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Molecular and Cellular Studies Examining the Biological Significance of Different Isoforms of the Receptor Tyrosine Kinase' C-Kit

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Molecular and Cellular Studies Examining the Biological Significance of Different Isoforms of the Receptor Tyrosine Kinase' C-Kit Molecular and cellular studies examining the biological significance of different isoforms of the receptor tyrosine kinase' c-Kit Antony Charles Cambareri B. Sc. (Hons) Institute of Medical and Veterinary Science Department of Medicine University of Adelaide South Australia A thesis submitted to the University of Adelaide in candidature for the degree of Doctor of Philosophy October 2004 Declaration This thesis contains no material that has been accepted for the award of any other degree or diploma in any other university. To the best of my knowledge and belief' this thesis contains no material previously published or written by another person' except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopylng. p? Antony C atefl 4 ,(? ll Acknowledgments I would like to sincerely thank my supervisors, Professor Leonie Ashman and professor Bik To. I thank them both for their encouragement, persistence and support over the period of this work' Special thanks to Steve Fitter, who was pivotal in "retraining" me in the art of science upon my return to the lab from "the dark side". He is both a great friend and a legendary molecular biologist (it is true that it will work if you add a microlitre!)' I also thank Andrew Zannettino for spending the time to critique drafts of this thesis, and for valuable input to experimental design. I acknowledge the valuable technical assistance of Ly Nguyen and Peter Konstantopolous from the Leukaemia Haemopoiesis Laboratory. Thanks also to Sue Branford and Rebecca Lawrence for their assistance with Quantitative PCR. Thankyou to Alan Bishop and Sandy Maclntyre, who provided valued assistance in the operation of the flow cytometers and sorters, Dr Jo Woodcock for assistance with saturation binding assays and Paul Sincock, who was a valuable resource in educating me in the finer points of confocal microscopy. I also thanks all members of the previous Leukaemia Haemopoiesis Laboratory headed by Leonie Ashman, as well as current members of the Mathew Roberts Laboratory headed by Andrew Zamettino and Stan Gronthos, for their support and assistance, and making work an enjoyable place to be! Finally, I thank my family. My parents made many sacrifices to equip me with the skills and education to achieve this, and I am forever indebted to them for this. My lll darling wife, Bron, has provided the most crucial support throughout this study' Her patience, encouragement and faith have never wavered and are the driving force behind any success I achieve. Thanks also to my daughter, Tiana' She is too young to realise this yet, but has been a model child with a beautiful nature. This allowed me the time to devote more efforts than would normally be possible in family time toward completion of this thesis. lv Errata Insert - Abstract: page xiii, lst Para - additional sentence: The c-Kit isoforms analysed throughout this thesis arise by differential splicing at 1 site in the c-Kit gene resulting in the insertion/deletion of l2bp encoding the amino acids GNNK. These are referred to as GNNK* and GNNK- throughout the thesis. Chapter 1, Section 1.1.2, p5, para 2: Replace "epinephrin: with "ephrin" Chapter 3, Table Legend, Table 3.3: Add "4, B, C represent separate pools of NIH3T3 infectants with different levels of surface expression c-Kit." Chapter 3, Section 3.2,p97r last sentence: Change to "The findings of that study are discussed in this section." Chapter 3, Figure 3.9 Replace "12-s+ PMAPK" with "GNNK-" and "12*sf PMAPK" with "GNNK+" Replace "0","I5" and "30" with "0 min", "15 min", and "30 min" respectively Chapter 4,Introduction, pl18, Para2 after l't sentence: Insert "Previous studies by Ferrao et al. (Fenao et a1., 1997) had demonstrated that stimulation of MIHC expressing c-Kit could differentiate in response to exogenous SCF." Chapter 4,Introduction, p119, Para 1 after 1't sentence: Insert at end "and is activated by SCF stimulation (Liang et a1.,2002*; Ueda et al., 2002**)." Chapter 4, Section 4.1.5rp125, Para lr last sentence: Replace "AMV" with "Myeloproliferative Sarcoma Virus (MPSV)" Chapter 5, Section 5.4.2,last sentence, pL68: Add "To confirm this, nuclear run on assays or Actinomycin D experiments to assess mRNA stability are required." Chapter 5, Section 5.5, l't para, 2nd sentence on p173: Replace sentence \Mith "It has often been noted that c-Kit is down-modulated in mobilisation of CD34 cells, however this did not appear to be at the level of mRNA transcription or stability, although experimentation such as nuclear run on assays is required to confirm this." x Liang, X., Wisniewski, D., Shife, 4., Shivakrupa, Clarkson, B. and Resh, M.D. (2002).Phosphatidylinositol 3- kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-l in c-Kit signaling. J Biol Chem277 (16),13732-8 ** Ueda, S., Mizuki, M. Ikeda, H., Tsujimura, T. Matsumura, L, Nakano, K., Daino, H., Honda, Z.Z.,Sonoyama,J., Shibayama, H., Sugahara, H., Machii, T., and Kanakura, Y. (2002) Critical roles of c-Kit tyrosine residues 567 and 719 n stem cell factor-induced chemotaxis: contribution of src family kinase and Pl3-kinase on calcium mobilization and cell migration. Blood 99(9),3342-9 Abbreviations 6FAM 6-Carboxyfluorescein O-MEM Alpha modifrcation of minimum essential medium ATP Adenosine tripho sPhate BM MNCS Bone marrow mononuclear cells bp Base pair(s) BSA Bovine serum albumin CD C luster o f differentiation CDIIT c-Kit cDNA Complimentary DNA CFU Colony Forming Unit Chi c-Kit Chimaeric human/mouse c-Kit c-Kit CDl77, Stem Cell Factor RecePtor COOH Carboxy (terminus) CSF Colony stimulating factor DEPC Diethylpyrocarbonate DMEM Dulbecco's modified eagles medium DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid ECF Enhanced Chemifluorescence EGF Epidermal Growth Factor EGFR Epidermal Growth Factor RecePtor ELISA Enz¡rme-linked immuno sorbent assays FACS Fluorescence activated cell sorting FCS Foetal calf serum FITC Fluorescein isothiocYanate G-CSF Granulocyte-CSF GIST Gastrointestinal Stromal cell Tumour GM-CSF Granulo c yt e I macr oPhage - C S F HBSS Hank's balanced salt solution ICC Úrterstitial Cells of Cajal Ig Immunoglobulin isotype IgGr, IgGzu, IgGzu Immuno globulin gamma- 1, gamma-2a and gamma-2b IgM Immunoglobulin M IMDM Iscove's modified Dulbecco's medium IMVS Institute of Medical and Veterinary Science ru International units JM Juxtamembrane KD Kilodalton LPS Lipopolysaccaride Mab Monoclonal antibodY MAPIIERK Mitogen-activated protein kinase/extracellular signal-regulated kinase MCS Multiple cloning site M-CSF Macrophage colony-stimulating factor MFI Mean fluorescence intensitY MIHC Myb Immortalised Haemopoietic Cells mRNA Messenger RNA NH2 Amino (terminus) vl NP-40 Nonidet P40 PB MNCS Peripheral blood mononuclear cells PBS Pho sphate-buffered s aline PBS/BSA/Az PBS + 0.lo/owlv BSA + O.Io/owlv Sodium Azide PCR Polymerase chain reaction PDGF Platelet Derived Growth Factor PDGFR Platelet Derived Growth Factor Receptor PE Phycoerythrin PH Pleckstrin HomologY PI3-K Phosphatidyl Inositol-3 Kinase PTB Pho sphotyro sine Binding PTP Protein Tyrosine PhosPhatase PVDF Poly Vinyl Di Fluoride RNA Ribonucleic acid RPMI-1640 Rosewell Park Memorial Institute Medium 1640 RTK Receptor Tyrosine Kinase RT-PCR Reverse transcription- polymerase chain reaction Factor, Steel SCF Stem Cell Factor, Kit Ligand, Mast Cell Growth Factor SCLC Small cell Lung Carcinoma SEM Standard error of mean SH2 Src llomology 2 SH3 Src llomology 3 TEMED N, N, N' N' -tetramethyl-ethylenediamine TM Transmembrane vll Ub Ubiquitin UV Ultra violet vlv Volume per volume w/v Weight per volume vlll Contents 1 INTRODUCTION 1 I.I. C-KN- AMEMBER oF THE Gnowrs F¡,croRRBcspronTvnosnqe KINASE FAMITy """""""' 1 1.1.1. The Growth Factor Receptor Tyrosine Kinases"""""""" 1.1.2. Signalling through RTKs ........ -.. 1.t.3. c-Kit in Cancer I.2. HISTORY OF C-KIT...... 1 .2. I . - The discovery of the c-Kit proto-oncogene"" " " " 1.2.2. Association of c-Kitwith the LI/ locus 13 1,3. THE LIGAND TO C-KIT: THB LTNT EETWEEN W ¡,NO SI- IT¡UTRNT MICE ..'"...."' I.4. BIOCHEMICAL ANALYSIS OT C-KN. LIGAND BINDINGDOMAIN .'. 1.5. LIGANDINoUcT'oRscBpronDIMERISATIoN 1.6. LIcAND INDUCED R¡ceproR SlcN¡'rlrxc 1 .6.1 . r.6.2. 1.6.3. 1.6.4. 23 1.6.5. 24 1.7 . StcN¡.lLruc rHRouGH c-Ktr....'.....' 25 1.7.1 . PI3-Kinase.. 26 1.7.2. P hospholipase C-Y 1 .......... 27 L7.3. RAS-MAPK. 28 1.7.4. Protein Kinase C...............'.. 29 1.7.5. Src FamilY Kinases 29 1 .7 .6. Other .. ......... 1.8. DowNMoDULATIoNorc-KnsxpRnssloN/¡'crIVATIoN"""" 30 1.8.1. PKC............ 31 1.8.2. Extracellular domain shedding 32 32 1.8.3 33 1.8.4. Phosphatases .34 1.8.5. Others .35 1.9. MECFIANISMS INDUCING SIGNALLING DIVERSITY EY THg RTK .35 L9.1. c-Kit Isoþrm,s '.''........ .36 1.9.2. Heterodimerisation.... .39 1.9.3. Receptor levels .41 2 MATERIALS AND METHODS .43 .43 2.1. TIssun,CuLTURE..... .43 2.1.I Tissue Culture Medis and Solutions 46 2.1.2 Cytokines and growth factors ............ 4',7 2.2. Culrune MnINTENANCE oF CELLS ....'. 47 2.2.I. Psi 2 cell line maintenance............... 48 2.2.2. FDC-PI cell line maintenance 2.2.3. NIH3T3 cell line maintenqnce lines .., 2.2.4. Maintenance of Myb Immortalised Haemopoietic cell (MIHC) 2.2.5. Maintenance of MOTe 2.2.6. Cryopreservation of Cells ." 2.2.7. Thawing of Cryopreserved Cells 5 2.2.8. Cytolog,t, Cytochemistry and Histolo gt' 50 2.2.9. Morphological characteris ation of cells'.... ............,,,..' 1 2.2.10. Phenotypic characterisation of cells - ester(tse staining ................
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