Histone Deacetylase Regulation of ATM-Mediated DNA ......Cancer Therapeutics Insights Histone...

Cancer Therapeutics Insights

Histone Deacetylase Regulation of ATM-Mediated DNADamage Signaling

K. Ted Thurn, Scott Thomas, Paromita Raha, Ian Qureshi, and Pamela N. Munster

AbstractAtaxia–telangiectasiamutated (ATM) is amajor regulator of the DNAdamage response. ATMpromotes the

activation of BRCA1, CHK2, and p53 leading to the induction of response genes such as CDKN1A (p21),

GADD45A, and RRM2B that promote cell-cycle arrest and DNA repair. The upregulation of these response

genes may contribute to resistance of cancer cells to genotoxic therapies. Here, we show that histone

deacetylases (HDAC) play a major role in mitigating the response of the ATM pathway to DNA damage.

HDAC inhibition decreasedATMactivation and expression, and attenuated the activation of p53 in vitro and in

vivo. Select depletion of HDAC1 and HDAC2 was sufficient to modulate ATM activation, reduce GADD45A

and RRM2B induction, and increase sensitivity to DNA strand breaks. The regulation of ATM by HDAC

enzymes therefore suggests a vital role for HDAC1 and HDAC2 in the DNA damage response, and the

potential use of the ATMpathway as a pharmacodynamicmarker for combination therapies involvingHDAC

inhibitors. Mol Cancer Ther; 12(10); 2078–87. �2013 AACR.

IntroductionAtaxia–telangiectasia mutated (ATM) is a major regu-

lator of the DNA damage response. ATM is a member ofthe phosphatidylinositol-3 kinase-related kinases family,whose auto-phosphorylation is promoted by the MRN(MRE11, Rad50, and NBS1) complex in response to DNAdouble-strand breaks (1). DNA damage signaling is thenpropagated byATM through activation of BRCA1, CHK2,and p53, leading to the induction of response genesinvolved in growth arrest, DNA repair, and/or apoptosis(1–3). The repair of double-strand breaks occurs primarilythrough homologous recombination and nonhomologousend joining (NHEJ).

Cells with defective ATM exhibit enhanced chromatindecondensation, increased genomic instability, and greatersensitivity to DNA damaging agents (4, 5). These featuresare similar to those observed in cells treated with histonedeacetylase (HDAC) inhibitors (6–9). Emerging evidencesuggests a role for HDACs in regulating ATM. ATM andHDAC1 associate in fibroblast cells, and their interactionincreases after g-irradiation (10). HDAC2 regulates theexpressionofchromatin remodelinggenes includingSMC1

(11), which is phosphorylated by ATM after the inductionof double-strand breaks (12).

HDACs catalyze the removal of acetyl groups fromhistone and nonhistone proteins alike, altering gene exp-ression and protein stability/function, respectively (13).The 18 human HDAC proteins are divided into 4 groupsincluding the Znþ-dependent class I, IIa, IIb, and IV, andthe NADþ-dependent class III HDACs. HDAC1 andHDAC2 are members of class I HDACs. HDAC expres-sion is frequently deregulated in human cancers, andseveral pharmacologic inhibitors of HDACs are under-going clinical testing (14, 15). The HDAC inhibitors vor-inostat and romidepsin have been approved by the USFood andDrugAdministration for the treatment of refrac-tory cutaneous T-cell lymphoma.

HDAC inhibitors sensitize cancer cells to DNA damag-ing therapies (e.g., irradiation and various chemotherapeu-tics) by altering chromatin structure and downregulatingDNA repair. HDAC inhibition reduced homologous rec-ombination in several cell lines (16, 17). A clear synergisticeffect has been showed in vivo when combining HDACinhibitors and anthracyclines (8, 18). Depletion of HDAC1andHDAC2by siRNA targeting also reducedhomologousrecombination, but had a greater effect on NHEJ (19).Furthermore, HDACs promote the stability and functionof proteins involved in the DNA damage response such asKu70 and p53 (20–22). Inhibition of class I/IIa HDACs byvalproic acid attenuated the activation of theMec1 [ataxia–telangiectasia and Rad3-related (ATR) ortholog] pathwayin the presence of DNA damage (23).

Clinical studies have showed a benefit in some patientsby adding HDAC inhibitors to therapeutic regimens thatinduce DNA damage (24). Epigenetic modulation isbelieved to play a role in therapy resistance, and these

Authors' Affiliation: Division of Hematology andOncology, Department ofMedicine, University of California, San Francisco, California

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

Corresponding Author: Pamela N. Munster, Division of Hematology andOncology, Department of Medicine, University of California, 1600 Divisa-dero, Room A719, Box 1711, San Francisco, CA 94143. Phone: 415-885-7810; Fax: 415-353-7779; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-1242

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 12(10) October 20132078

on May 19, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-1242

http://mct.aacrjournals.org/

clinical trials have showed responses in some patientswho have previously progressed on treatment (24). Theexact mechanisms governing HDAC inhibitor potentia-tion of DNA damage and their optimal use in the clinicalsetting are not yet fully understood (7, 25, 26). Therefore,we set out to investigate the role ofHDACs in the responseof cancer cells to chemotherapeutic induction of DNAdamage.Results from this study show that treatment with an

HDAC inhibitor caused reduced activation of ATM-mediated DNA damage signaling in various tumor celltypes. ATM downregulation via HDAC inhibition res-ulted in diminished DNA damage signaling and atten-uated the induction of p53 response genes. The inabilityto initiate a robust DNA damage response was associ-atedwith increased sensitivity to DNAdamaging agentsand persistence of DNA damage. Select depletion ofHDAC1 andHDAC2 (HDAC1/2) was sufficient to mod-ulate ATM expression and confer sensitivity to DNAdamage. Genetic depletion of ATM by siRNA mirroredthe phenotypic effects of HDAC inhibition. In addition,the results were recapitulated in vivo showing an HDACinhibitor-mediated reduction of DNA damage signal-ing. The relationship between ATM and HDAC1/2supports further investigation of ATM-dependent DNAdamage signaling in combination treatments includingHDAC inhibitor treatment. The results suggest thisHDAC inhibitor effect on DNA damage signaling maybe applied to any DNA double-strand break inducingtherapy.

Materials and MethodsChemicalsEntinostat (MS-275) was obtained from Selleck Chemi-

cals LLC, epirubicin from Calbiochem (EMD Chemical),dimethyl sulfoxide (DMSO) from MP Biomedicals LLC.Vorinostat was provided by Aton Pharma Inc. All otherchemicals were obtained from Sigma-Aldrich unless oth-erwise noted.

Cell culture and treatmentMCF-7, T-47D, SK-MEL-28, Saos-2, and A549 cell lines

wereobtained fromtheAmericanTypeCultureCollection(ATCC) and maintained in Dulbecco’s Modified EagleMedium high glucose (25 mmol/L) supplemented with10%FBS, 4mmol/L L-glutamine, 100 units/mLpenicillin,and 100 mg/mL streptomycin in 5% CO2 at 37�C. Celllineswere authenticated by short tandemrepeat profiling.For experiments, cells were treated for 48 hours withan HDAC inhibitor or vehicle (DMSO) before epirubicin(0.5 mmol/L).

DNA damage detection assayExpression of g-H2AX was detected using the Accuri 6

flow cytometer (BD Biosciences). Cells were treated withvehicle orVPA (2mmol/L) for 48 hours before the additionof epirubicin (0.5 mmol/L) for 4 hours. Cells were either

collected immediately, or washed to remove epirubicin,and allowed to recover for 12 hours. Collected cells werewashed, fixed in 3% paraformaldehyde, permeablized(0.5% saponin, 10 mmol/L HEPES, 0.14 mmol/L NaCl,and 2.5 mmol/L CaCl2), and incubated with fluoresceinisothiocyanate (FITC)-conjugated immunoglobulinG (IgG)or anti-phospho H2AX (Ser139) antibodies (Millipore).

Colony-forming assayApproximately 200 cells were seeded per well in a 12-

well plate. Cells were then treated with vehicle or vorino-stat (1.0 mmol/L) and epirubicin (50 nmol/L) for up to 14days. Cells were fixed in methanol and stained with 2%crystal violet. Colonies measuring at least 50 cells werecounted and normalized to plating efficiency (27).

Western blottingProtein was extracted in lysis buffer (0.1% SDS, 1%

triton X-100, 50 mmol/L Tris-HCl pH 7.4, 150 mmol/LNaCl, 10% glycerol), Halt protease and phosphataseinhibitor cocktail (Thermo Scientific), separated by SDS-PAGE, and transferred to Immobilon-P polyvinylidenefluoride microporous membranes (Milllipore). Mem-branes blocked in 5% nonfat milk were incubated withprimary antibodies p53 andATM (Abcam Inc.), phospho-p53 (S15), phospho-CHK2 (Thr68), and phospho-BRCA1(S1423; Cell Signaling Technology Inc.), p21 (Santa CruzBiotechnology), HDAC1, acetylated-Tubulin, and phos-pho-ATM (S1981; Millipore), E2F1 (BD pharmigen), andHDAC2, Acetyl-Histone H4, g-H2AX, GAPDH (UpstateBiotechnology). Membranes were incubated with horse-radish peroxidase–linked secondary antibodies and visu-alized using the ECL Plus Western Blotting DetectionSystem (GE Healthcare).

Immunofluorescence microscopyCells were washed and fixed in 4% paraformaldehyde

and permeabilized in 0.5% triton-X and blocked in 1%bovine serum albumin. Next they were incubated withprimary antibodies, washed, and incubated with FITC-labeled secondary antibodies. Cells were mounted inDAPI-supplementedmedia for imaging on the Zeiss AxioImager 2 (Carl Zeiss MicroImaging, LLC).

TransfectionFor siRNA experiments, cells were nucleofected

using the Amaxa Cell Line Nucleofector kit V (LonzaGroup Ltd.) in buffer containing 1 to 2 mmol/L of siRNApools or Silencer negative control #2 (Ambion, AppliedBiosystems). Pulsed cells were suspended in completemedia without antibiotics, and experiments were con-ducted 48 hours posttransfection. The pcDNA3.1(þ)Flag-His-ATM-wt plasmid was obtained from Addgeneand was originally created by the Kastan lab. Cells werenucleofected with 10 mg of plasmid. The followingday cells were treated with vehicle or vorinostat for48 hours before addition of epirubicin for an additional8 hours.

Histone Deacetylase Regulation of ATM Signaling

www.aacrjournals.org Mol Cancer Ther; 12(10) October 2013 2079




Quantitative real-time PCRRNA was extracted from cells using the RNeasy kit

(Qiagen). cDNA was generated using the iScript cDNAsynthesis kit (Bio-Rad Labs Inc.). Gene expression anal-ysiswas conducted using TaqMan qPCRgene expressionassays (MRE11, BRCA1, ATM, TP53, GADD45A, andRRM2B) on the ABI 7900 HT Thermocycler (AppliedBiosystems), and normalized to b-glucuronidase (h.Gus).

In vivo tumor xenograft studiesAthymic, nude, female mice (Taconic Farms Inc.) with

implanted estrogen pellets were injected with 1 � 107

MCF-7 breast cancer cells into the right flank.Whenmeantumor volumes reached 300 mm3, mice were randomizedinto 4 separate cohorts (n¼ 5–6) to be treatedwith vehicle,vorinostat, epirubicin, or vorinostat–epirubicin. Micewere treated by intraperitoneal injection with vehicle(10% DMSO, 45% PEG400, 45% H2O) or vorinostat (150mg/kg/day) for 2 days. On day 3, mice were treated withvehicle, vorinostat, and/or epirubicin (5mg/kg). Tumorswere harvested 8 hours after epirubicin administration

and flash frozen in liquid N2. Care of animals was inaccordance with institutional guidelines.

StatisticsData are expressed as means � SEM. A two-sided

nonpaired Student t test was used to determine differ-ences between 2 groups with P < 0.05 considered statis-tically significant. For multiple groups, the differenceswere measured by ANOVA using SPSS software.

ResultsHDACs regulate p53-dependent DNA damagesignaling

In the presence of DNA double-strand breaks, DNAdamage signaling includes p53 phosphorylation byATM,BRCA1, and CHK2. Phosphorylation of p53 at varioussites leads to enhancedprotein stability, increased nuclearretention, and greater DNA binding (28). The subsequentinduction of p53 response genespromotes cell-cycle arrestto permit DNA repair, and their upregulation may play a

Figure 1. HDACs regulate the DNA damage response. MCF-7 cells were treated with vehicle (DMSO) or vorinostat (Vor.) for 48 hours before adding epirubicin(0.5 mmol/L) for an additional 0 to 8 hours. Total cell lysate was processed for Western blotting examining the activation and stabilization of p53 due toincreasing lengths of exposure to epirubicin (A), and the concentration effect of vorinostat (B). C, cells grown on glass coverslips were treated as in Abefore fixation and immunofluorescence imaging of FITC-p53 and DAPI. D, cells treated as in A were analyzed for induction of p21 and acetylation of histoneH4 byWestern blotting. E, quantitative reverse transcription-PCR analysis for the induction ofGADD45A and RRM2B normalized to b-glucuronidase (h.Gus)for total RNA in cells treated as in A (�P < 0.05 vs. epirubicin-treated).

Thurn et al.

Mol Cancer Ther; 12(10) October 2013 Molecular Cancer Therapeutics2080




role in conferring chemotherapy resistance (29, 30).Because HDAC inhibitors sensitize tumor cells to DNAdamage andmayplay a role in overcoming chemotherapyresistance, we investigated the effect of pharmacologicHDAC inhibition on the DNA damage response.MCF-7breast cancer cells expressingwild-typep53were

exposed to 0.5 mmol/L of the topoisomerase II inhibitor,epirubicin, to induce DNA double-strand breaks. Thedamage caused by epirubicin triggered robust and time-dependent DNA damage signaling showed by increasedp53 Serine 15 (S15) phosphorylation and enhanced p53protein stability (0–8 hours; Fig. 1A). In contrast, cellspretreated with an HDAC inhibitor before the addition ofepirubicin showed a significantly altered DNA damageresponse. Exposure to therapeutic doses of the HDACinhibitor vorinostat (1 mmol/L) for 48 hours considerablyreduced the activation of p53 (Fig. 1A); whereas epirubicincaused p53 activation within 4 hours of treatment, cellspretreatedwithvorinostat hadundetectable levelsofphos-phorylated p53 at the same time point and significantlyreduced p53 activation after 8 hours (Fig. 1A). In addition,vorinostat pretreatment attenuated the stabilization oftotal p53protein inducedbyepirubicin exposure. Reducedp53 activation was detected in cells pretreated with vor-inostat concentrations as low as 0.2 mmol/L (Fig. 1B),which is well within the range of therapeutic concentra-tions (31).The attenuation of p53 phosphorylation caused by

HDAC inhibition in response to DNA damage was sup-ported by immunofluorescence microscopy (Fig. 1C).Nuclear accumulation of p53 significantly increasedafter exposure to epirubicin. Pretreatment of cells withvorinostat, however, significantly inhibited this epiru-bicin-induced nuclear localization of p53 (Fig. 1C).Activation and nuclear stabilization of p53 caused byDNA damage leads to induction of its response genesthat contribute to cell-cycle arrest and/or DNA repair.The cell-cycle checkpoint regulator CDKN1A (p21) canbe induced in a p53-dependent and independent man-ner. p21 was induced by exposure to vorinostat and toa greater extent by epirubicin (Fig. 1D). Interestingly,treatment of cells with the combination resulted inlower p21 induction compared to treatment with epir-ubicin alone. The acetylation of histone H4 inducedby vorinsotat was strongly inhibited by the additionof epirubicin (Fig. 1D). This is likely due to inhibition ofN-terminal lysine acetylation caused by binding ofepirubicin to histone proteins (32).The reduced activation of p53 caused by HDAC inhi-

bition attenuated the induction of p53 response genes,GADD45A andRRM2B. Exposure to epirubicin increasedthe expression of GADD45A and RRM2B mRNA in atime-dependent manner (Fig. 1E). Pretreatment withvorinostat, however, partially abrogated DNA damage-induced expression of GADD45A and RRM2B (Fig. 1E).This suggests anHDAC inhibitor-dependent attenuationof DNA damage signaling in the presence of genotoxicstress.

HDAC inhibitors attenuateATM-pathway activationTo identify the factors upstream of p53 that may be

influenced by HDACs, we investigated the activation ofATM, BRCA1, and CHK2 in the presence of vorinostat.Pretreatmentwith therapeutic doses of vorinostat reducedthe activation of ATM in the presence of DNA double-strand breaks (Fig. 2A). Phosphorylation of BRCA1 atS1423 and CHK2 at T68 was also reduced in the presenceof epirubicin after pretreatment with an HDAC inhibitor(Supplementary Fig. S1A and Fig. 2A). These sites aremodified byATM in the presence of double-strand breaks.ATM expression was reduced by HDAC inhibitor treat-ment in a dose (Fig. 2B) and time dependent (Fig. 2C andSupplementary Fig. S1B and S1C) manner. The effect ofvorinostat on ATM mRNA and protein expression wasalso observed in lung adenocarcinoma (A549), melanoma(SK-MEL-28), breast adenocarcinoma (T-47D), and osteo-sarcoma (Saos-2) cells (Fig. 2D and E). Reduced expressionof ATM in T-47D breast cancer cells also inhibited DNAdamage induced activation of p53 (Supplementary Fig.S1D).

HDAC inhibitors potentiate the effects of epirubicinand reduce DNA repair

Next we determined if the HDAC inhibitor–mediatedreduction of DNA damage signaling led to increased

Figure 2. HDAC inhibitors attenuate ATM pathway activation. A, cellspretreated with vehicle or vorinostat were exposed to epirubicin for 8hours before collection and analysis of whole-cell lysate by Western blotfor ATM and CHK2 phoshporylation. B, MCF-7 cells were treated withincreasing concentrations (48 hours) and time points of vorinostat(1 mmol/L). C, ATM expression in cell lines treated with vehicle orvorinostat were determined by quantitative reverse transcription-PCR formRNA normalized to b-glucuronidase (h.Gus; D) and for total proteinby Western blot (E; �P < 0.05 vs. 0.0 mmol/L vorinostat).






sensitivity to DNA damage. In agreement with previousresults, exposure to an HDAC inhibitor significantlyenhanced the cell-killing effects of topoisomerase II inhi-bition (20, 24, 25). Cells pretreated with vorinostat beforeepirubicin exhibiteddecreased cell survival and increasedapoptosis compared to cells treated with epirubicinalone (Fig. 3A–C). Vorinostat alone had little effect oncell viability at therapeutically relevant concentrations(1 mmol/L; Fig. 3A and C).

Vorinostat can itself affect chromatin stability andinduce DNA damage in breast cancer cells by inhibitionof HDAC3 (Supplementary Fig. S2A and S2B; ref. 33). Toseparate the effects of HDAC inhibitor–mediated induc-tion of DNA damage from its effect on DNA repair, cellswere treated with valproic acid that causes only minimalDNA damage (34). With a small increase in detectableDNA strand breaks after treatment with valproic acidalone, cells treated with epirubicin (0.5 mmol/L) for 4hours exhibited a significant induction of g-H2AX expres-sion (Fig. 3D). When epirubicin was removed to permitDNA repair, DNA damage persisted to a significantlygreater extent in cells that were treated with valproic acidcompared to vehicle-treated cells (Fig. 3D). Importantly,g-H2AX expression can occur in the absence of ATM dueto the overlapping function of DNA-PK (35).

HDAC1/2 regulate the DNA damage responseTo identify the HDAC enzymes that regulate the

expression of DNA repair genes, cells were treated withpan- or class-specificHDAC inhibitors. Breast cancer cellswere exposed to DNA damage after 48 hours of HDAC

inhibitor pretreatment and evaluated for DNA damagesignaling. At therapeutic doses, vorinostat acts as a pan-HDAC inhibitor targeting classes I, II, and IV (HDACs 1–11), whereas valproic acid inhibits classes I and IIa(HDACs 1–5 and 8), and entinostat (MS-275) inhibits classI HDACs (HDAC1–3), as well as HDAC9 (36). Epirubicintreatment elicited robust p53 phosphorylation (Fig. 4A)that was attenuated in the presence of all 3 HDAC inhi-bitors (Fig. 4A). Entinostat pretreatment was sufficient todelay epirubicin-induced p53 phosphorylation, suggest-ing the involvement of class I HDACs in DNA damagesignaling (Fig. 4A).

The DNA damage response activated by DNA double-strand breaks is promoted by the MRN complex (MRE11,Rad50, NBS1), ATM, BRCA1, and p53. To determine ifclass I HDACs affect the expression of these genes,MCF-7cells were transfectedwith siRNAs targeting HDAC1 andHDAC2, alone or simultaneously due to some functionalredundancy (37).WhenHDAC1 andHDAC2were simul-taneously depleted (HDAC1/2), there was a minor butreproducible, compensatory effect on HDAC2 comparedto knockdown of HDAC2 alone (Fig. 4B).

Previous studies have showed reductions in MRE11and Rad50 protein after vorinostat treatment in LNCaPand A549 cells (38). Similarly we observed decreasedMRE11 and Rad50 protein in MCF-7 breast cancer cellsfollowing vorinostat treatment (Supplementary Fig.S3A). Vorinostat treatment only caused a minimal dec-rease in MRE11 mRNA levels and had no effect onRad50 mRNA (Supplementary Fig. S3B). Select deple-tion of HDAC1/2 by RNAi had no effect on MRE11,

Figure 3. HDAC inhibitorspotentiate the effects of epirubicinand downregulate DNA repair. A,MCF-7 cells were treated withvorinostat (1 mmol/L) and/orepirubicin (50 nmol/L). After 10 to14 days, colonies (>50 cells) werecounted and normalized tocontrols. Surviving fractions werecalculated based on colony-forming capacity. B, images ofcolonies from A. C, cells weretreated with vehicle or vorinostatbefore the addition of epirubicinand scored by microscopy forapoptotic nuclei counting at least100 cells per condition. D,induction of g-H2A-FITCexpression as determined by flowcytometry for cells pretreated withvehicle or valproic acid (4 mmol/L)before the addition of epirubicin (4hours). Cells were either collectedimmediately and fixed, or washedto remove epirubicin to permitrecovery for 12 hours at 37�C.Cells were gated based on controlIgG-FITC treated cells analyzing atleast 0.8 � 105 to 1 � 105 percondition (�, P < 0.05 vs. epirubicintreated).

Thurn et al.





Rad50, or TP53 mRNA (Fig. 4C). In contrast, both ATMmRNA and protein levels were affected by HDAC1/2depletion (Fig. 4C and D). ATM mRNA was decreasedapproximately 32% after HDAC1/2 knockdown. BRCA1mRNA was only slightly reduced (�13%; Fig. 4C).Depletion of HDAC3, another class I HDAC, did notaffect the expression of any of the DNA damage res-ponse genes examined (data not shown). The reductionof ATM mRNA by pharmacologic HDAC inhibitionwas comparable to that detected after select depletionof HDAC1/2 by RNAi (Fig. 4C and SupplementaryFig. S1C).Next we tested whether the reduced ATM expression

caused by HDAC1/2 siRNA led to abrogated DNA dam-

age signaling. In fact, cells transfected with scrambledsiRNAs and exposed to epirubicin showed significantlyhigher levels of activated ATM and CHK2 comparedto those treated with HDAC1/2 siRNA pools (Fig. 4E).In addition, depletion of HDAC1/2 also reduced theinduction of p53 response genes GADD45A and RRM2B(Fig. 4F) and increased sensitivity to DNA damage(Fig. 4G).

Rescue of diminished DNA damage signaling byATM overexpression

To provide mechanistic insight into the HDAC inhib-itor-mediated reduction of DNA damage signaling, p53phosphorylation was evaluated after siRNA-mediated

Figure 4. HDAC1/2 regulate ATMexpression. A, after pretreatmentwith the HDAC inhibitorsvorinostat (1 mmol/L), valproic acid(1mmol/L), or entinostat (1mmol/L),cells were treated with epirubicin(Epi.) and analyzed byWestern blotfor activation and stabilization ofp53. B, cells nucleofected withsiRNA pools targeting HDAC1,HDAC2, both HDAC1/2 or controlscrambled sequences for 48 hourswere examined by quantitativereverse transcription-PCR for genemRNA expression normalized to h.Gus examining HDAC (C) and DNArepair genes (�, P < 0.05 vs.scramble. D, Western blot ofHDACs, ATM, and acetylatedtubulin expression from cells in B.Cells transfected as in B weretreated with vehicle or 0.5 mmol/Lepirubicin (8 hours) and examinedfor activation of ATM and CHK2 byWestern blot (E) and p53 responsegenes induction by quantitativereverse transcription-PCR (�, P <0.05 vs. epirubicin-treated; F). G,cell death was assayed by trypanblue inclusion in cells with depletedHDAC expression and treated withepirubicin (�, P < 0.05 vs.epirubicin-treated).






depletion of ATM. Concentrations of siRNAs were usedthat reduced ATM to levels comparable to those detect-ed with HDAC inhibitor treatment (Fig. 5A). Inductionof DNA damage by epirubicin was associated with signi-ficant phosphorylation of p53 in cells transfected withscrambled siRNA sequences, but not in those treatedwithsiRNAs against ATM (Fig. 5B).

To determine if the attenuated DNA damage signalingcaused by HDAC inhibitors could be rescued, cells weretransfected with wild-type ATM DNA whose constitu-tive expression was driven by the cytomegalovirus(CMV) promoter. The overexpression of ATM was suf-ficient to rescue the reduction in epirubicin-inducedDNA damage signaling detected after pretreatment withan HDAC inhibitor (Fig. 5C). Unexpectedly, in the ATMoverexpressing cells, the induction of CHK2 activationwas reduced in the presence of epirubicin despite robustphosphorylation of p53. The abundant ATM levels mayhave increased direct activation of p53, reducing thedependency on CHK2. Loss of DNA repair gene expres-sion caused byHDAC inhibitors is due in part to reducedE2F1-dependent transcription (16, 39). Finally, treatmentwith vorinostat (Fig. 5D) and HDAC1/2 depletion (Fig.5E) significantly reduced the expression of the E2F1 tra-nscription factor in the presence of damage. E2F1 pro-motes the expression ofATM(40). Thus, the expressionofATM was necessary and sufficient to promote robustDNA damage signaling in the context of combinationtherapy treatment.

HDAC inhibition mitigates DNA damage responseactivation in vivo

To confirm the regulation of DNAdamage signaling byHDACs in vivo, tumors from an MCF-7 breast cancerxenograft model were examined for their response toepirubicin in the presence or absence of an HDAC inhib-itor (Fig. 6A). Tumor bearingmice were treated for 2 dayswith 150mg/kg of vorinostat or vehicle. On the third day,mice were treated with vehicle, vorinostat, and/or epir-ubicin (5 mg/kg). Expression of p21 protein, an indicatorof HDAC inhibition (41), was increased 2.3-fold in thetumors of mice treated with vorinostat, versus thosetreatedwith vehicle (Fig. 6B). The level ofATMexpressionwas lower in the group treated with vorinostat than thecontrol-treated group (Fig. 6C). Vorinostat pretreatmentsignificantly attenuated p53 stabilization in the presenceof epirubicin (Fig. 6D). In fact, tumors from mice treatedwith the vorinostat–epirubicin combination had levels ofp53 similar to those of control treated mice (Fig. 6D).Vorinostat treatment alone did not increase p53 activationin vivo. These results emphasize the in vitro findings thatthe ATM-mediated DNA damage response is targeted byHDAC inhibition in the presence of genotoxic stress.

DiscussionAcquired and de novo resistance of tumor cells to DNA-

damaging modalities (e.g., irradiation, chemotherapeu-tics) can be the result of alterations to DNA damagesignaling and repair. Treatments targeting components

Figure 5. Rescue of diminishedDNA damage signaling by ATMoverexpression. A, nucleofectionof cells with siRNA pools againstATM or scrambled controlsequences were examined byquantitative reverse transcription-PCR for ATM/h.Gus expression(left) and by Western blot (right).B, DNA damage signaling in thepresence of epirubicin. C, cellswere transfectedwith plasmidDNAencoding constitutively expressedgreen fluorescent protein (GFP) orwild-type ATM (left). The followingday cells were treated with vehicleor vorinostat before the addition ofepirubicin (right). D, cells treatedwith vorinsotat or siRNAs againstHDAC1/2 (E) were compared tothose treated with vehicle orscrambled sequences,respectively, for DNA damagesignaling.

Thurn et al.





of these pathways such as poly (ADP-ribose) polymerase(PARP) and the CHK1/CHK2 proteins have beenexplored extensively (42, 43). Preclinical and clinical stud-ies have showed that HDAC inhibitors potentiate theeffects of DNA damage inducing therapies and maycontribute to overcoming therapy resistance (20, 44). Assingle agents, however, HDAC inhibitors have limitedtherapeutic efficacy against solid tumors, despite theireffects on chromatin stability and transcription. BaselineHDAC2 expression correlates with response to HDACinhibitor-anthracycline based regimens and plays animportant role in chromatin regulation (11, 31). Themech-anism of potentiation by HDAC inhibitors is not yet fullyunderstood. Furthermore, the lack of predictive markersin the clinical setting has hindered identification of opti-mal drug combinations and patient populations for noveltreatment regimens.Results from this study showed that inhibition of

HDACs significantly reduced the initiation of DNA dam-age signaling following epirubicin treatment in severaltumor types.We found that HDAC inhibition diminishedthe phosphorylation of ATM, BRCA1, CHK2, and p53 inresponse to DNA damage (Fig. 2). The reduced activationof these proteins occurred at early time points (0–6 hours)before expected contribution of ATR (45). Pharmacologic

or siRNA-mediated inhibition of HDAC1/2 caused asignificant deficiency in the induction of crucial p53response genes that regulate cell-cycle arrest and promoteDNA repair (i.e., GADD45A and RRM2B; ref. Fig. 4). Thisprevented cells from sufficiently inducing late-actingATM-mediated DNA repair pathways in the presence ofchemotherapeutically induced genotoxic stress. RRM2Bencodes p53R2, an enzyme that catalyzes the creation ofdeoxyribonucleoside diphosphates (dNTP precursors)required for DNA synthesis and repair (46). Consistentwith other reports, downregulation of RRM2B in MCF-7cells coincided with reduced DNA repair and increasedsensitivity to DNA damage (29).

The reduced DNA damage response caused by HDACinhibition seemed to be HDAC6 and HDAC8-indepen-dent because class I HDAC inhibitor, entinostat (MS-275),treatment was sufficient to mitigate epirubicin-inducedp53 activation in the presence of damage (Fig. 4A). Fur-thermore, select HDAC1/2 depletion attenuated ATMactivation and GADD45A and RRM2B induction. Theeffect on p53 response genes in these cells is likely indirectas HDAC1/2 knockdown did not significantly alter theirexpression in the absence of DNA damage (Fig. 4F).

Although vorinostat reduced MRE11 and Rad50 pro-tein levels, select HDAC1/2 depletion had no effect on

Figure 6. Vorinostat inhibits DNAdamage signaling in vivo. A, usingan MCF-7 breast cancer mousexenograft model, animals withsimilar mean tumor volumes wererandomized and separated intogroups to be treated for 2 dayswithvehicle or vorinostat (150 mg/kg),before treatment with vehicle,HDAC inhibitor, and/or epirubicin(5 mg/kg) on day 3. Tumors wereharvested and prepared foranalysis. B, mean levels ofp21/GAPDH protein levels intumors from mice in the vehicleversus vorinostat-treated groups.C, the expression of ATM in tumorsdetermined by Western blot. D,mean levels of stabilized p53protein in tumors quantified relativeto GAPDH.






their mRNA expression (Fig. 4C). Only ATM mRNA andproteinwere reducedbypharmacologic and siRNA-medi-ated inhibitionofHDAC1/2 in these cells.HDAC inhibitortreatment affected BRCA1 expression, but HDAC1/2knockdown had only a minor effect on its mRNA levels.HDAC inhibition reducedATMlevels, andpotentiated theeffects of DNA damaging agents. Importantly, HDACinhibitor treatment did not have a significant impact oncell viability at the therapeutically relevant concentrations(Fig. 2A and C). The loss of ATM was deleterious to cellsurvival only after induction of DNA damage. Thesefindings are consistent with the clinical observations thatHDAC inhibitormonotherapy is not an effective treatmentagainst solid tumors (47, 48).

Results from our group and others suggest that HDACinhibition downregulates DNA repair by modulating theexpression of DNA repair genes (16, 25, 49). Other reportshave showed that HDAC inhibitors can activate the DNAdamage response within 2 to 4 hours of treatment bycausing replication and transcription-associated damageinvolving HDAC3 (33, 50). Prolonged pharmacologicand siRNA-mediated inhibition of HDAC1/2, however,downregulates the expression of ATM (Fig. 2C), which isconsistent with reports showing reduced expression ofDNA repair and chromatin remodeling genes at compa-rable time points (8, 11, 38). This may explain the need forsequence-specific administration of HDAC inhibitorsbefore DNA-damaging therapies to achieve synergisticcell death (25). Reduced expression of DNA repair genesby HDAC inhibitors has been reported to be because ofdiminished recruitment of E2F1 to promoter regions(16, 39). Importantly, E2F1 also promotes the expressionof ATM (40) and we show a clear reduction of E2F1 byvorinsotat and HDAC1/2 knockdown in the presence ofgenotoxic stress (Fig. 5D and E).

Taken together, these results show that HDAC inhibi-tion in vitro and in vivo attenuates ATM-dependentDNA damage signaling in response to induction of DNAstrand breaks (Supplementary Fig. S4). Treatmentwith anHDAC inhibitor targets E2F1 andHDAC1/2 reducing theexpression of ATM and other DNA repair genes. Sub-

sequent treatments inducing DNAdouble-strand breaksthen induce an insufficient DNA damage response,resulting in sustained DNA damage, and increased celldeath. This suggests the need for further explorationinto ATM pathway activation as a potential pharmaco-dynamic marker for identifying patients most likely tobenefit from novel therapeutic approaches combiningHDAC inhibitors with DNA damage inducing modal-ities. Furthermore, this underlying mechanism suggeststhat the therapeutic addition of HDAC inhibition shouldnot be limited to topoisomerase II inhibitors, and shouldbe explored with other agents that induce DNA double-strand breaks.

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

Authors' ContributionsConception and design: K.T. Thurn, P.N. MunsterDevelopment of methodology: K.T. Thurn, S. Thomas, P.N. MunsterAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.T. Thurn, S. Thomas, I. Qureshi, P.N. MunsterAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K.T. Thurn, I. Qureshi, P.N. MunsterWriting, review, and/or revision of the manuscript: K.T. Thurn,S. Thomas, P. Raha, P.N. MunsterAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.T. Thurn, P.N. MunsterStudy supervision: K.T. Thurn, P.N. Munster

AcknowledgmentsThe authors thank the University of California, San Francisco Genome

and Laboratory for Cell Analysis Cores for their assistance, and alsoStephanie Chen and Nevetha Ganesan for their help.

Grant SupportThis work was supported by grants from the National Institutes of

Health (R01 CA122657-01) awarded to P.N. Munster. Work conducted byK.T. Thurn, S. Thomas, P. Raha, and I. Qureshi were supported by the R01CA122657 grant.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 3, 2013; revised July 24, 2013; accepted July 31, 2013;published OnlineFirst August 12, 2013.

References1. Lee JH, Paull TT. Direct activation of the ATM protein kinase by the

Mre11/Rad50/Nbs1 complex. Science 2004;304:93–6.2. Gatei M, Scott SP, Filippovitch I, Soronika N, Lavin MF, Weber B, et al.

Role for ATM in DNA damage-induced phosphorylation of BRCA1.Cancer Res 2000;60:3299–304.

3. Kurz EU, Douglas P, Lees-Miller SP. Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in partthrough the generation of reactive oxygen species. J Biol Chem 2004;279:53272–81.

4. Hittelman WN, Pandita TK. Possible role of chromatin alteration in theradiosensitivity of ataxia-telangiectasia. Int J Radiat Biol 1994;66:S109–13.

5. Lavin MF, Shiloh Y. The genetic defect in ataxia-telangiectasia. AnnuRev Immunol 1997;15:177–202.

6. Eot-Houllier G, Fulcrand G, Magnaghi-Jaulin L, Jaulin C. Histonedeacetylase inhibitors and genomic instability. Cancer Lett 2009;274:169–76.

7. Munshi A, Kurland JF, Nishikawa T, Tanaka T, Hobbs ML, Tucker SL,et al. Histone deacetylase inhibitors radiosensitize human melanomacells by suppressing DNA repair activity. Clin Cancer Res 2005;11:4912–22.

8. Marchion DC, Bicaku E, Daud AI, Sullivan DM, Munster PN. Valproicacid alters chromatin structure by regulation of chromatin modulationproteins. Cancer Res 2005;65:3815–22.

9. Ji P, Yeh V, Ramirez T,Murata-HoriM, LodishHF.Histone deacetylase2 is required for chromatin condensation and subsequent enucleationof cultured mouse fetal erythroblasts. Haematologica 2010;95:2013–21.

10. KimGD,Choi YH,DimtchevA, JeongSJ, Dritschilo A, JungM.Sensingof ionizing radiation-inducedDNAdamage byATM through interactionwith histone deacetylase. J Biol Chem 1999;274:31127–30.

11. Marchion DC, Bicaku E, Turner JG, Schmitt ML, Morelli DR, MunsterPN. HDAC2 regulates chromatin plasticity and enhances DNA vulner-ability. Mol Cancer Ther 2009;8:794–801.

Thurn et al.





12. KitagawaR, Bakkenist CJ,McKinnonPJ, KastanMB. Phosphorylationof SMC1 is a critical downstream event in the ATM-NBS1-BRCA1pathway. Genes Dev 2004;18:1423–38.

13. Thurn KT, Thomas S, Moore A, Munster PN. Rational therapeuticcombinations with histone deacetylase inhibitors for the treatment ofcancer. Future Oncol 2011;7:263–83.

14. Osada H, Tatematsu Y, Saito H, Yatabe Y, Mitsudomi T, Takahashi T.Reduced expression of class II histone deacetylase genes is associ-ated with poor prognosis in lung cancer patients. Int J Cancer2004;112:26–32.

15. Weichert W, Roske A, Gekeler V, Beckers T, Stephan C, Jung K, et al.Histone deacetylases 1, 2 and 3 are highly expressed in prostatecancer and HDAC2 expression is associated with shorter PSA relapsetime after radical prostatectomy. Br J Cancer 2008;98:604–10.

16. Kachhap SK, Rosmus N, Collis SJ, Kortenhorst MS, Wissing MD,Hedayati M, et al. Downregulation of homologous recombination DNArepair genesbyHDAC inhibition in prostate cancer ismediated throughthe E2F1 transcription factor. PLoS One 2010;5:e11208.

17. Adimoolam S, Sirisawad M, Chen J, Thiemann P, Ford JM, Buggy JJ.HDAC inhibitor PCI-24781 decreases RAD51 expression and inhibitshomologous recombination. Proc Natl Acad Sci U S A 2007;104:19482–7.

18. Marchion DC, Bicaku E, Daud AI, Sullivan DM, Munster PN. In vivosynergy between topoisomerase II and histone deacetylase inhibitors:predictive correlates. Mol Cancer Ther 2005;4:1993–2000.

19. Miller KM, Tjeertes JV, Coates J, Legube G, Polo SE, Britton S, et al.Human HDAC1 and HDAC2 function in the DNA-damage response topromote DNA nonhomologous end-joining. Nat Struct Mol Biol2010;17:1144–51.

20. Chen CS, Wang YC, Yang HC, Huang PH, Kulp SK, Yang CC, et al.Histone deacetylase inhibitors sensitize prostate cancer cells toagents that produce DNA double-strand breaks by targeting Ku70acetylation. Cancer Res 2007;67:5318–27.

21. Li D,MarchenkoND,Moll UM. SAHA shows preferential cytotoxicity inmutant p53 cancer cells by destabilizingmutant p53 through inhibitionof the HDAC6-Hsp90 chaperone axis. Cell Death Differ 2011;18:1904–13.

22. Harms KL, Chen X. Histone deacetylase 2 modulates p53 transcrip-tional activities through regulation of p53-DNAbinding activity. CancerRes 2007;67:3145–52.

23. Robert T, Vanoli F, Chiolo I, Shubassi G, Bernstein KA, Rothstein R,et al. HDACs link the DNA damage response, processing of double-strand breaks and autophagy. Nature 2011;471:74–9.

24. Munster P, Marchion D, Bicaku E, Schmitt M, Lee JH, DeConti R, et al.Phase I trial of histone deacetylase inhibition by valproic acid followedby the topoisomerase II inhibitor epirubicin in advanced solid tumors: aclinical and translational study. J Clin Oncol 2007;25:1979–85.

25. Marchion DC, Bicaku E, Daud AI, Richon V, Sullivan DM, Munster PN.Sequence-specific potentiation of topoisomerase II inhibitors by thehistone deacetylase inhibitor suberoylanilide hydroxamic acid. J CellBiochem 2004;92:223–37.

26. Singh S, Le H, Shih SJ, Ho B, Vaughan AT. Suberoylanilide hydro-xyamic acid modification of chromatin architecture affects DNA breakformation and repair. Int J Radiat Oncol Biol Phys 2010;76:566–73.

27. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C.Clonogenic assay of cells in vitro. Nat Protoc 2006;1:2315–9.

28. Dumaz N, Meek DW. Serine15 phosphorylation stimulates p53 trans-activation but does not directly influence interaction with HDM2.EMBO J 1999;18:7002–10.

29. Devlin HL, Mack PC, Burich RA, Gumerlock PH, Kung HJ, Mudryj M,et al. Impairment of the DNA repair and growth arrest pathways byp53R2 silencing enhances DNA damage-induced apoptosis in a p53-dependent manner in prostate cancer cells. Mol Cancer Res 2008;6:808–18.

30. Sato J, Kimura T, Saito T, Anazawa T, Kenjo A, Sato Y, et al. Geneexpression analysis for predicting gemcitabine resistance in humancholangiocarcinoma. J Hepatobiliary Pancreat Sci 2011;18:700–11.

31. Munster PN, Marchion D, Thomas S, Egorin M, Minton S, Springett G,et al. Phase I trial of vorinostat and doxorubicin in solid tumours:

histone deacetylase 2 expression as a predictive marker. Br J Cancer2009;101:1044–50.

32. KhanSN,DanishuddinM,VarshneyB, Lal SK,KhanAU. InhibitionofN-terminal lysines acetylation and transcription factor assembly byepirubicin induced deranged cell homeostasis. PLoS One 2012;7:e51850.

33. Conti C, Leo E, Eichler GS, Sordet O,MartinMM, Fan A, et al. Inhibitionof histone deacetylase in cancer cells slows down replication forks,activates dormant origins, and induces DNA damage. Cancer Res2010;70:4470–80.

34. Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G,et al. Interplay between oncogene-induced DNA damage responseand heterochromatin in senescence and cancer. Nat Cell Biol2011;13:292–302.

35. Stiff T, O'DriscollM, Rief N, Iwabuchi K, LobrichM, JeggoPA.ATMandDNA-PK function redundantly to phosphorylate H2AX after exposureto ionizing radiation. Cancer Res 2004;64:2390–6.

36. Khan N, Jeffers M, Kumar S, Hackett C, Boldog F, Khramtsov N, et al.Determination of the class and isoform selectivity of small-moleculehistone deacetylase inhibitors. Biochem J 2008;409:581–9.

37. Jurkin J, Zupkovitz G, Lagger S, Grausenburger R, Hagelkruys A,Kenner L, et al. Distinct and redundant functions of histone deacety-lases HDAC1 and HDAC2 in proliferation and tumorigenesis. CellCycle 2011;10:406–12.

38. Lee JH, Choy ML, Ngo L, Foster SS, Marks PA. Histone deacetylaseinhibitor inducesDNAdamage,which normal but not transformed cellscan repair. Proc Natl Acad Sci U S A 2010;107:14639–44.

39. Lopez G, Liu J, RenW,Wei W,Wang S, Lahat G, et al. Combining PCI-24781, a novel histone deacetylase inhibitor, with chemotherapy forthe treatment of soft tissue sarcoma. Clin Cancer Res 2009;15:3472–83.

40. Berkovich E,GinsbergD. ATM is a target for positive regulation byE2F-1. Oncogene 2003;22:161–7.

41. Gui CY, Ngo L, Xu WS, Richon VM, Marks PA. Histone deacetylase(HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1. Proc Natl Acad Sci U S A2004;101:1241–6.

42. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al.Inhibition of poly(ADP-ribose) polymerase in tumors fromBRCAmuta-tion carriers. N Engl J Med 2009;361:123–34.

43. Ashwell S, Zabludoff S. DNA damage detection and repair pathways–recent advances with inhibitors of checkpoint kinases in cancer ther-apy. Clin Cancer Res 2008;14:4032–7.

44. Nome RV, Bratland A, Harman G, Fodstad O, Andersson Y, Ree AH.Cell cycle checkpoint signaling involved in histone deacetylase inhi-bition and radiation-induced cell death. Mol Cancer Ther 2005;4:1231–8.

45. Siliciano JD, CanmanCE, Taya Y, Sakaguchi K, Appella E, KastanMB.DNA damage induces phosphorylation of the amino terminus of p53.Genes Dev 1997;11:3471–81.

46. Kimura T, Takeda S, Sagiya Y, Gotoh M, Nakamura Y, Arakawa H.Impaired function of p53R2 in Rrm2b-null mice causes severe renalfailure through attenuation of dNTP pools. Nat Genet 2003;34:440–5.

47. Blumenschein GR Jr, Kies MS, Papadimitrakopoulou VA, Lu C, KumarAJ, Ricker JL, et al. Phase II trial of the histone deacetylase inhibitorvorinostat (Zolinza, suberoylanilidehydroxamic acid, SAHA) in patientswith recurrent and/or metastatic head and neck cancer. Invest NewDrugs 2008;26:81–7.

48. Luu TH, Morgan RJ, Leong L, Lim D, McNamara M, Portnow J, et al. Aphase II trial of vorinostat (suberoylanilide hydroxamic acid) in meta-static breast cancer: a California Cancer Consortium study. ClinCancer Res 2008;14:7138–42.

49. Zhang Y, Carr T, Dimtchev A, Zaer N, Dritschilo A, JungM. AttenuatedDNA damage repair by trichostatin A through BRCA1 suppression.Radiat Res 2007;168:115–24.

50. Lee JS. Functional link between DNA damage responses and tran-scriptional regulation by ATM in response to a histone deacetylaseinhibitor TSA. Cancer Res Treat 2007;39:116–24.






2013;12:2078-2087. Published OnlineFirst August 12, 2013.Mol Cancer Ther K. Ted Thurn, Scott Thomas, Paromita Raha, et al. SignalingHistone Deacetylase Regulation of ATM-Mediated DNA Damage

Updated version

10.1158/1535-7163.MCT-12-1242doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/08/12/1535-7163.MCT-12-1242.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/12/10/2078.full#ref-list-1

This article cites 50 articles, 26 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/12/10/2078.full#related-urls

This article has been cited by 10 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/12/10/2078To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-12-1242

http://mct.aacrjournals.org/content/suppl/2013/08/12/1535-7163.MCT-12-1242.DC1

http://mct.aacrjournals.org/content/12/10/2078.full#ref-list-1

http://mct.aacrjournals.org/content/12/10/2078.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/12/10/2078


Histone Deacetylase Regulation of ATM-Mediated DNA ......Cancer Therapeutics Insights Histone...

Documents

Transcript of Histone Deacetylase Regulation of ATM-Mediated DNA ......Cancer Therapeutics Insights Histone...