthanatos. Exp Neurol 2009;218:193–202.

28. Moubarak RS, Yuste VJ, Artus C, Bouharrour A, Greer PA, Menis-sier-de Murcia J, et al. Sequential activation of poly(ADP-ribose)polymerase 1, calpains, and Bax is essential in apoptosis-inducingfactor-mediated programmed necrosis. Mol Cell Biol 2007;27:4844–62.

29. Okuno S, Shimizu S, Ito T, Nomura M, Hamada E, Tsujimoto Y, et al.Bcl-2 prevents caspase-independent cell death. J Biol Chem 1998;273:34272–7.

Dutta et al.





30. Kane DJ, Ord T, Anton R, Bredesen DE. Expression of bcl-2 inhibitsnecrotic neural cell death. J Neurosci Res 1995;40:269–75.

31. Del Gaizo Moore V, Brown JR, Certo M, Love TM, Novina CD, Letai A.Chronic lymphocytic leukemia requires BCL2 to sequester prodeathBIM, explaining sensitivity to BCL2 antagonist ABT-737. J Clin Invest2007;117:112–21.

32. Arai F, OhnedaO,Miyamoto T, Zhang XQ, Suda T.Mesenchymal stemcells in perichondrium express activated leukocyte cell adhesionmolecule and participate in bone marrow formation. J Exp Med2002;195:1549–63.

33. Elstrodt F, Hollestelle A, Nagel JH, Gorin M, Wasielewski M, van denOuweland A, et al. BRCA1 mutation analysis of 41 human breastcancer cell lines reveals three new deleterious mutants. Cancer Res2006;66:41–5.

34. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, et al. Poly(ADP-ribose) (PAR) polymer is a death signal. Proc Natl Acad Sci U S A2006;103:18308–13.

35. Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, et al.Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci U S A 2006;103:18314–9.

36. Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, MooreVdel G, et al. Pretreatment mitochondrial priming correlates withclinical response to cytotoxic chemotherapy. Science 2011;334:1129–33.

37. Saintigny Y, Dumay A, Lambert S, Lopez BS. A novel role for the Bcl-2protein family: specific suppression of the RAD51 recombinationpathway. EMBO J 2001;20:2596–607.

38. Dumay A, Laulier C, Bertrand P, Saintigny Y, Lebrun F, Vayssiere JL,et al. Bax and Bid, two proapoptotic Bcl-2 family members, inhibithomologous recombination, independently of apoptosis regulation.Oncogene 2006;25:3196–205.

39. Laulier C, Barascu A, Guirouilh-Barbat J, Pennarun G, Le Chalony C,Chevalier F, et al. Bcl-2 inhibits nuclear homologous recombination bylocalizing BRCA1 to the endomembranes. Cancer Res 2011;71:3590–602.

40. Wiese C, Pierce AJ, Gauny SS, Jasin M, Kronenberg A. Gene conver-sion is strongly induced in human cells by double-strand breaks and ismodulated by the expression of BCL-x(L). Cancer Res 2002;62:1279–83.

41. Liu Y, Naumovski L, Hanawalt P. Nucleotide excision repair capacity isattenuated in humanpromyelocyticHL60cells that overexpressBCL2.Cancer Res 1997;57:1650–3.

42. KuoML,ShiahSG,WangCJ,ChuangSE.Suppression of apoptosis byBcl-2 to enhance benzene metabolites-induced oxidative DNA dam-age and mutagenesis: a possible mechanism of carcinogenesis. MolPharmacol 1999;55:894–901.

43. Youn CK, Cho HJ, Kim SH, Kim HB, Kim MH, Chang IY, et al. Bcl-2expression suppresses mismatch repair activity through inhibition ofE2F transcriptional activity. Nat Cell Biol 2005;7:137–47.

44. PaddockMN,BaumanAT,HigdonR,Kolker E, TakedaS, ScharenbergAM. Competition between PARP-1 and Ku70 control the decisionbetween high-fidelity and mutagenic DNA repair. DNA Repair (Amst)2011;10:338–43.

45. Paddock MN, Buelow BD, Takeda S, Scharenberg AM. The BRCTdomain of PARP-1 is required for immunoglobulin gene conversion.PLoS Biol 2010;8:e1000428.

46. Pasqualucci L,NeumeisterP,GoossensT,NanjangudG,Chaganti RS,Kuppers R, et al. Hypermutation of multiple proto-oncogenes in B-celldiffuse large-cell lymphomas. Nature 2001;412:341–6.

47. ZongWX, Ditsworth D, Bauer DE,Wang ZQ, Thompson CB. AlkylatingDNA damage stimulates a regulated form of necrotic cell death. GenesDev 2004;18:1272–82.






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