Publikationsserver der Universitätsbibliothek Marburg

Titel:Analyse biologischer Effekte eines Fibroblasten-Wachstumsfaktor-bindenden Proteins (FGF-BP) in Tumoren über verschiedene Knockdown-Strategien
Autor:Schulze, Daniel
Weitere Beteiligte: Kissel, Thomas (Prof. Dr.)
Veröffentlicht:2011
URI:https://archiv.ub.uni-marburg.de/diss/z2011/0123
DOI: https://doi.org/10.17192/z2011.0123
URN: urn:nbn:de:hebis:04-z2011-01232
DDC: Medizin
Titel (trans.):Analysis of biological effects of fibroblast growth factor-binding protein (FGF-BP) through different knockdown strategies
Publikationsdatum:2011-08-08
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Rezeptor-Tyrosinkinasen, Krebs, RNS-Interferenz, Fibroblastenwachstumsfaktor, fibroblast growth factor-binding protein (FGF-BP), Colonkrebs, Fibroblasten-Wachstumsfaktor-bindendes Protein (FGF-BP), Apoptosis, Hammerkopf-Ribozym

Zusammenfassung:
Fibroblasten Wachstumsfaktoren (FGFs) spielen eine bedeutende Rolle bei der Zellproliferation und -differenzierung. Bei neoplastischem Wachstum fungieren FGFs als potente Mitogene. Einige der tumorrelevanten FGFs binden jedoch mit hoher Affinität an eine oder mehrere Komponenten der extrazellulären Matrix (EZM) und die Freisetzung der FGFs aus der EZM stellt einen kritischen Schritt in der FGF-Bioaktivierung dar. Das FGF-bindende Protein FGF-BP ist ein sekretiertes Heparin-bindendes Protein, welches mit mehreren FGFs, wie FGF1, FGF2, FGF7, FGF10 sowie FGF22, spezifisch interagiert und diese aus ihrem extrazellulären Speicherort freisetzt. FGF-BP ist in verschiedenen Tumoren, insbesondere in der frühen Phase der Karzinogenese, hochreguliert. Die (Über-)expression von FGF-BP in Tumorzellen stellt einen limitierenden Faktor für die Etablierung solider Tumore dar. Im Rahmen dieser Dissertation wurden verschiedene Knockdown-Strategien exploriert um FGF-BP hinsichtlich seines Beitrags zu Proliferation und Apoptose sowie zum in vivo-Tumorwachstum zu untersuchen. Unter Verwendung von vektorbasierter RNAi wurden isogene Kolonkarzinom-Zelllinien mit permanent vermindertem FGF-BP-Level generiert. Es wurde gezeigt, dass die Reduktion der FGF-BP-Level sowohl antiproliferative Effekt als auch pro-apoptotische Effekte hervorruft. Die Herunterregulation von FGF-BP ging einher mit komplexen zellulären und molekularen Veränderungen, u.a. in der MAPK- und Apoptose-Signaltransduktion. Im Vergleich zu den parentalen Zellen wies der knockdown-Phänotyp erhöhte Spiegel an aktiviertem Bad und Bax auf, was zu einer gesteigerten Aktivität der Effektorcaspasen 3 und 7 führte. Gleichzeitig wurde in FGF-BP-herunterregulierten Zellen eine Suppression von Akt Kinasen, eine Aktivierung von SAPK/JNK und eine Induktion der GSK3β detektiert. Klone mit vermindertem FGF-BP-Gehalt akkumulierten vermehrt in der G0/G1-Phase des Zellzyklus und wiesen erhöhte Konzentrationen an aktiviertem p21WAF1/CIP auf. Darüber hinaus zeigten diese loss-of-function-Mutanten Alterationen im Redoxstatus auf, was sich in einer erniedrigten Katalasekonzentration sowie erhöhten Konzentrationen an HIF1α widerspiegelte. In einem Zervixkarzinom-Modell wurde mit Hilfe eines Ribozym-Targetings gezeigt, dass FGF-BP die Sensitivität gegenüber Chemotherapeutika beeinflusst. Synergistische Effekte eines Knockdowns und einer Antitumorbehandlung waren nachweisbar bei dem Topoisomerase I Inhibitor Irinotecan, nicht aber bei den Spindelgiften Docetaxel und Vinorelbin. In einem in vivo-Experiment gelang es, mit Hilfe einer auf Polyethylenimin (PEI) basierenden Applikation von therapeutischen Nukleinsäuren (FGF-BP-siRNAs) einen spezifischen FGF-BP-Knockdown sowie einen antitumoralen Effekt im Kolonkarzinom-Nacktmausmodell zu erzielen. Um neue Interaktionspartner für FGF-BP aufzufinden, wurden unterschiedliche full length- sowie Deletionsmutanten des Proteins kloniert und rekombinant exprimiert. Durch Protein-Protein-Interaktions-Analysen konnten weitere Liganden für FGF-BP innerhalb der FGF-Familie ermittelt werden (FGF4, FGF8). Darüber hinaus wurde das DNA-assoziierte High-Mobility Group A-Protein (HMGA) als neuer Bindepartner für FGF-BP identifiziert. Ferner zeigten in vitro-Untersuchungen eine Affinität von FGF-BP zu Nukleinsäuren. In Anwesenheit von genomischer DNA wies FGF-BP eine erhöhte Proteinstabilität auf. Diese Daten unterstützen die Hypothese eines erst kürzlich ermittelten und bislang noch nicht näher untersuchten intrazellulären Mechanismus von FGF-BP. Insgesamt konnte gezeigt werden, dass FGF-BP BP bei proliferativen sowie zytoprotektiven Vorgängen in ein komplexes molekulares Netzwerk eingebunden ist. Die in dieser Arbeit ermittelten Daten demonstrieren, dass eine gezielte Verminderung der FGF-BP-Expression das Ausmaß der zellulären Apoptose erhöht, eine anti-proliferative Wirkung hervorruft und das Tumorwachstum in vivo vermindert. Die beschriebenen Untersuchungen zur Chemoresistenz erlauben eine Abschätzung der selektiven Sensitivität von Tumorzellen gegenüber verschiedenen Chemotherapeutika in Abhängigkeit von der FGF-BP-Expression. Es kann somit gefolgert werden, dass FGF-BP ein vielversprechendes Zielmolekül (target) in der Antitumortherapie darstellt.

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