Publikationsserver der Universitätsbibliothek Marburg
Titel:Targeting p53 and its domains for cancer gene therapy
Autor:Karina Julia Matissek
Weitere Beteiligte: Thomas Kissel (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0226
URN: urn:nbn:de:hebis:04-z2014-02265
DOI: https://doi.org/10.17192/z2014.0226
DDC: Biowissenschaften, Biologie
Titel (trans.):Targeting von p53 und seinen Domänen zur Krebsgentherapie
Publikationsdatum:2014-04-30
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Bak, Bcl-XL, mitochondria, Mitochondrium, Krebs, cancer, Protein p53, Bak, Bcl-XL

Summary:
Der Tumorsuppressor p53 ist eines der am häufigsten mutierten Proteine in humanen Krebsarten und wird daher umfassend für seinen Nutzen in der Krebstherapie erforscht. Dies führte in China zur Markteinführung von Wildtyp-p53 zur Therapie von Kopf-Hals-Karzinomen. p53 fungiert in der Zelle hauptsächlich als Transkriptionsfaktor und stimuliert eine Vielzahl von Genen, die im intrinsischen und extrinsischen Apoptosemechanismus involviert sind. In Krebszellen treten Mutationen normalerweise in der DNA-Bindungsdomäne von p53 auf, wohingegen die Tetramerizationsdomäne (TD) des Tumorsuppressors intakt bleibt. Dies führt intrazellular zur Bildung von Heterotetrameren von Wildtyp-p53 und seiner mutierten Form, was die Transkriptionsaktivität erheblich beeinträchtigt und einen dominant negativen Effekt ergibt. Während transkriptionell aktives p53 als Gentherapeutikum genutzt wird, ist das therapeutische Potential für den Einsatz von mitochondrialem p53 noch nicht vollständig ermittelt. Wenn p53 zum Mitochondrium getargeted wird, interagiert es mit pro- und anti-apoptotischen Proteinen, die sich in der mitochondrialen Auβenmembran befinden. Für diese Interaktion reicht die monomere Form von p53 aus, was bedeutet, dass es nicht durch mutiertes p53 inaktiviert werden kann. In dieser Arbeit wurde die Funktion von mitochondrialem p53 charakterisiert, indem es zu verschiedenen mitochondrialen Kompartimenten getargeted wurde: der mitochondrialen Auβenmembran, der Innenmembran und der Matrix. Es konnte nachgewiesen werden, dass mitochondriale Targeting Sequenzen (MTS) in der mitochondrialen Auβenmembran optimal für eine p53-spezifische Aktivierung geeignet sind. Auch konnte gezeigt werden, dass als minimalste Domäne von p53 die DNA-Bindungsdomäne (DBD) ausreicht, um Apoptose zu induzieren. Weitere Untersuchungen haben ergeben, dass das Vereinigen von p53 oder nur seiner DBD mit der MTS von Bcl-XL, eine Bcl-XL spezifische Apoptose hervorruft, während eine Vereinigung der Segmente mit Bak auf p53/Bak spezifische Apoptose zurückzuführen ist. Dies hebt hervor, dass mitochondriales Targeting von p53 stark von der benutzten MTS abhängig ist. Auβerdem haben in vitro-Studien gezeigt, dass die Bindung von p53 oder DBD an die MTS von Bcl-XL eine dominant negative Inhibition überwinden kann, aber auβerstande ist dominant negative MDA-MB-468 Tumore in einem orthotopischen Maus-Tumor-Model für das gewählte Dosisschema zu reduzieren. Die Thematik dieser Dissertation war die Entwicklung Apoptose-induzierender Proteine basierend auf p53 und seinen Bindungsdomänen, um eine modifizierte Version von p53 zu generieren. Der Schwerpunkt lag sowohl auf der Optimierung eines mitochondrial getargeten p53 für mögliche Krebstherapien als auch in der Neugestaltung der TD von p53, um den dominant negativen Effekt der Transkriptionsaktivität zu überwinden. Dafür wurde die Oligomerizationsdomäne von p53 mit der Coiled-Coil (CC) Domäne von BCR ersetzt, damit der dominant negative Effekt von mutiertem p53 ausgeschaltet wird. Experimente zeigen, dass das chimäre p53 (p53-CC) in den Nukleus transloziert, Gene transaktiviert und Apoptose in ähnlicher Form wie Wildtyp-p53 auslöst. In vitro- und in vivo-Studien haben gezeigt, dass im Gegensatz zu Wildtyp-p53, das neu generierte p53-CC nicht mit endogen mutiertem p53 interagiert und seine apoptotische Aktivität in Krebszellen, die dominant negatives mutiertes p53 enthalten, beibehält. Zusammenfassend liegt der Forschungsschwerpunkt dieser Dissertation in der Entwicklung neuartiger p53-Gentherapeutika, die das Potential haben derzeitige Einschränkungen einer Wildtyp-p53 Therapie zu überwinden.

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  71. Molecular Pharmaceutics Article dx.doi.org/10.1021/mp400379c | Mol. Pharmaceutics 2013, 10, 3922−3933 ■ EXPERIMENTAL SECTION Subcloning and Construction of Plasmids. pEGFP-Bcr- Abl was constructed as previously described. 3 Bcr-Abl DNA was also cloned into pmCherry-C1 (Clonetech, Mountain View, CA, USA) and pTagBFP-C (Evrogen, Moscow, Russia) at the EcoR1 site on both vectors creating pmCherry-Bcr-Abl and pBFP-Bcr-
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