Depletion des Myc-interagierenden Zinkfingerproteins 1 mittels RNA Interferenz und Die Rolle von Miz-1 in der zellulären DNA Schadensantwort auf UV-Licht

Die vorliegende Dissertationsarbeit beschäftigte sich zum einen methodisch mit dem Gen-Knock-out des mit Myc interagierenden Proteins Miz-1 mittels der kürzlich erschlossenen Technik der RNA Interferenz in Säugerzellen. Zum anderen wurde unter Nutzung der RNA Interferenz ein Zusammenhang aus dem B...

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Bibliographic Details
Main Author: Rogosch, Dorothee
Contributors: Eilers, Martin (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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On the one hand, this thesis deals with the gene knock-out of the Myc interacting protein Miz 1 by means of the technique of RNA interference in mammalian cells. On the other hand, an aspect of cellular damage control mechanisms, especially the regulation of UV irradiation induced DNA damage response by the Myc interacting protein Miz-1 was clarified by the use of RNA interference. The first part deals with an evolutionarily conserved cellular defence mechanism against foreign genes, the RNA interference. Knock-out by RNA interference replaces increasingly the conventional gene knock-out technologies. A significant decrease of plasmid induced luminescence was achieved by cotransfaction of a plasmid coding for firefly luciferase and a sequence homologous double stranded RNA. The suitability of the mammalian cell lines HaCaT, a humane keratinocyte cell line, HeLa, humane cervix carcinoma cells and MEF, murine embryo fibroblasts, for RNA interference was shown by that specific gene knock out. The posttranscriptional gene silencing of the transcription factor Miz-1 was realized. Like in previous RNA interference experiments in mammalian cells only a partial knock-out of the target-gene could be achieved. But also a partial knock-out of a gene has effects on the resulting phenotype. The possibility of a partial Miz-1 knock-out opens up extensive possibilities for the functional analysis of the gene and its interaction partners as well as the analysis of signal transduction pathways that are dependent on Miz-1. The second part deals with the cellular response to UV induced DNA damages. Therefore a DNA damage checkpoint that is dependent on p53 as well as a p53 independent one was investigated and the role of Miz-1 was examined. For the analysis of the p53 dependent cellular response to UV irradiation induced DNA damages ARF-/--mouse embryo fibroblasts were used. In wild-type mouse embryo fibroblasts, Myc alone can induce apoptosis through ARF. By p53 numerous genes which cause cell cycle arrest or apoptosis are activated, e.g. the CdK inhibitor p21cip1. Beside p53 the transcription factor Miz-1 is essential for the expression of p21cip1 after UV irradiation. We could show that treatment of cells with siRNA directed against Miz-1 attenuated the expression of p21cip1 in UV-irradiated cells. An overexpression of the onkogen Myc prevents an activation of the expression of p21cip1 after UV-irradiation in MEF cells. Consequently an abbreviated duration of cell cycle arrest and an early re-entry of the damaged cell into cell cycle occurs. In response to UV irradiation, Myc inhibits UV-induced cell cycle arrest by p53 through Miz-1. The p53 independent damage response to different DNA damaging agents could be examined in HaCaT cells lack functional p53 and therefore do not induce p21Cip1. UV irradiation downregulates expression of TopBP1 and releases Miz-1 from an inhibitory complex with TopBP1 and causes transcriptional activation of p15INK4B. Expression analyses by RT-PCR show that DNA damages caused by cytostatic drugs like Zeozin and Hydroxyurea have no influence on the expression of Myc, Miz-1, TopBP1 and p15INK4B. These results plead for the damage specificity of the p53 independent UV-response. No involvement of the examined Miz1 dependent signal transduction pathway could be shown in the cellular response to cytostatic drug induced DNA defects. Our data support the hypothesis that the Miz-1/TopBP1 complex is part of a checkpoint exclusively triggered by UV induced DNA damage.