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AcknowledgementsWethankS.H.Ahn,D.Allis,M.Reyland,U.Siebenlist,theRockefellerUniversityGenotypingResourceCenterandtheMSKCCGenomicsCoreLaboratoryfor

providingcells,vectors,reagentsandtechnicalassistance.WealsothankE.Besmerforhelpwithmanuscriptpreparation,andA.PatkeandmembersoftheTarakhovskylaboratoryfordiscussions.ThisworkwassupportedbyTheIreneDiamondFund/ProfessorshipProgram(A.T.),theNIH(A.T.)andTheS.L.E.Foundation(I.M.).

CompetinginterestsstatementTheauthorsdeclarethattheyhavenocompeting nancialinterests.

CorrespondenceandrequestsformaterialsshouldbeaddressedtoA.T.(tarakho@mail.rockefeller.edu).

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NF-kBfunctionsasatumourpromoterin

in ammation-associatedcancer

EliPikarsky1*,RinnatM.Porat1*,IlanStein1,2*,RinatAbramovitch3,SharonAmit2,Sha kaKasem1,ElenaGutkovich-Pyest2,SimchaUrieli-Shoval4,EithanGalun3&YinonBen-Neriah2

DepartmentofPathology,Hadassah-HebrewUniversityMedicalCenter,Jerusalem91120,Israel2

TheLautenbergCenterforImmunology,HebrewUniversity-HadassahMedicalSchool,Jerusalem91120,Israel3

GoldyneSavadInstituteofGeneTherapy,Jerusalem91120,Israel4

HematologyUnit,HadassahUniversityHospital,MountScopus,Jerusalem91120,Israel

*Theseauthorscontributedequallytothiswork

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1

Thecausesofsporadichumancancerareseldomrecognized,butitisestimatedthatcarcinogenexposureandchronicin am-mationaretwoimportantunderlyingconditionsfortumourdevelopment,thelatteraccountingforapproximately20%ofhumancancer1.Whereasthecausalrelationshipbetweencarcinogenexposureandcancerhasbeenintenselyinvestigated2,themolecularandcellularmechanismslinkingchronicin am-mationtotumorigenesisremainlargelyunresolved1.Wepro-posedthatactivationofthenuclearfactorkB(NF-kB),ahallmarkofin ammatoryresponses3thatisfrequentlydetectedintumours4,5,mayconstituteamissinglinkbetweenin ammationandcancer.Totestthishypothesis,westudiedtheMdr2-knock-outmousestrain,whichspontaneouslydevelopscholestatichepatitisfollowedbyhepatocellularcarcinoma6,aprototypeofin ammation-associatedcancer7.WemonitoredhepatitisandcancerprogressioninMdr2-knockoutmice,andhereweshowthatthein ammatoryprocesstriggershepatocyteNF-kBthroughupregulationoftumour-necrosisfactor-a(TNFa)inadjacentendothelialandin ammatorycells.SwitchingoffNF-kBinmicefrombirthtosevenmonthsofage,usingahepatocyte-speci cinducibleIkB-super-repressortransgene,hadnoeffectonthecourseofhepatitis,nordiditaffectearlyphasesofhepatocytetransformation.Bycontrast,suppressingNF-kBinhi-bitionthroughanti-TNFatreatmentorinductionofIkB-super-repressorinlaterstagesoftumourdevelopmentresultedinapoptosisoftransformedhepatocytesandfailuretoprogresstohepatocellularcarcinoma.OurstudiesthusindicatethatNF-kBisessentialforpromotingin ammation-associatedcancer,andisthereforeapotentialtargetforcancerpreventioninchronicin ammatorydiseases.

Hepatocellularcarcinoma(HCC),thethirdleadingcauseofcancermortalityworldwide,commonlydevelopsinthebackgroundofchronichepatitis8.Wecon rmedandextendedprevious

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results6,9,accordingtowhichtumourdevelopmentintheMdr2-knockoutmouse,similarlytohumanHCC8,progressesthroughdistinctphases:in ammation,dysplasia,dysplasticnodules(ade-noma-like),carcinomaandmetastasis(SupplementaryFig.1a,b).HepatitisistheearliestsignofdiseaseintheMdr2-knockoutmice,andischaracterizedbyanextensiveperiductularandperiportalmixedin ammatoryin ltrate,richinCD3-positivecells(Sup-plementaryFig.1c).

NF-kBactivationisoftenobservedinhumanHCC,particularlyfollowinghepatitis8.ThepossibilitythatNF-kBactivationisinvolvedinMdr2-knockouthepatocarcinogenesiswasinvestigatedbyRelA(p65)immunostaining(Fig.1a).Nuclearstaining,indica-tiveofNF-kBactivation,wasevidentinknockoutliversofmiceatallages,butnotinnormal,age-matchedmice;thispromptedasearchforthesourceofNF-kBactivationintheknockoutmice.InsomeneoplasmssuchasHodgkin’sdiseaseandcertainlymphomas,NF-kBactivationisanintrinsic,tumourautonomousfeature,possiblyrelatedtomutationsinitssignallingpathway10.IfasimilarmechanismoccursinMdr2-knockoutHCC,onewouldobservenuclearNF-kBactivationinallneoplastichepatocytes.However,thiswasnotthecase;NF-kBactivationappearedscatteredattheparenchyma,predominantlyadjacenttoin amedportaltracts(Fig.1e,leftpanel),suggestinganin ammation-inducedphenom-enon.Toevaluatethispossibility,wefedknockoutandwild-typemicewiththenon-steroidalanti-in ammatorydrug(NSAID)ibuprofenfor10days.Thistreatmentresultedindecreasedin am-mation,evidentbyhistologicalanalysis(Fig.1b),decreasedserumalanineaminotransferase(ALT),amarkerforhepatocytedamage,(Fig.1c)andfewerneutrophils(Fig.1c,d).p65immunostainingshowedthatthedegreeofhepatocyteNF-kBactivationintheNSAID-treatedgroupwassigni cantlylowerthaninthecontrols(Fig.1e).Incontrast,ibuprofenhadnoeffectonhepatocyteNF-kBactivationfollowingintraperitonealTNFaadministration(datanotshown).Hence,itseemsthatNF-kBactivationintheknockouthepatocytesissecondarytoparenchymalin ltrationbyin amma-torycells.

OnelikelymediatorofNF-kBactivationinthein amedliverisTNFa,particularlyitsmembraneform,whichisupregulatedinknockoutlivers(Fig.2a).Tostudythispossibility,wetreatedknockoutmicewithneutralizinganti-TNFaantibodiesforthreedaysandanalysedhepatocyteNF-kBactivation.Anti-TNFatreat-mentabolishedhepatocytep65nuclearstaining(Fig.2b),con rm-ingthepredictionthatNF-kBactivationisprimarilyinducedbyTNFa.TodeterminethesourceofTNFainthein amedliver,wefractionatedwild-typeandknockoutliverstohepatocytes(.80%puritybyhaematoxylin-and-eosin(H&E))andothercells(,2%hepatocytes).AnalysisofthetwofractionsforTNFaexpressionbyreal-timepolymerasechainreaction(PCR)con rmedthatthesourceofTNFawasthenon-hepatocytefraction,whichexpressed vefoldmoreTNFathanthecorrespondingfractionofwild-typemice(Fig.2c).

TostudytherelationshipbetweentheTNFa-producingcellsandNF-kBactivationinthehepatocytes,liversectionswerestainedforbothTNFaandp65(Fig.2d,f).TNFaexpressionwasdetectedprimarilyatportalspaces,bothinendothelialandin ammatorycells(Fig.2d).Hepatocytenuclearp65stainingwasparticularlyabundantclosetoTNFa-positiveportalspaces(Fig.2f),indicatingthathepatocyteNF-kBisactivatedthroughparacrineTNFastimu-lation.Tocon rmtheidentityoftheTNFaproducersanddis-tinguishthemfromTNFa-bindingcells,liversectionsofwild-type,knockoutandibuprofen-treatedmicewereanalysedbyinsitumessengerRNAhybridization.Similartotheimmunostainingdata,TNFamRNAwasfoundtobehighlyexpressedbyendothelialandin ammatorycellsofknockoutmice,butfarlessinwild-typeandibuprofen-treatedknockoutmice.TNFaexpressionwashardlydetectedineitherknockoutorwild-typehepatocytes(Fig.2e).ThefrequentactivationofNF-kBinknockouthepatocytes

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suggestedthatin ammation-associatedNF-kBactivationpromotesneoplasticgrowth4,5.TodirectlyassesstheroleofNF-kBinhepato-carcinogenesis,webredMdr2-knockoutmicewithDN-IkBhepmice,whichcarrytwotransgenes:anon-degradableDN-IkBcontrolledbyatetracycline(tet)-regulatedpromoterandthetetracyclinetrans-activatorundercontrolofthehepatocytespeci cC/EBPbpromoter(TALAP1)11.Thetet-regulatedpromoteralsodirectstheexpressionofluciferase;consequently,transgeneexpressionlevelsanditstissuespeci citycanbedeterminedusingalive-mouserecordingofluciferaseactivity11.Crossingthebi-transgenicDN-IkBhepmicewithMdr2-knockoutmicegeneratesMdr22/2DN-IkBhep(doublemutant)miceamenabletoNF-kBmodulation.Immunohisto-chemicalstainingofliversfromthesameanimalswithanti-luciferaseantibodiesdemonstratedthatonlyhepatocytes,butnotthebileductepithelium,expressthetransgene(SupplementaryFig.2a).Asexpected,p65immunostainingdetectedmanypositivehepatocytenucleiinknockoutanddoxycycline(Dox)-treated(transgene-suppressed)mice,butnoneintheuntreateddoublemutantmice(SupplementaryFig.2b,c).Positively-stainedbiliaryandKupffercellsweredetectableinallknockoutanddoublemutantmiceirrespectiveofDoxtreatment(notshown),attestingtothespeci cityandef ciencyofthetransgeneinblockingNF-kBactivation.

Becausetheoriginofliverin ammationintheMdr2-knockoutisthebiliarysystem6,itshouldnotbeaffectedbythehepatocyte-speci cDN-IkBheptransgene.Nevertheless,beforeassessingtheroleofNF-kBintumorigenesis,wewantedtoruleoutanypossibleeffectofNF-kBinhibitiononthein ammatoryprocess.Boththeknock-outandthedoublemutantmicehadhigherALTlevelsthanthoseofage-matchedwild-typemice(Fig.3b).Histologicalanalysisofliversfrombothgroupsatallagesrevealedcomparablenon-suppurativecholangitiswithportalexpansionduetoductularproliferation,amixedportalin ammatoryin ltrateandmildtomoderate brosis(Fig.3a).BothstrainshadhighnumbersofCD3-postiveandmyeloperoxidase(MPO)-positivecellsintheportaltractsandtheliverparenchyma.Thus,itseemsthatthefundamentalin amma-toryprocessinMdr2-knockoutmiceismaintainedinthedoublemutantmiceandisindependentofhepatocyteNF-kBactivity.Mdr2-knockouthepatocytesaredistinguishablefromwild-typecellsbyseveralabnormalfeatures:highproliferationrate,acceler-atedhyperploidyanddysplasia.Whereasthe rstarealsocharac-teristicsofliverdamageandpartialhepatectomy12,dysplasiaisapremalignantcondition13.Hepatocyteproliferationwasevaluatedbybromo-deoxyuridine(BrdU)incorporationandKi-67antigenstaining.Bothmarkerswereclearlyenhancedinknockoutanddoublemutantmicecomparedwithwild-typemice(Fig.3b).Inagreementwithpreviousstudies14,15,NF-kBde ciencydoesn’tcompromisehepatocyteproliferation:therewasnosigni cantdifferenceineitherhepatocyteBrdUincorporationorKi-67stain-ingbetweentheknockoutandthedoublemutantmiceatany

age.

Figure1NF-kBactivation,aprominentcomponentofMdr2-knockouthepatitis,isabrogatedbyNSAIDtreatment.a,Tissuesectionsfromwild-typeandknockoutmicewereimmunostainedwithantibodiesagainstp65/RelA.TNFa-treatedwild-typeliverservesasapositivecontrolforp65staining.HCC:asectionfromahepatocellularcarcinomathatdevelopedinaknockoutmouse.b,H&E-stainedsectionsfromliversof2.5-month-oldwild-type,knockoutandibuprofen-treatedknockoutmice.Notedecreasedin ammatoryin ltrateafteribuprofentreatment.c,MPOcellcountsandserumALTlevelsfrom

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wild-type,knockoutandibuprofen-treatedknockoutmice(mean^s.e.m.).

d–e,ImmunohistochemicalstainsforMPO(d)andp65(e)wereperformedonsectionsfromuntreatedandibuprofen-treatedknockoutmice.NotetheNF-kBnuclearstaininginthehepatocytesandbileductsofknockoutmiceandthedecreasedstaininginibuprofen-treatedmice.Allscalebars,50mm.Knockout(KO)¼Mdr22/2;WT,wildtype.Arrowspointtoexamplesof:Kupffercells(redarrows),NF-kB-positivehepatocytes(yellowarrows)andbileepithelium(turquoisearrows).

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Hepatocytedysplasiahastwomajorfeatures,architecturaldis-organizationandcytologicalatypia.Histologicalanalysisrevealedthatanisocytosiswasaprominentfeatureofbothknockoutanddoublemutantmice;dysplasiawasevidentat4monthsandincreasedinseveritywithageinbothanimalstrains.Wecouldnotdetectanydifferenceintheextentordegreeofdysplasiabetweenknockoutanddoublemutantmiceatanyagegroup(Fig.3c).Inanattempttoquantifythepremalignantchangesweanalysedhepato-cyteploidyinbothstrainsincomparisontoage-matchedwild-typemice.Isolatednucleifromliverswerestainedwithpropidium-iodideandsubjectedto uorescence-activatedcellsorting(FACS)analysis.Bothgroupshadahighdegreeofhyperploidy,andwenotednosigni cantdifferencebetweenthecell-cyclepro lesoftheknockoutanddoublemutantmiceat4or7months(Fig.3b).The ndingthatknockoutanddoublemutantmicedisplaycomparabledegreesofproliferation,hyperploidyanddysplasiaimpliesthatNF-kBisnotrequiredforearlyneoplasticevents.

WhereasNF-kBinhibitionhadnoeffectatthetumourinitiationphases,magneticresonanceimaging(MRI)andhistologicalanaly-sisofoldermiceclearlydiscernedtheknockoutandDox-treatedmicefromtheuntreated(NF-kBde cient)doublemutantmice.At10months,60%and78%ofknockoutandDox-treateddoublemutantmice,respectively,hadlivertumours,comparedwithonly10%ofuntreateddoublemutantmice(P,0.01,Fig.4).Thus,ablationofNF-kBactivityinthehepatocytesledtoadramaticdecreaseintumourprogression.AsimilartumoursuppressioneffectintheNF-kB-de cientanimalswasnotedwhencomparingthemeannumberoftumoursperanimalforthetwomousegroups.

Histologicalanalysisoftheliverscon rmedtheMRIdata(datanotshown).

AtwhichstageoftumourdevelopmentdoesNF-kBinhibitionexertitsanti-tumorigeniceffect?By7monthsnodifferenceinearlytumourdevelopmentwasnoticedbetweenknockoutanddoublemutantmice:neithergrouphaddetectabletumours;onlydysplasticparenchyma.Wethereforeswitchedoffthetransgeneat7months,allowingNF-kBactivationinthehepatocytes.TumourdevelopmentwasmonitoredusingMRIfor3months.Surprisingly,7of9micefedonDox(NF-kBpro cient)developedMRI-detectabletumourswithinthis3-monthperiod.Thesewereindistinguishableinsize(Fig.4b)andhistologicalappearance(datanotshown)fromknockouttumoursatacomparableage.Hence,itseemsthatwhereasNF-kBisdispensablefortheearlypremalignantphase,(thatis,tumourinitiation)itisessentialforsubsequenttumourpromotion.

Finally,howdoesNF-kBsupporttumourpromotion?promisingtheanti-apoptoticfunctionofNF-kBcouldcontributetothetumour-suppressingeffectoftheIkB-super-repressorinthedoublemutantmice.Tostudythispossibility,weassessedapoptosisusingimmu-nostainingforactivatedcaspase-3.Knockoutliversat7months(inwhichthemajorityofhepatocytesaredysplastic;Fig.3c)showedmoreapoptosisthanwild-typelivers—probablyduetothetoxiceffectsofin ammation(Fig.5a).However,blockinghepatocyteNF-kBinducedafurtherthreefoldincreaseinhepatocyte

apoptosis.

Figure2NF-kBactivationinMdr2-knockoutmiceisinducedbyparacrineTNFa.

a,WesternblotanalysisofTNFainknockoutversuswild-typeliversamples.Anti-TNFaantibodiesdetectonlythetransmembraneTNFa(mTNFa;26kD),theexpressionofwhichisenhancedintheknockoutsamples.n.s.,nonspeci cband.b,Anti-p65immunostainingofknockoutliversfrom7-month-oldmicetreatedwithanti-TNFaneutralizingantibodiesorcontrolIgGasindicated.Quantitativeanalysisrevealeda vefoldreductioninthenumberofp65-positivehepatocytenuclei.c,Real-timePCRanalysisofTNFainhepatocyte(H)andnon-hepatocyte(R)cellularfractionsofknockoutandwild-typelivers.TNFa-treated(TNF)andnon-treated(NT)mouseembryo broblasts

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(MEF)serveascontrols.ValuesgivenareTNFamRNAlevelsnormalizedtoaribosomalstandard(mean^s.e.m.).d,ImmunostainingforTNFa;notethemarkedendothelialandmononuclearcellstaininginknockoutmice.e,InsitumRNAhybridization(ISH)forTNFa;notetheabundantsignalsinendothelial,mononuclearandKupffercells,particularlyinuntreatedknockoutsamples.f,DoubleimmunostainingofknockoutliversectionsforTNFa(AEC,redprecipitate)andp65(NBT/BCIP,purple-blackprecipitate).Allscalebars,50mm.Arrows:red,Kupffercells;yellow,hepatocytes;turquoise,endothelium;purple,in ammatorycells.

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Figure3InhibitionofhepatocyteNF-kBactivityin uencesneitherhepatitisnordevelopmentofthepremalignantfeaturesinMdr2-knockoutmice.a,H&E-stainedsectionsof4-month-oldwild-type,knockoutanddoublemutantmice.b,SerumALTlevels,averagenumberofCD3-positivecellsperonehighpower eld(HPF),numberofKi-67-positivehepatocytesper20HPFsandpercentageofhyperploidnuclei.Allbar-graphsindicatemean^s.e.m.c,H&E-stainedsectionsfromliversof7-month-oldmiceoftheindicatedgenotypes,showingcomparabledysplasiaevidentbyarchitecturaldisorganizationandprominentnuclearpleomorphisminbothknockoutsanddoublemutants,incontrasttothenormalnucleiinwildtypes.Allscalebars,100mm.m.o.,monthsold;DM,doublemutant.

Figure4NF-kBisdispensableforearlystagesofHCCdevelopment,butiscriticalfortumourpromotion.a,MRIsfromtheindicatedgenotypesweretakenat10monthsofage;eachimageshownisfromadifferentanimal.Reddashedlinesoutlinetheliver

circumference.Thecolouredarrowheadsindicatethetumours;differentcoloursindicateindividualtumours.b,Livertumourvolumewascalculatedbyanalysingallcoronaland

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axialimagesofeachanimal;nodulessmallerthan100mm3wereoftenfoundtorepresentdysplasticnodulesratherthanHCCinhistologicalanalysisandwerethereforenotscoredastumours.DMþDox¼micefedonDoxbetween7–10monthsofage.BothknockoutanddoublemutantþDoxgroupsdiffersigni cantly(P,0.01,Mann–Whitneyanalysis)fromthedoublemutantgroup,butnotfromeach

other.

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Notably,asimilareffectwasachievedbyanti-TNFatreatment.Takentogether,ourresultssuggestthatNF-kBinhibitionexertsitstumour-suppressiveeffectbypromotingapoptosisoftransformedhepatocytes.

NF-kBregulatesmultipleanti-apoptoticgenes,someofwhichcouldberelevanttohepatocytetransformation.Toidentifyrelevanttargetgenesandcorroboratethepro-tumorigenicroleoftheTNFa–NF-kBactivationaxis,expressionofcandidategeneswasdeterminedbysemiquantitativePCRwithreversetranscription(RT–PCR)andwesternblotanalyses.Whereassomeexamined

targets(forexample,XIAP)weresimilarlyexpressedinNF-kB-pro cientand-de cientlivers,A1/B 1,c-IAP1andGADD45bweresigni cantlyenhancedintheknockoutlivers(SupplementaryFig.3a,b).Concordantly,anti-TNFatreatmentpreventedtheinductionofGADD45bandA1/B 1inknockoutmice(Fig.5b).

Itisconceivablethatthein ammatoryprocessinthelivercontributestotumorigenesisthroughtwoarmsthatcomplementoneother(Fig.5d).Onearmwouldberesponsibleforinducinghepatocyteproliferationinknockoutlivers.AcceleratedDNAreplicationisbyitselferrorproneandagroundforfurtherpro-tumorigenicalterationsbygenotoxicagents18,manyofwhicharespeci callyactivatedintheliver(forexample,a atoxin)19.Inaddition,tumourpromotionrequiresasecondarmprovidingapoptosisprotectionthroughNF-kBactivation.OurdataindicatethatTNFaisamajordriveroftheanti-apoptoticarm,necessaryforactivationofNF-kB-dependentanti-apoptoticgenes,suchasA1/B 1,c-IAP1andGADD45b.AlthoughNF-kBisactivatedearlyinthecourseofthedisease,itisdispensableforaprolongedperiodduringwhichpremalignantfociaccumulateinthein amedliver,butiscriticalatthestageduringwhichthesefociturnmalignant.Perhapsacquisitionofoncogenicmutationsduringthetumourpromotionphaserenderspremalignanthepatocytesparticularlysensitivetoapoptoticfactors,whichmustbeneutralizedthroughNF-kBactivity.Putativeapoptosis-regulatingfactorsatthisvulner-abletumourdevelopmentphasearep53andcJun(ref.20),TNFreceptorfamilymembers21andcJunamino-terminalkinase(JNK)22.JNKactivationandaugmentedcJunexpressionwereobservedinknockouthepatocytesat7months(Fig.5candSupplementaryFig.3a),aroundthepeakperiodofpremalignantchanges(Fig.3).SimilarlytoNF-kB,theseweredependentoncontinuousTNFasignalling,asanti-TNFatreatmentofknockoutmicesuppressedliverJNKactivationandtheexpressionofhepato-cytecJun(Fig.5b,c).JNKactivationissubjecttoNF-kBinhibitionviatheNF-kBtargetGADD45b(refs23,24).NF-kBinhibitioninknockouthepatocytescouldthereforeenhanceJNK-mediatedapoptosisthroughinhibitionofGADD45b(Fig.5b),andpromotecaspase-mediatedhepatocyteapoptosisthroughsuppressionofmultipleNF-kB-inducedanti-apoptoticgenes(SupplementaryFig.3).Inparallel,cJuninhibitionbyanti-TNFatreatment(Fig.5c)couldpromotep53-mediatedapoptosis20.Thus,bysup-pressingmultipleanti-apoptoticpathways(Fig.5d),anti-TNFatreatmentmayserveasapotentialmodeofHCCprophylaxisinchronichepatitispatients.

Ourconclusionsaresupportedbyrecentstudiesinacolonin ammationmodel,inwhichcolonicIkBkinaseb(IKKb)in-activationresultsinepithelialapoptosisduringtumourpro-motion25.Whereasin ammationreleasesmanygrowthfactorsandcytokines1,26,ourdatasuggestthatTNFahasacentralroleinactivatingNF-kBandprotectingtransformedhepatocytesagainstapoptosis.Ithasbeenproposedthatanticancerresearchmightbemoreeffectiveifaimedateradicatingthecauseorthesignallingcontextofabnormalityratherthanjusttreatingtheendresult27.Whereaseradicatingtheprimarycause,forexamplechronichepa-titis,iscurrentlyunattainable,disruptingthesignallingcontextoftheevolvingtumourmaybeamorerealisticobjective.Our ndingthatshort-termablationofepithelialNF-kBactivationissuf cienttoinducepremalignantcellapoptosisandhalttumourprogressionsupportsthisconjecture.Intermittentsuppressionofamajorsignallingfactor,suchasNF-kBorTNFa,couldthusbeatooltoprotractthepremalignantphaseandinhibittumourprogressioninchronicin ammatorydiseaseswithhighcancerrisk.A

Figure5NF-kBinhibitionthroughtheIkB-super-repressororanti-TNFatreatment

resultsinsuppressionofanti-apoptoticfactors.a,Caspaseactivitywasquanti edbycountingthenumberofhepatocytesstainedwithantibodiesagainstactivatedcaspase-3in10high-power elds(mean^s.e.m.).b,Liverproteinextractsfromknockoutanimalstreatedfor3dayswithanti-TNFaantibodies(anti-TNF),orIgGweresubjectedtowesternblotanalysiswiththeindicatedantibodies.c,cJunimmunostainingofwild-typeandknockoutliversectionsfromIgG-andanti-TNFa-treatedmice.TNFa-treatedwild-typeliverisapositivecontrolforcJunstaining.Allscalebars,50mm.d,Aproposedmodelforin ammation-orchestratedsignallingatthepremalignantphaseofHCCdevelopment:TNFa,amajormediatorofthein ammatoryprocess,tiltsthebalanceinfavourofhepatocyteproliferation.Carcinogenicmetaboliteswouldpushtumourprogressionfurthertowardsmalignancy.

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Methods

ThefollowingdetailscanbefoundintheSupplementaryInformation:reagents,transgenicmouseconstructionandgenotyping,semiquantitativeRT–PCR,real-timePCR,insitumRNAhybridization,livercellfractionation,westernblotandhepatocyteploidy

analyses.

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Animalstudiesandtissuepreparation

Allanimalexperimentswereperformedinaccordancewiththeguidelinesofthe

institutionalcommitteefortheuseofanimalsforresearch.Micewerekilledbyalethaldoseofanaesthesia.Invivoluciferaseimaginganddoxycyclinetreatmentwereperformedaspreviouslydescribed11.Animalswereinjectedintraperitoneally(i.p.)with20nghumanrecombinantTNFa(Roche)andkilledafter1hforliverp65immunostainingandRNAandproteinextraction.Foranti-TNFatreatment,micewereinjectedwith22mgofgoatanti-mouseTNFaneutralizingantibodiesi.p.(R&DSystems;AF-410-NA)for3consecutivedays,orwithcontrolgoatIgG.Allmicewereinjectedi.p.withBrdU(Amersham;100mlper10gbodyweight)3and24hbeforetheywerekilled.Insomeanimals,liversampleswereremovedandsnap-frozenforproteinandRNAanalyses.Theanimalwasthenperfusedthroughtheleftventriclewith10mlofcoldheparinizedPBS,followedwith25mlof4%bufferedformalin.Liverswereremoved,weighed,photographedand xedinformalinovernight.Thenextdaytheentireliverwas

submittedforparaf nembeddinginthreeto vecassettes.5-mMsectionswerestainedwithH&Eandevaluatedbyapathologisttowhomthegeneticmakeuportreatmentgroupwerenotknown.

15.Maeda,S.etal.IKKbisrequiredforpreventionofapoptosismediatedbycell-boundbutnotby

circulatingTNFa.Immunity19,725–737(2003).

16.Beg,A.A.,Sha,W.C.,Bronson,R.T.,Ghosh,S.&Baltimore,D.Embryoniclethalityandliver

degenerationinmicelackingtheRelAcomponentofNF-kB.Nature376,167–170(1995)von,I.etal.Nuclearfactor-kBprotectstheliveragainstgenotoxicstressandfunctions

independentlyofp53.CancerRes.63,25–30(2003).

18.Friedberg,E.C.,McDaniel,L.D.&Schultz,R.A.TheroleofendogenousandexogenousDNAdamage

andmutagenesis.Curr.Opin.Genet.Dev.14,5–10(2004).

19.Wang,J.S.&Groopman,J.D.DNAdamagebymycotoxins.Mutat.Res.424,167–181(1999).

20.Eferl,R.etal.Livertumordevelopment.c-Junantagonizestheproapoptoticactivityofp53.Cell112,

181–192(2003).

21.Ehrhardt,H.etal.TRAILinducedsurvivalandproliferationincancercellsresistanttowards

TRAIL-inducedapoptosismediatedbyNF-kB.Oncogene22,3842–3852(2003).

22.Kennedy,N.J.&Davis,R.J.RoleofJNKintumordevelopment.CellCycle2,199–201(2003).23.Tang,G.etal.InhibitionofJNKactivationthroughNF-kBtargetgenes.Nature414,313–317(2001).24.Papa,S.etal.Gadd45bmediatestheNF-kBsuppressionofJNKsignallingbytargetingMKK7/

JNKK2.NatureCellBiol.6,146–153(2004).

25.Greten,F.R.etal.Ikkblinksin ammationandtumorigenesisinamousemodelofcolitis-associated

cancer.Cell118,285–296(2004).

26.Wilson,J.&Balkwill,F.Theroleofcytokinesintheepithelialcancermicroenvironment.Semin.

CancerBiol.12,113–120(2002).

27.Radisky,D.,Hagios,C.&Bissell,M.J.Tumorsareuniqueorgansde nedbyabnormalsignalingand

context.Semin.CancerBiol.11,87–95(2001).

MRIanalysis

MRIwasperformedonahorizontal4.7TBiospecspectrometer(BrukerMedical),usingabirdcagecoil.Micewereanaesthetized(30mgkg21pentobarbital,i.p.)andplacedsupinewiththeliverlocatedatthecentreofthecoil.Liverandtumourvolumesweredeterminedfrommulti-slicecoronalandaxialT1weightedfastspinechoimagescoveringtheentireliverbothcoronallyandaxially(repetitiontime,400ms;echotime,17ms;slicethickness,1mm; eldofview,5cm(coronal)and3.4cm(axial)usinga256£256matrix).Toestimatetumourandlivervolumes,theliverandtumourboundariesvisualizedineachslicewereoutlinedbyusingimageprocessingsoftware(NIHimage),byanobservertowhomthegeneticmakeupwasnotknown.Thenumberofpixels(forbothliverandtumour)wereconvertedtoanareabymultiplyingbythefactor(( eldofview)2£(matrix)2).

/nature.

AcknowledgementsMdr2-knockoutmicewereagiftfromR.P.OudeElferinkandtheTALAP1micewerereceivedfromH.Bujard.WearegratefultoN.Berger,T.GolubandN.Kidess-Bassirfortechnicalassistance,toA.Hatzubai,O.Mandlboim,N.Lieberman,G.Kojekaro,M.DavisandH.HarelforhelpingwithMRI,FACS,mRNAandproteinanalysis.WethankK.MeirandM.Orenfordiscussionsandacriticalreadingofthemanuscript.ThisresearchwassupportedbygrantsfromtheIsraelScienceFoundationfundedbytheIsraelAcademyforSciencesandHumanities(CenterofExcellenceProgram),ProstateCancerFoundationIsrael—CenterofExcellence,

German-IsraeliFoundationforScienti cResearchandDevelopment(GIF,incollaborationwithH.Bujard),agrantinmemoryofH.andF.BrodyfromH.M.KruegerastrusteeofacharitabletrustandtheIsraelCancerResearchFoundation(ICRF).I.S.issupportedbytheLadyDavisFellowshipTrust.

CompetinginterestsstatementTheauthorsdeclarethattheyhavenocompeting nancialinterests.

CorrespondenceandrequestsformaterialsshouldbeaddressedtoE.P.(peli@.il)andY.B.-N.(yinon@cc.huji.ac.il).

Immunohistochemistry

Forp65immunostains,5-mMsections,cutonthesameday,werede-waxedandhydratedthroughgradedethanols,cookedin25mMcitratebufferpH6.0inapressurecookerat1158Cfor3min(decloakingchamber,BiocareMedical),transferredintoboiling

deinoizedwaterandlefttocooldownfor20min.After5mintreatmentin3%H2O2,slideswereincubatedwithrabbitpolyclonalp65antibodiesdiluted1:100inCAS-Block(Zymed)for3hatroomtemperature,washedthreetimeswithOptimax(Biogenex),incubatedfor30minwithantirabbitEnvisionþ(DAKO)anddevelopedwithDABfor15min.AntigenretrievalforTNFaandp65doubleimmunostainingwasperformedin100mMglycinebufferpH9.0.After5mintreatmentin3%H2O2,slideswereincubatedwithgoat

polyclonalanti-TNFaantibodiesdiluted1:50inCAS-Blockfor1hatroomtemperature,washedinOptimax,incubatedwith(1:100)rabbitanti-goatantibodies(JacksonLaboratories)for30min,washedagainwithOptimaxandincubatedwithanti-rabbitEnvisionþ(DAKO)anddevelopedwithAECfor15minat378C.Followingthis,slideswerereboiledfor7minin100mMglycinebufferinamicrowaveovenandcooleddowntoroomtemperature.Slideswereincubatedwithrabbitpolyclonalanti-p65antibodiesdiluted1:100inCAS-Blockfor3hatroomtemperature,washedthreetimeswith

Optimax,post- xedfor5minin4%formaldehydeinTrisbufferedsaline,washedwithOptimax,incubatedfor30minwithalkalinephosphatase-conjugatedpolymeranti-rabbitIgG(Zymed)anddevelopedwithBCIP/NBT.Slideswerecounterstainedwithnuclearfastred.AntigenretrievalforCD3,cJun,BrdU,activatedcaspase-3,luciferase,MPOandKi-67wereperformedin25mMcitratebufferpH6.0.AntigenretrievalforTNFawasperformedin100mMglycinebufferpH9.0.Detailedprotocolsforimmunostainingwitheachantibodyareavailableonrequest.

Received14June;accepted10August2004;doi:10.1038/nature02924.Publishedonline25August2004.

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Mrf4determinesskeletalmuscleidentityinMyf5:Myoddouble-mutantmice

`s1,LinaKassar-Duchossoy1,BarbaraGayraud-Morel1*,DanielleGome

DidierRocancourt2,MargaretBuckingham2,VasilyShinin1*&ShahragimTajbakhsh1

StemCellsandDevelopment,2MolecularGeneticsofDevelopment,

DepartmentofDevelopmentalBiology,CNRSURA2578,25rueduDrRoux,75724ParisCedex15,France

*Theseauthorscontributedequallytothework

.............................................................................................................................................................................

1

Invertebrates,skeletalmuscleisamodelfortheacquisitionofcellfatefromstemcells1.Twodeterminationfactorsofthebasichelix–loop–helixmyogenicregulatoryfactor(MRF)family,Myf5andMyod,arethoughttodirectthistransitionbecausedouble-mutantmicetotallylackskeletalmuscle bresandmyoblasts2–4.Intheabsenceofthesefactors,progenitorcellsremainmulti-potentandcanchangetheirfate5,6.GenetargetingstudieshaverevealedhierarchicalrelationshipsbetweentheseandtheotherMRFgenes,Mrf4andmyogenin,wherethelatterareregardedasdifferentiationgenes7.Hereweshow,usinganallelicseriesofthreeMyf5mutantsthatdifferentiallyaffecttheexpressionofthegeneticallylinkedMrf4gene,thatskeletalmuscleispresentinthe

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