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Gaedtke, Lars (2006): Cell culture models and novel gene therapeutic strategies for colorectal cancer. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The development of relevant cellular model systems for colorectal cancer is of utmost importance for an improved in vitro assessment of therapeutic strategies against colorectal cancer. Recently published low passage colon cancer cell lines that closely reflect the characteristics of the respective parental in vivo tumor cells represent very promising cell culture models and were therefore used for the investigations in the present thesis. To provide an in vitro model system that also recapitulates the three-dimensional structure of in vivo tumors, these low passage cell lines were cultivated as multicellular spheroids. Compared to monolayer cultures the multicellular spheroids exhibited a wide variety of changes in their expression patterns. The differential expression includes proteins that are involved in growth signaling (15-hydroxyprostaglandin dehydrogenase), protein biosynthesis (acidic ribosomal protein P0), and regulation of the cyto- or nucleoskeleton (acidic calponin and LMNA protein). These proteins were identified by 2D electrophoresis and subsequent MALDI-TOF mass spectrometry. Both methods were established in the lab in the context of this work. Chemotherapy with 5-fluorouracil (5-FU) represents the traditional treatment of colorectal cancer. However, in many patients the efficiency of this therapeutic strategy is often limited by the development of chemoresistance against 5-FU. Therefore, it was an aim of this thesis to detect novel proteins involved in 5-FU chemoresistance that were previously not ascribed to resistance against this chemotherapeutic drug. A chemoresistant subline of a colon cancer cell line was generated by long-term treatment with 5-FU and served as a model for the investigation of 5-FU chemoresistance. This subline exhibited resistance against both 5-FU-induced inhibition of proliferation and apoptosis. Differences in the expression of cytokeratin 18, heat shock protein 27 and aldehyde dehydrogenase 1B1 between the chemoresistant subline and parental cells were detected by 2D electrophoresis. These findings imply that the cytoskeleton plays a role in the development of chemoresistance against 5-FU. Furthermore, processes located to the mitochondria seem to be involved in this resistance, since heat shock protein 27 and aldehyde dehydrogenase 1B1 are associated with this subcellular organelle. The biological relevance of the findings made in the present PhD thesis has to be determined in further studies. Gene therapy represents a promising alternative strategy for the treatment of colorectal cancer. A novel nonviral gene transfer system was developed by combination of DNA with the polycation PEI25br and the cationic lipids DOCSPER or DOSPER to form lipopolyplexes. These lipopolyplexes enabled enhanced gene transfer in vitro and are promising for in vivo applications, since the established lipopolyplexes preserved their small size at physiological conditions; a property essential for a successful in vivo application. Furthermore, the lipopolyplexes exhibited the capability to efficiently transfect three-dimensional multicellular spheroids. The potential of lipopolyplexes for therapeutic applications was further increased by the utilization of the artificial promoter CTP4 which enables highly specific gene expression in cancer cells with mutations in the Wnt signaling pathway by transcriptional targeting. In addition to its high specificity, this promoter enabled high gene expression levels that were comparable to expression levels obtained by the strong, but unspecific CMV promoter. The efficiency of the CTP4 promoter was demonstrated in seven low passage colon cancer cell lines and also in multicellular spheroids. The transcriptional targeted lipopolyplexes not only enabled high tumor specific expression of reporter genes like luciferase or EGFP but also the expression of a therapeutic gene, interleukin-2 (IL-2). Furthermore, tumor specific expression of cytotoxic protease 2A in combination with IL-2 was possible by using a novel bicistronic construct. The expression of the rhinoviral protease 2A led to efficient reduction of overall cap-dependent gene expression levels and therefore also the proliferation of the transfected cells, while continued IL-2 expression was guaranteed by an IRES element enabling cap-independent gene expression in the presence of protease 2A. In summary, the present results provide a promising basis for the development of novel potent strategies in the treatment of colorectal cancer.