Identification of Genes Involved in Leukaemia and Differentiation Induced by Activated Mutants of the GM-CSF Receptor β subunit Brenton James Reynolds A thesis submitted for the degree of Doctor of Philosophy to the University of Adelaide, School of Medicine (Discipline of Medicine) November, 2005 Table of Contents Table of Contents ........................................................................................... i Abstract.........................................................................................................ix Declaration....................................................................................................xi Acknowledgements......................................................................................xii Publications.................................................................................................xiii Conference Presentations..........................................................................xiii Abbreviations..............................................................................................xiv Chapter 1: Introduction ............................................................................... 1 1.1 Haematopoiesis .............................................................................................1 1.2 Leukaemias ...................................................................................................3 1.2.1 Animal models of leukaemia – retrovirus mediated leukaemia....................6 1.2.2 Human myeloid leukaemias..........................................................................8 1.2.2.1 Chronic myelogeneous leukaemia (CML)..................................................11 1.2.2.2 Acute myeloid leukaemia (AML) ...............................................................12 1.3 Cytokines and their receptors......................................................................14 1.3.1 Structure ......................................................................................................15 1.3.2 Generation of intracellular signals by cytokine receptors...........................17 1.3.2.1 The JAK/STAT pathway ............................................................................18 1.3.2.2 The Ras/Raf/MAP kinase pathway .............................................................19 1.3.2.3 The PI3K/Akt pathway ...............................................................................19 1.4 Growth factor receptors in haematopoiesis and leukaemia.........................21 1.4.1 Tyrosine kinase receptors – FLT3...............................................................21 1.4.2 Cytokine receptor mutations in leukaemia..................................................22 1.4.2.1 Erythropoietin receptor ...............................................................................22 1.4.2.2 c-Mpl...........................................................................................................23 1.4.2.3 Granulocyte colony-stimulating factor receptor .........................................24 1.5 GM-CSF, IL-3 and their shared receptor (hβc) in leukaemia.....................25 1.5.1 Biology of GM-CSF, IL-3 and IL-5............................................................25 1.5.1.1 IL-3/IL-5/GM-CSF system deficient mice..................................................25 1.5.2 Involvement of GM-CSF and IL-3 in human diseases ...............................27 i 1.5.2.1 Autocrine production and over-expression of GM-CSF and IL-3 in leukaemia ....................................................................................................27 1.5.2.2 Involvement of GM-CSF in juvenile myelomonocytic leukemia (JMML) 28 1.5.3 βc receptor activated mutants ......................................................................29 1.5.3.1 Overview.....................................................................................................29 1.5.3.2 Isolation and in vitro properties of the hßc mutants in haematopoietic cells.. ……………………………………...……………………………………..31 1.5.3.3 Signalling and biochemical properties of the hßc activated mutants..........36 1.5.3.4 Leukaemogenic potential of activated hβc mutants– in vivo models.........38 1.6 Large scale gene expression profiling techniques.......................................39 1.6.1 Overview of membrane arrays and microarrays .........................................39 1.6.2 Overview of data acquisition and image analysis.......................................41 1.6.2.1 Data acquistion............................................................................................42 1.6.2.2 Processing the scanned image.....................................................................42 1.6.3 Overview of statistical methods used in the analysis of microarray experiments .................................................................................................44 1.6.3.1 Handling variation in a microarray experiments.........................................44 1.6.3.2 Determination of differential gene expression............................................45 1.6.3.3 Single-slide methods of statistical analysis.................................................46 1.6.3.4 Multiple-slide methods of statistical analysis .............................................47 1.7 Project rationale ..........................................................................................51 1.7.1 Project aims.................................................................................................52 Chapter 2: Materials and Methods ........................................................... 53 2.1 Materials......................................................................................................53 2.1.1 Chemicals, reagents and consumables ........................................................53 2.1.2 Solutions and Buffers – commonly used ....................................................53 2.1.2.1 DNA and RNA Loading Buffers.................................................................54 2.1.2.2 Bacterial culture media ...............................................................................55 2.1.3 Restriction endonucleases ...........................................................................55 2.1.4 Growth factors.............................................................................................55 2.1.5 Radiochemicals (Radionucleotides)............................................................55 2.1.6 Bacteria .......................................................................................................55 2.1.6.1 Preparation and transformation of competent Escherichia coli (E. coli)....56 ii 2.1.7 Tissue culture solutions and media .............................................................56 2.1.8 Molecular weight standards ........................................................................57 2.1.9 Vectors and cDNAs ....................................................................................57 2.2 Methods for manipulating DNA .................................................................58 2.2.1 Small scale plasmid preparations................................................................58 2.2.2 Large scale plasmid preparations................................................................59 2.2.3 Agarose gel electrophoresis ........................................................................59 2.2.4 Restriction endonuclease digestion.............................................................59 2.2.5 Preparation of inserts and cloning vectors ..................................................59 2.2.5.1 Phosphatase treatment of cloning vectors...................................................60 2.2.6 Ligation of DNA into plasmid vectors........................................................60 2.2.7 Bacterial transformation..............................................................................60 2.2.7.1 Heat shock method......................................................................................60 2.2.8 RNase A treatment and phenol chloroform extraction of plasmid mini- preparations for sequencing ........................................................................60 2.2.9 DNA sequencing.........................................................................................61 2.2.10 Polymerase Chain Reaction (PCR) .............................................................61 2.2.10.1 Standard PCR conditions: ...........................................................................61 2.2.10.2 cDNA synthesis...........................................................................................62 2.2.10.3 Cloning........................................................................................................62 2.2.10.4 Primers ........................................................................................................62 2.3 Methods for manipulating RNA..................................................................65 2.3.1 Total RNA isolation ....................................................................................65 2.3.2 Poly A+ RNA preparation from FDC-P1 cells for Northern Blotting.........66 2.3.3 RNA preparation from FDB-1 cells for Northern Blotting and Microarrays . …………………………………………………………………………….67 2.3.4 Reverse Transcription Polymerase Chain Reaction (RT –PCR).................67 2.3.5 Northern Blotting ........................................................................................67