Browsing by Person "Graeve, Lutz"

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Aktivierung eines neuartigen Apoptose-Signalweges durch den Proteinkinaseinhibitor Staurosporin
(2009) Daubrawa, Merle; Graeve, Lutz
The protein kinase inhibitor staurosporine induces apoptosis via the activation of the intrinsic pathway. First staurosporine was described as a specific PKC inhibitor. Today it is known as a broad range kinase inhibitor and is used as a potent apoptosis inductor. However, the mechanism of the apoptotic effect remains elusive. Furthermore, staurosporine obviously exhibit the potential to eliminate chemotherapy resistant tumors by the induction of a novel intrinsic apoptotic signaling pathway. Different derivatives of staurosporine, e.g. UCN-01, PKC-412 or Enzastaurin are already tested in clinical trials phase I-II for cancer therapy. In the present work it could be shown that overexpression of Bcl-2 does not impede the caspase-dependent induction of apoptosis in J16- and JE6.1-Jurkat T-lymphocytes or in DT40 B-lymphocytes following staurosporine treatment . After generation of apaf-1 -/- DT40 cells it was demonstrated that staurosporine induces apoptosis despite the absence of Apaf-1 and therefore independently of the apoptosome. Together with the generated caspase-9 -/- DT40 cells, caspase-9 was identified as the central effector protein of both staurosporine-induced apoptotic pathways. The involvement of published and putative caspase-9 kinases could not be confirmed by the usage of specific inhibitors. Using phospho-mimicking and phospho-deficient caspase-9 variants, S183 could be identified as an essential phosphorylation site during staurosporine-induced apoptosis. In addition, after treatment with anticancer drugs apoptosome formation was blocked by an N-terminal tag of caspase-9. However, this tag could not prevent staurosporine-induced apoptosis. In further studies the potential role of cathepsines for this novel apoptosis signaling pathway could be analysed by their specific inhibition. In order to investigate the involvement of multiple kinases in this novel apoptotic signaling pathway, combination experiments with specific inhibitors of the respective kinases should be accomplished. Further investigations should clarify whether the influence of S183 on staurosporine-induced apoptosis is based on conformational alteration or on phosphorylation of caspase-9. The generation of additional caspase-9 variants including deltaCARD-caspase-9 or non-cleavable caspase-9 could lead to a deeper understanding of the role of caspase-9 for staurosporine-induced apoptosis. For this purpose caspase-9 -/- DT40 cells and cells reconstituted with different caspase-9 variants could be employed. The phosphorylation pattern of caspase-9 could be determined by mass spectrometric analysis. Xenograft or chorio allantois membrane models were used to investigate if the staurosporine derivative UCN-01 is also able to induce this novel apoptosis signaling pathway in vivo. The identification of both the mechanisms and the effector proteins of this staurosporine-induced apoptotic signaling pathway should provide the opportunity to develop novel agents for the elimination of chemotherapy-resistant tumors.
Charakterisierung von Interaktionspartnern des Kernrezeptors CAR ("Constitutive Androstane Receptor") mittels MALDI-TOF Massenspektrometrie
(2010) Melgar, Clint; Graeve, Lutz
The human nuclear receptor "constitutive androstane receptor" (CAR; NR1I3) plays a pivotal role in the induction of drug metabolism and transport by phenobarbital-type inducers. The hepatic expression of phase I and phase II drug metabolizing enzymes and of transporters is activated by CAR in response to structurally diverse chemicals. In addition to xenobiotic detoxification, activation of CAR is also involved in other hepatic functions like fatty acid oxidation, gluconeogenesis, clearance of steroid hormones and bilirubin. In primary hepatocytes, CAR resides predominantly in the cytoplasm associated with other proteins in a multimeric complex of which some components still remain to be identified. Upon exposure to inducers CAR dissociates from the already identified proteins of the complex, the cytosolic CAR retention protein (CCRP) and HSP 90 resulting in its translocation into the nucleus, where it heterodimerizes with the retinoid X receptor (RXR). The CAR-RXR heterodimer binds to its respective response elements in the regulatory region of target genes and recruits coactivators like SRC-1 (steroide receptor co-aktivator), GRIP1 (glutamate receptor interacting protein) and PGC 1 (peroxysom-proliferator-aktivated receptor γ co-activator 1) to induce gene transcription. To better understand the function of CAR, this study was focused on the identification of proteins which associate with CAR. The aim of this work was to identify putative interaction partners of the nuclear receptor CAR which are expressed in liver to get additional information on structure and regulation of the native protein multimer complex und to obtain a better understanding of the functionality of CAR. Hence, we have established an in vitro pulldown assay with liver homogenate to analyze protein-protein interactions of CAR. As a first step we generated GST and GST-CAR fusion proteins which were expressed in E. coli followed by affinity purification. Then we incubated the fusion proteins with total liver homogenate and separated bound proteins with SDS-PAGE. After visualization with silver staining, the protein bands were excised and prepared for mass spectrometry. For identification of new interaction partners of CAR in liver, the Matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF) was used. After optimization of the pulldown assay we could identify several proteins like cytoskeletal proteins (e.g. lamin A), enzymes (e.g. pyruvate carboxylase, GAPDH) and chaperones (e.g. HSP 70) as binding to CAR. As an especially interesting interaction partner of CAR we decided to further investigate the interaction of CAR with BRG1-associated factor (BAF) 155. This protein is a component of the mammalian SWI / SNF chromatin remodeling complex that plays an important role in fundamental cellular processes such as transcription, replication, and the repair of chromatin. Interaction of GST-CAR-LBD fusion protein was confirmed by additional methods like pulldown assay of GST-CAR with (35S)-methionine labeled BAF 155 in vitro and co-immunoprecipitation of the two proteins. Additionally, we could confirm BAF 155 interaction with CAR by Western blotting of the original pulldown samples. Analyzing the interaction of CAR and BAF 155 with RNA interference was not successful, since the method could not be optimized and validated appropriately, due to time constraints. In conclusion, we were able to establish a highly reliable and reproducible assay to investigate protein interactions resulting in the significant identification of new interaction partners of CAR. Regarding the identified CAR interaction partner BAF 155, this protein or the complete SWI / SNF complex could play a functional role in CAR-mediated transcriptional activation, however further research is needed to establish the role of BAF 155 in CAR function.
Der Einfluss verschiedener Ölemulsionen und der IL-6-Typ-Zytokine CNTF und LIF auf die Differenzierung von 3T3-L1-Präadipozyten zu Fettzellen
(2009) Stäbler, Antje; Graeve, Lutz
Type 2 diabetes is a disease of civilization spreading all over the world like an epidemic. Among other things it is characterized by increased glucose and triglyceride levels of the blood. Thus the differentiation of preadipocytes into adipocytes that are specialized in storing large fat depots is essential for the removal of excess nutrients from the circulation. This process can be simulated through the differentiation of 3T3-L1 cells by treatment with insulin, IBMX, and dexamethasone. In this study a compensative effect of olive oil as well as soy bean oil on the differentiation of 3T3-L1 cells was found. The differentiation triggered by insulin, IBMX, and dexamethasone was somewhat inhibited by incubating the cells with one of the two oils, whereas both olive and soy bean oil had a certain potential to act as differentiating agents them-selves when insulin, IBMX, and dexamethasone were not added. This points to benefits of certain oils contained in food compared to thiazolidinediones that are used as antidiabetics and lead to weight gain in the long term. Furthermore the inhibitory effect of the proinflammatory cytokine TNFα on the differentiation process could be compensated to some degree by either oil. The transcription factor PPARγ was used to measure the state of differentiation. Caveolin1 is involved in both buildup and degradation of intracellular fat stores and was induced by oil incubation in differentiated as well as in undifferentiated 3T3-L1 cells. Moreover the signal transduction of the IL-6-related cytokine CNTF in 3T3-L1 cells and its influence on the differentiation process of these cells was investigated. In recent years CNTF was spotlighted because of its leptin-like properties that persist even in leptin resistant states. In this study an increasing sensitivity to CNTF during the differentiation of 3T3-L1 cells was shown. Furthermore the positive effects of the cytokine on fatty acid metabolism and adipocyte development were verified. CNTF favored the emergence of many small adipocytes compared to fewer, but larger adi-pocytes under different conditions. Thereby the functionality of the cells is influenced in a positive way and the protection from the development of insulin resistance is increased. The effect of LIF - another IL-6-related cytokine - was not similar to that of CNTF but was more comparable to the effect of the oils.
Eliminierung apoptotischer Zellen durch professionelle Phagozyten: Generierung, Freisetzung und Erkennung des monozytären Attraktionssignals Lysophosphatidylcholin und Bedeutung von Annexin I als Brückenprotein in der phagozytotischen Synapse
(2007) Waibel, Michaela; Graeve, Lutz
The efficient elimination of apoptotic cells by neighbouring cells or professional phagocytes is essential for tissue homeostasis in multicellular organisms. Therefore, the apoptotic cell displays different so-called ?eat-me?-signals on its cell surface that are crucial for its recognition and engulfment. Especially in higher organisms, where the dying cell and the phagocyte are usually not located in immediate proximity, the release of soluble attraction signals is of special importance. Only recently, the phospholipid lysophosphatidylcholine (LPC) could be identified as a central ?find-me?-signal that is generated by the calcium-independent phospholipase A2 (iPLA2)-mediated hydrolysis of phosphatidylcholine. During apoptosis iPLA2 is processed in a caspase-3-dependent fashion. In the present thesis it could be demonstrated that iPLA2 is cleaved directly by caspase-3 and that this processing leads to its activation. The active iPLA2 is essential for the production of the phospholipid-?find-me?-signal LPC in apoptotic cells. However, the observation that overexpression of the active form of iPLA2 alone was not sufficient for the release of the attraction signal from vital cells implied that other apoptotic events might contribute to the generation and export of the ?find-me?-signal LPC. It turned out that the reactive oxygen species-driven oxidation of membrane lipids like phosphatidylcholine is an additional factor that leads to the enhanced production of LPC, probably because oxidized lipids are more susceptible for PLA2-mediated hydrolysis than non-oxidized lipids. Further studies about the detailed export mechanism of LPC revealed that the ATP-binding cassette transporter (ABC)-family member ABCA1 is essential for the release of the attraction signal during apoptosis. Thus, the oxidation of membrane lipids and the ABCA1-driven export of LPC could be identified as important elements of LPC-generation and its subsequent release during apoptosis. After the generation and the release of the attraction signal LPC could be demonstrated in more detail the consequent question arose which receptors might mediate the effects of LPC on the phagocytes. In the present thesis it could be demonstrated that the G-protein-coupled receptor G2A is responsible for the LPC-stimulated migration of monocytic cells. However, the molecular mechanisms that ultimately lead to the LPC-driven, G2A-mediated migration, are not known so far. Accordingly, a participation of other receptors or the existence of further chemotactic signals cannot be ruled out at this point. Moreover, there are some hints for chemotactically active proteins in literature. If these or other factors contribute to the LPC-mediated chemotaxis of monocytic cells is completely unknown and needs to be clarified in future studies. The recognition and internalization of dying cells is mediated by the interaction between different ?eat-me?-signals that are displayed on apoptotic cells, and specific surface receptors on phagocytes. In this scenario, the interaction can be of a direct nature ore rather indirect via bridging molecules. In this context, here it could be demonstrated that the calcium- and phospholipid-binding protein annexin I gets externalized by dying cells independently of the apoptotic stimulus applied, but dependent on the cell type. On the surface of the apoptotic cell, annexin I binds in a calcium-dependent fashion via its annexin-boxes to externalized phosphatidylserine, which represents a central ?eat-me?-signal. Thereby, annexin I is able to stimulate the elimination of these cells by professional phagocytes and thus fulfills the function of a bridging molecule in the phagocytic synapse. In contrast, the receptors that are responsible for the binding of annexin I to phagocytes are not known so far. As a conclusion it can be stated that the phenomena studied in this thesis represent important steps in the process of apoptotic cell elimination. The physiological relevance of apoptotic cell clearance is the fact that apoptosis, in contrast to necrotic cell death, is highly regulated at all stages and usually turns out without any harmful consequences to the organism. If this complex, multistep process is disturbed, non-cleared apoptotic cells can become a source for inflammatory reactions. In different animal models it could be demonstrated that defects in the attraction of phagocytes as well as deficiencies in the recognition and internalization via ?eat-me?-signals and the subsequent degradation of the apoptotic prey can be a reason for the onset of severe autoimmune disorders. In this context, the development of human systemic lupus erythematosus and of chronic arthritis is discussed to be initiated by the inefficient elimination of apoptotic cells.
Entwicklung und Charakterisierung des rekombinanten, bifunktionalen Proteins SDF1-GPVI und dessen Einfluss auf die myokardialen Reparaturmechanismen
(2012) Ziegler, Melanie; Graeve, Lutz
Cardiovascular diseases like myocardial infarction, arteriosclerosis and coronary heart disease are the leading cause of death worldwide. Because of this, development of novel and innovative biopharmaceutical approaches is very important to improve the therapy of cardiovascular diseases. Especially after myocardial infarction, promoting cardiac repair mechanisms is of vital importance to preserve cardiac function and decrease scar size. The chemokine ?stromal cell-dervied factor-1? (SDF-1) plays a crucial role in stem cell homing and is upregulated after myocardial infarction. It is already known that the SDF-1/ CXCR4 axis promotes myocardial repair. In this study we connected SDF-1 with an anchor structure to accumulate this chemokine specifically at the infarcted myocardium. The anchor structure consists of the soluble glycoprotein VI (GPVI). GPVI is the major collagen receptor on platelets and binds to nearly all collagen types and other proteins of the extracellular matrix like fibronectin and vitronectin. Hence, SDF1-GPVI specifically accumulates at the injured tissue and attracts progenitor cells from the blood stream to the lesion site. During the course of this study, the recominant, bifunctional protein SDF1-GPVI, consisting of human SDF-1 and soluble human GPVI, was generated. Subsequently, the functional efficacy of the SDF-1 and GPVI domains were proven by several functional assays and the bifunctionality of SDF1-GPVI was determined by dynamic adhesion in a flow chamber system. Further, in vitro studies showed that SDF1-GPVI triggers chemotaxis of haematopoetic progenitor cells (HPCs), enhances and accelerates endothelial differentiation of HPCs and preserves survival of HPCs. Furthermore, using the ?Chorioallantoic Membrane? (CAM) assay SDF1-GPVI revealed proangiogenic effects in ovo. Prior to in vivo analysis, a pharmacokinetic study showed that SDF1-GPVI has a biological half-life >48 h. After induction of myocardial infarction, treatment with SDF1-GPVI and G-CSF lead to a significantly decrease in infarct size and to a significantly preserved myocardial function 28 days after transient ischemia/reperfusion. Additional positive effects were enhanced homing of GFP-labelled ?bone marrow cells? (BMCs), increased recruitment of CXCR4-positive cells and enhanced neovascularisation in the infarcted myocardium. Therefore, the bifunctional protein SDF1-GPVI bears the potential to serve as a promising biopharmaceutical therapeutic to promote myocardial repair especially after myocardial infarction.
Functional validation of genetic changes discovered in high-throughput mutational and copy number screens with respect to carcinogenesis and treatment sensitivity for Head and Neck Cancer
(2015) Keck, Michaela-Kristina; Graeve, Lutz
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease regarding both anatomic location and molecular characteristics. It is comprised of two distinct disease entities - human papillomavirus (HPV)-positive and HPV-negative HNSCC - that differ regarding primary site of disease as well as aetiology/pathogenesis. Persistent infection with high-risk HPV is causally related to oropharyngeal HNSCC, whereas tobacco and alcohol consumption are linked to the development of HPV-negative head and neck cancers. HPV(+) and HPV(-) HNSCC differ regarding their clinical behavior as well as prognosis with HPV(+) tumors being associated with a more favorable prognosis than HPV(-) tumors. However, besides Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), which is used with modest success as no predictive biomarkers have been identified, no targeted treatments are available that consider the biologic heterogeneity and no classification is used in clinical practice that reflects the underlying biology of the disease. As a consequence, all patients are treated uniformly with a combination of surgery, radiation, and chemotherapy depending on stage and location of the tumor. In this study, a homogeneous and clinically annotated cohort of 134 treatment-naive HNSCC specimens, including 59 HPV(+) samples (44%), were processed, subjected to Agilent 4X44Kv2 gene expression arrays and Nanostring copy number assays, and analyzed together with all available HNSCC cohorts (n=938). As a result, a clinically meaningful classification was identified for HNSCC - which was hampered in previous studies due to a limited inclusion of HPV(+) samples, unknown HPV status, small sample size, or missing clinical annotation. Three HNSCC supergroups were identified - basal (BA), classical (CL), inflamed/mesenchymal (IMS) - and further subdivided by HPV status into five HNSCC subtypes - BA, CL-HPV, CL-nonHPV, IMS-HPV, IMS-nonHPV - that differ regarding survival and show different expression profiles and copy number (CN) aberrations. Importantly, two HPV-associated subtypes with different molecular profile and prognosis were identified - CL-HPV and IMS-HPV. The IMS subtypes show a strong immune phenotype with expression of immune response genes and may benefit from immune therapies such as programmed cell death protein 1 (PD-1) inhibitors. The BA subtype shows markedly increased expression of EGFR and hypoxia related genes, which might render these tumors susceptible to EGFR-targeting and hypoxia-targeting therapies. Besides differences regarding their biological and clinical behavior, HPV(+) and HPV(-) HNSCC show a distinct mutational landscape. The incidence of HPV(+) tumors is rising, however, studies are missing that investigate a sufficient number of HPV(+) tumors to derive meaningful information and assign targetable genomic changes to either disease entity. 120 (51 HPV(+), 69 HPV(-)) out of the cohort of 134 samples were further used to build DNA sequencing libraries and processed to next generation sequencing (NGS) on the Illumina platform (Illumina HiSeq 2000/2500 sequencers) to detect novel or targetable genomic changes. The overall mutational burden is similar in both disease entities, however, the mutational landscape differs. In addition to known aberrations, novel targetable aberrations with predicted driver potential are identified in various genes, among which are EGFR, CCND1, and FGFR1 in HPV(-) tumors, and FGFR2/FGFR3, KRAS, BRCA1/2 in HPV(+) tumors, as well as PIK3CA and PI3K pathway genes in both and rare aberrations in DDR2 and EPHA2. The fibroblast growth factor receptor 3 (FGFR3) S249C mutation is recurrently found in six HPV(+) tumors, which makes FGFR3 the second most mutated gene in HPV(+) tumors - after the well-known oncogene PIK3CA. Therefore, functional validation of FGFR3 S249C was performed in vitro in the two HNSCC cell lines SCC47 and 93VU147T. Two different FGFR3 isoforms were identified in all six FGFR3 mutated HNSCC tumor samples - FGFR3IIIb and the soluble FGFR3Delta8-10. Both isoforms were used to stably transfect the S249C mutation into SCC47 and 93VU147T and to study its effects on proliferation and migration as well as FGFR3 signaling. No effect on proliferation or migration rate is seen beyond an increase in proliferation rate in SCC47 cells transfected with wild type (WT) FGFR3IIIb. No constitutive activation of downstream pathways is seen in unstimulated SCC47 cells. Investigation of different downstream signaling pathways identifies the MAPK and the PI3K/Akt pathway as the two important signaling pathways downstream of FGFR3 in HNSCC cell lines. FGFR3 S249C cannot be confirmed as an oncogenic driver in SCC47 and 93VU147T. However, its possible role as an oncogenic co-driver besides other receptor tyrosine kinases and its function in drug resistance mechanisms - especially resistance to EGFR inhibitors - remain to be investigated.
Health enhancing traditional foods in Brazil : an interdisciplinary approach to food and nutritional security
(2012) Abadio Finco, Fernanda; Graeve, Lutz
The Brazilian nutritional profile is currently characterized by the so-called "nutrition transition process" i.e. the population presents nutritional status characteristics of both developing and developed countries. Therefore, malnutrition is present not only in the form of undernutrition but increasingly also presents as overweight and obesity. Some studies suggest that this is not only a particular problem of urban societies but also of rural communities. Recently, Brazil has impressively advanced on issues which address nutrition, agriculture and health within a sustainable framework. One of the recent initiatives encompasses the Brazilian Food and Nutrition Security Policy, which could be considered as the vanguard of this theme by covering different dimensions of nutritional issues, as defined hereunder: ?Food and Nutrition Security is the achievement of the right of all people to access food regularly and permanently, with quality and enough quantity, without compromising the access to other basic needs, based on food practices to promote health, with respect to cultural differences and being social, economic and environmentally sustainable?. Since the Brazilian Food and Nutritional Policy is characterized by a broad view on food and nutrition, different components related to food and nutrition have to be considered. Therefore, the health side of the food, in a pluralistic vision has to be taken into account. Thus, food and their consumers are unavoidably connected. Beyond classical nutrients, much attention has recently been focused on bioactive compounds and their preventive role on non-communicable diseases such as diabetes, cardiovascular diseases and cancer. Therefore researchers are increasingly interested to unfold the preventive biochemical processes of these compounds. Hence, the current research aimed to investigate health enhancing properties of traditional Brazilian fruits within the Food and Nutrition Security definition of the country. Given the interdisciplinary feature of the topic Food and Nutrition Security, the work was performed in two stages. The first one encompasses a nutritional survey with two rural communities in APA ? Cantão, Tocantins State, Brazil and the second part comprises experimental laboratory research. The outcome from the nutrition survey showed a high level of food insecurity among the families (84.2%). The nutritional profile of the study population expressed a high prevalence of overweight for the adults (53.7%). Regression analysis showed that the high Body Mass Index (BMI) is influenced by the consumption of an imbalanced diet and the physical activity level. Furthermore, women had a higher prevalence of overweight and obesity in comparison to men. Another observation is that rural communities have a monotonous diet with very low consumption of fruits and vegetables. Besides the negative effect on their body composition, this last result points to the risk of developing micronutrient deficiencies, i.e. hidden hunger. Based on the outcome and the demand presented by the participants in the nutrition survey, two fruits available in the region were chosen to investigate their possible biofunctional properties. Different assays were performed with Bacaba (Oenocarpus bacaba Mart.) and Jenipapo (Genipa americana L.) phenolic extracts. Extracts from both fruits showed antioxidant and antiproliferative capacities. Since bacaba displayed higher activities than Jenipapo, this fruit was chosen for a more detailed investigation of the biochemical mechanisms involved. The results showed that bacaba phenolic extracts induced apoptosis in MCF-7 breast cancer cells through the mitochondrial pathway. Caspase-6, -8 and -9 were activated when compared to the untreated control in a dose dependent manner (p<.05). However, caspase-9 showed the highest activation. Since MCF-7 cells do not express caspase-3 and based on additional investigations on PARP (Poly (ADP-ribose) polymerase) - cleavage, the experiments suggest that caspase-9 plays an important role in the observed apoptotic effect. The laboratory work thus emphasizes the potential healthy properties of traditional fruits from the Brazilian biodiversity with high antioxidant activities. Altogether, the results indicate the need of a better nutritional education with the involved communities in order to promote healthy eating practices and to increase the consumption of fruit and vegetables. Based on this, it is suggested for government and policy makers to take action in rural communities. Indeed, it is undeniable that the biodiversity available in Brazil is a huge treasure and source of novel ?superfruits?. Therefore, the current work reinforces the development of research in this area in order of identify health enhancing neglected traditional fruits and to promote their consumption, add value and generate income to small farmers and traditional communities with not only the improvement of their economic power, but also of their diets and health respecting their tradition and culture. Not to mention the contribution to biodiversity preservation since plants that were merely discarded could now have a multifactor value in line with the Brazilian Food and Nutritional Security policy.
Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas
(2007) Aichler, Michaela; Graeve, Lutz
Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers in humans. It is the fourth leading cause of cancer related deaths in Germany and in the United States. Most PDA occurs sporadically, but there are also approximately 5-10% of patients with a family history of pancreatic cancer. The high mortality of PDA is attributed to a lack of early detection methods and poor efficacy in therapies for advanced disease. As an alternative, preventive strategies in individuals with familial pancreatic carcinoma should be considered. Several epidemiological studies showed an inverse correlation between selenium-intake and mortality of certain types of cancer and particularly in gastrointestinal cancers. To this end, in the first part of this study, the influence of selenium as a preventive nutritional additive was investigated in a genetically defined pancreatic cancer mouse model, the EL-TGFatg/+;p53+/- mouse strain. As a major finding, the differentiation grade of the pancreatic carcinomas was heavily influenced by the selenium status. In the selenium-deficient group there were more non-differentiated pancreatic carcinomas than in the selenium-adequate group, which highlighted the implication of selenium or selenoproteins in tumour differentiation. Unexpectedly, however, there was no protective effect of selenium on total or pancreatic tumour latency. Within the selenoproteins, the thioredoxin reductases are strong candidates which may influence cell death and differentiation in pancreatic carcinogenesis. Their function is generally associated with tumour proliferation and also linked to the activation of the tumour suppressor p53. Consequently, the role of the thioredoxin reductases in the pancreas was studied in the second part of this thesis. The enzymatic activity of cytosolic (TXNRD1) and mitochondrial (TXNRD2) thioredoxin reductase in the pancreas and other organs was determined in relation to the selenium-status. TXNRD1 activity in the pancreas was moderate and decreased under selenium deficiency. TXNRD2, instead, showed very high pancreatic activity in relation to other organs and its activity was even increased under selenium-deficiency emphasising its special role in this organ. To further investigate the function of Txnrd1 and Txnrd2 in the pancreas, tissue-specific knockout mice were created and characterized. The Txnrd1 knockout mice did not show an overt phenotype. Interestingly although, pancreatic acinus cells in one year old mice showed a disturbed rough endoplasmic reticulum and alterations in serum amylase and lipase. These mice also had an impaired glucose tolerance. The pancreas of Txnrd2 knockout mice showed severe chronic pancreatitis and pancreatic atrophy at the end of an observation period of one year. The progressive pathogenic process started with mild pancreatitis, developing spontaneously at an age of four weeks. The chronic stage was characterized by the formation of different types of acinar-to-ductal metaplastic lesions, which could be classified in part as early precursor lesions of pancreatic carcinomas. The endocrine pancreas was not affected. The pancreas-specific Txnrd2 knockout mouse strain is the first genetically modified mouse model spontaneously developing acute and chronic pancreatitis. This strain constitutes a unique and powerful tool to model pancreatic pathogenesis, especially the yet unresolved process of transformation from inflammatory to malignant disease.
Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.
Posttranslationale Modifikationen der IL-6-Typ-Zytokin-Rezeptoren gp130 und LIFR und ihr Einfluss auf die Assoziation mit Detergenz-resistenten Membranmikrodomänen (DRM)
(2008) Ziegler, Inna; Graeve, Lutz
Post-translational modification of proteins is an important event in the regulation of cellular functions. Glycosylation or palmitoylation, but also ligand binding can affect the localization of proteins in membrane microdomains and thus affect signal transduction. The aim of this study was to analyze how posttranslational modifications of LIFR and the common signal transducer gp130 impact the translocation to detergent resistant membranes (DRMs, lipid rafts). Palmitoylation of cysteine residues within the transmembrane domain of a protein is considered to be one process that assists in the localization of proteins to DRMs. Gp130 has two cysteine residues C711 and C725 in its transmembrane domain. My studies indicate that these cysteine residues have no significant influence on lipid raft association of gp130. Contrary to our expectations, after isolation of DRMs with Brij 58 and Triton X-100 an increase of raft association of the C->A-mutants was detected. Partial DRM association of LIFR was confirmed by using Brij 58 and Triton X-100 protocols. Furthermore, two different N-glycosylation types of that receptor could be detected. The mannose-rich (precursor) species is preferentially found in non-DRMs and is degraded by Endo-Glycosidase Hf. The hybrid-type (mature) tends towards an association with DRMs. My results indicate that only the mature-type of LIFR was phosphorylated after LIF binding to the receptor complex in 3T3-L1 and HepG2 cells. Combined with other data from our workgroup these findings suggest that only the mature-type of LIFR is expressed at the plasma membrane surface and involved in signal transduction. After stimulation with LIF an increase of LIFR tyrosine phosphorylation was observed in DRMs in HepG2 cells. However, phosphorylation of gp130 was detected only in non-DRMs fractions after stimulation with LIF. The inconsistency of these results can be explained with methodical problems. Furthermore, the translocation of phosphorylated receptors described above could not confirmed in 3T3-L1 cells. In this cell line, the activation of gp130 and LIFR occurs in detergent-resistant membranes. These findings indicate differences between cell lines with respect to receptor activation and translocation within the plasma membrane on the one hand and demonstrate a differential sensitivity of raft subdomains to extraction by different detergents on the other hand.
Rolle der GPCR-Signaltransduktion bei der Peptidhormonsekretion in neuroendokrinen Zellen im Darm und im Pankreas
(2008) Leicht, Stefanie; Graeve, Lutz
The insulinotropic peptide hormone Glucagon-like peptide-1 (GLP-1) led to intense interest in the use of this peptide for the treatment of patients with type 2 diabetes. The molecular mechanisms of GLP-1 in the β-cells are examined and well understood, whereas the mechanisms leading to GLP-1 secretion in the L-cells are not understood in detail. However the regulation of GLP-1 secretion from intestinal L-cells seems to be similar to the regulation of the insulin secretion in pancreatic β-cells. In the β-cells a number of G-protein coupled receptors influence the insulin secretion and other signal transduction cascades. Due to the fact, that the three G-protein coupled receptors GPR40, GPR119 and GPR120 are expressed in pancreatic β-cells as well as in the intestinal L-cells, the present studies concentrated on the expression and importance of the three receptors and on their intracellular effects in the L-cells and in the β-cells. GPR40, GPR119 and GPR120 are colocalized with GLP-1 in the enteroendocrine L-cells in the rat ileum and colon between the enterocytes. Moreover GPR119 is colocalized with insulin in the pancreatic β-cells. GPR40 and GPR120 are Gαq-coupled receptors, ligands are longchain unsaturated free fatty acids. GPR119 is a Gαs-coupled receptor being activated by lipids like oleylethanolamide. Activation of the three receptors by selective and unselective agonists stimulates GLP-1 secretion and the glucose induced insulin secretion in vitro and ex vivo, whereas GPR119 amplifies the Gαq-induced GLP-1 secretion. Synthetic agonists were able to enhance the fatty acid induced GLP-1 secretion in an additive manner. Glucose also stimulated the GLP-1 secretion in vitro and ex vivo. In L-cells and β-cells it has been shown that GPR40, GPR119 and GPR120 stimulate cell proliferation and inhibit cell apoptosis via different signal transduction pathways in vitro. Hence the present studies make a contribution to the understanding of the importance of GPR40, GPR119 and GPR120 and their signal transduction pathways for the function of the L-cell and the β-cell.
Variabilität und Induzierbarkeit von Cytochrom P450 Monooxygenasen in humanen Leberproben und Hepatozyten : Untersuchungen mittels LC-MS/MS Cocktail-Assay und RNA-Interferenz
(2010) Feidt, Diana M.; Graeve, Lutz
The variability of the expression and function of the P450 enzymes (CYPs) is a cause for having different intensities and lengths of effects as well as side effects when patients are given the same dosage of medication. Up to this day, one cannot allover explain this inter individual variability of expression and activity of the enzymes. In general, there are several factors that may affect the variability, including biological factors (such us age, gender, hormonal status), environmental factors (such as nutrition, smoking, medication) as well as genetic factors like polymorphisms In order to solve this problem and to analyze relatively easy and fast activity differences, we developed an LC-MS/MS based P450 activity cocktail assay to quantify and detect simultaneously the seven most important CYPs as judged by their roles in the metabolism of clinically used drugs. The assay was established for use in in human hepatocytes as well as in recombinant and microsomal enzymes. We used the newly developed model-substrate cocktail assay to analyze the time-dependent induction of seven drug metabolizing cytochrome P450 activities as response to treatment of primary human hepatocytes with different statins. The strongest induction was observed for amodiaquine N-desalkylation of CYP2C8, which was induced up to 20-fold by atorvastatin and approximately 10-fold by simvastatin and lovastatin. Enzymes CYP3A4, CYP2B6 and 2C9 showed lower, but also significant induction after treatment with atorvastatin and simvastatin (4-11-fold). lovastatin and rosuvastatin demonstrated minor effects. Quantitative RT-PCR confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450 expression and activity than previously known, thus further emphasizing their drug-drug interaction potential, especially for CYP2C8. Based on correlation analysis with P450 enzyme activity to their specific protein amount in human liver samples (liverbank IKP) were different functional results observed. Enzymes like CYP3A4 with atorvastatin hydroxylation or CYP1A2 with formation of acetaminophen showed very good correlations. Others like i.e. CYP2C9 with specific substrate diclofenac correlated much lower. Besides biological and environmental factors could these differences based on variability of the enzymes NADPH:P450 oxidoreductase (POR), cytochrome b5 or the two progesterone receptor-membrane components PGRMC1 and PGRMC2. After all, they would take active part in the metabolism of the xenobiotica as possible electron donors of the CYP enzymes. In order to analyze what kind of influence these proteins have on CYP enzyme activity, we developed a lentiviral based RNA-interference (RNAi) method in human hepatocytes and determined P450 activity with cocktail-assay after knocking down these genes. For the P450 reductase, we achieved a successful gene silencing of about 85% on mRNA level. The expression of cytochrome b5 was reduced by 51%, PGRMC1 about 30%. So far, it has not been possible to prove a significant knock down of PGRMC2. After silencing of reductase, an average light decline of about 10-30% of P450 enzyme activities was observed after 4 days. After a period of 7 days, a rest activity of CYP3A4 of only about 5% was detected. For both other potential electron donators cytochrome b5 and PGRMC1 was a reduced activity of 85% and 75% determined. These first results indicate a clear interaction of the enzymes POR, cytochrome b5 and PGRMC1 with the drug metabolizing enzymes.