TY - THES A1 - Hochheimer, Vanessa Christine T1 - Of cells and enzymes: How dermal fibroblasts can impact pain in Fabry Disease and Why looking at the 3D-structure of α-Galactosidase A may be worthwhile for clinical management of Fabry patients T1 - Über Zellen und Enzyme: Wie Hautfibroblasten Schmerz bei Morbus Fabry beeinflussen können und Warum sich die Betrachtung der 3D-Struktur der α-Galaktosidase A für die klinische Versorgung von Fabry Patienten lohnt N2 - Fabry Disease (FD) is a genetic lysosomal storage disorder based on mutations in the gene encoding α-Galactosidase A (α-GalA) leading to accumulation of globotriaosylceramide (Gb3). Missense mutations induce an amino acid exchange (AAE) in the α-GalA. Pain is a predominant symptom in FD and the pathophysiology is unclear. Skin punch biopsies were obtained from 40 adult FD patients and ten healthy controls and dermal fibroblast cultures were generated for cell culture experiments to investigate Gb3 load, gene and protein expression patterns and ion channel activity. The 3D-structure of α-GalA was downloaded into Pymol Graphics System and the AAE was depicted and located in order to investigate the correlation between the AAE location type in the α-GalA and the clinical FD phenotype. FD dermal fibroblasts showed high Gb3 load depending on treatment interval and expressed Kca1.1 channels. Activity was reduced in FD cells at baseline, but increased over-proportionately upon Gb3-cleavage by enzyme replacement therapy. Gene and protein expression of Kca1.1 was increased in FD cells. FD dermal fibroblasts showed higher gene expression of Notch1 and several cytokines. Further, it was shown that three different AAE location types can be differentiated: mutations in the active site (‘active site’), those buried in the core of α-GalA (‘buried’) and those at another location, mostly on the protein surface (‘other’). FD patients carrying active site or buried mutations showed a severe clinical phenotype with multi-organ manifestation and early disease onset. Patients with other mutations were less severely affected with oligo-organ manifestation sparing the nervous system and later disease onset. These results show that dermal fibroblasts may be involved in FD-associated pain and that stratification of FD patients carrying missense mutations by AAE location type may be an advantageous parameter that can help in the management of FD patients. N2 - M. Fabry ist eine genetisch bedingte lysosomale Speichererkrankung aufgrund von Mutationen im Gen der α-Galaktosidase A (α-GalA) mit Ablagerung von Globotriaosylceramid (Gb3). Missense-Mutationen führen zum Austausch einer Aminosäure (ASA) in der α-GalA. Schmerz ist ein häufiges Symptom, dessen Pathophysiologie unklar ist. Bei 40 Patient*innen mit M. Fabry sowie zehn Kontrollprobanden wurde eine Hautstanzbiopsie durchgeführt und zur Kultivierung von dermalen Fibroblasten verwendet, um den Gb3-Gehalt, Gen- und Proteinexpressionsmuster und Ionenkanalaktivität zu untersuchen. Zudem wurde die 3D-Struktur der α-GalA in Pymol Graphics System geladen und der Ort des ASA dargestellt, um den Zusammenhang zwischen dem ASA in der α-GalA und dem klinischen Phänotypen zu untersuchen. Es zeigte sich, dass Fabry-Fibroblasten erhöhte Gb3-Ablagerungen beinhalten, abhängig von der Zeit zwischen Enzymersatztherapie (ERT) und Biospieentnahme, sowie Kca1.1 Kanäle, deren Funktion in Patientenzellen unter Normalbedingungen reduziert war, der jedoch eine überproportionale Aktivitätszunahme nach Gb3-Abbau mittels ERT zeigte. Die Gen- und Proteinexpression des Kanals war in Fabry-Zellen erhöht. Fabry-Zellen wiesen eine erhöhte Genexpression von Notch1 sowie mehrerer Zytokine auf. Zudem zeigte sich, dass es drei verschiedene ASA Gruppen gab: Mutationen im aktiven Zentrum („active site“), in der Tiefe des Enzyms („buried“) und an anderen Orten, meist an der Oberfläche („other“). Patient*innen mit active site- oder buried-Mutationen zeigten einen schweren Phänotypen mit Multi-Organbeteiligung und frühem Krankheitsbeginn. Patient*innen mit other-Mutationen zeigten eine Beteiligung von wenigen Organen ohne Nervensystem und späteren Krankheitsbeginn. Es zeigt sich, dass dermale Fibroblasten zu Schmerz bei M. Fabry beitragen können und die Einteilung von Patient*innen mit M. Fabry-Missense Mutationen anhand des Ortes des ASA ein lohnender Parameter bei der Betreuung der Patient*innen sein kann. KW - Fabry-Krankheit KW - Hautzelle KW - Schmerz KW - Fabry KW - Fibroblasten KW - 3D-Struktur KW - Fabry disease KW - Pain KW - Fibroblasts KW - 3D structure Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-296607 ER - TY - JOUR A1 - Nietzer, Sarah A1 - Baur, Florentin A1 - Sieber, Stefan A1 - Hansmann, Jan A1 - Schwarz, Thomas A1 - Stoffer, Carolin A1 - Häfner, Heide A1 - Gasser, Martin A1 - Waaga-Gasser, Ana Maria A1 - Walles, Heike A1 - Dandekar, Gudrun T1 - Mimicking metastases including tumor stroma: a new technique to generate a three-dimensional colorectal cancer model based on a biological decellularized intestinal scaffold JF - Tissue Engineering Part C-Methods N2 - Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of beta-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions. KW - Multicenter randomized-trial KW - Carcinoma cells KW - Tissue KW - Fluorouracil KW - Matrix KW - 1st-line treatment KW - Beta-catenin KW - Invasion KW - 5-Fluorouracil KW - Fibroblasts Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188202 VL - 22 IS - 7 ER - TY - JOUR A1 - von Bueren, André O. A1 - Oehler, Christoph A1 - Shalaby, Tarek A1 - von Hoff, Katja A1 - Pruschy, Martin A1 - Seifert, Burkhardt A1 - Gerber, Nicolas U. A1 - Warmuth-Metz, Monika A1 - Stearns, Duncan A1 - Eberhart, Charles G. A1 - Kortmann, Rolf D. A1 - Rutkowski, Stefan A1 - Grotzer, Michael A. T1 - c-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients JF - BMC Cancer N2 - Background: To study whether and how c-MYC expression determines response to radio-and chemotherapy in childhood medulloblastoma (MB). Methods: We used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio-and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio-and chemotherapy as examined by neuroradiological imaging. Results: In DAOY -and to a lesser extent in UW228 -cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation-and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio-(14 responders (CR, PR) vs. 5 non-responders (SD, PD)) or chemotherapy (23 CR/PR vs. 20 SD/PD) was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively). Conclusions: c-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment. KW - Induced apoptosis KW - Down-regulation KW - Childhood medulloblastoma KW - Melanoma-cells KW - Cisplatin KW - Lines KW - Gene KW - Radiotherapy KW - Fibroblasts KW - Activation Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134185 VL - 11 IS - 74 ER -