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Mechanisms of Skeletal Disease Mediated by Haematological

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Mechanisms of Skeletal Disease Mediated by Haematological OF z a ) n Mechanisms of Skeletal Disease Mediated by Haematolo gical Malignancies Beiqing Pan B. Med. M. Med. Sc. Matthew Roberts Laboratory, Hanson Institute, Institute of Medical and Veterinary Science Department of Medicine, The University of Adelaide, South Australia A thesis submitted to the University of Adelaide in candidature for the degree of Doctor of Philosophy August 2004 TABLE OF CONTENTS DEGLARAT|ON......... """' l AGKNOWLEDGMENT.............. """' ll A8STRACT............. """'lv ABBREVIATIONS """""v1 PUBLICATIONS """"""'xl CHAPTER 1 GENERAL INTRODUCTION ......1 TO BONE 1.1 HAEMATOLOGICAL MALIGNANCIES WHICII GIBE RISE 1 LESIONS 2 1.1.1 Osteolytic Bone Disease Mediated by Multiple Myeloma 2 1.1.1.1 General Description...'.. 4 t.l.l.2 P athophysiolo gy of tvttvt 5 1.1.1.3 Osteolytic Bone Disease Lt.2 Osteoblastic Bone Disease Mediated by POEMS" 6 t.l.2.1 General DescriPtion...... 6 l.l.2.2 Pathophysiology of POEMS Syndrome. 1 8 1.1.2.3 Osteoblastic Bone Disease 1.2 BONE PHYSIOLOGY.............. 8 1.2.1 PhysiologicalBoneRemodelling 9 1.2.2 Bone ResorPtion 9 1.2.2.1 Osteoclasts 9 1.2.2.2 Osteoclast Stem Cells 1.2.3 BoneFormation.......'......... 1.2.3.1 Osteoblast Cells 1.2.3.2 OsteoblastStemCells..... 1.2.3.3 OsteocYtes '.".....' 1.2.3.4 Lining Cells FACTORS INVOLVED IN BONE LESIONS... t4 1.3 l6 1.3.1 Osteoclast Activating Factors (OAFÐ """"' 1.3.1.1 Interleukin-1P 16 1.3.1.2 Interleukin-6 t7 l8 1 .3.1 .3 Tumour Necrosis Factor-cx'...... 1.3.1.4 ParathyroidHormone Related Protein""""""' t9 1.3.2 RANKL/RANIIOPGSYStem 20 1.3.2.1 GeneralDescriPtion 20 1.3.2.2 RANKL/OPG Ratio and Bone Lesion 22 1.3.2.3 Incrçased RANKL/OPG Ratio in MM"' 22 1.3.2.4 The Regulation of RANKL/OPG Ratio in MM 23 1.3.3 Endothelin-1 (ET-l)'..'.'.... 26 1.3.3.1 ET-1Structure.... 26 26 1.3.3.2 Endothelin ConveÍing Enzyme-1 (ECE-1) .... 27 1 .3.3.3 ET- 1 Receptors ...............' t.3.3.4 The Expression and Function ofET-1 and Its Receptors"""' 30 l.3.3.5 ET-l and PathologY. 32 t.3.3.6 ET-l and Bone Cells 34 1.3.3.7 Application of ET-1 in Clinic 38 1.4 THE ROLE OF ANGIOGENESIS IN MM 38 t.4.1 Angiogenesis and Tumour Growth 39 1.4.2 Angiogenesis and Bone Remodelling 39 1.4.3 IL- 1 B and Angiogenesis.. :.........'...'..'... 40 1.4.4 RANKL and Angiogenesls'.......'....'.'.. 40 1.4.5 ET-l and Angiogenesis 1.5 1.6 1.7 GHAPTER 2 MATERIALS AND METHODS ............. ....44 .......44 2.1 CELL CULTURE. 44 2.r.1 Buffers and Media """"""""""" 2.1.1.l 2-Mercaptoethanol (2-ME) 44 2.1.1.2 Double-Strength Iscove's Modifred Dulbecco's Medium (2xrMDM)..44 2.r.1.3 10% (dv) Bovine Serum Albumin (BSA) 45 45 2.1.1.4 ThawMedium.. 45 2.1.1.5 Dulbecco' Modihed Eagles Medium-10 (DMEM-10) """"""""""""" 45 2.1.1.6 Roswell Park Memorial Institute Medium (RPMI-1640)-I0""""""""' 2.r.1;7 Alpha Modification of Minimum Essential Medium-10 (cr-MEM-l0).46 46 2.1.1.8 Serum Deprived Medium (SDM) ................ 46 2.7.2 Human Myeloma Cell Lines .............,., A7 2.1.3 Retroviral Packaging Cell Line PT67 "....'.'." 47 2.1.4 Murine Stromal Fibroblastic Cell Line, Swiss NIH-3T3 "' ..................,.,.. 47 2.1.5 Bone Marrow Mononuclear Cells (BMMNCs) """"""""' 48 2.1.6 BM CFU-F Clonogenic AssaY 48 2.1.7 Normal Human Bonc Cells (NHBC) 48 2.1.8 Normal Osteoblast Donor (NOD) 2.t.9 Co-culture of OBJike Cells with Myeloma Cells ' 49 2.1.10 2.t.tl 2.r.12 2.1.13 ', I CELL PROLIFERATION ASSAYS................... 50 50 2.2.1 Reagents Used in This StudY '.' 50 2.2.2 Cell Proliferation AssaY...' 2.3 EXAMINATION OF' GENE EXPRESSIONIN OSTEOBLAST-LIKE CELLS........ 2.3.1 TRIzolrM Isolation of Total RNA 2.3.2 Determination of RNA Concentration'...'.'...... " " " (PCR) 2.3.3 Reverse Transcription (RT) Polymerase Chain Reaction Amplifrcation of DNA 52 2.3.3.1 Synthesis of Complementary DNA (cDNA) ...'."' 53 2.3.3.1 P olymerase Chain Reaction (P CR) Amplifrcation of cDNA 2.3.4 Primers Used in This StudY.. 2.4IMMUNOLOGICALSTUDIESEXAMININGCELLSURF'ACE 54 54 2.4.1 Buffers and Fixatives 2.4.1.1 Blocking Buffer for Flow Cytometric Analysis" 2.4.1.2 Magnetic Activated Cell Sorting Buffer (MACS Buffers).. 54 2.4.r.3 Flow Cytometry Fixative (FACS Fix) ' 54 2.4.2 Single-Colour Flow Cytometric Analysis 55 2.4.3 Two-Colour Flow Cytometric Analysis 55 2.4.4 Three-Colour Flow Cytometric Analysis 56 2.4.5 Detection of Intracellular Antigens......'.. of OB- 2.4.6 Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) Labelling like Cells 57 57 2.4.7 Fluorescence-Activated Cell Sorling (FACS)...."" 2.4.8 Magnetic Activated Cell Sorting (MACS)'..'." 2.4.9 Immunofluorescence Staining for Confocal Microscopy' " " " " 2.4.10 Antibodies Used in This Study......'... 2,s PREPARATION oF A RETROVIRAL CONSTRUCT HARBOURING THE HUMAN ET-l cDNA 2.5.1 Buffers and Reagents 2.5.1.1 Luria-Bertani (LB) Broth' 2.5 .l .2 L-agat Plates ... '.. '... ' 2.5.1.3 SOC Medium'... 2.5.2 DNA SYnthesis bY PCR using Pfu DNA PolYmerase 2.5.3 Restriction Digestion of pRUFneo Vector and PCR Product 2.5.4 Extraction of DNA from Agarose Gels 2.5.5 Ligation of Insert to Vector 2.5.6 Prèparation of Electrocompetent DHI0B Cells for Transformations ...... 2.5.7 Transformation of Competent Cells. "... 2.5.8 Isolation and Analysis of Plasmid DNA"""""" 2.5.9 Automated DNA Sequence Analysis.' 2.5.10 Transfection of the Retroviral Packaging cell Line PT67 Using 63 2.5.ll Retroviral Infcction of OB-Like Cells and Myeloma Cells """"""""""' 64 2.6 IN VITRO ASSAY OF BONE FORMING POTENTIAL """'64 2.7 IN VIVO ASSAYS OF BONE FORMTNG POTENTrAL"""""""""""""" 65 66 2.8 ELISA MEASUREMENT OF SOLUBLE PROTEIN 66 2.8.1 ET-l Level in BM and PB Plasma and Cell Supematant 2.8.2 Determination of IL-lB, IL-6, OPG and TNF-cr Levels in Culture Supernatants... 6',7 2.9 WESTERN BLOT A¡IALYSIS 68 2.9.1 Buffers and Reagents 68 2.9.1.1 Tris-Saline-EDTA(TSE). 68 2.9.r.2 t% (vlv) NP4O-TSE. 68 2.9.r.3 0.1% (v/v) NP4O-TSE..' 68 2.9.1.4 25x Protease Inhibitor Cocktail "' 69 69 2.9.1.5 1x Reducing SamPle Buffer 69 2.9.1.6 1 x Non-reducing Sample Buffer.'.'.........'..'. 2.9.t.1 Electrophoresis "Rururing" buffer....'........... 69 69 2.9.1.8 Transfer Buffer... 69 2.9.1.9 Separating Buffer 69 2.9.1.10 Stacking Buffer............'...... 69 2.9.1.11 PBST.. 69 2.9.2 Preparation of Cell Lysates....'.... 70 2.9.3 Immunoprecipitaiton (IP) with Dlmabeads 70 2.9.4 Western Blot AnalYsis .'.. CHAPTER 3. THE MULTIPLE MYELOMA.DERIVED PRO- TNFLAMMATORY GYTOKINE, lL'1p, INHIBITS BONE FORMATION AND ENHANCES OSTEOCLATOGENESIS BY INCREASING THE NUMBER OF RANKL-EXPRESSING STRO'1 POSITIVE OSTEOPROGENITOR CELLS 72 3.1 INTRODUCTION """'72 3.2 3.2.r 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 Exposure... 19 of OB-Like 3.2.',7 IL-lB Does not Impair the Bone Mineral Forming Capacity Cells under Osteoinductive Condition 80 * I Cells Possess an Enhanced CapacitY 3.2.8 IL- 1 B -upregulated STRO- OB-like to Support Osteoclastogenesis' 80 CHAPTER 4. THE POTENT OSTEOGLASTOGENIC FACTOR' RANKL, IS A MARKER OF IMMATURE OSTEOPROGENITOR GELLSANDBoNEMARRoWSTRoMALSTEMGELLS...........92 4.1 INTRODUCTION """'92 94 4.2 RESULTS.. 94 4.2.1 Human OB-like Cells Co-express STRO-1 and TM-RANKL"""""""""' 4.2.2 HumanBMMNCsCo-expressSTRo-landTM-RANKLontheCell Surface. 94 4.2.3 Distribution of STRO-I and RANKL on BMMNCs 95 to a Population 4.2.4 colony Forming units-Fibroblastic (cFU-F) are Restricted orsMMNCs Wtti"n Express RANKL and STRO-I at High Levels......... 95 96 4.2.5 RANKL and STRO-I aie Highly Associated Molecules """"""""""""" 98 GHAPTER 5. ELEVATED SERUM LEVELS OF THE VASOPEPTIDE, ET.1, MAY ACCOUNT FOR THE SUPPRESSI ON OF BONE FORMATION IN MYELOMA PATIENTS........... 102 5.2 5.2.1 \)') 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.3 DrscussroN................. """"""""' 111 CHAPTER 6. GENERAL DISGUSSION AND FUTURE CONSTDERATTONS .....120 BIBLIoGRAPHY...... ""126 Errata Errata o Table of contents, 1.1 and Page 2, section 1.1 : "GIBE" should be "GIVE" o Page VI, BMPs: "marphogenetic" shOUld be "mOrphogenetic" o Page 3, line 10: ">3/dl" should be ">3gldl" o Page 4, 12 lines up: "lyphocyte" should be "lymphocyte" "a o page 9, line 5-6: "a calcified connective tissue matrix (hydroxyapatite)" should be calcified (highly-substituted hydroxyapatite) connective tissue matrix" physiology" o page 9, 14 lines up: "In the normal physiology" should be "In normal o page 11, l't paragraph: "Osteoblasts are derived from the stromal or mesenchymal cell the stromal or system and represent..." should be "Osteoblast lineage is derived from mesenchymal cell system and represented by' "" o Page 15, 15 lines up: "ostolytic" should be "osteolytic" o Page 76, 14 lines up: "cycloocygenase" should be "cyclooxygenase" o Page 18, 7 lines up: "macrophage" should be "macrophages" o Page 20,line 13: "an" should be "and" o Page 35, line 2: "localises to in" should be "localises in" lines up o page 40, 13lines up: "Collin, 2001 #51 1" should be "Collin-Osdoby et a1.,2001";3 "Le Brun, l99g # 765" should be "Le Brun et al',1999" o Page 43,line 2: "...one of major..." should be "...one of the major.
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