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SUMMARY GABP is a heterodymeric member of Ets-family transcription factors. It consists of two subunits – GABPa which contains DNA binding domain and GABPb, which provides transcriptional activation domain and nuclear localization signal. GABPa/b complex is essential for transcriptional activation of multiple lineage-restricted and housekeeping genes, several viral genes, and in some cases might function as transcriptional repressor. Large variety of data indicates involvement of GABP in the complex regulation of cell growth, specified by quiescence, stimulation/proliferation, apoptosis and senescence. Expression level of GABPa subunit is rapidly increased when resting cells enter S-phase, and GABPa/b complex is critical to promote the continuity of the cell cycle. Conditional inactivation of GABPa expression in mouse embryonic fibroblasts results in a complete block of proliferation and acquisition of senescence-like phenotype. However, the influence of GABP on the other cell growth determinant – the apoptosis – remains largely obscure. Therefore we aimed to investigate the influence of GABPa/b expression level on the cell growth in vitro. Using siRNA approach we achieved efficient but only transient down-regulation of GABPa expression which precluded further cell growth studies. Persistent increase of the expression of GABPb subunit only resulted in a positive effect on the cell growth speed. Simultaneous conditional overexpression of both GABPa and GABPb subunits though, strongly reduced the growth of the affected cell cultures in reversible and in expression level dependent manner. Interestingly, GABPa/b overexpressing cells did show neither cell cycle arrest nor massive induction of apoptosis. However, more detailed analyses revealed that dampened apoptotic processes were taking place in GABPa/b−overexpressing cells, starting with a prominent activation of caspase-12. Interestingly, activation of downstream effector caspases was rather suppressed explaining a weak increase of apoptotic cells in GABPa/b overexpressing cultures. This effect suggests that the activation of caspase-12 by elevated amounts of exogenous GABPa/b reflects the normal physiological mechanism of caspase-12 regulation. / ZUSAMMENFASSUNG GABP ist ein heterodimerisches Mitglied aus der Familie der Ets- Transkriptionsfaktoren. Es besteht aus zwei Untereinheiten – GABPa, welche die DNA-Bindedomäne enthält, sowie GABPb, welche sowohl die Transkriptions-Aktvierungsdomäne als auch das Kernimportsignal umfasst. GABPa/b ist für die Transkriptions-Aktivierung mehrerer differenzierungstypischer als auch sog. Housekeeping Gene, sowie einiger viraler Gene essentiell und kann, in einigen Fällen, auch als Transkriptionsrepressor fungieren. Eine Vielzahl von Daten deutet darauf hin, dass GABP in der komplexen Kontrolle des Wachstums von Zellen ein wichtige Rolle zukommt. Dies zeigt sich z. B. im Einfluss von GABP auf zelluläre Vorgänge wie der Stimulation/Proliferation, Apoptose und Seneszenz. So steigt z. B. der Spiegel der GABPa Untereinheit rapide an, nachdem ruhende Zellen die G0-Phase verlassen und in die S-Phase eintreten. Der aus beiden Untereinheiten gebildete Komplex ist dann für die Progression der Zellen durch den gesamten Zellzyklus von entscheidender Bedeutung. Die Unterdrückung der Expression der GABPa Untereinheit in embryonalen Mausfibroblasten hingegen führt zu einem vollkommenen Proliferations-Stopp dieser Zellen und induziert in diesen einen Seneszenz-artigen Phänotyp. Andererseits ist über den Einfluss von GABP auf andere wichtige das Zellwachstums beeinflussende Faktoren wie z. B. der Apoptose bislang noch recht wenig bekannt. Daher lag es im Fokus dieser vorliegenden Arbeit, den Einfluss der GABPa/b-Spiegels auf das Zellwachstum in vitro näher zu untersuchen. Mithilfe von siRNA-Ansätzen gelang uns die effiziente Herunterregulierung von GABPa. Diese war jedoch nur von vorübergehender Natur, so dass weitere Studien zum Zellwachstum nicht möglich waren. Die stabile Überexpression der GABPb Untereinheit führte dagegen nur zu einem Anstieg der Zellwachstumsgeschwindigkeit. Wurden jedoch sowohl beide Untereinheit gleichzeitig überexprimiert, so resultierte dies in einer deutlichen, Expressionsspiegel-abhängigen und reversiblen Wachstumshemmung der Zellen. Bemerkenswerterweise zeigte die GABPa/b-überexprimierende Zellpopulation weder einen erhöhten Anteil an G0-Phase noch war eine deutlich ausgeprägte Zunahme der Apoptose-Rate zu verzeichnen. In weiteren Experimenten konnte dennoch eine leichte Erhöhung der Apoptose-Rate in den überexprimierenden Zellen gezeigt werden, was sich durch die deutliche Aktivierung von Caspase-12 belegen ließ. Die Aktivierung von Effektor-Caspasen der Caspase-12 schien allerdings nicht zu erfolgen, was den nur schwach ausgeprägten Charakter der Apoptose zu erklären vermag. Diese Beobachtungen suggerieren, dass die Aktivierung der Caspase-12 durch erhöhte Mengen von exogenem GABPa/b den normalen physiologischen Mechanismus der Caspase-12 Regulation widerspiegelt.

Bei Melanomen handelt es sich um die gefährlichste Form von Hautkrebs mit der höchsten Mortalitätsrate. Deshalb sind Untersuchungen dieser Hautkrebsart von immenser Bedeutung. Es ist bekannt, dass der AP-1-Transkriptionsfaktorkomplex eine große Rolle für Melanomentstehung und -progression spielt. In der vorliegenden Arbeit wurde die Funktion der AP-1 Komponente FOSL1 in Melanomen untersucht. Hierbei konnte zunächst ermittelt werden, dass die FOSL1 Expression im humanen Melanom durch den MAPK-Signalweg vermittelt wird und von den Onkogenen BRAF und NRAS abhängig ist. Dies wird auch durch die Tatsache unterstützt, dass die Stabilität von FOSL1 durch MAPK reguliert wird. Des Weiteren konnte gezeigt werden, dass FOSL1 in vielen Melanomzellen die Proliferation verstärkt und auch an Migration beteiligt ist. Da diese Prozesse zur Krebsprogression beitragen, deutet dies darauf hin, dass FOSL1 bei der Melanomentwicklung eine wichtige Funktion besitzt. Weiterhin konnten SLUG, SNAI3, IL6 und MMP14 als FOSL1-Zielgene identifiziert werden, deren Regulierbarkeit durch FOSL1, jedoch abhängig von der jeweiligen Zelllinie war. Somit konnte mit dieser Arbeit gezeigt werden, dass FOSL1 nicht nur, wie zuvor für Brustkrebszellen beschrieben, an Migration beteiligt ist, sondern auch zur Proliferation humaner Melanome beiträgt. Zukünftige Arbeiten werden zeigen, ob die identifizierten Gene für die FOSL1-vermittelte Migration und Proliferation verantwortlich sind. / Melanoma is the most aggressive type of skin cancer with the highest mortality rate. Hence, the investigation of this type of cancer is of great importance. The AP-1 transcription factor complex is known to play a major role during initiation and progression of melanoma. This research was aimed at investigating the role of the AP-1 component FOSL1 in melanoma. Initial determinations showed that in human melanoma the FOSL1 expression is mediated by the MAPK-signaling pathway and dependent on the oncogenes BRAF and NRAS. This finding is supported by the fact that the stability of FOSL1 is regulated by MAPK. Furthermore, this research revealed that FOSL1 enhances proliferation in several cell lines and also contributed to migration. Since these processes contribute to cancer progression, FOSL1 even seems to play an important role in melanoma development. Moreover, SLUG, SNAI3, IL6 and MMP14 were identified to be target genes of FOSL1. However, the FOSL1-dependent regulation of these genes differed between the cell lines. Thus, this investigation provides evidence that FOSL1 does not only promote migration, which was described previously in breast cancer, but it also contributes to proliferation of human melanoma. Experiments in the future will unravel if the identified genes are responsible for the FOSL1-mediated migration and proliferation.

During embryonic development, the Hedgehog (Hh) signaling pathway plays an important role in the growth and patterning of numerous tissues and organs. In the developing retina, Hh signaling regulates the proliferation and differentiation of retinal progenitor cells (RPC) through mechanisms that are not completely understood. The principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3), which regulate the expression of target genes responsible for the effects of the Hh pathway on RPC. The network of genes targeted by this pathway in neural progenitor cells however, remains unknown. The objective of this thesis was to identify and characterize novel targets of Hh/Gli during retinal development. Using a computation approach, 390 genes were identified as having at least one conserved Gli binding motif within the vicinity of the coding sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5 retinal explants and that the induction of 25 of these were significantly different between the two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp, two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to determine a role for Sox8 during retinal development which could reflect compensation by the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient and required for Hh mediated induction in progenitor cell proliferation and cell fate determination. Therefore, in this thesis Hh target genes have been identified which could provide some insight into the mechanisms that are responsible for the cellular outcome of a response to the pathway.

Genome-wide association studies (GWAS) have identified up to 30 loci that associate with increased risk of coronary artery disease or myocardial infarction. Here, I tested the function of one locus that changed the amino acid sequence of a mitochondrial matrix protease called paraplegin (SPG7) that performs critical quality assurance functions. Loss-of-function mutations in this protease are associated with hereditary spastic paraplegia. Here, I show that this variant that changes an arginine to a glutamine at position 688 within the protease domain is a gain-of-function. Cells bearing this variant have increased mitochondrial fusion and number, produce higher levels of reactive oxygen species and have increased cellular proliferation. Importantly, when expressed in yeast, the Q688 variant of SPG7 rescues the growth arrest caused by a protease-deficient mutation in AFG3L2. My study identifies a novel functional variant of SPG7 and highlights the need to go beyond the GWAS paradigm.

Urinary bladder urothelial carcinoma is the most common cancer of the urinary tract. About 70% of the diagnosed tumors classified as Non-invasive tumor, which is usually multiple. Despite surgical removal and perioperative chemotherapy, tumor recurrence is not uncommon. However, the chance for such non-invasive tumors to advance to the muscle-invasive stage is relatively small and the 5-year survival rate approaches 95%. The rest 30% are classified as invasive tumors which usually pursue aggressive clinical course. In spite of radical cystectomy in conjunction with debilitating chemotherapy and/or radiotherapy, more than 50% of invasive tumors eventually spread to distant organs. The 5-year survival rate for patients with distant metastasis is only about 6%. The current challenge in the management of urinary bladder carcinoma is the lack of powerful prognostic marker and promising therapeutic agents. Accordingly, to identify novel biomarks to adjust therapeutic strategy is mandatory. The BCL6 proto-oncogene encodes a nuclear transcriptional repressor, it¡¦s inhibits DNA repair pathways and TP53. Perturbation of both these pathways may contribute to normal cell function by repressing DNA damage responses and permitting somatic hypermutation but , in the context of malignancy, this could lead to mutations promoting aggressive tumor. Several studies have demonstrated that BCL6 play a role in different cancer types, however, the function of BCL6 in bladder cancer is understood. Therefore, in this study, we will analyze the endogenous BCL6 mRNA and total/activated BCL6 protein in various bladder cancer cell lines, including BFTC905, and J82. Then we will knockdown of the BCL6 gene by shRNA interference and analyze how it implicates various cellular processes essential to cancerous states. And then we will be analyzed the affection of cell survival, migration and invasion. Conversely, Overexpression of BCL6 in bladder cancer cell lines will be assessed cell proliferation, migration and invasion. Finally, studying it¡¦s affection in vivo. We demonstrate that BCL6 is correlated with bladder cancer.

Hepatocellular carcinoma (HCC) or hepatoma is the fifth most common cancer and the third most common cause of cancer-related deaths worldwide. Early detection of HCC still remains challenge. Patients with late-stage HCCs have very limited therapeutic options and relatively poor prognosis. Therefore, recognition of useful tumor marker for the diagnosis of HCC may aid in the management with this lethal malignancy, and designing effective therapies. In our previous study, through in silico data mining of numerous available cDNA microarray databases, the CDC28 protein kinase regulatory subunit 1B (CKS1B) transcript was found to be frequently upregulated in HCCs and the gene is strongly associated in clinical aggressiveness. Moreover, gain of chromosome 1q copy is one of the most frequently detected alterations in HCC and 1q21 is the most frequent minimal amplifying region. Mammalian CKS1B gene, a member of the highly conserved cyclin kinase subunit 1 protein family, mapped to 1q21.2-q22, which interacts with cyclin-dependent kinases and frequently up-regulated in human HCC specimens, and this gene also has been reported to play oncogenic roles in other human cancers. Furthermore, this gene was identified as an essential cofactor of S-phase kinase-associated protein 2 (SKP2) in the SKP2-containing SKP1-Cullin1-F-box ubiquitin ligase complex¡Vmediated ubiquitination of the cell cycle inhibitor protein cyclin-dependent kinases 1A (p27kip1) that leads to the proteasomal degradation of p27kip1. In the present study, our results indicate that CKS1B overexpression may contribute to hepatocarcinogenesis by enhancing the cellular tumorigenic potential. In addition, CKS1B was correlated with the HCC-derived cells proliferation, and CKS1B knockdown significantly inhibited the tumorigenic potential of HCC-derived cells. In this study, we examined that CKS1B knockdown and overexpressed in HCC cells significantly changed the expression of genes known to participate in various cellular processes, including, apoptosis, cell cycle, proliferation, viability, invasion, and migration, implying that CKS1B may play considerable and diverse roles in hepatocarcinogenesis.

Hepatocyte growth factor (HGF) is a multifunctional mitogen, stimulating cell proliferation, motility, angiogenesis and morphogenesis via activating its receptor, c-Met tyrosine kinase. Overexpression of HGF and c-Met has been shown as a characteristic of cancer transformation and metastasis. Inhibition of HGF/c-Met signaling may abrogate the malignant and metastatic states of cancer cells and offer a useful therapeutic approach for treating cancers. Thus with the aim of creating inhibitors to HGF-cMet signaling, we constructed plasmids containing truncated N-terminus of HGF, NK1, NK2, NK3 and NK4, respectively, and transfected into MDA MB 435S cells by electroporation. After selection with antibiotics, stable transfectants were obtained. Proliferation assay showed that the truncated NKs significantly inhibited the growth of the cells. Moreover, wound healing assay showed that migration of the NK-transfected cells were also significantly inhibited in comparison with GFP-transfected and nontransfected cells. These results therefore suggest that truncated NKs may be good inhibitors to HGF/c-Met signaling in the proliferation and migration of human breast cancer cells in vitro. In the future, the in vivo animal model is needed to be carried out to further clarify the clinical values of truncated NKs for application to cancer therapy.