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Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes.
A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells.
The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.
In a variety of established tumour cell lines, but also in primary mammary epithelial cells metalloprotease-dependent transactivation of the EGFR, and EGFR characteristic downstream signalling events were observed in response to stimulation with physiological concentrations of GPCR agonists such as the mitogens LPA and S1P as well as therapeutically relevant concentrations of cannabinoids. Moreover, this study reveals ADAM17 and HB-EGF as the main effectors of this mechanism in most of the cancer cell lines investigated. However, depending on the cellular context and GPCR agonist, various different members of the ADAM family are selectively recruited for specific ectodomain shedding of proAR and/or proHB-EGF and subsequent EGFR activation. Furthermore, biological responses induced by LPA or S1P such as migration in breast cancer and HNSCC cells, depend on ADAM17 and proHB-EGF/proAR function, respectively, suggesting that highly abundant GPCR ligands may play a role in tumour development and progression. Moreover, EGFR signal transactivation could be identified as the mechanistic link between cannabinoid receptors and the activation of mitogen activated protein kinases (MAPK) ERK1/2 as well as pro-survival Akt/PKB signalling. Depending on the cellular context, cannabinoid-induced signal cross-communication was mediated by shedding of proAmphiregulin and/or proHB-EGF by ADAM17. Most importantly, our data show that concentrations of THC comparable to those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby may contribute to cancer progression in patients.
In this thesis, the development of a phylogenetic DNA microarray, the analysis of several gene expression microarray datasets and new approaches for improved data analysis and interpretation are described. In the first publication, the development and analysis of a phylogenetic microarray is presented. I could show that species detection with phylogenetic DNA microarrays can be significantly improved when the microarray data is analyzed with a linear regression modeling approach. Standard methods have so far relied on pure signal intensities of the array spots and a simple cutoff criterion was applied to call a species present or absent. This procedure is not applicable to very closely related species with high sequence similarity because cross-hybridization of non-target DNA renders species detection impossible based on signal intensities alone. By modeling hybridization and cross-hybridization with linear regression, as I have presented in this thesis, even species with a sequence similarity of 97% in the marker gene can be detected and distinguished from related species. Another advantage of the modeling approach over existing methods is that the model also performs well on mixtures of different species. In principle, also quantitative predictions can be made. To make better use of the large amounts of microarray data stored in public databases, meta-analysis approaches need to be developed. In the second publication, an explorative meta-analysis exemplified on Arabidopsis thaliana gene expression datasets is presented. Integrating datasets studying effects such as the influence of plant hormones, pathogens and different mutations on gene expression levels, clusters of similarly treated datasets could be found. From the clusters of pathogen-treated and indole-3-acetic acid (IAA) treated datasets, representative genes were selected which pointed to functions which had been associated with pathogen attack or IAA effects previously. Additionally, hypotheses about the functions of so far uncharacterized genes could be set up. Thus, this kind of meta-analysis could be used to propose gene functions and their regulation under different conditions. In this work, also primary data analysis of Arabidopsis thaliana datasets is presented. In the third publication, an experiment which was conducted to find out if microwave irradiation has an effect on the gene expression of a plant cell culture is described. During the first steps, the data analysis was carried out blinded and exploratory analysis methods were applied to find out if the irradiation had an effect on gene expression of plant cells. Small but statistically significant changes in a few genes were found and could be experimentally confirmed. From the functions of the regulated genes and a meta-analysis with publicly available microarray data, it could be suspected that the plant cell culture somehow perceived the irradiation as energy, similar to perceiving light rays. The fourth publication describes the functional analysis of another Arabidopsis thaliana gene expression dataset. The gene expression data of the plant tumor dataset pointed to a switch from a mainly aerobic, auxotrophic to an anaerobic and heterotrophic metabolism in the plant tumor. Genes involved in photosynthesis were found to be repressed in tumors; genes of amino acid and lipid metabolism, cell wall and solute transporters were regulated in a way that sustains tumor growth and development. Furthermore, in the fifth publication, GEPAT (Genome Expression Pathway Analysis Tool), a tool for the analysis and integration of microarray data with other data types, is described. It consists of a web application and database which allows comfortable data upload and data analysis. In later chapters of this thesis (publication 6 and publication 7), GEPAT is used to analyze human microarray datasets and to integrate results from gene expression analysis with other datatypes. Gene expression and comparative genomic hybridization data from 71 Mantle Cell Lymphoma (MCL) patients was analyzed and allowed proposing a seven gene predictor which facilitates survival predictions for patients compared to existing predictors. In this study, it was shown that CGH data can be used for survival predictions. For the dataset of Diffuse Large B-cell lymphoma (DLBCL) patients, an improved survival predictor could be found based on the gene expression data. From the genes differentially expressed between long and short surviving MCL patients as well as for regulated genes of DLBCL patients, interaction networks could be set up. They point to differences in regulation for cell cycle and proliferation genes between patients with good and bad prognosis.
Bispecific T cell engager (BiTE) display a novel design among the class of bispecific antibodies and hold great promise to fight diverse cancers. BiTE molecules consist of two different binding entities derived from two human IgG antibodies connected by a short peptide linker. Their binding arms are directed against the CD3e chain of the T cell receptor on T cells and against an antigen that is specific for (e.g., CD19 for lymphoma in MT103) or over-expressed on (e.g., EpCAM for epithelial cancer in MT110) tumor cells. Without requirement for pre- or co-stimulation, BiTE molecules efficiently redirect CD3+ T cells towards tumor cells expressing the relevant target antigen. Only a BiTE molecule simultaneously bound to both tumor cell and T cell activates the T cell to exert its cytolytic function resulting in tumor cell death. In T cells stimulated with both BiTE and target cells, elevated levels of caspase activation and increased expression of cytotoxic and signaling proteins are observed. These include cytolytic proteins granzyme B and perforin, activation markers CD69 and CD25 and adhesion molecules CD2 and LFA-1. Activated T cells secrete the usual mix of cytokines, among them pro-inflammatory cytokines IFN-g and TNF-a. The membrane of tumor cells expressing the relevant target antigen is perforated during the attack of BiTE-stimulated effector cells as can be concluded from adenylate kinase release from the cytosol of tumor cells. Ca2+-chelator EGTA completely blocked BiTE-mediated activation of caspases and tumor cell lysis. As perforin is strictly Ca2+-dependent, a major role for this pore-forming protein is assumed for the elimination of tumor cells via BiTE-stimulated T cells. Granzyme B and caspases are main players in BiTE-mediated elimination of tumor cells. Inhibitors of granzyme B or caspases reduce or block, respectively the activation of caspases. However, other signals of apoptosis (cleavage of PARP and fragmentation of DNA) were only reduced by granzyme B inhibitor or caspase inhibitor. Most interestingly, the lytic capacity of BiTE molecules was not impaired by granzyme B inhibitor or caspase inhibitor. It seems that there is no requirement for granzyme B and caspases to be present simultaneously. Instead the data presented provide evidence that they can be replaced one at a time by related proteins. Pre-incubation of effector cells with the glucocorticoids dexamethasone or methylprednisolone resulted in markedly decreased secretion of cytokines by T cells yet only a small reduction in the expression of activation markers and adhesion molecules on T cells and specific lysis of tumor cells upon BiTE stimulation. Soluble factors secreted in an undirected manner by BiTE-stimulated T cells do not mediate tumor cell death by themselves. Bystander cells negative for the antigen that is recognized by the BiTE molecule will not be compromised by BiTE activity. The cytokine TGF-b reduced proliferation as well as granzyme B and perforin expression of BiTE-stimulated T cells. Redirected lysis by BiTE-activated T cells was also decreased under the influence of TGF-b, however lysis was still performed at a reasonable rate (72 % of target cells). TGF-b does not exert a deleterious effect on lytic potential of BiTE-stimulated T cells. The minimal anticipated biological effect level for the BiTE MT110 was determined for the entry of MT110 into phase I clinical studies. Experiments analyzing redirected lysis of tumor cells, expression of activation marker CD25 and cytokine release by T cells revealed a MABEL value of 50 pg/ml for MT110.
Development of novel Listeria monocytogenes strains as therapeutic agents for targeted tumor therapy
(2010)
Despite marked progress in development and improvement of cancer therapies the rate of cancer related death remained stable over the last years. Especially in treating metastases alternative approaches supporting current therapies are required. Bacterial and viral vectors have been advanced from crude tools into highly sophisticated therapeutic agents detecting and treating neoplastic leasions. They might be potent enough to fill in this therapeutic demand. In this thesis Listeria monocytogenes was investigated as carrier for targeted bacterial cancer therapy. One part of the study focussed on modification of a functional bacterial mRNA delivery system. Genomic integration of T7 RNA polymerase driving mRNA production allowed reduction to an one-plasmid-system and thereby partially relieved the growth retardation exerted by mRNA delivery. Importantly the integration allowed metabolic attenuation of the mRNA delivery mutant potentially enabling in vivo applications. Further expansion of the bacterial RNA delivery system for transfer of shRNAs was examined. Bacterial mutants producing high amounts of RNA containing shRNA sequences were constructed, however a functional proof of gene silencing on delivery in eukaryotic cell lines was not achieved. The second part of this thesis focussed on increasing tumor colonization by Listeria monocytogenes in vivo. Coating bacteria with antibodies against receptors overexpressed on distinct tumor cell lines enabled specific bacterial internalization into these cells in vitro. Optimization of the bacterial antibody coating process resulted in an up to 104-fold increase of intracellular bacteria. Combination of this antibody-mediated targeting with the delivery of prodrug-converting enzymes showed a cytotoxic effect in cell lines treated with the corresponding prodrug. Since incubation in murine serum completely abrogated antibodymediated bacterial internalization the antibodies were covalently linked to the bacteria for application in xenografted tumor mice. Bacteria coated and crosslinked in this manner showed enhanced tumor targeting in a murine tumor model demonstrating antibodymediated bacterial tumor targeting in vivo. Independent of antibody-mediated tumor targeting the intrinsic tumor colonization of different Listeria monocytogenes mutants was examined. Listeria monocytogenes ΔaroA ΔinlGHE colonized murine melanoma xenografts highly efficient, reaching up to 108 CFU per gram of tumor mass 7 days post infection. Taken together the presented data shows highly promising aspects for potential bacterial application in future tumor therapies. Combination of the delivery systems with antibodymediated- and intrinsic bacterial tumor targeting might open novel dimensions utilizing Listeria monocytogenes as therapeutic vector in targeted tumor therapy.
Ungeachtet der enormen Entwicklung in Krebsdiagnostik und -Therapie in den letzten Jahren, sind vollständige Heilungsaussichten weiterhin gering und die aktuellen Behandlungsmethoden oftmals mit schwerwiegenden Nebeneffekten verbunden. Aufgrund dessen sind alternative Behandlungsmethoden unbedingt erforderlich und führten zu einer zunehmenden Bedeutung des Vaccinia-Virus als onkolytisches Virus in der Krebstherapie. In der vorliegenden Arbeit wurden zwei mögliche Therapieansätze zur Verstärkung der onkolytischen Effekte in humanen Tumormodellen untersucht. Die Kombination einer gene-directed enzyme prodrug Therapie (GDEPT) mit dem onkolytischen Vaccinia-Virus GLV 1h68 sollte zur Selektivitätssteigerung eines ß-Galaktosidase-aktivierbaren, cytotoxisch-aktiven Drugs führen. Darüber hinaus diente das für MCP-1 codierende Vaccinia-Virus GLV-1h80, zielend auf eine Cytokin-vermittelten Immuntherapie, als Vektor zur spezifischen Beeinflussung des intratumoralen Chemokin-Netzwerks. Im Zuge der GDEPT wurde in dieser Arbeit ein, durch enzymatische Deglykosylierug aktivierbares Prodrug, basierend auf dem cytotoxischem Antibiotikum Duocarmycin SA verwendet. Durch eine Infektion mit GLV-1h68 und einer resultierenden Expression des aktivierenden Enzyms ß-Galaktosidase, sollte eine Umwandlung des Prodrugs in ein cytotoxisches Drug erfolgen. In vitro Infektionsstudien zeigten ein nahezu identisches Replikationsverhalten des Vaccinia-Virus GLV-1h68 und des als Kontrollvirus dienenden rVACV GLV-1h43 in humanen GI-101A-Brustkrebszellen. Die Expression der beiden Reporter-Gene Ruc-GFP sowie ß-Galaktosidase konnten auf Protein-Ebene und mittels RT-PCR nach Infektion mit GLV-1h68 nachgewiesen werden. GLV-1h43-Infektion von GI-101A-Zellen führte zu GFP-Expression, jedoch nicht zur Expression des Enzyms ß Galaktosidase. Untersuchung der Enzym-Aktivität in Zelllysaten und Zellkultur-Überständen zeigten nach Infektion mit GLV 1h68 steigende Menge zellulär assoziierter und freier ß-Galaktosidase. Des Weiteren wurde durch Koinkubation von GI-101A-Zellen mit Virus-freien, ß Galaktosidase-haltigen Zelllysaten bzw. –überständen und Prodrug eine Aktivierung des Prodrugs durch das Virus codierte Enzym nachgewiesen. Diese Koinkubation führte zur Abtötung der Zellen. Nach Inkubation mit Proben mock- oder GLV 1h43-infizierter Zellen konnte keiner Veränderung der Proliferationsrate von GI-101A-Zellen gefunden werden. Kombinierte Behandlung von GI 101A-Zellen mit Viren des Stammes GLV 1h68 und Prodrug führte zu starken Synergieeffekten bei der Abtötung der Zellen und wies einen Bystander Effekt der Kombinationstherapie nach. Dieser konnte in 4 weiteren humanen und 2 Hunde-Brustkrebszellen bestätigt werden. Der erzielte Bystander-Effekt zeigt, dass es nach Virus-induzierter ß-Galaktosidase-Expression in GLV 1h68-infizierten Zellen zu einer enzymatischen Spaltung des Prodrugs in das cytotoxische seco-Analogon des Antibiotikums Duocarmycin SA kommt. Durch die Membrangängigkeit des Drugs konnte auch in angrenzenden uninfizierten Zellen eine Wirkung erzielt werden. Anhand von Expressionsanalysen an Apoptose-assoziierten Proteinen, wie PARP und Caspasen, wurde eine Wirkung des Prodrugs über den intrinsischen Apoptose-Signalweg nachgewiesen. In athymischen Nude-Mäusen durchgeführte Replikationsanalysen und X-Gal-Färbungen GLV 1h68 infizierter Tumore nach Prodrug-Behandlung zeigten, dass GLV-1h68 ungeachtet der simultanen Behandlung mit Prodrug im Tumorgewebe repliziert und es nicht zur Anreicherung lacZ-negativer Virusmutanten kommt. Es konnten, durch Prodrug-Behandlung und einer simultanen Expression aktiver ß Galaktosidase, starke synergistische Effekte und eine signifikante Steigerung der Tumorregression erzielt werden. Da die Kombinationstherapie zu keinerlei Unterschieden in Gewicht und Gesundheitszustand behandelter Versuchstiere führte, konnte eine systemische Toxizität außerhalb des Tumorgewebes ausgeschlossen werden. Verschiedene Zelllinien weisen Unterschiede in ihrer Sensitivität gegenüber der onkolytischen Aktivität von Vaccinia-Virus GLV-1h68 auf. Während einige Zelllinien trotz Virus-Behandlung unverändertes Proliferationsverhalten zeigen (non- oder poor-responder), führt diese Behandlung in anderen Zelllinien zu einer vollständigen Tumorregression (responder). In Anbetracht dieser Unterschiede wurden in dieser Arbeit die Effekte einer induzierten Expression des murinen Chemokins MCP-1 in GI-101A-Tumoren (responder) und HT29-CBG-Tumoren (poor-responder) untersucht. MCP-1 zeichnet sich durch seine chemotaktischen Eigenschaften gegenüber mononukleärer Zellen aus und führt zu pleiotropen Tumor-Effekten. Replikationsstudien am Virus GLV-1h80 und des als Kontrollvirus dienenden rVACV GLV-1h68 zeigten, dass aus der Expression des Fremd-Gens mcp-1 sowohl in vitro als auch in vivo keinerlei negativen Effekte auf das Replikationsverhalten in humanen GI-101A- und HT29-CBG-Zellen resultieren. Durch Real-time Monitoring der GFP-Expression im Tumorgewebe lebender Tiere konnte zunächst eine mit dem Infektionsverlauf zunehmende Signalstärke beobachtet werden, welche dann 42 dpi an Intensität verlor. Toxizität und schädliche Nebeneffekte durch Infektion mit den beiden rVACV konnten anhand der viralen Titer in den Organen der Maus ausgeschlossen werden. Die Titer wiesen auf eine ausschließlich auf das Tumorgewebe begrenzte Replikation der Viren nach Injektion in Tumor-tragende Tiere hin. Die Expression des Chemokins MCP-1 wurde sowohl auf transkriptioneller als auch auf translationeller Ebene in GLV-1h80-inifzierten Zellen und im Tumorgewebe GLV 1h80-injizierter Mäuse nachgewiesen. Nach Infektion mit GLV-1h80 konnte eine mit dem Infektionsverlauf zunehmende MCP-1-Expression gezeigt werden. Dabei wurde zudem deutlich, dass nicht nur eine GLV-1h80-Infektion in vivo zu einer Zunahme der intratumoralen MCP-1-Expression führte, sondern eine Vaccinia-Virus-Infektion allein einen Anstieg des Chemokins zu bewirken vermag. Eine Quantifizierung durch ELISA machte Konzentrationsunterschiede von MCP-1 zwischen den Tumormodellen GI-101A und HT29-CBG deutlich. Sowohl in vitro als auch in vivo führte ein GLV-1h80-Infektion zu deutlich niedrigeren Konzentrationen im HT29-CBG-Kolon-Adenokarzinommodell. Ein Nachweis murinen MCP-1 in Blutseren Tumor-tragender Tiere zeigte eine für therapeutische Effekte erwünschte systemische Freisetzung des intratumoral durch die Infektion mit GLV-1h80 gebildeten Chemokins MCP-1. Durch immunhistologische Untersuchungen GLV-1h80-infizierter Zellen und Tumoren konnte diese, mit dem Infektionsverlauf zunehmende MCP-1-Expression bestätigt werden. Die funktionelle Aktivität des rekombinanten Proteins wurde anhand TNF-α-spezifischer ELISA-Analysen überprüft. Dabei zeigte sich eine erhöhte Expression dieses proinflammatorischen Cytokins in GI-101A-Tumoren nach Infektion mit GLV-1h80. Dagegen konnte keine Steigerung der Expression im HT29-CBG-Tumorgewebe nachgewiesen werden. Ein Nachweis des durch proinflammatorische Immunzellen exprimierten Oberlflächenproteins CD14 zeigte ebenfalls einen Anstieg nach Infektion mit GLV-1h80. Auch diese veränderte Expression blieb im poor-Responder-Modell HT29-CBG aus. Die steigende intratumorale Expression der beiden Proteine in GI-101A-Tumoren nach GLV 1h80-Infektion lässt auf eine Zunahme pro-inflammatorischer Immunzellen, basierend auf einer Virus-induzierten MCP-1-Expression schließen. Ein Monitoring der Tumorprogression nach Implantation von GI 101A-Zellen und Injektion der rVACV GLV-1h80 und GLV-1h68 bzw. einer PBS-Injektion führte nach einer anfänglichen Zunahme des Tumorwachstums schließlich bei beiden Viren zu einer Tumorregression. Jedoch konnte durch die GLV-1h80-vermittelte MCP-1-Expression eine Verstärkung der onkolytischen Effekte erzielt werden, welche sich durch eine signifikante Abnahme des Tumorvolumens zeigte. Im HT29-CBG-Modell führten die therapeutischen Effekte durch rVACV GLV-1h80 zwar zu keiner Regression des Tumors, jedoch zeigte sich auch in diesem humanen Tumormodell eine Verstärkung der onkolytischen Effekte nach GLV-1h80-Infektion im Vergleich zu einer GLV 1h68-Behandlung. Durch die GLV-1h80-induzierte Expression des Chemokins MCP-1 konnte somit eine Hemmung des Tumorwachstums auch im poor-Responder-Modell HT29-CBG erzielt werden. Sowohl die Verwendung eines ß-Galaktosidase-aktivierbaren Prodrugs im Zuge einer GDEPT, als auch die Beeinflussung des intratumoralen Chemokin-Netzwerks durch Expression des Chemokins MCP-1 führten in dieser Arbeit zu positiven Synergismus-Effekten in der onkolytischen Virustherapie. Durch künftige Konstruktion eines rVACV, welches sowohl die Expression des Chemokins MCP-1, als auch des prodrug-aktivierenden Enzyms ß-Galaktosidase im Tumorgewebe induziert, könnte in Kombination mit einer Prodrug-Behandlung eine zusätzliche Verstärkung der Effekte erzielt und möglicherweise eine erfolgreiche Virustherapie in bisher schwach ansprechenden poor- bzw. non-Responder-Modellen ermöglicht werden.
Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.