Zellbasierte Hochdurchsatzanalysen identifizieren eine zentrale, wachstumsregulierende Rolle von Cofilin-1 im humanen Pankreaskarzinom

Aufgrund der ungünstigen Prognose des humanen duktalen Adenokarzinoms des Pankreas ist es von großer Bedeutung, neue Zielgene zu identifizieren, die Ansatzstellen sowohl für neue Therapien als auch für eine frühere Diagnose bieten können. In der hier vorgestellten Arbeit wurden deshalb insgesamt...

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Bibliographic Details
Main Author: Kirchhoff, Sandra
Contributors: Buchholz, Malte (PD Dr. ) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
Online Access:PDF Full Text
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Due to the poor prognosis of the human ductal adenocarcinoma of the pancreas (PDAC) it is of major importance to identify novel target genes with high functional relevance to provide a better diagnosis or new therapeutic approaches. In this study, 91 potential candidate genes identified as differentially expressed in carcinogenic pancreatic tissues in previous high-throughput analyses were functionally characterized. To this end, the technology of “reverse transfection“ was established in our lab to perform the analyses in a highly parallelized microarray approach. Overexpression and knockdown experiments were carried out in transformed and nontransformed cell lines for all candidates to screen for subcellular localisation of the gene products and their impact on cellular functions like apoptosis, proliferation and differentiation. Data evaluation led to the selection of 10 genes which showed significant and reproducible effects in the parallelized experiments. One of them was Cofilin-1 (CFL1), a member of the ADF/Cofilin family of actin-binding proteins. Parallelized microarray analyses revealed a previously undocumented growth-regulatory effect of this gene in pancreatic cancer. Subsequent detailed characterization demonstrated a strong overexpression of CFL1 in PDAC on mRNA as well as on protein level. RNAi-mediated knockdown of CFL1 in 4 different cell lines led to a significant inhibition of cell growth whereas apoptotic cascades were not affected. Furthermore, a significant decrease of anchorage-independent growth was observed in soft agar assays. Flow cytometric analyses indicated an attenuation of the G1 to S phase transition. Aside from its growth-regulatory function, migration assays revealed an additional inhibitory effect of CFL1 on cell motility identified in “time lapse” and wound healing assays. In contrast to this, a directional migration analyzed in Boyden Chamber analyses remained unaffected. Based on these data and the current state of the literature, the results of this study present for the first time a direct growth-regulatory effect of CFL1 in pancreatic cancer.