Bedeutung Und Charakterisierung Der 6Q-Deletion Beim Prostatakarzinom

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Bedeutung Und Charakterisierung Der 6Q-Deletion Beim Prostatakarzinom Bedeutung und Charakterisierung der 6q-Deletion beim Prostatakarzinom Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) am Department Biologie der Fakultät Mathematik, Informatik und Naturwissenschaften der Universität Hamburg Vorgelegt von Martina Kluth Heide-Holstein Hamburg 2015 Gutachter der Dissertation Prof. Dr. Guido Sauter Prof. Dr. Thomas Dobner Datum der Disputation: 22.01.2016 Inhaltsverzeichnis 1Einleitung 1 1.1 Epidemiologie und Ätiologie des Prostatakarzinoms .............. 1 1.2 Früherkennung und Primärdiagnostik des Prostatakarzinoms .............................. 2 1.2.1 Digitale rektale Untersuchung (DRU) .................. 3 1.2.2 Prostataspezifisches Antigen (PSA) ................... 3 1.2.3 Stanzbiopsie der Prostata ........................ 5 1.3 Pathologie und Stadieneinteilung des Prostatakarzinoms ................................ 5 1.4 Therapie des Prostatakarzinoms ......................... 7 1.5 Prognosemarker beim Prostatakarzinom .................... 11 1.6 Genomische Veränderungen beim Prostatakarzinom .............. 12 1.6.1 Punktmutation .............................. 12 1.6.2 Chromosomale Zugewinne ........................ 12 1.6.3 Translokationen .............................. 13 1.6.4 Deletionen ................................. 14 1.7 Die 6q-Deletion beim Prostatakarzinom ..................... 15 1.7.1 Prävalenz der 6q-Deletion beim Prostatakarzinom ........... 15 1.7.2 Assoziation der 6q-Deletion mit dem Tumorphänotyp und der Progno- se des Prostatakarzinoms ......................... 15 1.7.3 Architektur der 6q-Deletion ....................... 16 1.7.4 Potentielle Tumorsuppressorgene der 6q-Deletion ........... 19 1.8 Zielsetzung der Arbeit .............................. 20 2Material 23 2.1 Gewebekollektiv .................................. 23 i INHALTSVERZEICHNIS 2.2 Prostata-Prognose-Array ............................. 23 2.3 Prostata-Heterogenitätsarray ........................... 26 2.3.1 Multifokaler Prostata-Heterogenitätsarray ............... 26 2.3.2 Unifokaler Prostata-Heterogenitätsarray ................ 27 2.4 DH10B Escherichia coli (E. coli) Klone für die Herstellung von Fluoreszenz- markierten Sonden ................................ 28 2.5 Humane Zelllinien ................................. 29 2.6 shRNA-pLKO.1 transformierte E. coli Klone .................. 29 2.7 Plasmide zur Produktion von lentiviralen Partikeln .............. 31 2.8 TaqMan®-Sonden ................................. 31 2.9 Western Blot Antikörper ............................. 34 2.10 Zellkulturmedien für humane Zelllinien ..................... 34 2.11 Reagenzien und Kits ............................... 35 2.12 Verwendete Lösungen ............................... 38 2.13 Geräte ....................................... 39 2.14 Materialien .................................... 41 2.15 Software und Datenbanken ............................ 41 3Methoden 42 3.1 Fluoreszenz-in-situ-Hybridisierung (FISH) ................... 42 3.1.1 Kultivierung von pBACe3.6 transformierten DH10B E. coli ..... 42 3.1.2 Isolierung von Plasmid-DNA aus DH10B E. coli ............ 43 3.1.3 Bestimmung der Konzentration und Reinheit von DNA-Proben ... 44 3.1.4 Nick Translation zur Herstellung von FISH-Sonden .......... 44 3.1.5 Isolierung und Aufreinigung von FISH-Sonden ............. 45 3.1.6 Durchführung der FISH ......................... 46 3.1.7 Mikroskopische Auswertung ....................... 48 3.1.8 Statistische Auswertung ......................... 49 3.2 Zellkulturmethoden ................................ 49 3.2.1 Kultivierung von humanen Zelllinien .................. 50 3.2.2 Kryokonservierung von humanen Zelllinien ............... 50 3.2.3 Bestimmung der Zellkonzentration ................... 51 3.2.4 Kultivierung von shRNA-pLKO.1 transformierten E. coli ....... 51 3.2.5 Isolierung von shRNA-Konstrukten aus E. coli ............. 52 ii INHALTSVERZEICHNIS 3.2.6 Transfektion von Prostata-(karzinom)Zelllinien mit Lipofectamin®2000 ............................ 52 3.2.7 Produktion von lentiviralen Partikeln in 293T-Zellen ......... 53 3.2.8 Transduktion von Prostata-(karzinom)Zelllinien mit lentiviralen Par- tikeln .................................... 55 3.2.9 Colony-Formation-Assay ......................... 55 3.2.10 Softagar-Colony-Formation-Assay .................... 57 3.2.11 Invasion-Assay .............................. 58 3.3 Expressionsanalysen ............................... 59 3.3.1 RNA-Präparation ............................. 59 3.3.1.1 RNA-Isolierung aus mikrodissezierten Prostataepithelzellen 60 3.3.1.2 RNA-Isolierung aus kryokonservierten Gewebegroßflächen . 61 3.3.1.3 RNA-Isolierung aus Prostata-(karzinom)zellen nach Qiagen . 62 3.3.1.4 RNA-Isolation aus Prostata-(karzinom)zellen nach Machery- Nagel .............................. 62 3.3.2 Bestimmung der Konzentration und Reinheit von RNA-Proben ... 63 3.3.3 Bestimmung der RNA-Integritätzahl (RIN) ............... 63 3.3.4 Reverse Transkription .......................... 64 3.3.5 TaqMan®-PCR .............................. 64 3.3.6 Proteinpräparation aus Prostata-(karzinom)zellen ........... 65 3.3.7 Bestimmung der Proteinkonzentration ................. 66 3.3.8 Western Blot ............................... 66 4Ergebnisse 69 4.1 Untersuchungen des 6q15 (MAP3K7 ) Locus mittels FISH am Prognose-TMA 69 4.1.1 Auswertbarkeit der 6q15 FISH-Analyse ................. 69 4.1.2 Prävalenz und Assoziation der 6q15-Deletion zum Tumorphänotyp .............................. 70 4.1.2.1 Prävalenz der 6q15-Deletion .................. 70 4.1.2.2 Assoziation der 6q15-Deletion mit den klinisch-pathologischen Parametern des Prostatakarzinoms .............. 70 4.1.2.3 Assoziation der 6q15-Deletion mit der Zellproliferation (Ki67 Labeling Index) ......................... 73 iii INHALTSVERZEICHNIS 4.1.3 Assoziation der 6q15-Deletion mit der Prognose des Patienten beim Prostatakarzinom ............................. 75 4.1.3.1 Assoziation der 6q15-Deletion mit dem Auftreten eines PSA- Rezidivs ............................. 75 4.1.3.2 Assoziation der 6q15-Deletion mit biologisch-klinischen End- punktgruppen .......................... 79 4.1.4 Assoziation der 6q15-Deletion mit häufigen genomischen Veränderungen des Prostatakarzinoms ................. 82 4.1.4.1 Assoziation der 6q15-Deletion mit der TMPRSS2 :ERG Fusion 82 4.1.4.2 Assoziation der 6q15-Deletion mit weiteren häufigen Deletio- nen des Prostatakarzinoms ................... 83 4.2 Untersuchungen der 6q-Deletionsgröße mittels FISH am Prognose-TMA ... 85 4.2.1 Auswertbarkeit der 6q FISH-Analyse .................. 85 4.2.2 Prävalenz und Assoziation der unterschiedlichen 6q-Deletions-größen zum Tumorphänotyp ........................... 86 4.2.2.1 Prävalenz der unterschiedlichen 6q-Deletionsgrößen ..... 86 4.2.2.2 Assoziation der unterschiedlichen 6q-Deletionsgrößen mit den klinisch-pathologischen Parametern des Prostatakarzinoms . 87 4.2.2.3 Assoziation der unterschiedlichen 6q-Deletionsgrößen mit der Zellproliferation (Ki67 Labeling Index) ............ 89 4.2.3 Assoziation der unterschiedlichen 6q-Deletionsgrößen mit der Prognose des Patienten beim Prostatakarzinom .................. 91 4.2.3.1 Assoziation der unterschiedlichen 6q-Deletionsgrößen mit dem Auftreten eines PSA-Rezidivs ................. 91 4.2.3.2 Assoziation der 6q-Deletionsgröße mit biologisch-klinischen Endpunktgruppen ....................... 93 4.2.4 Assoziation der 6q-Deletionsgröße mit der TMPRSS2 :ERG Fusion .. 94 4.3 Untersuchungen des 6q15 (MAP3K7 )LocusmittelsFISHammultifokalen Heterogenitäts-TMA ............................... 95 4.3.1 Auswertbarkeit der 6q15-Heterogenitätsanalyse mittels FISH ..... 95 4.3.2 Heterogenität der 6q15-Deletion ..................... 96 4.3.3 Chronology der Entstehung einer 6q15-Deletion und der TMPRSS2 :ERG Fusion ......................... 97 iv INHALTSVERZEICHNIS 4.4 Untersuchungen zur 6q-Deletionsgröße mittels FISH am unifokalen Heterogenitäts- TMA ........................................ 98 4.4.1 Auswertbarkeit der 6q-FISH-Analyse und Prävalenz der 6q-Deletion . 98 4.4.2 Ausdehnung der 6q-Deletion während der Tumorprogression ..... 99 4.5 Identifizierung potentieller Tumorsuppressorgene in vitro ....................................... 102 4.5.1 Expression und Auswahl von Kandidatengenen innerhalb der 6q12- q22-Deletion ................................ 102 4.5.1.1 Expression von 43 Genen innerhalb der 6q14-q16 Region in 6 Prostata-Zelllinien ...................... 102 4.5.1.2 Expression von 43 Genen innerhalb der 6q14-q16 Region in mikrodissezierten benignen Prostatadrüsengewebe ...... 103 4.5.1.3 Expression von 13 Genen außerhalb der 6q14-q16 Region in 4 Prostata-Zelllinien ...................... 104 4.5.2 Auswahl der optimalen shRNA-Konstrukte für die Suppression der Kandidatengene innerhalb der 6q12-q22-Deletion ........... 105 4.5.3 Einfluss der lentiviralen Transduktion auf die shRNA-vermittelte Sup- pression der mRNA (Depletionseffizienz) ................ 107 4.5.4 Einfluss der lentiviralen Transduktion auf die shRNA-vermittelte Sup- pression auf Proteinebene (Depletionseffizienz) ............. 108 4.5.5 Quantifizierung des Suppressions-bedingten Einflusses der Kandida- tengene auf das Zellwachstum im Colony-Formation-Assay ...... 109 4.5.6 Validierung der potentiellen Zielgene der 6q12-q22-Deletion im Colony- Formation-Assay ............................. 119 4.5.7 Auswirkungen der Ko-Depletion der vier potentiellen Tumorsuppres- sorgene auf das Wachstumsverhalten von Prostatazellen ........ 121 4.5.7.1 Nachweis
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