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Analysis of Genomic Alterations in Malignant Melanoma” Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften der Fakultät für Biologie der Ludwig-Maximilians-Universität München Analysis of genomic alterations in malignant melanoma vorgelegt von Henrike Körner am 02.05.2007 Die Arbeit wurde am Max-Planck Institut für Biochemie (Martinsried) in der NWG Molekulare Onkologie angefertigt. First examiner: PD Dr. Heiko Hermeking Second examiner: Prof. Dr. Thomas Cremer Additional examiners: Prof. Dr. Michael Boshart Prof. Dr. Heinrich Leonhardt Date of the oral examination: 05.12.2007 Erklärung Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig verfasst habe und keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt habe. Sämtliche Experimente sind von mir selbst durchgeführt worden, außer wenn explizit auf dritte verwiesen wird. Ferner erkläre ich, dass ich nicht anderweitig versucht habe, eine Dissertation oder Teile einer Dissertation einzureichen bzw. einer Prüfungskommission vorzulegen oder mich einer Doktorprüfung zu unterziehen. München, den 07.12.07 Henrike Körner TABLE OF CONTENT TABLE OF CONTENT 1 INTRODUCTION..................................................................................................1 1.1 Cutaneous malignant melanoma...................................................................1 1.1.1 Germ line mutations in melanoma ..........................................................3 1.1.1.1 CDKN2A ..........................................................................................3 1.1.1.2 CDK4 ...............................................................................................5 1.1.2 Pigmentary traits.....................................................................................6 1.1.3 Somatic mutations in melanoma.............................................................7 1.1.3.1 Mitogen-activated protein kinase signaling pathway........................7 1.1.3.2 HGF/SF-MET signaling pathway .....................................................8 1.1.3.3 PTEN-AKT signaling pathway..........................................................8 1.2 Methods for cytogenetic and whole-genome analyses..................................9 1.2.1 Fluorescence in situ hybridization.........................................................10 1.2.2 Comparative genomic hybridization......................................................11 1.2.3 Digital karyotyping ................................................................................12 1.3 Dystrophin ...................................................................................................15 1.3.1 DMD gene and isoforms.......................................................................15 1.3.2 Dystrophin protein.................................................................................17 1.3.3 Duchenne and Becker Muscular Dystrophies.......................................18 1.3.4 Mutations in DMD .................................................................................19 1.3.5 Dystrophin-glycoprotein complex..........................................................20 2 AIM OF THE STUDY .........................................................................................22 3 MATERIALS ......................................................................................................23 3.1 Chemicals ...................................................................................................23 3.2 Enzymes .....................................................................................................24 3.3 Commercial kits and other materials ...........................................................24 3.4 Buffers and stock solutions .........................................................................25 3.5 Antibodies ...................................................................................................28 3.5.1 Primary antibodies................................................................................28 3.5.2 Secondary antibodies ...........................................................................29 3.6 DNA constructs ...........................................................................................29 3.7 Oligonucleotides..........................................................................................30 I TABLE OF CONTENT 3.8 Bacteria strains ...........................................................................................31 3.9 Cell lines and cell culture media..................................................................32 3.10 Equipment...................................................................................................32 4 METHODS .........................................................................................................33 4.1 Chromosome metaphase spreads ..............................................................33 4.2 Comparative genomic hybridization ............................................................33 4.3 7-Fluor M-FISH ...........................................................................................34 4.4 Digital karyotyping.......................................................................................35 4.5 Isolation of genomic DNA from cell lines and paraffin-embedded tissue sections.......................................................................................................40 4.6 RNA isolation and RT reaction ....................................................................40 4.7 RT-PCR analysis.........................................................................................40 4.8 qPCR analysis.............................................................................................41 4.9 PCR analysis of genomic DNA....................................................................41 4.10 DNA sequencing .........................................................................................42 4.11 Western blot analysis ..................................................................................42 4.12 Generation of stable cell lines .....................................................................42 4.13 Cell growth in matrigel.................................................................................43 4.14 Cell migration assays ..................................................................................44 4.15 Cell proliferation assay................................................................................44 4.16 Analysis of apoptosis by flow cytometry......................................................45 4.17 Senescence-associated β-galactosidase staining.......................................45 4.18 Immunofluorescence staining and confocal microscopy .............................46 4.19 Statistical analysis.......................................................................................46 5 RESULTS ..........................................................................................................48 5.1 Characterization of malignant melanoma cell lines .....................................48 5.1.1 General characteristics.........................................................................48 5.1.2 M-FISH analysis of malignant melanoma cell lines...............................49 5.1.3 CGH analysis of malignant melanoma cell lines...................................58 5.2 Digital karyotyping analysis of malignant melanoma cell lines ....................63 5.2.1 Validation of the digital karyotyping results...........................................63 5.3 Analysis of DMD in malignant melanoma....................................................67 II TABLE OF CONTENT 5.3.1 Deletion of DMD in malignant melanoma cell lines...............................67 5.3.2 Expression of DMD isoforms in primary melanocytes...........................68 5.3.3 Expression of dystrophin isoforms in melanoma cell lines....................70 5.3.4 Functional analysis of dystrophin..........................................................75 5.3.4.1 Analysis of the components of the dystrophin-glycoprotein complex .......................................................................................................75 5.3.4.2 Knockdown of DMD in melanoma cell lines...................................81 5.3.4.3 Re-expression of dystrophin in melanoma cell lines ......................83 6 DISCUSSION.....................................................................................................86 6.1 Cytogenetic characterization of melanoma cell lines...................................86 6.2 Digital karyotyping of melanoma cell lines...................................................89 6.3 Down-regulation of DMD in melanoma cell lines.........................................90 6.4 The dystrophin-glycoprotein complex in melanocytes and melanoma cell lines.............................................................................................................93 7 SUMMARY.........................................................................................................96 8 REFERENCES...................................................................................................98 9 ABBREVIATIONS ...........................................................................................130 10 APPENDIX.......................................................................................................132 11 PUBLICATION LIST........................................................................................137
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