Cell culture models and novel gene therapeutic strategies for colorectal cancer

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

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency

Surface expression of mutant prion proteins in prion protein knockout cell line HpL3–4.

<p>Flow cytometry analysis confirmed surface expression of mutant and wild-type mPrP. The number of PrP positive cells as well as the mean fluorescent intensity of positive cells is given.</p

<i>In vitro</i> conversion of isoleucine mutants.

<p>A) <i>In vitro</i> fibrillization of wild-type (▪), V175I (⋄), T187I (▴), and V209I (▿) mPrPs followed by fluorescence intensity of thioflavin T. A representative of four experiments is shown. B) Presence of fibrils was confirmed by TEM. Bar represents 250 nm. C) Final thioflavin T fluorescence intensity of T187I is significantly higher than in the samples of other mPrPs (p<0.0005). The differences among wild-type, V175I and V209I are not significant.</p

Thermal stability of mutant prion proteins.

<p>Thermal stability of mutant prion proteins.</p

AFM reveals the presence of thin fibrils in addition to mature fibrils in conversion reactions of T187I.

<p>Conversion reactions of wild-type (WT), V175I, V209I (upper row) and T187I (middle, bottom row) were observed under the atomic force microscope. In all samples fibrils with approximate height around 7 nm were observed. Cross-section of such fibril from sample T187I is shown (bottom row right, full line). In the T187I sample in addition to such fibrils, fibrils with less than 1 nm in height were also present (middle row). Image 187B shows a close-up of selected part in image 187A. Cross-section of thin fibril is shown in the bottom row (bottom row right, broken line). Bar represents 500 nm. Arrows in images 187B and 187C indicate positions where cross-sections were taken. Images have not been corrected for the width and shape of the AFM tip.</p

Globular Domain of the Prion Protein Needs to Be Unlocked by Domain Swapping to Support Prion Protein Conversion*

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the β-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention