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Charakterisierung Interindividueller Strahlenempfindlichkeit in Zellen Aus Tumorpatienten

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Charakterisierung Interindividueller Strahlenempfindlichkeit in Zellen Aus Tumorpatienten Charakterisierung interindividueller Strahlenempfindlichkeit in Zellen aus Tumorpatienten Anne Maria Dietz Charakterisierung interindividueller Strahlenempfindlichkeit in Zellen aus Tumorpatienten Dissertation an der Fakultät für Biologie der Ludwig-Maximilians-Universität München zur Erlangung des Doktorgrades der Naturwissenschaften Angefertigt an der Klinik und Poliklinik für Strahlentherapie und Radioonkolgie in der Arbeitsgruppe Molekulare Strahlenbiologie unter der Leitung von PD. Dr. Anna Friedl Vorgelegt von Anne Maria Dietz München, den 03. Dezember 2014 Erstgutachter: PD Dr. Anna A. Friedl Zweitgutachter: Prof. Dr. Peter Becker Tag der mündlichen Prüfung: 05.05.2015 Meinem Vater Inhaltsverzeichnis Inhaltsverzeichnis 1 Zusammenfassung ........................................................................................................................... 5 2 Einleitung ......................................................................................................................................... 7 2.1 Individuelle Strahlenempfindlichkeit ...................................................................................... 7 2.2 Das Chromosomeninstabilitätssyndrom Ataxia Telangiectasia .............................................. 9 2.3 Biomarker-Entdeckung im Zeitalter der Omics-Technologien .............................................. 12 2.3.1 Charakterisierung eines validen Biomarkers ................................................................. 12 2.3.2 Biomarker für Strahlenempfindlichkeit ......................................................................... 12 2.3.3 Verschiedene Omics-Technologien und ihre Anwendungen ........................................ 14 2.4 Protein Biomarker-Entdeckung ............................................................................................. 17 2.5 Pathway-Analyse zur Biomarker-Detektion .......................................................................... 17 2.6 Interindividuelle Strahlenempfindlichkeit in Zelllinien aus Tumorpatienten ....................... 18 2.7 Ziel der Arbeit ........................................................................................................................ 19 3. Ergebnis ......................................................................................................................................... 20 3.1 Patientenproben der LUCY-Studie ........................................................................................ 20 3.2 Etablierung der Versuchsbedingungen für das Screening .................................................... 20 3.2.1 Screening des Überlebens lymphoblastoider Zelllinien nach Bestrahlung ................... 23 3.3 Karyotypisierung der unterschiedlich strahlenempfindlichen Zelllinien ............................... 26 3.4 Proteomanalyse zur Identifizierung deregulierter Proteine der Strahlenantwort in LCLs .... 27 3.4.1 Hochauflösende Gele für die 2D-Gelelektrophorese .................................................... 29 3.4.2 Ergebnisse der differentiellen Analyse .......................................................................... 29 3.4.3 Vergleich der differentiell exprimierten Proteine innerhalb einer Zelllinie zu drei unterschiedlichen Zeitpunkten nach Bestrahlung mit 1 Gy und 10 Gy ......................................... 31 3.4.4 Vergleich der differentiell exprimierten Proteine zwischen allen Zelllinien zu einem Zeitpunkt (0,5 h, 4 h und 24 h) nach Bestrahlung mit 1 Gy und 10 Gy ......................................... 39 3.5 Bioinformatische Analyse der differentiell exprimierten Proteine aus 2D-DIGE mit der Ingenuity Pathway Analysis Software (IPA) ....................................................................................... 43 3.5.1 Zelluläre Funktionen der differentiell exprimierten Proteine ....................................... 47 3.5.2 Netzwerke der differentiell exprimierten Proteine ....................................................... 53 3.6 Auswahl einzelner Proteine zur Validierung der 2D-DIGE Regulationsfaktoren ................... 58 3.7 Validierungen der 2D-DIGE Regulationsfaktoren durch quantitativen Western Blot ........... 59 3.7.1 Validierung der Regulationsfaktoren durch Normalisierung mit einer Ladekontrolle .. 59 1 Inhaltsverzeichnis 3.7.2 Validierung der Regulation von RAD50 mit der Stain-Free-Methode (BioRad) ............ 67 3.8 Analyse der miRNA-Expression nach ionisierender Strahlung .............................................. 71 3.8.1 Vergleich der differentiell exprimierten miRNAs zwischen allen Zelllinien zu einem Zeitpunkt (4 h und 24 h) nach Bestrahlung mit 10 Gy .................................................................. 84 3.9 Bioinformatische Netzwerkanalyse der deregulierten Proteine und miRNAs nach Bestrahlung in den Kontrollinien Linien GM03323(+) und GM03189(-) ........................................... 88 3.10 Korrelation der WST-1 Viabilitätsdaten 24 h und 48 h nach Bestrahlung mit 10 Gy mit 4 ausgewählten SNPs ........................................................................................................................ 93 4 Diskussion ...................................................................................................................................... 95 4.1 Eignung von EBV-immortalisierten Zelllinien zur Analyse der Strahlenempfindlichkeit....... 95 4.2 Zellüberleben nach Bestrahlung ........................................................................................... 97 4.3 Differentielle Proteom-und MALDI-TOF/MS-Analyse ........................................................... 99 4.4 Western Blot Analyse ausgewählter Kandidatenproteine .................................................. 104 4.4.1 SND1 ............................................................................................................................ 104 4.4.2 SerpinB9 ...................................................................................................................... 105 4.4.3 MCM7 .......................................................................................................................... 105 4.4.4 RAD50 .......................................................................................................................... 106 4.5 Differentielle Proteinexpression nach Bestrahlung in unterschiedlich strahlenempfindlichen LCLs ............................................................................................................................................. 108 4.6 Bioinformatische Analyse der differentiell exprimierten Proteine aus 2D-DIGE mit der Ingenuity-Software .......................................................................................................................... 109 4.7 Analyse der miRNA-Expression nach ionisierender Strahlung ............................................ 113 4.8 Bioinformatische Netzwerkanalyse der deregulierten Proteine und miRNAs 24 h nach Bestrahlung mit 10 Gy in den Linien GM03323(+) und GM03189(-) .............................................. 115 4.9 Biomarker für Strahlenempfindlichkeit ............................................................................... 116 5. Material ....................................................................................................................................... 118 5.1 Geräte, Chemikalien und Computerprogramme ................................................................ 118 5.1.1 Geräte .......................................................................................................................... 118 5.1.2 Chemikalien ........................................................................................................................ 119 5.1.3 Computerprogramme.................................................................................................. 121 5.2 Verwendete Lösungen und Puffer (Lösung/Puffer-Zusammensetzung/ Hersteller) .......... 121 5.3 Kommerzielle Kits ................................................................................................................ 126 5.4 Verbrauchsmaterialien ........................................................................................................ 127 5.5 Verwendete Antikörper und Verdünnungen ...................................................................... 128 5.6 Zelllinien und Zellkulturmedien ........................................................................................... 129 5.7 Kontroll-Zelllinien ................................................................................................................ 129 2 Inhaltsverzeichnis 5.8 Zellkulturmedium ................................................................................................................ 129 6 Methoden .................................................................................................................................... 130 6.1 Zellbiologische Methoden ................................................................................................... 130 6.1.1 Kultivieren, Vermehren und Passagieren einer Suspensionszellkultur ....................... 130 6.1.2 Kryokonservierung von Säugerzellen .......................................................................... 130 6.1.3 Auftauen von humanen Zellen .................................................................................... 130 6.1.4 Zellzahlbestimmung lymphoblastoider Zelllinien ........................................................ 131 6.1.5 Anzucht und
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