Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions

ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s

Erythroid

induction

of

K562

cells

treated

with

mithramycin

is

associated

with

inhibition

of

raptor

gene

transcription

and

mammalian

target

of

rapamycin

complex

1

(mTORC1)

functions

Alessia

Finotti,

Nicoletta

Bianchi,

Enrica

Fabbri,

Monica

Borgatti,

Giulia

Breveglieri,

Jessica

Gasparello,

Roberto

Gambari

DepartmentofLifeSciencesandBiotechnology,SectionofBiochemistryandMolecularBiology,UniversityofFerrara,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29September2014 Receivedinrevisedform 21November2014 Accepted24November2014 Availableonline3December2014

Chemicalcompoundstudiedinthisarticle: Mithramycin(PubChemCID:457831)

Keywords: Raptor mTOR Sp1 Mithramycin Erythroidinduction Fetalhemoglobin

a

b

s

t

r

a

c

t

Rapamycin,aninhibitorofmTORactivity,is apotentinducer oferythroiddifferentiationandfetal hemoglobinproductionin␤-thalassemicpatients.Mithramycin(MTH)wasstudiedtoseeifthisinducer ofK562differentiationalsooperatesthroughinhibitionofmTOR.Wecanconcludefromthestudythat themTORpathwayisamongthemajortranscriptclassesaffectedbymithramycin-treatmentinK562 cellsandasharpdecreaseofraptorproteinproductionandp70S6kinaseisdetectableinmithramycin treatedK562cells.ThepromotersequenceoftheraptorgenecontainsseveralSp1bindingsiteswhichmay explainitsmechanismofaction.WehypothesizethattheG+C-selectiveDNA-bindingdrugmithramycin isabletointeractwiththesesequencesandtoinhibitthebindingofSp1totheraptorpromoterdueto thefollowingresults:(a)MTHstronglyinhibitstheinteractionsbetweenSp1andSp1-bindingsitesof theraptorpromoter(studiedbyelectrophoreticmobilityshiftassays,EMSA);(b)MTHstronglyreduces therecruitmentofSp1transcriptionfactortotheraptorpromoterinintactK562cells(studiedby chro-matinimmunoprecipitationexperiments,ChIP);(c)Sp1decoyoligonucleotidesareabletospecifically inhibitraptormRNAaccumulationinK562cells.Inconclusion,raptorgeneexpressionisinvolvedin mithramycin-mediatedinductionoferythroiddifferentiationofK562cellsandoneofitsmechanismof actionistheinhibitionofSp1bindingtotheraptorpromoter.

©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction

Themammaliantargetofrapamycin(mTOR)formstwo

com-plexes,namedmTOR complex1 (mTORC1)and mTORcomplex

2 (mTORC2) which are regulatedby phosphorylation, complex

formationandlocalizationwithinthecells[1–5].mTORC1is

com-posedofmTOR,theregulatoryassociatedproteinofmTOR(raptor),

Abbreviations: Raptor, regulatory associated protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; mTOR, mammalian target of rapamycin;mTORC1,mTORcomplex1;m-TORC2,mTORcomplex2;Sp1,specific protein1;MTH,mithramycin;RAPA,rapamycin;ChIP,chromatin immunoprecip-itation;EMSA,electrophoreticmobilityshiftassay;FBS,fetalbovineserum;PBS, phosphate-bufferedsaline;TBS,tris-bufferedsaline;HbF,fetalhemoglobin;ODN, oligonucleotide.

∗ Correspondingauthorat:DepartmentofLifeSciencesandBiotechnology, Molec-ularBiologySection,UniversityofFerrara,ViaFossatodiMortara74,44121Ferrara, Italy.Tel.:+39532974443;fax:+39532974500.

E-mailaddress:gam@unife.it(R.Gambari).

mammalianLST8/G-protein␤-subunit likeprotein(mLST8/G␤L)

and the recentlyidentified partners PRAS40 and DEPTOR [6,7].

Raptorbinds directlytomTORsignaling(TOS)motifson

down-stream targets,includingS6K1 (ribosomal S6protein kinase 1)

and 4EBP1 (eukaryotic initiation factor 4E-binding protein 1)

as well as PRAS40 and Hif1␣,thus linking them to themTOR

kinase [2,3]. mTORC1 senses and integrates diverseextra- and

intracellularsignalstopromoteanabolicandtoinhibitcatabolic

cellularprocesses.Thiscomplexischaracterizedbytheclassic

fea-turesofmTORbyfunctioningasanutrient/energy/redoxsensor

andcontrollingproteinsynthesis [1,6].Theactivityofthis

com-plexisstimulatedbyinsulin,growthfactors,serum,phosphatidic

acid,aminoacids(particularlyleucine),andoxidativestress[6,8].

mTORC1inyeastandmammalsalsopromotes“ribosome

biogen-esis”,aprocess wherebymTORC1increasesthetranscriptionof

ribosomalRNAsandproteinstoaugmentcellularprotein

biosyn-theticcapacity[9–11].mTORComplex2(mTORC2)iscomposedof

mTOR,rapamycin-insensitivecompanionofmTOR(Rictor),G␤L,

and mammalianstress-activatedproteinkinaseinteracting

pro-tein1(mSIN1)[2,3,12,13].mTORC2hasbeenshowntofunction

http://dx.doi.org/10.1016/j.phrs.2014.11.005

asanimportantregulatorofthecytoskeletonthroughits

stimula-tionofF-actinstressfibers,paxillin,RhoA,Rac1,Cdc42,andprotein

kinaseC␣(PKC␣)[14–17].mTORC2alsoappearstopossessthe

activityofapreviouslyelusiveproteinknownas“PDK2.”mTORC2

phosphorylatestheserine/threonineproteinkinaseAkt/PKBata

serineresidueS473.PhosphorylationoftheserinestimulatesAkt

phosphorylationatathreonineT308residuebyPDK1andleads

tofullAktactivation[13,16,17];mTORC2appearstoberegulated

byinsulin,growthfactors,serum,andnutrientlevels[6,7].

Origi-nally,mTORC2wasidentifiedasarapamycin-insensitiveentity,as

acuteexposuretorapamycindidnotaffectmTORC2activityorAkt

phosphorylation.However,subsequentstudieshaveshownthat,

atleastinsomecelllines,chronicexposuretorapamycin,while

notaffecting pre-existingmTORC2s, promotesrapamycin

bind-ingtofreemTORmolecules,thusinhibitingtheformationofnew

mTORC2[14].

Despite the fact that the involvement of mTOR in all these

andotherbiologicalprocesseshasbeenfirmlyestablished,little

informationisavailableontheroleofmTORonerythroid

differ-entiation.Ithasbeendemonstratedthatrapamycin,aninhibitor

ofmTORactivity,isapotentinduceroferythroiddifferentiationof

humanleukemicK562cells[18]andfetalhemoglobinproduction

by␤-thalassemicpatients[19].Accordingly,otherinducersofK562

differentiationmightoperatethroughinhibitionofmTOR[20–22].

Inordertoaddressthisissue,mithramycin(MTH)wasstudied.This

DNA-bindinglowmolecularweightmoleculeisselectiveforG+C

richregions[23–25].ItbindstotheminorgrooveofDNA

generat-ingunstableMTH-DNAcomplexes[20].Itisoneofthemostpotent

inducersofK562differentiation[21]andHbFproductionby

ery-throidprecursorcellsfromnormaldonorsaswellas␤-thalassemia

patients[26].

Themainobjectiveofthepresentstudywastoverifywhether

inductionofdifferentiationbyMTHisassociatedwithinhibitionof

mTORactivity.TheeffectsofMTHonraptor,rictorandmTORgene

expressionwerefirstanalyzedbyq-RT-PCR.Secondly,we

deter-minedbyWesternblotting,theproductionofmTOR,raptorand

rictorproteinsinMTHtreatedcellswiththeaimofdetermining

whethermithramycinaffectsmTORC1,mTORC2orboth.Thirdly,

weverifiedthepossibleeffectofMTHontheinteractionofthe

regulatorytranscriptionfactorSp1withtheraptorpromoter.This

wasapproachedbyelectrophoreticmobilityshiftassay,chromatin

immunoprecipitationandtreatmentoftargetcellswithSp1decoy

molecules.

Materialsandmethods

HumanK562cellcultures

The humanleukemia K562 [27,28]cells were cultured in a

humidified atmosphere of 5% CO2/air in RPMI 1640 medium

(SIGMA,St.Louis,MO,USA)supplementedwith10%(vol/vol)fetal

bovineserum(FBS;Biowest,Nuaille,F),100U/mlpenicillin,and

100␮g/mlstreptomycin.Cellgrowthwasstudiedbydetermining

thecellnumberpermlwithaZ2CoulterCounter(BeckmanCoulter,

Fullerton,CA,USA).

Antibodies

TheprimaryantibodiesforWesternblottingwere:anti-Raptor

cat.2280,anti-Rictorcat.2114,anti-mTORcat.2983,anti-p-mTOR

(Ser2448)cat.2971,anti-p-mTOR(Ser2481)cat.2974,anti-p70S6

kinasecat.2708,anti-p-p70S6kinase(Thr389)cat.9234.All

anti-bodieswerepurchasefromCellSignaling(EurocloneS.p.A.,Pero,

MI,Italy).

RNAextraction

Cellswereisolatedby centrifugationat1500rpm for10min

at 4◦C, washed in PBS, lysed in Tri-reagentTM (Sigma–Aldrich,

St. Louis, MO, USA), according to the manufacturer’s

instruc-tions.TheisolatedRNAwaswashedoncewithcold75%ethanol,

driedanddissolvedindiethylpyrocarbonatetreatedwaterbefore

use.

Reversetranscriptionandquantitativereal-timePCR(RT-qPCR)

Thereagentsforgeneexpressionanalysisbyreal-timeRT-PCR

wereobtained fromApplied Biosystems (FosterCity, CA,USA).

500ngoftotalRNAwerereversetranscribedusingrandom

hex-amers.RT-qPCRassaywascarriedoutusinggene-specificdouble

fluorescentlylabeledprobes.Theprimersandprobesusedtoassay

theexpressionof␥-globin,␣-globinandmTORmRNAwere

pur-chasedfromIDT(IntegratedDNAtechnologies,SanJose,CA,USA).

Thenucleotidesequencesusedforreal-timeqPCRanalysisof

␥-and ␣-globinmRNAs andmTOR wereas follows:␣-globin

for-wardprimer,5-CAC GCGCACAAGCTT CG-3,␣-globinreverse

primer,5-AGGGTCACCAGCAGGCAG T-3,_␣-globinprobe,5

-FAM-TGGACCCGGTCAACTTCAAGCTCCT-TAMRA-3;␥-globin

forwardprimer,5-TGGCAAGAAGGTGCTGACTTC-3,␥-globin

reverse primer, 5-TCA CTC AGC TGG GCA AAG G-3, ␥-globin

probe,5-FAM-TGGGAGATGCCATAAAGCACCTGG-TAMRA-3;

mTORforwardprimer5-GCTGTACGTTCCTTCTCCTTC-3,mTOR

reverseprimer 5-CAA GAACTC GCTGATCCA AAT G-3,mTOR

probe,5-FAM-TGCATTCCG-ZEN-ACC TTCTGCCTT

CA-IABkFQ-3.Thenucleotidesequencesusedforreal-timeqPCRanalysisof

transferrinreceptorandglycophorinAmRNAswere:transferrin

receptorforwardprimer,5-TCAGAGCGTCGGGATATCG-3,

trans-ferrin receptor reverse primer, 5-TGA ACTGCC ACA CAG AAG

AACA-3,transferrin receptorprobe5-FAM-TGGCGGCTCGGG

ACG GA-TAMRA-3; glycophorin A forward primer, 5-CGG TAT

TCGCCGACTGATAAA-3,glycophorinAreverseprimer,5-AAA

GGCAGTCTGTGTCAGGT-3,glycophorinAprobe,5-FAM-AAA

GCCCATCTG ATG TAA AACCTC TTC CCC T-TAMRA-3.The kit

for quantitativeRT-PCRfor ␨-globinmRNAand ␧-globin mRNA

werefromAppliedBiosystems(␨-globinmRNA:Hs00923579m1;

␧-globinmRNA:Hs00362216m1).Theprimersandprobesusedto

assaytheexpressionofraptormRNA(AssayIDHs00977502m1)

and forRictor (AssayIDHs.PT.56a.40621153.g) werepurchased

from Applied Biosystems and from IDT, respectively. Relative

expression wascalculated using the comparativecycle

thresh-old method and the endogenous controls human 18S rRNA

(AssayID4310893E, AppliedBiosystems)andRPL13A(AssayID

Hs03043885g1,Applied Biosystems)asreference genes.

Dupli-catenegative controls(no template cDNA)were also run with

every experimental plate to assess specificity and to rule out

contamination.

Westernblotting

Forextractpreparation,MTHtreatedoruntreatedK562cells

werelysedinaicecoldRIPAbuffer(10mMTris–HCl,pH8.0,0.5mM

EDTA,150mMNaCl,1%NP40,0.1%SDS,5mg/mlDeoxyCholicacid,

1mMDTT,2mMPMSF,2mMNa3VO4,10mMNaF,1␮g/ml

Leu-peptin,1␮g/mlAprotinin).Briefly,K562cells(8×106_cells)were

collectedand washedtwice withcoldPBS (Phosphate-Buffered

Saline, Lonza-Biowhittaker, Basel, Switzerland). Cellular pellets

werethensuspendedwith400␮lofcoldRIPAbuffer,incubated

onicefor20minandsubjectedtofivecyclesoffreeze–thawing.

Sampleswerefinallycentrifugedat14,000_×gfor3minat4◦Cand

thesupernatantcytoplasmicfractionswerecollectedand

usingPierceTM _{BCA ProteinAssayKit (Thermo FisherScientific,}

Rockford,USA).Ten␮gofcytoplasmicextractsweredenaturedfor

5minat98◦Cin1×SDSsamplebuffer(62.5mMTris–HClpH6.8,

2%SDS,50mMDithiotreithol(DTT),0.01%bromophenolblue,10%

glicerol)andloadedonSDS-PAGEgel(10cm×8cm)inTris-glycine

Buffer(25mMTris,192mMglycine,0.1%SDS).Abiotinylated

pro-tein ladder(sizerangeof 9–200kDa)(Cat. 7071,Cell Signaling,

Euroclone S.p.A.,Pero,MI, Italy) and/ora prestainedmulticolor

proteinladder(sizerange10–260kDa)(Cat26634,ThermoFisher

Scientific, Rockford,USA) were usedas standardsto determine

molecularweight.Theelectrotransferto0.2_␮mporesize

nitro-cellulosemembrane(Pierce,EurocloneS.p.A.,Pero,Milano,Italy)

wasperformedover-nightat360mAand 4◦Cin electrotransfer

buffer(25mM Tris, 192mM Glycine, 5% methanol). The

mem-braneswereprestainedwithPonceauSSolution(Sigma,St.Louis,

MO,USA)toverifythetransfer,washedwith25mlTBS(10mM

Tris–HClpH7.4,150mMNaCl)for 10minatroomtemperature

andincubatedin25mlofblockingbufferfor2hatroom

temper-ature.Themembraneswerewashedthreetimesfor 5mineach

with 25ml of TBS/T (TBS, 0.1% Tween-20) and incubated with

theprimary rabbit monoclonal antibody(1:1000) in 15ml

pri-maryantibodydilutionbufferwithgentleshaking over-nightat

4◦C.Thenextday,themembraneswerewashedthreetimesfor

5mineachwith20mlofTBS/Tandincubatedin15mlof

block-ingbuffer,withgentleshakingfor2hatroomtemperature,with

anappropriateHRP-conjugatedsecondaryantibody(1:2000)and

anHRP-conjugatedanti-biotin antibody(1:1000) usedtodetect

biotinylatedproteinmarker.Finally,afterthreewasheseachwith

20ml of TBS/T for 5min,the membraneswere incubated with

10mlLumiGLO®(0.5ml20xLumiGLO®,0.5ml20×Peroxideand

9.0ml Milli-Qwater) (CellSignaling,Euroclone S.p.A., Pero,MI,

Italy) with gentle shaking for 5min at room temperature and

exposedtox-rayfilm(Pierce,EurocloneS.p.A.,Pero,MI,Italy).In

ordertore-probethemembranes,theywerestrippedusingthe

RestoreTM_{WesternBlotStrippingBuffer(Pierce,EurocloneS.p.A.,}

Pero,MI,Italy)andincubatedwithotherprimaryandsecondary

antibodies.ThechemiluminescentsignalwasvisualizedonX-ray

filmsandtheintensityoftheimmunopositivebandswasanalyzed

byGelDoc2000(Bio-RadLaboratoires,MI,Italy)usingQuantity

Oneprogramtoelaboratetheintensitydataofourspecifictarget

protein.

Preparationofnuclearextractsforbandshiftandsupershiftassays

NuclearextractswerepreparedasdescribedbyAndrewsand

Faller[29].Briefly, cellswerecollected, washedtwicewith

ice-coldphosphate-bufferedsaline,andsuspendedin0.4ml/107_cells

ofhypotoniclysisbuffer(10mMHepes/KOH,pH7.9,10mMKCl,

1.5mMMgCl2,0.5mMdithiothreitol,and0.2mM

phenylmethane-sulfonylfluoride).Afterincubationonicefor10min,themixture

wasvortexedfor10s,andnucleiwerepelletedbycentrifugation

at 12,000×g for 10s, then nuclear proteinswere extracted by

incubationofthenucleifor20minonicewithintermittent

gen-tlevortexingin20mMHepes/KOH,pH7.9,25%glycerol,420mM

NaCl,1.5mMMgCl2,0.2mMEDTA,0.5mMdithiothreitol,0.2mM

phenylmethanesulfonylfluoride,1␮gmL−1 aprotinin,1␮gmL−1

leupeptin,2mMNa3VO4, and10mM NaF(Sigma,St Louis,MO,

USA);celldebriswasremovedbycentrifugationat12,000×gfor

5minat4◦C.TheBCAmethodwasusedtomeasuretheprotein

concentrationintheextract,whichwasthenstoredinaliquotsat

−80◦_C.

Electrophoreticmobilityshiftassays(EMSA)

The double-stranded oligonucleotides (ODN) used in the

EMSA are reported in Table 1 [30]. 3pmol of ODN were

32_{P-labeledusingOptiKinase(GEHealthcare,ChalfontStGiles,UK),}

annealedtoanexcessofcomplementaryODNandpurifiedfrom

[␥-32_{P]ATP(PerkinElmer,Wellesley,MA,USA).Bindingreactions}

wereperformedbyincubating2␮gofnuclearextractand16fmol

of32_{P-labeleddouble-strandedODN,withorwithoutcompetitor}

inafinalvolumeof20␮Lofbindingbuffer(20mMTris–HCl,pH

7.5, 50mM KCl, 1mM MgCl2, 0.2mM EDTA, 5% glycerol,1mM

dithiothreitol, 0.01% TritonX100, 0.05_␮g␮L−1 of poly dI-dC,

0.05␮g␮L−1_{ofasingle-strandedODN)[31].Competitor(100fold}

excessofunlabeledODNs)andnuclearextractmixturewere

incu-batedfor15minandthenprobewasaddedtothereaction.After

afurtherincubationof30minatroomtemperaturesampleswere

immediatelyloadedontoa6%nondenaturingpolyacrylamidegel

containing0.25×Tris/borate/EDTA(22.5mMTris,22.5mMboric

acid,0.5mMEDTA,pH8)buffer.Electrophoresiswascarriedoutat

200V.Gelswerevacuumheat-driedandsubjectedto

autoradiog-raphy.Toverifytheeffectsofmithramycin,DNAprobesornuclear

extracts,werepreincubatedfor1hat4◦Cwithdifferent

concen-trationofthecompoundbeforetheEMSAincubation.Supershift

assayswereperformedasdescribedpreviously[31,32]byusing

2_␮gofcommerciallyavailableantibodiesspecificforSp1

(cat.07-645)transcriptionfactorandanti-NF-kB(cat.06–556)asacontrol

unrelatedantibody(UpstateBiotechnologyInc.,LakePlacid,NY,

USA).

Bio-Plexanalysis

The levels of multiple transcription factors within nuclear

extractsobtainedfromstimulatedcellswereanalyzedusingthe

Bio-Plextechnology(Bio-RadLaboratories,Hercules,CA)andthe

BioSource’s Transcription Factor (TF) Assays (Biosource

Inter-national, Inc., California USA). Nuclear extracts were prepared

usingBioSource’sNuclearExtractionKit(BiosourceInternational,

Inc., California USA) and quantified using the Bradford assay.

The assay was performed as reported by the manufacturer.

Briefly,biotinlabeledDNAprobesandcontrolsornuclearextract

samples were incubated for 20min at 25◦C in 96 well PCR

thermocycler-compatible microtiter plate. Subsequently

Diges-tionReagentcontainingnucleasewasaddedtosamples(except

positive control)for 20minat 37◦C.The fluorescentlyencoded

microspheresconjugatedtoDNAsequencescomplementarytothe

probeswerethenincubatedwithsamplesfor45minatroom

tem-perature.Finally,theindividualmixturesweretransferredtothe

wellsof afilterplate,washedand incubatedwith

streptavidin-RPE.AfterthewashingstepthesampleswereanalyzedinBio-plex

instrument.

Transcriptionfactordecoy(TFD)approachtargetingSp1protein

bindingonraptorpromoter

TheeffectoftheSp1econsensusoligonucleotidedecoy[33,34]

onraptortranscriptionwasevaluatedbyadding2␮g/mlofSp1e

double-strandedoligonucleotideorascrambledsequenceofthe

samelengthasacontrol(scrambleODN) (Table1), and4␮g/ml

␮g/mloflipofectamine2000toK562cellsseededat50–70%

conflu-encein16mmwells.Twenty-fourhourslater,RNAextractionand

reversetranscriptionwereperformed.Aliquots,1/20␮lofcDNA

wereusedforeachSYBRGreenrealtimePCRreactiontoquantify

thedepletionofraptortranscripts,usingtheRaptorFprimerand

theRaptorRreverseprimer(Table1)designedtoamplifya355bp

sequence.AmplificationofhumanGAPDHcDNAservedasan

inter-nalstandard(housekeepinggene).Real-timePCRreactionswere

performedforatotalof40cycles(95◦Cfor3min,66◦Cfor30s,

and72◦Cfor25s).TheCTmethodwasusedtocomparegene

Table1

DoublestrandedsynteticoligonucleotidesandPCRprimersemployed.

Name Method Amplifiedregion Sequences

Sp1mera _{EMSA N.A.}b ₅_{CCCTGGCCACGCCTCACTG3}

Sp1a EMSA N.A. 5_{TGCACCACCACGCCTGGCCTCG3}

Sp1b EMSA N.A. 5_{CACGCCACCACGCCCAGCTAAT3}

Sp1c EMSA N.A. 5_{CGCACCACCACGCCCAGCTAAT3}

Sp1d EMSA N.A. 5_{TTAGTAGGGACGGGGTTTCACC3}

Sp1e EMSAanddecoy N.A. 5TTTAATAACACGCCTCTACTGA3

Sp1f EMSA N.A. 5AGGTAACGGGGTCGGGGACTCTTTC3

Sp1g EMSA N.A. 5_{CTGTCGGCCACGCCGTAGGCCG3}

AspODN EMSA N.A. 5_{TGTCGAATGCAATCACTAGAA3}

ScrambleODN Decoy N.A. 5_{AGTCGTCACGTAAGTCGAGCAC3}

RaptorF DecoyrealtimePCR Raptortranscripts 5_{CTGCAAGCATTCCAGGTGTG3}

RaptorR DecoyrealtimePCR Raptortranscripts 5GTTCAGCTGGCATGTAGGGG3 GAPDHFc _{DecoyrealtimePCR GAPDHtranscripts 5}_{AAGGTCGGAGTCAACGGATTT3}

GAPDHR DecoyrealtimePCR GAPDHtranscripts 5_{ACTGTGGTCATGAGTCCTTCCA3}

RaptorChIPF ChIPrealtimePCR Raptorpromoter 5_{AACCGACAGTTTCATTTGTAGGATTG3}

RaptorChIPR ChIPrealtimePCR Raptorpromoter 5_{AAACTCATCTCATCAGCCCATCA3}

NegChIPF ChiPrealtimePCR Negativecontrolregion 5_{AGACAGGGTTTCACCATGTTGG3}

NegChIPR ChIPrealtimePCR Negativecontrolregion 5_{GCCATAGCTAACTGCAGAGGACA3} a_{Sp1consensussequence[30].}

b _{N.A.,notapplicable.}

c _{GAPDH,Glyceraldehyde3-phosphatedehydrogenase.}

ChromatinImmunoprecipitationAssays(ChIP)

Chromatinimmunoprecipitationassayswereperformedusing

theChromatinImmunoprecipitationAssayKit(Upstate

Biotech-nology)[31,35]. Briefly, a total of 6×107 _{K562 cells, untreated}

or treated with MTH for 24h, were incubated, for 10min at

roomtemperature,with1%formaldehydeculturemedium.After

washing in phosphate-buffered saline, glycine was added to a

finalconcentrationof0.125M.Thecellswerethensuspendedin

1.5mloflysis buffer(1%SDS,10mMEDTA,and 50mMTris–Cl,

pH 8.1) plus protease inhibitors(1␮g/ml pepstatin A, 1␮g/ml

leupeptin,1␮g/mlaprotinin,and1mMphenylmethylsulfonyl

flu-oride)andthechromatinsubjectedtosonication(usingaSonics

VibracellVC130sonicatorwitha2-mmprobe).Fifteen15-s

son-icationpulsesat30%amplitudewererequiredtoshearchromatin

to 200–1000bp fragments. 0.2-ml aliquots of chromatin were

dilutedto2mlinChIPdilutionbuffercontainingproteaseinhibitors

and then cleared with 75␮l of salmon sperm DNA/protein

A-agarose50%gelslurry(UpstateBiotechnology)for1hat4◦Cbefore

incubation on a rocking platform with either 6–10␮g of

spe-cificantiserumSp1(UpstateBiotechnology,cat.07-645)ornormal

rabbitserum. 20␮l of dilutedchromatinwas savedand stored

for subsequent PCR analysis as 1% of the input extract).

Incu-bationsoccurred overnightat4◦C and continuedan additional

1hafter theaddition of 60␮l protein A-agarose slurry.

There-aftertheagarosepelletswerewashedconsecutivelywithlowsalt,

highsalt and LiCl buffers. DNA/protein complexes were

recov-eredfromthepelletswithelutionbuffer(0.1MNaHCO3with1%

SDS), and cross-linkswerereversed byincubating overnightat

65◦C with0.2MNaCl.Thesampleswere treatedwithRNase A

andproteinaseK,extractedwithphenol/chloroformand

ethanol-precipitated.ThepellettedDNAswerewashedwith70%ethanol

anddissolvedin40␮lofTris/EDTA.2␮laliquotswereusedforeach

real-timePCRreactiontoquantitateimmunoprecipitatedpromoter

fragments.

Real-timePCRquantificationofimmunoprecipitatedpromoter

fragments

Forquantitativereal-timePCRreactionconditionseach25␮l

reactioncontained2_␮loftemplateDNA(fromchromatin

immuno-precipitations), 10pmol of primers (Table 1) and 1× iQTM

SYBR® GreenSupermix(Bio-Rad).Real-timePCRreactionswere

performedforatotalof40cycles(97◦Cfor15s,65◦Cfor30s,and

72◦Cfor30s)usinganiCyclerIQ®(Bio-Rad).Therelative

propor-tionsofimmunoprecipitatedpromoterfragmentsweredetermined

based on thethreshold cycle (Tc) value for each PCR reaction.

Real-time PCR data analysis followed the methodology

previ-ously described [35–37]. A TC value was calculated for each

sample by subtracting the Tc value for the input (to account

for differences in amplification efficiencies and DNA quantities

beforeimmunoprecipitation)fromtheTcvalueobtainedforthe

immunoprecipitated sample. Real-timePCR data analyseswere

obtained using the comparativeCt method: a  Ctvalue was

calculated for each sample by subtracting the Ctvalue for the

sampleamplifiedwithraptorpromoterprimersfromtheCtvalue

obtained for the same sample amplifiedwith negative control

primers.Foreachkindofimmunoprecipitation(IgGorSp1

anti-serum),aCtvaluewasthencalculatedbysubtractingthe

Ctvaluefor theuntreated cells sample fromtheCt valuefor

thetreatedcellsamples.Fold differenceswerethendetermined

bythe2−Ctmethod.Eachsamplewasquantifiedinduplicate

for atleastthree separateexperiments.Mean±SD valueswere

determined.

Cellcycleanalysis

ForflowcytometricanalysisofDNAcontent,1×106_K562cells

inexponentialgrowthweretreatedwithIC75concentrationsofthe

testedcompounds.After72h,thecellswerecentrifuged,washed

oncewithPBS,thentreatedwithlysisbuffercontainingRNAseA,

NP400,1%,andfinallystainedwithpropidiumiodideat50␮g/ml

(DNAQCparticles,BectonDickinson,Milan,Italy).Sampleswere

analyzedonaBectonCoulterEpicsXL-MCLflowcytometer.Forcell

cycleanalysis,DNAhistogramswereanalyzedusingMultiCycle®

forWindows(PhoenixFlowSystems,SanDiego,CA).

Statisticalanalysis

All the data were normally distributed and presented as

mean±SD. Statistical differences between groups were

com-pared using one-way ANOVA (ANalyses Of VAriance between

groups) software. P values were obtained using the Paired t

testoftheGraphPadPrismSoftware.Statisticaldifferenceswere

considered significant when *p<0.05, highly significant when

Fig. 1. Effects of mithramycin (MTH) on erythroid induction and gene expression of K562 cells. A,B. Effects of 30nM mithramycin on kinetics of the increase of the proportion of benzidine-positive cells (%) (average±SD; number of independent experiments: 5) (A) and on the expression of ␨-globin, ␣-globin, ␧-globin, ␥-globin, transferrin receptor and glycophorin A genes evaluated by RT-qPCR of RNA isolated from K562 cells treated with 30nM mithramycin for 5 days (B). MTH-treated samples of panel A are indicated with black circles). C,D. Effects of MTH on (A) ␣-globin and ␥-globinand(B)raptor,rictorandmTORgeneexpressioninMTH-treatedK562cells.RNAsampleswereobtainedfromK562cellsculturedintheabsence(white cir-cles)orinthepresence(blackcircles)of30nMMTHfortheindicatedlengthoftime.ThedatashowninpanelsB-Drepresentchangesingeneexpressionreferredtotime0, determinedbyquantitativeRT-PCR(average±SD;n=5).

Results

TreatmentofK562cellswithMTHleadstoinhibitionofraptor

mRNAaccumulation,butnotofrictorandmTORmRNAs

Torelateglobingeneexpressiontothatofgenesinvolvedin

m-TORC1andm-TORC2pathways,mRNAswerestudiedby

quan-titativeRT-PCRanalysisusingastemplatecytoplasmicRNAisolated

fromK562cellsculturedfordifferentlengthoftimeintheabsence

orinthepresenceof30nMMTH.Inordertodeterminetheextent

oferythroidinduction,theproportionofbenzidine-positivecells

wasfirstlymeasured(Fig.1A)andwasshowntobeincreasedfrom

1–5%(incontroluntreatedcells)to75–85%(inMTH-treatedcells),

inagreementwithpreviouslypublishedobservations[26].Fig.1B

(leftsideofthepanel)showsthatMTHtreatmentinduces,after5

daystreatment,alargeincreaseof(a)the␣-like␨-globinand

␣-globinmRNAsand(b) the␤-like␧-globinand␥-globinmRNAs.

Thesedataconfirm thattheMTHmediatederythroidinduction

wasfullyactivated, sustainingthestrongeffectof MTHas

ery-throidinducerofthiscellline.Theongoingoftheerythroidprogram

inMTH-inducedK562cellsis alsoconfirmedbytheincreaseof

othererythroidassociatedmarkers, includingthose encodedby

theCPOX(coproporphyrinogenoxidase), UROS

(uroporphyrino-genIIIsynthase),UROD(uroporphyrinogendecarboxylase),FECH

(ferrochelatase),NFE2L3nuclearfactorerythroid-derived2-like3),

EPB49(erythrocytemembraneproteinband4.9),SLC4A1(solute

carrierfamily 4,anionexchanger, member1 erythrocyte

mem-braneproteinband3,Diegobloodgroup)mRNAs(datanotshown)

andtransferrinreceptorandglycophorinAmRNAs(Fig.1B,right

Fig.2. MTHeffectsonraptor,rictorandmTORinK562cells.WesternblottinganalyseswereperformedonproteinextractsobtainedfromK562cellstreatedwith30nMMTH fortheindicatedlengthoftime,usingraptor(A),rictor(B)andmTOR(C,upperpanel),p-mTOR(Ser2448)(C,middlepanel),p-mTOR(Ser2481)(C,lowerpanel)monoclonal antibodies.TherelativeexpressionvaluesofsamplesfromMTHtreatedwithrespecttountreatedcellswereobtainedfromdensitometricmeasurementandreportedinthe lowerpartofthepanels.Datarepresenttheaverage±SD;n=3).

participatingto the mTOR pathway, such as raptor, rictor and

mTOR,werethen performedusing RT-PCRanalyses.Panel C of

Fig.1 showstheearlyincreaseofthelevelsof␣-globinand

␥-globinmRNAs.Fig.1Dshowsthat(a)thecontentofraptormRNA

sharplydecreasesfollowingMTHtreatment(leftsideofthepanel)

and(b)thatthisdecreaseoccursbeforetheMTH-inducedincrease

ofglobinmRNAs(seeFig.1,panelC).Onthecontrary,no

inhibi-tionofrictormRNAandmTORmRNAwasdetected(Fig.1D,middle

andrightsidesofthepanel).Thesefindingswerefullysupported

bytheWesternblottinganalysisofcytoplasmicextractsisolated

fromK562cellsculturedfor24,48and72hwithMTH,asreported

inFig.2.TheresultsshowninFig.2Ademonstrateasharp

inhi-bitionofraptorproteinproductionfollowingMTHtreatment.As

farasrictorexpressionisconcerned,nochangesinitsproduction

wereobservedat24and48h,whileafter72hoftreatment,aslight

increasewasdetectable(Fig.2B).Inaddition,nochangesinmTOR

content(Fig.2C,upperpartofthepanel)wasfoundinMTHtreated

cells.Finally,nomajorchangeswerefoundinmTOR

phosphoryla-tionatSer2448andSer2481(Fig.2C,middleandlowerpartsof

thepanel).

These data demonstrate for the first time strong inhibitory

effectsofmithramycinonmTORC1,butnotonmTORC2.

Accord-ingly, the possible effects of mithramycin on the raptor gene

promoterweredetermined.

Structureoftheraptorgenepromoter

Inordertoidentifyputativeregulatoryregionslocatedwithin

theraptorgenepromoter,computer-aidedanalysisoftheraptor

promotersequencewasperformedwiththeaimtoidentify

puta-tivebindingsites for transcription factors(Fig.3A). Withinthe

−1400/+1promotersequence,weidentifiedhomologytothe

fol-lowingtranscriptionfactorbindingsites:LyF-1,AP1,C/EBP,USF,

Oct,GATA-1andatleasttwelvesequences98–81%homologous

tothecanonical 5-CCT GGCCAC GCC TCACTG-3 Sp1 binding

site.Mithramycinhasbeenwidelyreportedasanantibioticthat

bindsDNAexhibitingselectiveinteractionswithG+Crichregions,

demonstratedbycomputermodeling[38],Biacoreanalysis[20],

and DNase Footprinting [21]. As MTH was expected to affect

Sp1-DNAinteractionsasalreadyreported[39–41],theinvitro

inter-actionsofSp1withtheraptorSp1bindingsitespresentwithinthe

raptorgenepromoterwereanalyzed.

InvitrointeractionofSp1transcriptionfactorwiththeSp1

bindingsitesoftheraptorgenepromoter:effectsofMTH

TodeterminewhethertheraptorSp1bindingsitesareableto

interactwiththeSp1nucleartranscriptionfactor,EMSA

experi-mentswerecarriedoutusing3␮gofK562nuclearextractsand

theraptor-Sp1probesdescribedinFig.3AandTable1.Theresults

obtainedarereportedinFig.3B–EandindicatethattheSp1probes

interactdifferentlywithK562nuclearextracts.Thespecific

com-plexesgeneratedbytheinteractionbetweennuclearextractsand

Sp1b,Sp1c,andSp1earearrowedinpanelsB-EofFig.3.The

consen-susSp1merprobesefficientlycompetewiththebindingofnuclear

extractstotheconsensus32_{P-labeledSp1merprobe(Fig.3B),while}

Sp1a, Sp1d,Sp1fand Sp1gwereless efficientin binding.Fig.3C

shows apreliminaryexperiment demonstrating thatSp1e

com-petes,asSp1mer,forSp1/DNAinteractions.Fig.3DshowsthatSp1e

isefficientincompetingwiththebindingofnuclearextractstothe

consensus32_{P-labeledSp1merprobe.Fig.3Eshowsthatthebinding}

activityofSp1eoccursalsowhenpurifiedSp1proteinisemployed.

SimilarresultswereobtainedwithSp1bandSp1coligonucleotides

(datanotshown).

Sp1transcriptionfactorisinvolvedinraptorgeneexpression

TodeterminewhethertheSp1 transcription factormightbe

involvedinraptorgeneexpressionwedeterminedtheextentof

Fig.3.SchematicrepresentationofraptorgenepromoterandcomparisonbetweenthebindingefficiencyofthreeSp1consensussites.(A)BoxesindicatetheSp1homology bindingsites.Nucleotidepositionandsequences(onlyforsitesdisplayinghomology≥81%)areindicated.Thelengthandpositionofthepromoterfragmentamplifiedin ChIPPCRreactionarealsoreportedtogetherwiththelocationoftheRaptorChIPFandRaptorChIPRPCRprimers.(B-E)Competitivebandshiftassaysperformedusingthe Sp1consensusbindingsiteSp1mer(B,C,D,E)andSp1e(C)oligonucleotideasprobes,nuclearextractsfromK562cells(B,C,D)orrecombinantSp1factor(E),andincreasing quantityofthecompetitoroligonucleotidesasindicated.

assays.Fig.4AshowshighcontentofSp1inK562cells.Itshould

bepointedouthowever,thatwehavenoevidenceforthedown

regulationofSp1inMTH-treatedK562cells(Fig.4B).Inorderto

obtainresultsclarifyingapossibleroleofSp1intheregulationof

raptorgeneexpressionadecoyexperimentwasperformed,usinga

previouslypublishedprotocol[34].Theresultsobtainedshowthat

thedecoytreatmentusingdoublestrandedSp1eoligonucleotide

inducesdecreaseofraptormRNAcontent,suggestingaroleofSp1

inthecontrolofraptorgeneexpression.

EffectofMTHoninvitrointeractionofSp1transcriptionfactor

withtheSp1bindingsitesoftheraptorgenepromoter

Fig.5demonstratesthatinteractionofMTHwithSp1eprevents

bindingofnuclearextractstothemolecule(Fig.5A,rightsideof

thepanel).Inaddition,Fig.5B(rightsideofthepanel)showsthat

theinhibitoryeffectsofMTHoccuralsoonpre-formedSp1-DNA

complexes.BoththeseMTH-mediatedeffectsweresimilartothose

obtainedusingSp1mer(Fig.5AandB,leftsideofthepanels).These

dataclearlyindicatethatMTHisabletoinhibitthedenovo

interac-tionoftheSp1transcriptionfactorwithintargetSp1sequences,as

wellastodisassemblepreformedSp1/DNAcomplexes.MTH

treat-mentofK562cellsmaythereforeleadtosharpdecreasesofSp1

transcriptionfactoroccupancyattheleveloftheraptorgene

pro-moter.Inordertoconclusivelydemonstratethatthetranscription

factorSp1binds toSp1esequence,supershiftexperimentswere

performedusingmonoclonalantibodiesagainstSp1andnuclear

extractsfromK562cells.Theadditionoftheantibodyinducesa

clearsupershift(indicatedinFig.5Cwithanarrowhead)showing

Fig.4. Sp1expressioninK562cellsandeffectsofdecoymoleculestargetingSp1transcriptionfactors.(A)Bio-Plexanalysisofmultipletranscriptionfactorswithinnuclear extracts(expressedasfluorescenceintensity)obtainedfromuntreatedK562cells.(B)LevelofSp1transcriptionfactorwithinnuclearextractsobtainedfromMTHtreated K562cellsfor0,24,48and72hbyBio-plextechnology.Datarepresenttheaverage±SD.(n=3).(C)EffectoftheSp1e“decoy”ODN(graybox)orascrambledunrelated oligonucleotide(whitebox),onraptormRNAlevels.ThecDNAobtainedfromtotalRNAwassubjectedtoquantitativereal-timeRT-qPCRfortheraptorspecifictranscript. RelativequantificationwascalculatedbytheCtmethodusinguntreatedcellsascontrolsample(value=1).Datarepresenttheaverage±SDoftriplicateexperiments.p valueswereobtainedusingthePairedttestoftheGraphPadPrismSoftware.Statisticalsignificance:*p<0.05,significant;**p<0.01,highlysignificant.

Fig.5.MTHinterfereswiththeinteractionsbetweenSp1transcriptionfactorandSp1-sitesoftheraptorpromotersequences.(A,B)Bandshiftassaywasperformed(A) pre-incubatingtheprobeswiththeindicatedconcentrationsofMTH,thenaddingnuclearextractsor(B)pre-incubatingtheprobeswithnuclearextracts,thenaddingthe indicatedincreasingconcentrationsofMTH.(C)SupershiftassaywasperformedusingSp1easprobeand4␮gofnuclearextractfromK562cells;theprobewasincubated withnuclearextractsintheabsence(/)ofantibodyorinthepresenceofantibodiesagainstSp1(Ab-Sp1)orNF-kB(Ab-NF-kB)factors.Arrowsindicatethespecificcomplexes andarrowheadindicatesthesuper-shiftedcomplexes.

Fig.6.MTHinhibitsinK562cellstherecruitmentofSp1totheraptorgenepromoter.(A,B)CharacterizationoftheMTH-mediatedeffectsonraptorgeneexpressionanalyzed byRT-qPCR(A)andbyWesternblotting(B).K562cellswereculturedfor24hwiththeindicatedconcentrationsofMTHforRT-qPCR,whileproteinexpressionwasanalyzed inextractsfromK562cellsculturedwith15and30nMMTHfor24and48h,asindicated.(C)Quantitativereal-timePCRprofilesfortheamplificationofraptorpromoterfrom arepresentativeChIPassayinwhichchromatinfromK562cellswasimmunoprecipitatedusingSp1antiserum.Input,genomicDNAwithoutantibodiesaddition;Sp1ChIP, ChIPperformedwiththeSp1monoclonalantibody;IggChIP,controlChIPwithnon-immuneserum.Thedatademonstratetheearlyexponentialincreaseinfluorescence asaresultofSYBRGreenIincorporationintotheamplifyingraptorpromoterfragment.(D)InvivoassociationofSp1transcriptionfactorwithraptorpromoterobtained fromChIPexperimentonK562cellstreatedwith15and30nMMTH.Theresults,obtainedfromChIPassayquantitativereal-timePCRusingnegativecontrolIgGandSp1 antiserum,wereanalyzedfollowingthemethodologydescribedinSection‘Materialsandmethods’.Thefolddecreasecomparesthevaluesobtainedbyraptorgenepromoter amplificationofuntreatedK562cellsandcellstreatedwith15or30nMmythramicin.DatareportedinpanelsAandDrepresenttheaverage±SDoftriplicateexperiments. pvalueswereobtainedusingthePairedttestoftheGraphPadPrismSoftware.Statisticalsignificance:*p<0.05,significant;**p<0.01,highlysignificant.

againstNF-kBfailedtoinducesupershift(Fig.5C,leftrow).

Chro-matinimmunoprecipitationwasperformedtoverifytheeffectsof

MTHonthisbindingactivityinaninvivocontext.

Chromatinimmunoprecipitation(ChIP)analysis

Chromatinimmunoprecipitation(ChIP)assayswereperformed

onK562cellseitheruntreated,ortreatedwithMTHtocharacterize

theMTHeffectsonSp1functioninintactcells.AnRT-qPCRbased

studyoftheeffectsonraptorgene expressionof differentMTH

concentrations(Fig.6A)wasconductedpriortoperformingChIP.

Thistreatmentwasperformedfor24handdemonstrateda

con-centrationdependentinhibitoryactivityofMTH,suggestingthat

15nMand30nMconcentrationsmightbesuitableforChIPassay.

Inparallel,theeffectsof15nMand30nMMTHtreatmentson

rap-torproductionwereassessedusingWesternblottingonprotein

extractsisolatedafter24and48hfromMTH-treatedcells(Fig.6B).

ChiPwasperformedonK562cellstreatedfor24hwith15nMand

30nMMTH.ChromatinwasimmunoprecipitatedusingSp1

anti-serumandquantitativeamplificationofraptorgenepromoterwas

performedonpurifiedDNA.ArepresentativeChIPassayisshownin

Fig.6C,inwhichitisclearlyevidentthatamplificationcurvesfrom

samplesimmunoprecipitatedwithSp1antisera(Sp1ChIP)reach

threshold5cyclesearlythanthosetreatedwithnonimmuneserum

(IggChIP).Fig.6Dshowsthatraptor-specificPCRislessefficient

whensamplesfromMTH-treatedcellsareemployed,suggesting

thatMTHdose-dependentlyinhibitstherecruitmentofSp1tothe

raptorgenepromoter.

MTHtreatmentisassociatedwithinhibitionofmTORC1regulated

biologicalprocess

Inordertoobtainafinalproofofprinciplesustainingthatthe

raptor/mTORnetworkisinvolvedinMTHeffects,biological

param-eters downstream from mTOR were considered [42–46]. K562

cellsweretreatedwith30nMMTHandtheexpressionlevelsof

mTORC1-regulatedp70S6kinaseanalyzed.Theresultsareshown

inFig.7A,andclearlysustaintheconceptthatMTHinhibitsthe

phosphorylationofp70S6kinase.Fig.7Bshowstheinvolvement

ofp70S6kinaseonthecellcycle[43,45],anddemonstratesthat

Fig.7.EffectsofMTHonp70S6kinaseandcellcycle.(A)WesternblottinganalysiswasperformedonproteinextractsobtainedfromK562cellstreatedwith30nMMTHfor theindicatedlengthoftime,usingp70andp-p70(Thr389)monoclonalantibody.Densitometricvaluesindicatingtherelativep-p70(Thr389)phosphorylationarereported inthelowerpartofpanelA.(B)RepresentativehistogramsofflowcytometrydataofuntreatedcontrolK562cells(upperpanel),K562cellstreatedfor3days(middlepanel) or4days(lowerpanel)with30nMMTH(IC50).After3or4daysofincubationthecellswerelabeledwithpropidiumiodideandanalyzedbyflowcytometryasdescribedin Section‘MaterialsandMethods’.DatareportedinpanelArepresentstheaverage±SD(n=3).PvalueswereobtainedusingthePairedt-testoftheGraphPadPrismSoftware (**p<0.01).

inassociationwithasharpdecreaseoftheproportionofS-phase

cells.

Discussion

Thisstudy investigated the possible effects of mithramycin,

knowntobeastronginduceroffetalhemoglobininerythroidcells

fromnormaland ␤-thalassmicpatients,onthemTORpathway.

TheinvolvementofthemTORpathwayinerythroid

differentia-tionis sustainedbythe findingthatrapamycin, aninhibitor of

mTORactivityisapotentinduceroferythroiddifferentiationof

humanleukemicK562cells[18]andfetalhemoglobinproduction

by␤-thalassemicpatients[19].

Ofthemajor classes ofmRNAs involved in themTOR

path-way(mTOR,rictorandraptor),theexpressionofraptormRNAis

stronglyreduced bymithramycintreatment.Themechanism of

actionleadingtothiseffectwasstudiedviathepromotersequence

oftheraptorgenerevealingseveralSp1bindingsites.These

tran-scriptionfactorsarehighlyexpressedinK562cellsandexpression

doesnotchangefollowingMTHtreatment.Itsroleinraptorgene

expression is sustained by the finding that treatment of K562

cellswithdecoymoleculesmimickingtheSp1-esiteofthe

rap-torgene promoter leads toraptor mRNAdecrease (see Fig.4).

ThehypothesisthatoneofthemechanismsofactionoftheG+

C-selectiveDNA-bindingdrugmithramycinistheinteractionwith

Sp1-likesequencescausinginhibitionofthebindingofSp1with

theraptorpromoterintargetcellsissupportedbythefollowing

results:(a)MTHstronglyinhibitstheinteractionsbetweenSp1and

Sp1-bindingsitesoftheraptorpromoter(EMSAassays);(b)MTH

stronglyreducestherecruitmentofSp1transcriptionfactortothe

raptorpromoterinintactK562cells(ChIP experiments.Asharp

decreaseofp70S6kinasephosphorylationandcellcyclealterations

werereproduciblydetectableinmithramycintreatedK562cellsin

agreementwiththehypothesisthatMTHinhibits,throughraptor

decrease,themTORC1activity.

Thisstudy does not allowus to concludethat inhibition of

mTOC1isrequiredforerythroiddifferentiation,butsimplythat

thisissuedeservestobefurtherinvestigated,asmTORisexpressed

athighlevelsinerythrocytes[47].Ontheotherhand,themTOR

inhibitorsrapamycin[18,19]anditsanalogeverolimus [48]are

stronginducersoferythroiddifferentiationofK562cellsandHbF

inductioninerythroidprecursorcellsof␤-thalassemiapatients.

One possible explanation of this apparent discrepancy is that

changes of mTOR activity can be associated to specific stages

oferythroiddifferentiation,withsignificantdifferencesbetween

earlyandlate/terminalstagesofdifferentiation.Furthermore,the

decreaseofmTORactivitymightbelinked,insteadtothe

over-allerythroidphenotype,onlytosomeerythroidfeaturesleading

tohighexpressionof embryo-fetalglobin genes.In additionto

changesatthetranscriptionalcontrollevel,translationalcontrol

mightalsobeinvolvedbothinactivationoferythroidpathways,

specificissuewasnotaddressedbythepresentstudy.However,

hypoxia,aconditionstimulatingHbFinerythroidcells[51],isalso

associatedwiththedecreaseofmTORC1activity[52]andalteration

ofthecontrolofproteinsynthesis[53].

Whilstwecannotexcludeadditionalmechanism(s)ofaction,

theresultsreportedheresuggestthatoneofthebiologicaleffects

ofmithramycinmightbetheinhibitionofSp1bindingtothe

rap-torgenepromoter.In ordertoextendthis observationtoother

erythroidcellularsystem,furtherresearchshouldbefocusedon

erythroidprecursorcellsfromnormalsubjectsand␤-thalassemia

patients.Mithramycinhasbeenproposednotonlyasinducerof

HbFinthalassemiccells[26],butalsoasanti-tumordrugin

can-cercells[54,55].Therefore,ourstudymightbeofinterestinthe

field ofmechanism ofaction ofanti-tumor compounds.Finally,

theunderstanding of themechanism of action of mithramycin

mightretainpracticalapplicationinappliedbiomedicine.Infact,

biochemicaltargetsofMTHaction(forinstanceraptorandSp1)

might bethemselves novelbiomolecular targetsof therapeutic

interventionbasedonmoreselectiveagents(suchasantisenseand

shRNAmoleculestargetingmRNAs,ordoublestrandedmolecules

targetingtranscriptionfactors).Interestingly,limitingthis

consid-erationstoerythroid cells, themTOR inhibitorsrapamycin and

everolimus are potent inducers of HbF in erythroid precursor

cellsfrom␤-thalassemiapatients[19,48]andRNAi-mediatedSp1

knockoutinduceshighlevelofhemoglobinizationin K562cells

[56].

Conclusions

This study presents evidence supporting the concept that

alterationofraptorgeneexpressionoccursduring

mithramycin-mediatedinductionoferythroiddifferentiationofK562cells.One

ofthemechanismsofactionofmithramycinistheinhibitionof

Sp1bindingtotheraptorpromoter,thereby stronglyinhibiting

thetranscriptionoftheraptorgene.Inassociationwiththe

MTH-mediateddown-regualtionofraptor,mTORC1associatedfunctions

suchasp70S6kinasephosporilationandcellcycleparameterswere

alsoaltered.

Authorscontributions

AFandRGparticipatedinresearchdesign;NB,AFandJG

con-ductedK562cellculture;AFandGBperformedEMSAexperiments

andconductedSp1decoyexperiments;AFconductedChromatin

Immunoprecipitationexperiments;MBperformedBio-plex

exper-iments;AF,NBandEFconductedRT-PCRanalyses;EF,GBandJG

conductedFACSexperiments;NBandAFperformedWestern

blot-tingexperiments;AF,NBand MBanalyzeddata;AF, RGand JG

wroteorcontributedtothewritingofthemanuscript.

Conflictofinterest

Theauthorsdeclarenocompetingfinancialinterests.

Acknowledgements

RobertoGambariisfunded byFondazioneCariparo(Cassadi

RisparmiodiPadovaeRovigo,grantn.2011),UEFP7THALAMOSS

Project(ThalassemiaModularStratificationSystemfor

Personal-izedTherapyofß-Thalassemia,grantn.306201),Telethon(grant

n.GGP10124).Thisresearchactivitywasalsosupportedby

Associ-azioneVenetaperlaLottaallaTalassemia(AVLT,grantn.2013)and

byAIRC2012(grantn.IG13575).

WewouldliketothankDrAmandaJulieNevilleMSBforher

invaluablehelpinrevisingthescientificEnglishofthemanuscript.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2014.11.005.

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