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 ß-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.

Proteomic analysis reveals differences in protein expression in spheroid versus monolayer cultures of low-passage colon carcinoma cells.

Spheroid cultures of cancer cells may better reflect characteristics of tumors than traditional monolayer cultures. Furthermore, low-passage cancer cell lines recapitulate the properties of the original tumor cells more closely than commonly used standard cell lines that experience artificial selection processes and mutations over years of passaging. Here we established spheroid cultures of the low-passage colon cancer cell line COGA-5 and stable COGA-12 aggregates with local areas of compaction. The proteomes of both three-dimensional cultures were analyzed versus their corresponding two-dimensional cultures. 2-D gel electrophoresis followed by peptide mass fingerprinting identified three differently expressed proteins in COGA-5 spheroids (acidic calponin, hydroxyprostaglandin dehydrogenase, and lamin A/C) and two in COGA-12 partly compact aggregates (two isoelectric variants of the acidic ribosomal protein P0) compared to the respective monolayer cultures. The lamin A/C spot showed a lower molecular weight in the 2-D gel (30 kDa) than expected for full-length lamin. Further Western blot analysis and immunocytochemistry identified the lamin protein as a caspase-6-cleavage product in apoptotic cells of the spheroid. Similar caspase-dependent lamin cleavage was observed in monolayer cultures after serum withdrawal and further increased under hypoxic conditions, suggesting cleaved lamin as an indicator for apoptotic stress. In conclusion, proteome analysis of multicellular spheroids versus monolayers cultures identifies differential protein expression relevant to tumor cell proliferation, survival, and chemoresistance and thus may reveal novel targets for cancer therapy.

Transcriptionally targeted nonviral gene transfer using a beta-catenin/TCF-dependent promoter in a series of different human low passage colon cancer cells.

Nonviral transfections of six low passage human colon cancer cell lines using the artificial beta-catenin/TCF-dependent promoter CTP4 demonstrated a high promoter activity which was 1000- to 70000-fold higher than in HeLa control cells. Luciferase gene expression levels obtained with CTP4 in epithelial-like tumor cell cultures were only slightly lower than with the strong viral CMV promoter/enhancer, whereas in less differentiated tumor cultures CTP4 expression levels exceeded the CMV expression levels up to 28-fold. Three cell lines representing different morphology typical of the original tumors, more differentiated epithelial-like (COGA-5), piled-up (COGA-12), and poorly differentiated rounded-up (COGA-3), were selected for further investigation. Gene transfer was optimized using lipopolyplex formulation of cationic lipid DOSPER and polycation PEI25br. Lipopolyplexes enabled up to 1300-fold or 400-fold higher luciferase expression compared to the corresponding lipoplexes or polyplexes, respectively. Lipopolyfection of an interleukin-2 (IL-2) gene expression construct driven by the CTP4 promoter resulted in very high levels of up to 95 ng of secreted IL-2 per 105 cells and 24 h. The lipopolyplexes were also able to transfect multicellular spheroids that mimic the three-dimensional structure of real tumors.

Optimized lipopolyplex formulations for gene transfer to human colon carcinoma cells under in vitro conditions.

BACKGROUND: Polycation (PC, polyplex), cationic lipid (CL, lipoplex), and a combination of PC/CL (lipopolyplex) formulations were investigated for gene transfer to slow-proliferating human colon carcinoma cell lines (COGA). METHODS: The luciferase reporter gene was complexed with either PC, CL, or PC/CL. PCs included linear (PEI22lin, 22 kDa) and branched polyethylenimine (PEI2k, 2 kDa; PEI25br, 25 kDa) and poly-L-lysine (PLL18 with 18 lysine monomers). CLs included DOCSPER, DOSPER and DOTAP. Lipopolyplexes were formed by either sequentially first mixing DNA with PC or CL, followed by addition of CL or PC, respectively, or simultaneously with both PC and CL. Particle size and zeta-potential were determined and gene transfer and cytotoxicity were quantified on COGA-3, -5, -12, HeLa and Sw480 cells. RESULTS: The highest gene transfer was achieved when DNA was first complexed with PC followed by CL. At low ionic strength, particles were small (50-130 nm) with a zeta-potential of +20-40 mV. At physiological ionic strength, only lipoplexes of DOCSPER or DOSPER and their respective lipopolyplexes with PEI25br were stable to aggregation (140-220 nm). Lipopolyplexes of PEI25br were between 5- to 400-fold more efficient compared to the corresponding lipoplexes or polyplexes in all cases. Chloroquine did not significantly affect lipopolyplex-mediated gene transfer. CONCLUSIONS: Lipopolyplex formulations of PEI25br in combination with multivalent CLs (DOCSPER, DOSPER) are promising tools for in vitro and potentially also in vivo gene transfer to colorectal cancer cells.