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Methods in Molecular Biology 1465 Shaoguang Li Haojian Zhang Editors Chronic Myeloid Leukemia Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 Chronic Myeloid Leukemia Methods and Protocols Edited by Shaoguang Li Department of Medicine Division of Hematology/Oncology University of Massachusetts Medical School Worcester, MA, USA Haojian Zhang Medical Research Institute Wuhan University Wuhan, China Editors ShaoguangLi HaojianZhang DepartmentofMedicine MedicalResearchInstitute DivisionofHematology/Oncology WuhanUniversity UniversityofMassachusetts Wuhan,China MedicalSchool Worcester,MA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-4009-7 ISBN978-1-4939-4011-0 (eBook) DOI10.1007/978-1-4939-4011-0 LibraryofCongressControlNumber:2016943846 ©SpringerScience+BusinessMediaNewYork2016 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper ThisHumanaPressimprintispublishedbySpringerNature TheregisteredcompanyisSpringerScience+BusinessMediaLLCNewYork Preface Cancerstemcellsareresponsiblefor theinitiationandprogressionofmanytypesofcancer andareingenerallesssensitivetochemotherapy.Thereisanurgentneedtofullyunderstand the biology of cancer stem cells for developing effective therapies, and the success in this approachreliesonutilizingstemcelldiseasemodelsandadvancedtechnologiestostudythe molecular basis of cancer stem cells. It is generally believed that human chronic myeloid leukemia (CML) is derived from an abnormal hematopoietic stem cell harboring Philadel- phiachromosomethatresultsintheformationoftheBCR-ABLoncogene.Leukemiastem cells (LSCs) in CML provide an ideal cellular system for studying how LSCs survive, proliferate, and evade from treatment with tyrosine kinase inhibitors. Technically, various molecularandcellularmethodshavebeenutilizedtohelpunderstandthebiologyofLSCsin CML,andthesemethodshaveledtobreakthroughdiscoveriesusefulindevelopingeffective therapeutic strategies for CML. This book highlights the molecular and cellular methods usedinstudyingCMLpathogenesisandstemcellbiology. Massachusetts,USA ShaoguangLi Wuhan,China HaojianZhang v Contents Preface ..................................................................... v Contributors................................................................. ix 1 MolecularDetectionofBCR-ABLinChronicMyeloidLeukemia............. 1 Ya-ZhenQinandXiao-JunHuang 2 InductionofChronicMyeloidLeukemiainMice........................... 17 HaojianZhangandShaoguangLi 3 CMLMouseModelGeneratedfromLeukemiaStemCells................... 27 YiguoHu 4 ImmunologicalAnalysesofLeukemiaStemCells ........................... 37 KazuhitoNakaandYoshihiroTakihara 5 CellCycleAnalysisofCMLStemCellsUsingHoechst33342 andPropidiumIodide................................................... 47 NgocDeSouza,MeganZhou,andYiShan 6 HistologicalandInVivoMicroscopicAnalysisoftheBoneMarrow MicroenvironmentinaMurineModelofChronicMyelogenousLeukemia .... 59 EvaS.WeissenbergerandDanielaS.Krause 7 TheCultureRepopulationAbility(CRA)AssayandIncubation inLowOxygentoTestAntileukemicDrugsonImatinib-Resistant CMLStem-LikeCells ................................................... 73 GiuliaCheloniandMicheleTanturli 8 TumorSuppressorAnalysisinCML....................................... 87 OliverHerrmannandMirleSchemionek 9 DetectingAutophagyandAutophagyFluxinChronicMyeloid LeukemiaCellsUsingaCyto-IDFluorescenceSpectrophotometricAssay ..... 95 SujuanGuo,KevinJ.Pridham,andZhiSheng 10 DREAM:ASimpleMethodforDNAMethylationProfiling byHigh-throughputSequencing ......................................... 111 JaroslavJelinekandJozefMadzo 11 ChIP-seqAnalysisofHumanChronicMyeloidLeukemiaCells ............... 129 LarsAndersandZhaodongLi 12 QuantitativeProteomicsAnalysisofLeukemiaCells......................... 139 SebastianHalbach,JörnDengjel,andTilmanBrummer 13 AConvenientCellCultureModelforCMLAcquiredResistance ThroughBCR-ABLMutations........................................... 149 ZhiqiangWangandWenYongChen 14 High-ThroughputScreeningofTyrosineKinaseInhibitorResistant GenesinCML ......................................................... 159 LeyuanMa,JustineRoderick,MichelleA.Kelliher,andMichaelR.Green vii viii Contents 15 BiologicalAnalysisofHumanCMLStemCells;XenograftModel ofChronicPhaseHumanChronicMyeloidLeukemia....................... 175 SheelaA.Abraham 16 NewMouseModelstoInvestigatetheEfficacyofDrugCombinations inHumanChronicMyeloidLeukemia .................................... 187 HanyangLin,AdrianWoolfson,andXiaoyanJiang 17 miRNomeAnalysisofCMLCells......................................... 207 YadongYang,NanDing,XunongDong,andXiangdongFang 18 AnIntegrativeAnalysisofmicroRNAandmRNAProfiling inCMLStemCells ..................................................... 219 FarahJ.Nassar,RababElEit,andRihabNasr 19 Single-CellCytokineProfilingtoInvestigateCellularFunctional DiversityinHematopoieticMalignancies .................................. 243 JonathanJ.Chen,MinsukKwak,andRongFan Index ...................................................................... 255 Contributors SHEELAA.ABRAHAM (cid:1) PaulO’Gorman LeukaemiaResearchCentre,CollegeofMedical, Veterinary&LifeSciences,InstituteofCancerSciences,UniversityofGlasgow, Glasgow,UK LARSANDERS (cid:1) WhiteheadInstituteforBiomedicalResearch,Cambridge,MA,USA TILMANBRUMMER (cid:1) InstituteofMolecularMedicineandCellResearch(IMMZ),Faculty ofMedicine,UniversityofFreiburg,FreiburgimBreisgau,Germany;FacultyofBiology, UniversityofFreiburg,FreiburgimBreisgau,Germany;SpemannGraduateSchoolof BiologyandMedicine,UniversityofFreiburg,FreiburgimBreisgau,Germany GIULIACHELONI (cid:1) DepartmentofExperimentalandClinicalBiomedicalSciences“Mario Serio”,SectionofExperimentalandClinicalOncology,Universita` degliStudidiFirenze, Firenze,Italy WENYONGCHEN (cid:1) DepartmentofCancerBiology,BeckmanResearchInstitute,CityofHope, Duarte,CA,USA JONATHANJ.CHEN (cid:1) DepartmentofBiomedicalEngineering,YaleUniversity,NewHaven, CT,USA JO¨RNDENGJEL (cid:1) BIOSSCentreforBiologicalSignallingStudies,UniversityofFreiburg, FreiburgimBreisgau,Germany;FRIASFreiburgInstituteforAdvancedStudies, UniversityofFreiburg,FreiburgimBreisgau,Germany;DepartmentofDermatology, UniversityMedicalCenter,UniversityofFreiburg,FreiburgimBreisgau,Germany; ZBSACenterforBiologicalSystemsAnalysis,UniversityofFreiburg,FreiburgimBreisgau, Germany NGOCDESOUZA (cid:1) UniversityofMassachusettsMedicalSchool,Worcester,MA,USA NANDING (cid:1) CASKeyLaboratoryofGenomeSciencesandInformation,BeijingInstitute ofGenomics,ChineseAcademyofSciences,Beijing,China XUNONGDONG (cid:1) CASKeyLaboratoryofGenomeSciencesandInformation,Beijing InstituteofGenomics,ChineseAcademyofSciences,Beijing,China RABABELEIT (cid:1) DepartmentofAnatomy,CellBiologyandPhysiologicalSciences, FacultyofMedicine,AmericanUniversityofBeirut,Beirut,Lebanon RONGFAN (cid:1) DepartmentofBiomedicalEngineering,YaleUniversity,NewHaven,CT,USA XIANGDONGFANG (cid:1) CASKeyLaboratoryofGenomeSciencesandInformation,Beijing InstituteofGenomics,ChineseAcademyofSciences,Beijing,China MICHAELR.GREEN (cid:1) DepartmentofMolecular,CellandCancerBiology,University ofMassachusettsMedicalSchool,Worcester,MA,USA;HowardHughesMedicalInstitute, ChevyChase,MD,USA SUJUAN GUO (cid:1) VirginiaTechCarilionResearchInstitute,Roanoke,VA,USA SEBASTIANHALBACH (cid:1) InstituteofMolecularMedicineandCellResearch(IMMZ), FacultyofMedicine,UniversityofFreiburg,FreiburgimBreisgau,Germany;Faculty ofBiology,UniversityofFreiburg,FreiburgimBreisgau,Germany;SpemannGraduate SchoolofBiologyandMedicine,UniversityofFreiburg,FreiburgimBreisgau,Germany OLIVERHERRMANN (cid:1) DepartmentofHematology,Oncology,Hemostaseology,andStemCell Transplantation,FacultyofMedicine,UniversityHospitalRWTHAachen,Aachen, Germany ix