Die Rolle von HIF bei zellulären Transformationsprozessen [Elektronische Ressource] : I. HIF-2α im renalen Tubulus - ein transgenes Mausmodel ; II. Identifizierung und Charakterisierung von Lysyloxidasen als tumorfördernde HIF-Zielgene / vorgelegt von Ruth Elisabeth Schietke

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Die Rolle von HIF bei zellulären Transformationsprozessen: I. HIF-2 α im renalen Tubulus – ein transgenes Mausmodel II. Identifizierung und Charakterisierung von Lysyloxidasen als tumorfördernde HIF-Zielgene Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Ruth Elisabeth Schietke aus Tettnang Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 22. Januar 2009 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Thomas Winkler Zweitberichterstatter: PD Dr. Michael Wiesener für Mama und Papa The role of HIF for cellular transformation processes: I. HIF-2 α in the renal tubulus – a transgenic mouse model II. Identification and characterization of lysyl oxidases as tumour promoting HIF target genes Table of Contents I I. Table of Contents I II. Table of figures VII III.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 27
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2009/1243/PDF/DISSERTATION_RUTH_SCHIETKE_22.01.2009.PDF
Nombre de pages : 184
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Die Rolle von HIF bei zellulären Transformationsprozessen:
I. HIF-2 α im renalen Tubulus – ein transgenes Mausmodel
II. Identifizierung und Charakterisierung von Lysyloxidasen
als tumorfördernde HIF-Zielgene






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades










vorgelegt von
Ruth Elisabeth Schietke
aus Tettnang




Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Universität Erlangen-Nürnberg






















Tag der mündlichen Prüfung: 22. Januar 2009
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Thomas Winkler
Zweitberichterstatter: PD Dr. Michael Wiesener




für
Mama und Papa








The role of HIF for cellular transformation processes:
I. HIF-2 α in the renal tubulus – a transgenic mouse model
II. Identification and characterization of lysyl oxidases as
tumour promoting HIF target genes

















Table of Contents I
I. Table of Contents I

II. Table of figures VII

III. Abbreviations IX

1 INTRODUCTION..............................................................................................................1
1.1 Gene expression and regulation...............................................................................2
1.2 The hypoxia inducible factor (HIF)............................................................................3
1.3 Oxygen-dependent regulation of HIF .......................................................................6
1.4 Cellular transformation, cancer development and tumour progression.............10
1.5 Hypoxia in tumour biology ......................................................................................11
1.6 HIF in tumour biology ..............................................................................................13
1.7 Lysyl oxidases..........................................................................................................16
1.8 Aim of this study ......................................................................................................19
2 MATERIAL.....................................................................................................................21
2.1 Chemicals and disposable plastic materials .........................................................21
2.2 Solutions, buffer and media ....................................................................................21
2.2.1 Universal solutions .................................................................................................21
2.2.2 Solutions and media for cell culture........................................................................22
2.2.3 d media for bacterial cultivation ..........................................................23
2.2.4 Solutions for preparative plasmid-DNA isolation ....................................................23
2.2.5 nucleic acid analysis..........................................................................24
2.2.6 Agarose gel electrophoresis ...................................................................................25
2.2.7 Buffer for RNase Protection Assay (RPA) ..............................................................25
2.2.8 Solutions and buffer for Electromobility Shift Assay (EMSA)..................................26
2.2.9 Buffer for protein extraction ....................................................................................26
2.2.10 Solutions and buffers for protein gels .................................................................26 Table of Contents II
2.2.11 Solutions and buffer for protein transfer on PVDF-membrane („Western-Blot“)
and subsequent immunodetection .....................................................................................27
2.2.12 Staining solutions................................................................................................28
2.2.13 Solution for immunofluorescence analysis .........................................................28
2.2.14 Carrier material...................................................................................................29
2.3 Antibodies .................................................................................................................30
2.3.1 Primary antibodies..................................................................................................30
2.3.2 Secondary antibodies .............................................................................................31
2.4 Cell lines....................................................................................................................32
2.5 Bacterial strains........................................................................................................33
2.6 Commercial reagent kits..........................................................................................33
2.7 Enzymes34
2.8 Nucleic acids.............................................................................................................34
2.8.1 Cloning vectors.......................................................................................................34
2.8.2 Oligonucleotides .....................................................................................................35
2.8.2.1 Primers for polymerase chain reaction (PCR) ....................................................35
2.8.2.2 Primers for real-time RT-PCR ............................................................................36
2.8.2.3 Oligonucleotides for Electromobility Shift Assay (EMSA)...................................37
2.9 Size and molecular weight marker..........................................................................38
2.9.1 DNA size marker ....................................................................................................38
2.9.2 Protein weight marker.............................................................................................38
2.10 Equipment and instruments ....................................................................................39
2.11 Computer software...................................................................................................40
3 METHODS......................................................................................................................41
3.1 Cultivation of eukaryotic cells.................................................................................41
3.1.1 Passaging of eukaryotic cells42
3.1.2 Freezing and thawing of eukaryotic cells................................................................42
3.1.3 Transient transfection of eukaryotic cells43
3.1.3.1 Transient transfection with jetPEI™ (Polyplus) ..................................................43
®3.1.3.2 Transient Transfection with Fugene HD (Roche)...............................................44 Table of Contents III
3.2 Cultivation of bacteria..............................................................................................45
3.2.1 Cultivation of Escherichia coli.................................................................................45
3.2.2 Transformation of competent bacteria with circular plasmid DNA..........................45
3.3 Analysis of nucleic acids.........................................................................................46
3.3.1 Isolation of DNA......................................................................................................46
3.3.1.1 Analytical isolation of plasmid DNA from bacteria (mini preparation).................46
3.3.1.2 Isolation of plasmid DNA from bacteria (midi preparation).................................46
3.3.1.3 Isolation of genomic DNA from cultured eukaryotic cells ...................................47
3.3.1.4 Isolam mouse tail biopsies for PCR .............................47
3.3.1.5 Phenol/chloroform extraction of nucleic acids ....................................................48
3.3.1.6 Purification of DNA fragments from ethidium bromide agarose gels..................48
3.3.2 Enzymatic modification of DNA ..............................................................................49
3.3.2.1 DNA cleavage by restriction endonucleases......................................................49
3.3.2.2 Ligation of DNA fragments .................................................................................49
3.3.3 RNA extraction from cultivated cells.......................................................................50
3.3.4 cDNA synthesis by reverse transcription (RT)........................................................50
3.3.5 Agarose gel electrophoresis of double-stranded DNA............................................51
3.3.6 Sodium acetate precipitation of nucleic acids.........................................................51
3.3.7 Quantification of nucleic acid concentration ...........................................................52
3.3.7.1 Photometric quantification of nucleic acid concentration....................................52
3.3.7.2 Quantification of DNA amounts by intercalation of ethidium bromide ................52
3.3.8 Sequencing of DNA ................................................................................................52
3.4 Polymerase chain reaction (PCR) ...........................................................................53
3.4.1 Site-directed mutagenesis by the use of overlap-extension-PCR ..........................54
3.5 Protein biochemical procedures.............................................................................55
3.5.1 Extraction of total-protein from cultivated cells .......................................................55
3.5.2 Quantification of protein concentration ...................................................................55
3.5.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ..........................................55
3.5.4 Protein transfer on PVDF membranes („Western-Blot“).........................................56
3.5.5 Specific immunodetection of membrane bound proteins........................................57
3.6 Immunocytochemistry .............................................................................................58
3.6.1 Immunofluorescence staining of cultivated cells.....................................................58
3.6.2 Microscopic analysis of immunofluorescence stained cells....................................58
3.7 Immunohistology......................................................................................................59 Table of Contents IV
3.7.1 Fixation of tissues for immunohistochemistry.........................................................59
3.7.2 Immunohistological staining of tissues ...................................................................59
3.8 Gene expression analysis .......................................................................................60
3.8.1 Silencing of gene expression by RNA interference (RNAi).....................................60
3.8.1.1 Transient transfection of siRNA with Oligofectamine™......................................61
3.8.2 RNA expression analysis by RNase Protection Assay (RPA) ................................61
3.8.2.1 Cloning of plasmids for generating radiolabelled RPA probes ...........................62
3.8.2.2 Generating radioactive labelled RPA probes......................................................62
3.8.2.3 RNase Protection Assay.....................................................................................63
3.8.3 Quantitative real-time RT-PCR...............................................................................64
3.8.4 Luciferase reporter assay .......................................................................................65
3.9 Electrophoretic Mobility Shift Assay (EMSA) ........................................................66
3.9.1 Radioactive labelling of the DNA oligonucleotides .................................................66
3.9.2 In vitro transcription and translation........................................................................67
3.9.3 HIF-EMSA ..............................................................................................................67
3.10 Invasion assay..........................................................................................................69
4 RESULTS.......................................................................................................................70
4.1 Part A: A transgenic mouse model: Expression of stabilized and constitutive
active HIF-2α in renal tubular epithelial cells ....................................................................70
4.1.1 Construction of the expression vector Ksp/tmHIF-2 α.HA .......................................70
4.1.2 In vitro expression of tmHIF-2 α.HA under control of the Ksp-promoter..................73
4.1.3 Generation of a transgenic mouse with homozygous overexpression of tmHIF-
2 α.HA in renal tubular cells ................................................................................................77
4.1.4 Analysis of transgenic tmHIF-2 α.HA mRNA expression in different organs of the
three generated mouse strains...........................................................................................77
4.1.5 Analysis of transgenic tmHIF-2 α.HA protein expression in the kidney of the
generated mouse strains....................................................................................................80
4.1.6 HIF target gene activation in the kidneys of tmHIF-2 α.HA transgenic mice ...........82
4.1.7 Renal cyst formation in mice overexpressing stabilized and constitutive active HIF-
2 α 85
4.2 Part B: Role of the HIF target genes lysyl oxidase and lysyl oxidase-like 2 in
tumour progression .............................................................................................................88
4.2.1 EMT marker expression in RCC10 cells influenced by VHL and hypoxia ..............88 Table of Contents V
4.2.2 EMT marker expression in human primary tubular epithelial cells of the kidney ....91
4.2.3 Identification of the new HIF target genes LOX and LOXL2...................................93
4.2.3.1 Affymetrix array of Hep3B cells: Upregulation of LOX and LOXL2 expression ..93
4.2.3.2 Analysis of LOX and LOXL2 mRNA expression by RNase protection assay.....93
4.2.3.3 Analysis of HIF depended hypoxic upregulation of LOX and LOXL2 .................96
4.2.4 Expression of LOX and LOXL2 in renal clear cell carcinoma.................................99
4.2.5 Lysyl oxidases are involved in E-cadherin downregulation under hypoxia...........100
4.2.5.1 In vivo correlation between the loss of E-cadherin and the expression of HIF.100
4.2.5.2 In vitro analysis of E-cadherin expression under hypoxia in RCC10 and
RCC10/VHL cell lines...................................................................................................104
4.2.5.3 Analysis of LOX and LOXL2 involvement in the hypoxic repression of E-cadherin
......................................................................................................................................106
4.2.5.4 Analysis of Snail involvement in the LOX/LOXL2 dependent hypoxic repression
of E-cadherin ................................................................................................................110
4.2.6 Modulation of cellular invasiveness by hypoxia and VHL, which depends on lysyl
oxidases ...........................................................................................................................113
5 DISCUSSION ...............................................................................................................115
5.1 HIF-2 α in the renal tubulus – a transgenic mouse model...................................116
5.1.1 In vitro expression of pcKsp/tmHIF-2α.HA ...........................................................116
5.1.2 In vivo expression of tmHIF-2 α.HA in renal tubular epithelial cells leads to renal
cyst formation ...................................................................................................................117
5.2 HIF mediated hypoxic transformation processes involve LOX and LOXL 2 and
lead to loss of E-cadherin expression..............................................................................122
5.2.1 Hypoxia could induce the process of epithelial to mesenchymal transition (EMT)
122
5.2.2 Identification of the HIF-target genes LOX and LOXL2 ........................................123
5.2.3 LOX and LOXL2 in renal cancer...........................................................................123
5.2.4 Hypoxic repression of E-cadherin depends on LOX and LOXL2..........................124
5.2.5 LOX and LOXL2 dependent hypoxic E-cadherin repression might be mediated by
transcriptional repressors .................................................................................................125
5.2.6 Implication of lysyl oxidase expression for cellular transformation and
dedifferentiation processes ..............................................................................................126
5.2.7 Targeting lysyl oxidase activity as a therapeutic strategy against tumour
progression and metastasis .............................................................................................127 Table of Contents VI
6 SUMMARY...................................................................................................................129
7 ZUSAMMENFASSUNG ...............................................................................................131
8 REFERENCES.............................................................................................................133
9 APPENDIX ...................................................................................................................161



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