17 Hypomethylation of tumor suppressor genes in od

BrazDentJ22(5)2011422P.R.Moreiraetal.INTRODUCTIONOdontogenicmyxoma(OM)isanectomesenchymalbenignodontogenictumorcharacterizedbystellateandspindle-shapedcellsembeddedinanabundantmyxoidormucoidextracellularmatrix(1).Thislocallyaggressiveneoplasmisthesecondmostcommonodontogenicneoplasm(afterameloblastoma),withanincidenceofapproximately0.07newcasespermillionpeopleperyear(2).Despiteitsbenignnature,OMhasahighrateoflocalrecurrenceaftercurettagealone(3).Althoughmutationsoftheregulatorysubunit1Aofproteinkinase.AhavebeendescribedinOM(4),littleisknownaboutitsmolecularpathogenesis.DNAmethylationisanefficientepigeneticHypomethylationofTumorSuppressorGenesinOdontogenicMyxomaPaulaRochaMOREIRA¹FabianoPereiraCARDOSO²JoãoArturRicieriBRITO²AlineCarvalhoBATISTA³CarolinaCavaliériGOMES4RicardoSantiagoGOMEZ²1LaboratoryofCell-CellInteractions,DepartmentofMorphology,InstituteofBiologicalSciences,UFMG-FederalUniversityofMinasGerais,BeloHorizonte,MG,Brazil²LaboratoryofMolecularBiology,DepartmentofOralSurgeryandPathology,DentalSchool,UFMG-FederalUniversityofMinasGerais,BeloHorizonte,MG,Brazil³DepartmentofStomatology,DentalSchool,UFG-FederalUniversityofGoiás,Goiânia,GO,Brazil4DepartmentofPathology,UFMG-FederalUniversityofMinasGerais,BeloHorizonte,MG,BrazilOdontogenicmyxoma(OM)isanectomesenchymalbenignodontogenictumorcharacterizedbyspindleorstellate-shapedcellsembeddedinanabundantmyxoidormucoidextracellularmatrix.DNAmethylationischaracterizedbytheadditionofmethylgroupsincytosineswithinCpGislandsinthepromotergene.DNAmethylationcandecreasetheexpressionoftumorsuppressorgenesandcontributetothedevelopmentofneoplasticlesions.TheaimofstudywastoevaluatethemethylationpatternofthetumorsuppressorgenesP16(CDKN2A),P21(CDKN1A),P27(CDKN1B),P53(TP53)andRB1inOManddentalpulp.Methylationwasevaluatedusingmethylation-specificpolymerasechainreaction(PCR).ThetranscriptionwasstudiedinsomecasesbyusingreversetranscriptionquantitativePCR.AhigherfrequencyofunmethylatedP27,P53,andRB1sampleswasobservedintheOMwhencomparedwiththedentalpulp.OMexpressedmRNAofallthegenesevaluated.Consideringallthesamplestogether,theexpressionofRbwashigherintheunmethylatedsamplescomparedwiththepartiallymethylatedsamples.ThisinvestigationrevealedhypomethylationofthegenesP27,P53,andRB1inOM.Inaddition,methylationoftumorsuppressorgeneswasfoundtobeanusualeventinnormaldentalpulp.KeyWords:methylation,tumorsuppressorgenes,odontogenicmyxoma,dentalpulp.mechanismoftranscriptionalrepressionthatoccursincytosineswithinCpGdinucleotides.Thepresenceorabsenceofmethylgroupsincytosinespromotestheremodelingofchromatin,makingitlessormoreaccessibletotranscription(5).Epigeneticalterationsrelatedtotheregulationofgenesinvolvedinthecellcyclecontrolhavebeendescribedindifferentbenignandmalignantneoplasms(6-8).Recently,distinctmethylationprofilesintumorsuppressorgeneshavebeenreportedinepithelialodontogenictumors(9).ThepresentstudyisthefirstattempttoinvestigatethemethylationprofileofOM.MethylationpatternofthefollowingtumorsuppressorgeneswasinvestigatedinOManddentalpulps:P16(CDKN2A),P21(CDKN1A),P27(CDKN1B),P53(TP53)andRB1.InordertoCorrespondence:Profa.Dra.PaulaRochaMoreira,LaboratóriodeBiologiaMolecular,FaculdadedeOdontologia,UFMG,AvenidaAntônioCarlos,6627,Pampulha,31270-901BeloHorizonte,MG,Brasil.Tel:[phone].Fax:[phone].e-mailaddress:[e-mail]0103-6440BrazDentJ(2011)22(5):422-427BrazDentJ22(5)2011Hypomethylationinodontogenicmyxoma423validatetheimportanceofmethylationprofileintheregulationofgeneexpression,thetranscriptionoftumorsuppressorgeneswasalsostudiedinsomecases.MATERIALANDMETHODSSamplesTwenty-foursampleswereexaminedinthisstudy,whichincluded9samplesofOMand15samplesofdentalpulptissue.TheOMgroupwascomposedofsamplesof6femaleand3malesubjects(agerangingfrom13to34years),andthedentalpulpgroupwascomposedof12femaleand3malesubjects(agerangingfrom16to29years).Freshsamplesandparaffin-embeddedtissuesofOMwerecollectedduringincisionalbiopsy,enucleation,orresectionofthelesion.Pulptissuesampleswereobtainedfromthethirdmolarimpactedteethextractedfromhealthyvolunteers.ThefreshsampleswereimmediatelyincludedinTissue-Tek(SakuraFinetek,Torrance,CA,USA)andstoredat-80°C.Paraffin-embeddedtissueswerepreviouslyfixedin10%formalin.ForRNAextraction,apieceofthetissuewasstoredinRNAholder(BioAgencyBiotecnologia,SãoPaulo,SP,Brazil)at-80°C.ThisstudywasapprovedbyUFMG’sEthicsCommittee(Protocol#COEP266/11).DNAExtractionGenomicDNAfromtissuesectionsof24sampleswasextractedwiththeQIAampDNAMinikit(Qiagen,Hilden,Germany),asperthemanufacturer’sprotocol.BisulfiteModificationThemethylationpatternoftissueswasassessedusingDNAmodificationbybisulfitetreatment,similartothatreportedbyGoldenbergetal.(10).Withthebisulfitetreatment,unmethylatedcytosinesofDNAareconvertedtouracilwhilemethylatedcytosinesremainunmodified.Bisulfiteconversionwascarriedoutwith1µgofDNA,whichwasdenaturedbyincubationwith2µLofNaOH(3M)for20minat50°C.Then,theamplesweretreatedwithsodiumbisulfite(2.5M)andhydroquinone(1M)for3hat70°C.ModifiedDNAsampleswerepurifiedwithWizardD