Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine.

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LeukemiaResearch36 (2012) 607–618

ContentslistsavailableatSciVerseScienceDirect

Leukemia

Research

j ou rn a l h o m e pa g e : w w w . e l s e v i e r . c o m / l o c a t e / l e u k r e s

Proteomic

analysis

identiﬁes

differentially

expressed

proteins

in

AML1/ETO

acute

myeloid

leukemia

cells

treated

with

DNMT

inhibitors

azacitidine

and

decitabine

Francesca

Buchi,

Elena

Spinelli,

Erico

Masala,

Antonella

Gozzini,

Alessandro

Sanna,

Alberto

Bosi,

Germano

Ferrari,

Valeria

Santini

∗

FunctionalUnitofHematology,UniversityofFlorence,Florence,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received5August2011

Receivedinrevisedform28October2011 Accepted29November2011

Available online 9 January 2012

Keywords: AML Proteomics Azacitidine Decitabine

a

b

s

t

r

a

c

t

AzacitidineanddecitabineareDNAmethyltransferaseinhibitorsusedtotreatmyelodysplastic

syn-dromesandacutemyeloidleukemias.Tofurthercharacterizedifferentmechanismsbetweenthesetwo

agents,cellularextractsfromleukemiccellsuntreatedortreatedwitheitherdrugwereanalyzedusing

2Delectrophoresis.Numerousdifferentiallyexpressedproteinswereidentiﬁedwith

MALDI-TOF/TOF-MS.CyclophilinA,Catalase,NucleophosminandPCNAweredecreasedexclusivelybyazacitidine,TCP1

andhnRNPA2/B1bybothdrugs;alpha-EnolaseandPeroxiredoxin-1bydecitabine.Interestingly,the

expressionoftheproinﬂammatoryproteinCyclophilinA,alsosuggestedasmarkerofcellnecrosis,was

stimulatedbydecitabine.Finally,acomprehensivepathwayanalysisofdatahighlightedarelationship

betweentheidentiﬁedproteinsandpotentialeffectors.

1. Introduction

5-Azacitidineand 5-aza-deoxycytidine–decitabinearering analogsofcytosineanddeoxy-cytosine,differingfromthe natu-ralnucleosidebecauseofnitrogeninlieuofcarboninposition5of thepyrimidine.Theefﬁcacyofazacitidineanddecitabineas anti-neoplasticagents, widelyusedfor treatmentofmyelodysplastic syndromes(MDS)andacutemyeloidleukemia(AML)oftheelderly, isattributedtotwodistinctmechanisms:cytotoxicityand induc-tion of DNA hypomethylation [1,2]. Azacitidine is transformed intotheactivenucleotide,5-aza-2-deoxycytidine-5-triphosphate, through ATP-dependent phosphorylation by uridine-cytidine kinaseandsubsequentactivityofribonucleotidereductase. Previ-ousandrecentincorporationstudiesinleukemiccellshaveshown thatthedistributionofAZA innucleicacidsis65:35,RNA:DNA [3,4].Decitabine,quitedifferently,incorporatesprimarilyintoDNA after deoxycytidine kinase mediated phosphorylation. Regional hypomethylationisthoughttobetheprincipalandshared respon-siblefor theclinicalefﬁcacyof DNA methyltransferase(DNMT) inhibitors. In fact, once incorporated into DNA, by covalently trapping DNMT, both these drugs deplete the treated cells of functionallyactiveDNMT1,resultinginreversiblebutprofound

∗ Correspondingauthorat:FunctionalUnitofHematology,UniversityofFlorence, AOUCareggi,LargoBrambilla3,50141Florence,Italy.Tel.:+390557947296; fax:+390557947343.

E-mailaddress:santini@uniﬁ.it(V.Santini).

hypomethylation and re-expression of silenced oncosuppressor genes.

Therearefewdirectcomparisonsofthetwoagentsinvivoor invitro,buttheirdifferenceinmetabolismand DNA incorpora-tionsuggestspossiblediversemechanismsofactionandbiological effects[3,5,6].

WeandothershavepreviouslydemonstratedthatAML1/ETO positive leukemiccells arepreferentiallysensitivetoepigenetic drugs [7,8]and speciﬁcallytoDNMTinhibitors[9–11]. We uti-lized a two-dimensional electrophoresis system, followed by MALDI-TOF/MStoanalyzetheregulationofproteinexpressionin AML1/ETOpositivecells,tofurtherdeﬁnethemechanismofaction ofazacitidineanddecitabine,aswellaspossibledifferencesintheir biologicalactivityinthisparticularlysuitablecellularmodel. 2. Materialsandmethods

2.1. Cellsandcultureconditions

Kasumi-1cells(ahumanAML1/ETO-positivecelllinederivedfromanacute myeloblasticleukemia)wereplatedat3×105_cell/ml_in_RPMI₁₆₄₀_medium

sup-plementedwith10%FetalBovineSerum(FBS)(Gibco),4mMglutamine,50units/ml penicillinand50␮g/mlstreptomycinat37◦_C_in_a_humidiﬁed_atmosphere

contain-ing5%CO2.Cellsfromhumanbonemarrowaspirateswereobtainedafterinformed

consent(previouslyapprovedbythelocalEthicalCommittee),providedby2AML patientswitht(8;21),undergoingroutinediagnosticandinvestigationalprocedures. Inalltheexperiments,mononuclearcellsisolatedbydensitygradient centrifuga-tionwithLympholyte(CEDERLANE-Canada),andKasumi-1cellswereincubatedin thepresenceorabsenceoffreshlyprepared5-azacitidine(Sigma–Aldrich)1.0␮M anddecitabine(Sigma–Aldrich)0.5␮Mfor24h.Equipotentconcentrationsofdrugs werechosenonthebasisoftheirhypomethylatingactivityaspreviouslypublished

[3,12].

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608 F.Buchietal./LeukemiaResearch36 (2012) 607–618

2.2. Proteinextractionand2Delectrophoresis

Cellsafter24hincubationwerewashedtwiceincoldPBSplus100␮Msodium orthovanadateandProteaseInhibitorCocktail(Sigma).About20×106_cells_were

lysed,andafterultracentrifugation(100,000×g,1h,4◦_C),_protein_{concentration}

determinedbyBradfordassay(Roche).Two-dimensional(2D)electrophoresiswas performedaspreviouslydescribed[13].

2.3. Imageanalysisand2Dgelcomparison

Two-dimensionalgelimageswereacquiredbyamodiﬁedUMAXUtah.1100 ImageScanner(AmershamBiosciences,Uppsala,Sweden),at1200dpiof resolu-tion.GelswereanalyzedandcomparedusingthesoftwareImageMasterPlatinum Ed.(Version5.0,AmershamBiosciences,Uppsala,Sweden).Proteinabundancewas evaluatedbycomparingtheSpotStainingVolumeascalculatedbyImageMaster Platinumed.Gelswereruninduplicateforeachsampleandscatterplotv/vforthe matchingspotpoolswereobtainedtoverifythedegreeofreproducibilityamong tworeplicatesofeachsample.

2.4. Ingeldigestionandmassspectrometry

Spotstobeidentiﬁedwereexcisedfromthegels,de-stainedandsubjectedto trypticin-geldigestionwithSequencingGradeModiﬁedTrypsin(Promega,WI) at37◦_C_for₂_h._The_resulting_peptides_were_treated_by_Zip-Tip_C₁₈_(Millipore),

directlyelutedby2␮lofMatrixsolution(5g/l2,5-dihydroxybenzoicacidin50% ACN,0.1%TFA),on384position-400(m∅AnchorChipTarget(BrukerDaltonics, Bre-men,Germany).MassspectraforPeptideMassFingerprinting(PMF)wereacquired inreflectronpositive ionmodeonUltraflexMALDI-TOF/TOFMass Spectrome-ter(matrixassistedlaserdesorption/ionizationtime-of-flight,BrukerDaltonics, Bremen,Germany),withanaverageof100lasershots/spectrum.Spectrawere externallycalibratedusingacombinationoffourstandardpeptides:angiotensinII (1046.54Da),substanceP(1347.74Da),bombesin(1619.82Da)andACTH18-39Clip human(2465.20Da),spottedontopositionsadjacenttothesamples.Experimental PeptideMassFingerprintingintherangeof600–3500DawascomparedwithNCBI proteindatabasebythesoftwareMASCOT(www.matrixscience.org).Confirmatory fragmentationanalyses(MS/MS)wereperformedwhenneeded.

2.5. Westernblotting

Kasumi-1andhumanprimaryt(8;21)AMLcellswerewashedincoldPBS plus100␮MsodiumorthovanadateandProteaseInhibitorsCocktail(Sigma)and lysedbyincubatingat95◦CinLaemmlibuffer(62.5mMTris/HClpH6.8,10% glyc-erol,0.005%bluebromophenoland2%SDS).Proteinconcentrationwasdetermined byBCAassay(Pierce),30␮galiquotofeachsamplewasseparatedbySDS-PAGE (9,12.5or15%polyacrylamidegels)andthentransferredontoPVDFmembranes (Hybond-ECL;AmershamBiosciences)byelectroblotting.Membraneswere incu-batedwithprimaryantibodies(anti-calreticulin,anti-alpha-Enolase,anti-hnRNP A2/B1,anti-HSP60,anti-HSP70,anti-HSP90␣/␤,antiPRXI,antiTCP1␤,bySantaCruz Biotechnology;anti-cyclophilinA,anti-GAPDH,anti-PCNA,anti-␣-tubulin,byCell SignalingTechnology,anti-␤-tubulinbyUpstate,1:100016–18hat4◦_C_in_PBS

con-taining0.1%Tween20and5%BSA(T-PBS/5%BSA).Toverifyequalloadingofsample perlane,membraneswereincubatedinstrippingbuffer(62.5mMTris–HClpH6.7, 2%SDS,100mM2-mercaptoethanol;30minat50◦_C)_and_extensively_washed_with

T-PBS;thenthesamemembraneswereincubatedina1:1000dilutionofanti-p38 (CellSignalingTechnology)inT-PBS/%5BSA(16–18hat4◦C).Horseradish perox-idaseconjugatesecondaryantibodiesanti-rabbitIgG(Sigma)(1:5000inT-PBS/2% BSA;45minatroomtemperature)wereemployed.Immunoblotwerewashedin T-PBSandantibodycoatedproteinbandswerevisualizedbyECLchemiluminescence detection(AmershamBiosciences).

2.6. Dataanalysis

ScannedimageswereanalyzedbyImageMasterPlatinumEd.Version5.0 (AmershamBiosciences).Inordertoanalyzereproducibility,gelsimilaritiesor experimentalvariations,ScatterplotsanalysiswasperformedbysoftwareImage MasterPlatinumEd.whichcalculatesthelineardependencebetweenspotvaluesof onegelincomparisontoanothergel.Foreachproteinwecalculatedratiobetween spotvolumeinCTRLgelandspotvolumeintreatedgel.Aftermassspectrometry analysisandidentiﬁcationbysoftwareMASCOT(www.matrixscience.org),proteins wereclassiﬁedbyExPASyProteomicServer(http://expasy.org/)withUniProtKB database(http://www.uniprot.org).Functionalcategorieswereperformedusing DavidBioinformaticsResources6.7database(http://david.abcc.ncifcrf.gov/)and cutoffcriteriausedwereap-valueoflessthan0.001usingBenjamini–Hochberg correction.

Proteinnetworkanalyses– TheproteinsidentiﬁedbyMSwereincludedina pathwayanalysisusingtheMetaCoreTM_network_building_(tool_GeneGo_Inc.,_St.

Joseph,MI–http://www.genego.com/training.php).MetaCoreTM_is_formed_by_an

annotateddatabaseofproteininteractionsandmetabolicreactions.Genenamesof theallidentiﬁedproteinsandthecorrespondingmeanfold-changevaluesofsuch proteinswereimportedintoMetaCoreTM_and_processed_using_the_network_analysis

algorithm.Likethat,somenetworkswerebuilt.Pathwaysanddirectandindirect

associationsamongproteinswerealsoinvestigatedusingStringdatabases(Version 8.3,http://string-db.org/).

3. Results

3.1. ProteomiccomparisonaftertreatmentwithDNMTinhibitors Protein gels were reproducible, as veriﬁed by scatter plot v/v among the replicates of each condition of culture (Fig. S1, a and b – Supplementary data). Changes of protein expressionafter24hazacitidine anddecitabinetreatmentwere consideredsigniﬁcantwhentheirquantitydecreasedorincreased byatleast1.3-fold.

3.2. Treatmentwith5-azacitidine

Accordingtoimageanalysis,620spotswereconstantlydetected incontrolgeland486spotsintreatedcellgelandwerequantified, normalized,intergelmatched,yielding400pairsofproteins. Sixty-fourprotein spots had significantvolume differences occurring betweenuntreatedcontrolandazacitidinetreated,andweexcised 51proteinspotsfromcontrolgeland13spotsfromtreated-cellgel. MassspectrometryanalysisbyMALDI-TOFallowedusto success-fullyidentify43proteinspotswhoselevelschanged(Fig.1A,aand b)correspondingto30characteristicproteins,classifiedin func-tionalcategoriesbyDavidBioinformaticsdatabase.Resultswere reportedin Table1: Spot numberobtainedfrom ImageMaster correlatestocorrespondingspotsinFig.2A.

As shown in Fig. 1B, nine proteins (30%) identified by MS included molecules functionally classified as Heat Shock Pro-teins/Chaperons,nineproteins(30%)wereenzymes,mostofwhich metabolic,oneproteinwasinvolvedinstress/redoxhomeostasis, five(17%) werestructuralproteinsand sixproteins(20%)were involvedinsignaltransduction(Table1andSupplementarydata). Themajorityofthedetectedproteinsdecreasedafterazacitidine treatment,asshowedbytheratioofthevolumeofspotsintreated anduntreatedcells(Table1)and,afterStringanalysis,weobserved thatallproteinswerelinkedbydifferentcharacteristics,andwere involvedin severalneoplasticdiseases(Fig.S2,a– Supplemen-tarydata).

Differential expression of PCNA, TCP1, hnRNP A2/B1 and CyclophilinAwasconﬁrmedbywesternblot(Fig.1C, aandb). CyclophilinA,inparticular,wasidentiﬁedbymorethanonespotin 2Dmaps,becauseofitsdifferentisoformswhichcouldbepresents, anditsglobalexpressioninazacitidinetreatedcellswaslowerthan that inuntreated cells. Regardingbiological variation ofhnRNP A2/B1,westernblotanalysisshowedaninhibitionofexpression ofribonucleoproteinB1andanincreaseofA2,butglobalprotein expressiondecreased(ratioofdensitometryCTRL/AZA:1.3,respect toloadingcontrol)(Fig.1C,aandb).

We observed in 2D gels different spots of alpha-Tubulin, beta-Tubulin,alpha-Enolase,ACTB,HSP90,HSP70andCalreticulin (Table1–markedwithanasterisk),duetothepresenceofseveral isoforms.Forthisreason,globalexpressionoftheseproteinswas unmodiﬁedafterazacitidine(Fig.1C,c).Isoformsoftheseproteins, wellseparatedby2Delectrophoresis,in1Dwesternblotclustered inthesameband,precludingthevalidationofdifferencein expres-sion.

ThedecreaseofCyclophilinA,PCNAandTCP1betawas con-ﬁrmedbywesternblotwithspeciﬁcantibodiesalsoinprimary culturesof2AMLt(8;21)casesafterexposureofleukemiccells toazacitidine(Fig.1D,aandb).

3.3. Treatmentwithdecitabine

Afterimageanalysis,576spotsweredetectedincontrolcells and596spotsintreatedcells;twogelswerematchedand416pairs

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F. Buchi et al. / Leukemia Research 36 (2012) 607– 618 609 Table1

Differentiallyexpressedproteinsidentiﬁedbymassspectrometryafterazacitidinetreatment.

SpotCTRL SpotAZA Accession numbera

HeatShockProteins/Chaperons MWb _pIc _SCOREd _Vol._C/Vol._Ae _Induced_or

overexpressedbyAZA

Inhibitedor reducedbyAZA

C98 A69 P14625 Endoplasmin* 92,637 4.73 155 1.2 X

C96 A61 P14625 Endoplasmin* 90,351 4.73 32 0.4 X

C9 P07900 Heatshockprotein90kDaalpha,classBmember1* 83,638 4.94 75 CTRL X

C121 A82 P07900 Heatshockprotein90kDaalpha,classBmember1* 83,638 4.94 173 2.1 X

C139 A94 P07900 Heatshockprotein90kDaalpha,classBmember1* 83,638 4.94 69 0.5 X

C113 A83 P07900 Heatshockprotein90kDaalpha(cytosolic),classBmember1* 83,638 4.94 46 0.5 X

C181 A125 P11142 Heatshock70kDaprotein8* 71,138 5.37 186 2.5 X

C178 A123 P11142 Heatshock70kDaprotein8* 71,138 5.37 49 0.8 X

C216 A151 P10809 Heatshockprotein60 61,388 5.24 219 1.0

C249 A184 P78371 ChaperonincontainingTCP1,subunit2(beta) 57,878 6.02 153 1.8 X

C173 A119 P38646 Heatshock70kDaprotein9 74,089 5.97 117 1.2 X

C862 A528 P62937 Cypa(CyclophilinA)* 18,154 7.82 146 0.8 X

C263 A173 P27797 Calreticulin* 48,325 4.28 75 2.2 X

A306 P27797 Calreticulin* 48,325 4.28 46 AZA X

C235 P30101 Protien-disulﬁdeisomeraseA3 56,761 5.61 83 CTRL X

Enzymes

C295 A208 P06733 Alpha-Enolase* 47,538 6.99 61 0.4 X

C292 A213 P06733 Alpha-Enolase* 47,538 6.99 94 1.5 X

C268 A199 P07237 Prolyl4-hydroxylasebeta-subunit 57,578 4.69 211 1.4 X

C236 A163 P12268 Inosinemonophosphatedehydrogenase2 56,338 6.44 115 1.5 X

C470 A355 P04406 Glyceraldehyde-3-phosphatedehydrogenase 36,244 8.58 91 1.5 X

C546 A415 P60174 ChainB,TriosephosphateIsomerase(Tim) 26,877 6.51 191 1.2 X

C538 A406 P28066 Proteasomesubunit,alphatype,5 26,621 4.74 46 1.4 X

C815 P55072 Valosin-containingprotein 71,660 5.14 49 CTRL X

C665 A487 P15531 Non-metastaticcells1,protein(NM23A)isoforma 19,925 5.84 64 2.7 X

C226 A157 P04040 Catalase 59,947 6.95 93 1.8 X

Cellredoxhomeostasis

C596 A446 P09211 GlutathioneS-transferaseP 23,651 5.44 86 1.4 X

Structuralproteins

C253 A191 P68363 Alphatubulin* 50,972 4.94 149 1.9 X

C260 A194 P68363 Alphatubulin* 50,972 4.94 106 0.5 X

C280 A204 P68363 Alphatubulin* 50,972 4.94 60 0.9

C445 A334 P07437 Betatubulin* 48,233 4.78 182 1.6 X

C421 P07437 Betatubulin* 48,233 4.78 127 CTRL X

C284 P07437 Betatubulin* 48,233 4.78 70 CTRL X

C365 A274 P60709 ACTBprotein(Beta-actin)* 40,620 5.29 142 1.8 X

C330 A251 P60709 ACTBprotein(Beta-actin)* 40,620 5.29 48 0.8 X

C413 A314 P52907 F-actincappingproteinalpha1 33,115 5.45 118 2.7 X

C475 A362 Q8TCG3 Tropomysin,TPMsk3 28,934 4.72 74 0.9

DNAbinding/signaltransduction

C278 A201 Q09028 Retinoblastomabindingprotein4variant 47,910 4.74 67 1.1

C400 A299 P06748 Nucleophosmin 32,653 4.64 76 1.3 X

C463 A350 P12004 Pcna 29,156 4.57 154 2.5 X

C424 A321 P22626 Heterogeneousnuclearribonucleoproteins(hnRNP)A2/B1 37,464 8.97 95 2.2 X

A541 P62158 Calmodulin 17,152 4.09 48 AZA X

a_Accession_number_by_UniProtKB.

b _MW_–_theoretical_molecular_weigh_obtained_by_ExPASy_Proteomic_Server. c _pI_–_theoretical_isoelectric_point_obtained_by_ExPASy_Proteomic_Server. d _Score_–_index_calculated_by_{MASCOT-signiﬁcant}_when_greater_than₅₆_(p_<_0.05). e_Ratio_between_spot_volume_in_CTRL_and_spot_volume_in_treated_gels.

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610 F. Buchi et al. / Leukemia Research 36 (2012) 607– 618 Table2

Differentiallyexpressedproteinsidentiﬁedbymassspectrometryafterdecitabinetreatment.

SpotCTRL SpotDAC Accession numbera

HeatShockProteins/Chaperons MWb _pIc _Scored _Vol._C/Vol._De _Induced_or

overexpressed by DAC

Inhibitedor reduced by DAC

C1591 D43 P07900 Heatshockprotein90kDaalpha(cytosolic),classBmember1* 83,638 4.94 89 1.4 X

C1609 D66 P07900 Heatshockprotein90kDaalpha(cytosolic),classBmember1* 83,638 4.94 85 2.3 X

C1614 D73 P07900 Heat shock protein 90 kDa alpha (cytosolic), class B member 1* 83,638 4.94 93 0.2 X

C1689 D157 P10809 Heatshockprotein60 61,388 5.24 147 1.6 X

C1651 D119 P11142 Heatshock70kDaprotein8* 71,138 5.37 161 1.3 X

C2315 D71 P11142 Heat shock 70 kDa protein 8* 71,138 5.37 101 0.5 X

C1674 D155 P27797 Calreticulin 48,283 4.28 90 4.6 X

C1713 D184 P30101 Protein-disulﬁdeisomeraseA3* 56,747 5.61 82 1.5 X

C1768 D251 P30101 Protein-disulﬁde isomerase A3* 57,146 5.61 72 2.2 X

C1752 D225 P30101 Protein-disulﬁde isomerase A3* 57,146 5.61 88 1.2 X

C2007 D660 P30040 EndoplasmicreticulumproteinERp29 29,032 6.08 70 1.8 X

C2119 D565 P23284 Cyclophilin B 22,785 9.25 122 1.3 X

C2178 P62937 Cyclophilin A* 18,229 7.82 61 CTRL X

C2183 D606 P62937 CyclophilinA* 18,229 7.82 75 0.4 X

C2184 D612 P62937 Cyclophilin A* 18,229 7.82 91 1.4 X

C1732 D204 P78371 Chaperonin containing TCP1, subunit 2 (beta) 57,878 6.02 125 1.3 X

Enzymes

C1824 D283 P00558 Phosphoglyceratekinase1 44,985 8.30 100 3.9 X

C1875 D347 P06733 Alpha-Enolase* 47,481 6.99 121 2.4 X

C1778 D237 P06733 Alpha-Enolase* 47,481 6.99 195 2.1 X

D256 P07954 Fumaratehydratase,mitochondrial 54,773 6.99 55 DAC X

C1707 D160 P04040 Catalase 59,947 6.95 62 0.8 X

C1925 D403 Q15181 Inorganic pyrophosphatase 33,095 5.54 120 1.6 X

C1927 D402 Q15181 Inorganicpyrophosphatase 33,095 5.54 108 1.4 X

C1970 D424 P14207 Folatereceptorbeta 30,286 7.47 32 0.6 X

C2142 D593 Q8TAE6 Protein phosphatase 1 regulatory subunit 14 C 17,946 5.09 31 0.8 X

C1900 D363 P04406 Glyceraldehyde-3-phosphatedehydrogenase 36,201 8.58 59 1.5 X

C2037 D489 P60174 ChainB,TriosephosphateIsomerase(Tim) 26,877 6.51 155 1.2

C1693 D137 Q02318 Cytocrome P450 27, mitochondrial 60,595 8.43 27 0.8 X

Cellredoxhomeostasis

C2060 D522 P09211 Glutathione S-transferase P 23,569 5.44 58 1.4 X

C2072 D531 Q06830 Peroxiredoxin-1 22,324 8.27 81 2.3 X

C1848 D331 O76003 Glutaredoxin-3 37,693 5.31 83 2.0 X

Structuralproteins

C2323 D304 P60709 ACTBprotein(Beta-actin) 40,620 5.29 163 1.9 X

C1906 D375 P07437 Beta tubulin* 48,233 4.78 154 1.7 X

C1915 D391 P07437 Betatubulin* 48,233 4.78 58 1.1 X

C2269 D205 P68363 Alphatubulin 50,972 4.94 89 0.3 X

DNAbinding/signaltrasduction

C1995 D459 P35232 Prohibitin 29,843 5.57 142 1.6 X

C1933 D412 P12004 Pcna 29,156 4.57 127 0.8 X

C1888 D345 Q13347 Eukaryotictranslationinitiationfactor3subunitI 36,878 5.38 92 1.8 X

C1926 D394 P22626 Heterogeneous nuclear ribonucleoproteins A2/B1 37,464 8.97 112 1.4 X

C1940 D388 P09651 HeterogeneousnuclearribonucleoproteinA1 38,936 9.17 132 1.4 X

C1834 D307 P29992 Guaninenucleotide-bindingproteinsubunitalpha-11 42,382 5.51 54 2.0 X

C2035 D491 P52565 Rho GDP-dissociation inhibitor 1 23,250 5.03 80 1.4 X

C1862 D336 P06748 Nucleophosmin 32,653 4.64 64 0.9 X

C1841 D320 Q9Y3F4 Serine-threoninekinasereceptor-associatedprotein 38,756 4.98 51 0.6 X

C2160 P62158 Calmodulin 17,152 4.09 49 CTRL X

a_Accession_number_by_UniProtKB.

b _MW_–_theoretical_molecular_weigh_obtained_by_ExPASy_Proteomic_Server. c _pI_–_theoretical_isoelectric_point_obtained_by_ExPASy_Proteomic_Server. d _Score_–_index_calculated_by_{MASCOT-signiﬁcant}_when_greater_than₅₆_(p_<_0.05). e_Ratio_between_spot_volume_in_CTRL_and_spot_volume_in_treated_gels.

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F.Buchietal./LeukemiaResearch36 (2012) 607–618 611

Fig.1.Effectofazacitidine24honproteomeofKasumi-1cells.(A)Two-dimensionalgelsbefore(a)andafter(b)treatmentwereanalyzed.Spotswerethenpickedand identifiedbyMSanalysis.(B)Functionaldistributionofidentifiedproteins.(CandD)ConfirmatoryWesternblot;anti-p38isproteinloadingcontrol.CTRL,controluntreated cells;AZA,azacitidine;DAC,decitabine.

werefound;50proteinspotshadsigniﬁcantdifferencesbetween untreatedcontrolanddecitabinetreatedcells.Weexcised36 pro-teinspotsfromcontrolgeland14spotsfromtreatedgel;mass spectrometryanalysisMALDI-TOFallowedustoidentify45spots correspondingto35characteristicproteins(Fig.2A,aandb), clas-siﬁedinfunctionalcategoriesbyDavidBioinformaticsdatabase.

ResultswerereportedinTable2(spotnumberobtainedfromImage Master,correlatetocorrespondingspotsinFig.2A).

As shown in Fig. 2B, nine proteins (26%) identiﬁed by MS included molecules functionally classiﬁed as Heat Shock Pro-teins/Chaperons,tenproteins(29%)wereenzymes,mostofwhich metabolic, three proteins (9%) were involved in stress/redox

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Fig.2.Effectofdecitabine24honproteomeofKasumi-1cells.(A)Two-dimensionalgelsbefore(a)andafter(b)treatmentwereanalyzed.Spotswerepickedandidentified byMSanalysis.(B)Functionaldistributionofidentifiedproteins.(C)ConfirmatoryWesternblot;anti-p38isshownasproteinloadingcontrol.CTRL,controluntreatedcells; DAC,decitabine.

homeostasis, three (9%) were structural proteins and ten pro-teins (29%) were involved in signal transduction (Table 1 and Supplementarydata).Afterdecitabinetreatment,themajorityof identiﬁedproteins(thoseselectedonthebasisofasigniﬁcant mod-ulationinexpression)weredown-regulated,althoughproteinspot numberdidnotdecrease(Table2).

String analysisshowedthat the majority ofmodulated pro-teinswerecorrelated(FigS2,b–Supplementarydata).Decrease of hnRNPA2/B1, alpha-Enolase and theincrease of Cyclophilin A,HSP60 (ratioof densitometryCTRL/AZA: 0.4, respectto con-trolloadingp38) and alpha-Tubulinwereconﬁrmed (Fig.2C, a and b). Several proteins (HSP90, HSP70, PDIA3, Cyclophilin A,

beta-Tubulin, alpha-Enolase) had different migration sites into both2Dgels(Table2–markedwithanasterisk),becauseoftheir isoforms(seeabove),sosomeofthesedidnotchangetheglobal expression(Fig.2C,c).

CyclophilinAexpressionwasincreasedinprimaryt(8;21)AML cellsafter24hexposuretodecitabineinvitro(Fig.1D,b).

3.4. ComparisonbetweenAZAandDACtreatments

We matched gels after treatment with azacitidine and decitabine, in order to identify commonly modulated proteins and toevaluate possibledifferent effects of thetwo agents on

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Fig.3. ComparisonbetweenproteomeofKasumi-1afterazacitidineanddecitabinetreatments.(A)Two-dimensionalComassiestainedgelsafterazacitidine(a)anddecitabine (b)werecomparedandcommonproteinsidentiﬁed.(B)Tableofcommonproteins.AZA,azacitidine;DAC,decitabine.

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Fig.4.ProteomenetworksbyMetacoreanalysis.Metacoreanalysiswasperformedtocomparecommonproteinsidentiﬁedafterazacitidineanddecitabinetreatments.(A) Proteinsequallymodulatedbyeitherdrug:(a)proteinfoldingnetwork,(b)cellcyclenetwork.(B)Proteinsdifferentlymodulatedbytwodrugs:(a)gene-speciﬁctranscription network,(b)responsetostress-network.(C)CyclophilinApathwaynetwork.

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Fig.4.(continued)

proteinexpression(Fig.3A,a andb).Considering onlyproteins withasigniﬁcantMASCOTscore(Tables1and2;score>56),and thoseproteinswhoseglobalexpression(summingisoforms)was modulated,wefound14proteinsidentiﬁedbothinazacitidineand decitabinetreatedgels(Fig.3B).

3.5. Proteinnetworkanalysis

A pattern analysis was performed using the software MetaCoreTM_in_order_to_evaluate_the_possible_correlation_between

the2D-electrophoresis/MSidentiﬁedmodulatedproteinsand pos-sible cellular pathways affected by azacitidine and decitabine (Table3).Thisanalysisallowedalsotorelatethefunctionofsingle proteinstothecellularcontext.Allproteinsanalyzedwere pro-cessedbythe“networkanalysis”algorithmwithﬁftynodes,which generatessub-networksrankedbyp-values.

Firstofall,weinvestigatedthecorrelationbetweenproteins that were equally modulated by either drug, particularlyTCP1 beta,PDIA3,Calreticulin,Glyceraldehyde-3-phosphate dehydroge-nase(GAPDH),GlutathioneS-transferaseP,ACTBandhnRNPA2/B1 (Fig.3B).Theanalysisclusteredproteinsintwonetworks:theﬁrst, whichincludeTCP1,Calreticulin,ACTB,PDIA3andGSTP1regulates proteinfolding(Fig.4A,a)andthesecond,whichincludesGAPDH, Tcp1andhnRNPA2/B1,modulatescellcycle(Fig.4A,b).

Amongproteinsthathaddifferentmodulationaftertwodrugs treatments,wefocusedouranalysisonproteinsdecreased only

byazacitidine(Fig.3B),particularlyCyclophilinA,Catalase, Nucle-ophosminandPCNA.Thisanalysisleadtoclusterproteinsinthree network:two,includingPCNA,NucleophosminandCyclophilinA, areinvolvedingene-speciﬁctranscriptionandinresponsetostress (Fig.4B,aandb),thethirdoneisidentiﬁedviaCatalasepresence, involvedintriglyceridemetabolism.

SinceCyclophilinAshowedadecreaseofexpressionafter azac-itidineandanincreaseafterdecitabine,weanalyzeditspathway networkasaninterestingdifferencebetweenthetwo agents.It seemsparticularlyrelevantthatCyclophilinAisinvolvedinAML proliferationandmaintenance(Fig.4C–linksmarkedinblue). 4. Discussion

AzacitidineanddecitabineareusedfortreatmentofMDSand AML.Althoughthesetwoagentsshareasimilarchemical struc-ture and are often considered equivalent,they exertdivergent biologicaleffectsandhavesomedifferentclinicalactivity,asonly azacitidine hasbeen showntosigniﬁcantly improvesurvival in MDS[14]whiledecitabineseemstohavepreferentialactivityon CMML[15]andAML[11].Partofthedifferencesmaybedueto prevalentincorporationintoRNAofazacitidineandintoDNAof decitabine,but itappearsthatothereffects,beside hypomethy-lation,mayexplaintheirclinicalactivity[16].Characterizationof azacitidineanddecitabineeffects onproteinexpression,protein networksandpathwaysmayhelpinunderstandingtheirmolecular Table3

SummaryofproteomemodiﬁcationsafterazacitidineanddecitabinetreatmentofAML1/ETOpositiveleukemiccells.

Drug Numberofproteinspots Proteinsmodulated

AZA Decrease ↓5involvedinproteinfolding ↓3involvedingene-speciﬁctranscription

↓3involvedincellcycle ↓3involvedinresponsetostress ↓Catalase

DAC Nochange ↓5involvedinproteinfolding ↑CyclophilinA ↓alpha-Enolase

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mechanisms,whosediscrepancieshavestartedtobehighlighted inscientiﬁcliterature[3,5,6].AML1/ETOpositiveleukemiashave a particular good prognosisand are sensitive tochemotherapy [7–10].Mostinterestingly,theAML1/ETOcomplexbindingDNMTs andHDACformsaputativeidealtargetforepigeneticdrugs[7–10]. Weemployedasensitive,2D-electrophoresisapproachfollowed byMALDI-TOF/MStoinvestigateproteinsmodiﬁcationsaftershort terminvitrotreatmentwithazacitidineanddecitabine.This pro-teomic approach is increasingly used to implement studies of mechanismsofactionofdrugs[17,18].

We could demonstrate distinct expression pattern between proteinextractsfromuntreatedleukemiccellsandextractsfrom azacitidineanddecitabinetreatedcells.Speciﬁcproteinswere sig-niﬁcantlydown-regulated,althoughnotcompletely,bybothdrugs. Wefocusedourattentionbothonthecommonlyrepressed pro-teins,andonthosedecreasedexclusivelybyoneagent,toverify whetherthis differencecouldjustifythediverseclinical effects of thetwo DNMTinhibitors. Quite differentlyfrom azacitidine, decitabinedidnotdecreasetheglobalnumberofproteinspots, respecttountreatedsamples.Thisisconsistentwiththelackof inhibitionof proteinsynthesisbydecitabine,observedbyother authorswithdifferentanalyticalmethods[3].

Thetwopublishedreportsongeneexpressionmodificationby DNMTinhibitors[5,6]donotidentifythesamesetofgenes,and thereisnosignificantoverlapwithourproteinfindingseither.

Althoughthetwoagentsaresupposedtoactandbeefﬁcient inclinicsviahypomethylationofCpGislandsofgenepromoters, themajorityofmodulatedproteinsbyinvitroDNMTinhibitors treatment were repressed, similarly to what observed in gene expressionstudies[6].Moreover,wecouldnotdetectsigniﬁcant increase of tumor suppressorgenes, indicating, again similarly to what already observed by other authors, that although the genes of these proteins may be up regulated via hypomethy-lationto levelsdetectableby PCR-based methods,theyare not partofthegeneraldominantexpressionpatterninducedbythese drugs,andproteomicmethodsarenotsensitiveenoughtodetect them[5].

Inordertobetterunderstandtherelationshipsamongthe pro-teinsidentiﬁed,weusedStringdatabase,aswellasMetaCoreTM_.

Mostoftheproteinswhose expressionwasmodiﬁedby DNMT inhibitorswereinter-relatedinthepathwaymap,conﬁrmingthe biologicalreliability ofouranalysis.Inaddition,themajorityof modulatedproteinswereknowntobeinvolvedinneoplastic pro-gression,sothattheirdecreasedexpressionbyDNMTinhibitors mayhelptounderstandthemechanismsbywhichtheseagents exerttheiranticanceractivity.

IntheMetaCoreTM _produced_network,_the_identiﬁed_proteins

clusteredin twonetworks, onemodulating proteinfolding and polymerization(TCP1,PBAF,Calreticulin,PDIA3,GSTP1)andthe secondinvolvedinregulationofcellcycleandmitosisandRNA transport(hnRNPA2,BAF,G3P2,TCP1).

The Chaperonin Containing TCP1 in the eukaryotic cytosol ensuresthecorrectfoldingandassemblyofavarietyofproteins. Neoplastic cells show an overexpression of TCP1 beta [19]. In ourmodel,azacitidineanddecitabineprovokeddecreaseinTCP1 expression,andalsoadecreaseinitsbest-characterizedsubstrate beta-actin.Wecouldindeedconfirmthisfindingalsoinprimary AMLcells.Calreticulinisincreasedinseveralneoplasticdiseases [20],anditsexpressiondecreasedafterbothDNMTitreatments. The same inhibition is observed for Glutathione S-transferases (GSTs),whichareafamilyofenzymesthatcatalyzethe conjuga-tionofglutathione(GSH)tovariousxenobioticsandparticipatein cellularprotectionagainstoxidativestressorresistancetodrugs. Theseproteinsexhibitgeneticpolymorphismsintheirpopulation distribution,andthesevariantsarefrequentlyexpressedinAML patientandmayinfluencetheirprognosis[21,22].

Heterogeneousribonucleoproteins(hnRNPs)arecomplexesof RNA and protein present in thecell nucleus during gene tran-scriptionandsubsequentpost-transcriptionalmodiﬁcationofthe newlysynthesizedRNA(pre-mRNA).Theyareinvolvedinsplicing ofpre-mRNAandinitstransportoutofthenucleus.Overexpression ofhnRNPA2/B1iscorrelatedtocancerprogression[23], particu-larlyhnRNPB1isover-expressedinMDSandAML-MDS[24]while hnRNPA1isoverexpressedgenerallyinsolid[25]and hemato-logicalmalignances[26].Fromouranalysis,itemergedthatboth agents,inourexperimentalconditions,havethesameinhibiting activityoncellcycle/RNAmetabolismandproteinfolding.Effects ongeneexpressionandmiRNAexpressionhavebeendemonstrated tobetimedependent,butweprivilegedtheanalysisofearly modu-lationbythesetwodrugs,topossiblyexaminenoncytotoxiceffects [5].

Regardingproteinsselectivelymodulatedbyazacitidine,itlooks quite important that one cluster of decreased proteins (PCNA, CyclophilinA,NPM-1)isinvolvedinresponsetostress, suggest-ingaroleofazacitidineinimpairingreactionofleukemiccellsto DNAdamage.Thesameproteinsareregulatinggene-speciﬁc tran-scription,and azacitidinecouldinﬂuenceit, butnot necessarily byCpGislandhypomethylation.Besides,onlyazacitidineinhibits Catalase,which notonly protectscellsfromthetoxic effectsof hydrogenperoxide,butalsopromotesgrowthofmyeloidleukemia cells,thankstoERK-1activation[27].Inconclusions,azacitidine, butnotdecitabine,seeminglyexertsanimportantdownregulation ofanti-stressproteins.

CyclophilinA(CypA)modulationisquitepeculiar,becauseits expressionisreducedbyazacitidine,butincreasedbydecitabine, andtheseresultswereconfirmedalsoinprimaryhumant(8;21) AMLcells.RecentstudiesindicateddifferentpossiblerolesofCypA intumordevelopment,giventhatitsexpressionresultedincreased and inducedby c-Myc and ithasinhibitory activityonp53,as alsoshownbyouranalysiswithMetaCore.Thisdiscrepancyafter drug treatment could be consistent witha reported early and transientinductionof pro-carcinogenicgenesbydecitabine[5]. Althoughaspecificroleinnormalandmalignanthemopoiesishas not beendemonstrated for Cyclophilin A,it is possible thatits effectsoninflammation,neutrophilchemo-attractionand induc-tionofreactiveoxygenspeciesmaybecriticalforleukemiccells [28,29].Anotherinterestingfeatureofouranalysisisthatdecitabine isselectivelyabletodownregulatealpha-Enolase.Itcouldbethat thisoccursbecauseofdecitabinesite-specificDNAincorporation. Certainly,bydecreasingalpha-Enolaseactivity,decitabinecould actreducingleukemicproliferation.Moreover,onlytreatmentwith decitabinedown-regulatesHSP60,which indeedplaysa pivotal roleinleukemogenesis[30].

Differences between the mechanisms of azacitidine and decitabinewereobservedintheiractivitiesoncellviability, pro-teinsynthesis,cellcycle,miRNAandgeneexpression[3,5,6].The hypothesis that AZA and DAC have finely diversebiomolecular mechanismsissupportedbytheobservationthatthesetwoagents have a specific and selectivemodulation of thetarget proteins whichweidentified.Theireffectscouldbecausednotexclusively byDNAhypomethylation.Ourobservationsfurtherindicatethat azacitidineanddecitabinearenotinterchangeableandthattheir alternateorcombineduseinclinicsmaybeconceivable.

Conﬂictofinterest

Allauthorshavenoconﬂictofinteresttodeclare. Acknowledgments

Contributions.V.S.designedthestudyandwrotethemanuscript; F.B.performed experiments and wrote theresultssection; G.F.

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performedmassspectrometryanalysis;E.S.and E.M.performed Western blot experiments and A.S., A.G. and A.B. took part in designingthestudyandrevisedthemanuscript.Fundings.Regione Toscana,BandoSalute 2009;EnteCassa diRisparmiodiFirenze (ECR); and Ministero per l’Istruzione, l’Università e la Ricerca (MIUR).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.leukres.2011.11.024.

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