Institut für Ernährungswissenschaften

Permanent URI for this collectionhttps://hohpublica.uni-hohenheim.de/handle/123456789/5

Browse

Now showing 1 - 20 of 82

Role of reactive oxygen species in anti-cancer treatment: Investigations in 2-methoxyestradiol chemotherapy and 5-aminolevulinic acid based photodynamic therapy combined with hyperthermia
(2003) Lambert, Christine; Frank, Jürgen
The thesis deals with two different ROS-generating anti-cancer treatments: chemotherapy with the endogenous estrogen metabolite 2-methoxyestradiol and 5-aminolevulinic acid based photodynamic therapy. Both treatments were investigated with the rat DS-sarcoma model, which can be used in vitro and in vivo. It the first part, it could be shown that 2-methoxyestradiol induces apoptosis in DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to the mitochondria was identified as initial apoptotic event, followed by a decrease in mitochondrial transmembrane potential and the release of AIF out of the mitochondria. In addition, upregulation of FasL and TNFalpha by 2-ME, two death receptor ligands, was observed. Although, 2-ME administration resulted in activation of caspases, pan caspase inhibitor Z-VAD-FMK could not block 2-ME induced apoptotic cell death pointing to a caspase-independent mechanism. Furthermore, an increase in formation of reactive oxygen species was observed after 2-ME treatment. However, supplementation with different antioxidants could not decrease the toxic effect of 2-ME. This finding may indicate, that reactive oxygen species are not involved in apoptosis induction, rather they are a consequence of mitochondrial damage. In vitro and in vivo combination of 2-ME with another ROS-generating treatment resulted in a synergistic anti-tumour effect. In the second part of the thesis anti-tumour effects of 5-aminolevulinic acid based photodynamic therapy combined with simultaneous hyperthermia was investigated. Analysis of apoptosis associated nuclear changes clearly demonstrated the high efficiency of this treatment regime. Formation of reactive compounds (e.g. ROS, nitrogen monoxide, peroxynitrite) which is mainly responsible for toxicity of PDT, could be assessed in the shape of massive protein nitrosylation in tumours treated with PDT alone or the combined treatment. Detection of decreased amounts of heat shock proteins (HSP70 and HO-1) which protect tumour cells against damaging influences, lowered glutathione levels and reduced MMP-activity indicate an increase in degradation of proteins. This phenomenon may be caused by excessive generation of ROS. Taken together, the presented studies could demonstrate the high benefit of combining 2-ME resp. ALA-PDT with hyperthermia (or other ROS-generating therapies), which make them interesting candidates for future clinical applications.
Einfluss der Ernährung und von Genussmitteln auf Risikofaktoren für das Auftreten von ischämischem Herzinfarkt und Schlaganfall
(2005) Eckoldt, Joachim; Bode, Christiane
Background and aims of the study: Arteriosclerotic changes of blood vessels which contribute to coronary heart disease (CHD) and ischemic stroke are influenced by risk factors like cigarette smoking, overweight, hypertension, diabetes mellitus, missing physical exercise and nutritional factors, such as alcohol consumption. Beyond this, the concentrations of serum lipids, antioxidants, coagulation factors or other risk factors, such as C-reactive protein, and homocysteine are considered to be additional factors that indicate an enhanced or lowered risk of atherosclerosis. In this study we examined the effect of nutritional factors, in particular alcohol consumption, on various plasma components that are believed to play a role in the pathogenesis of atherosclerosis. Multiple epidemiologic studies suggest that moderate alcohol consumption reduces the mortality from cardiovascular diseases and that this effect is chiefly mediated by elevation of high-density lipoprotein (HDL). This cross-sectional study assessed the effect of moderate alcohol consumption and other life-style factors on the composition of HDL in healthy working males. An additional goal of the present study was to find out whether there is an association between alcohol consumption and the concentration of vitamins and cardio-protective substances. Methods: We included a collective of healthy men (n = 284, age 23-66 years), investigated with respect to cardiovascular risk factors. The average daily alcohol consumption, nutrient intake, smoking and other life-style factors was assessed by a computer based questionnaire. Group 1 (n = 62) comprised subjects with an average daily alcohol consumption of 0-5g, group 2 (n = 175): 5-30g, and group 3 (n = 47): 30-70g. In addition, the study design made it possible to subdivide groups 2 and 3 in a so called ?beer drinker group 2+3? (> 80 % beer), a ?wine drinker group 2+3? (> 80 % wine) and persons without preference of a certain alcoholic beverage. Results: The alcohol groups showed no significant differences in the nutritional profile (nutrients, energy intake, and metabolic rate). The markers for regular, higher alcohol consumption (g-GT and MCV) were positive correlated to the amount of alcohol consumption. There was no correlation between the great number of clinical laboratory parameters and the amount or kind of alcohol consumption. Thereby the groups are comparable in view of these laboratory parameters. Besides, there were no indications for the existence of diseases, which might influence blood lipid and vitamin concentrations. Antioxidative substances in the blood: Dietary assessment: The intake of vitamin B2, B6, B12, folic acid, retinol, ß-carotene and other carotenoids, as assessed by the computer interview was comparable in groups 1-3. The subjects in group 1 had a higher supply of the vitamins C, B1 and a-tocopherol, ?beer drinker Gr. 2+3? had a higher intake of vitamin B2, B6 and folic acid. Blood measurements: Antioxidative vitamins: The vitamin B2 status in erythrocytes (EGRAC) was lower in group 3 (vs. group 1 and 2). The plasma level of ß-carotene and ß-cryptoxanthin was lower in group 3 than in group 1. Vitamins that influence homocysteine metabolism (including homocysteine): Influence of beer and wine: The status of vitamin B6 and the concentration of free plasma pydridoxal phosphate in group 3 was significantly higher than in group 1. These results cannot explain the postulated positive influence of moderate or higher alcohol consumption through improvements of the vitamin status and the concentration of vitamins in the blood. The vitamins in beer improved the vitamin status only in case of vitamin B6, no effect was calculated in case of vitamin B2 and folic acid. Higher alcohol consumption (group 3) made the vitamin status respectively the plasma concentration of vitamin B2, ß-carotene and ß-cryptoxanthin lower compared with group 1 ? in spite of comparable supply. Coagulation factors, markers of inflammation: The coagulation factors prothrombin time and fibrinogen as well as the ?newer? risk factors C-reactive protein and homocysteine were not correlated to the amount or the kind of alcohol. Lipoproteins: The serum concentration of total cholesterol, cholesterol ester, phospholipids, apolipoprotein A-1 and A-2 was higher in group 3. Moderate and higher alcohol consumption raises the concentration of cholesterol in the high-density lipoproteins (HDL) (including subfractions) ? independent of the sort of alcoholic beverage. The concentration of cholesterol in the low- (LDL) (including subfractions), very-low (VLDL) and intermediate-density lipoproteins (IDL) in the blood was not influenced by alcohol consumption. Composition of HDL: The induced increase of HDL cholesterol was lower in the subfraction HDL3 as in the subfractions HDL2b und HDL2a. Besides we found qualitative changes of the HDL-components: the phospholipid component increased more than the other HDL-components. This phenomenon might play a beneficial role in the mechanism of atherosclerosis. Conclusions: Vitamins: The changes of antioxidative vitamins and vitamins influencing homocysteine metabolism observed in persons with moderate and increased alcohol consumption do not explain the antiatherogenic effect of alcohol. On the other hand, our study confirmed a positive association of moderate alcohol consumption with HDL plasma levels ? independent of other nutritional factors. In addition, alcohol might induce qualitative alterations of HDL composition (more pronounced increased of HDL2 relative increase of the phospholipid component). The pathophysiological significance of this phenomenon remains unclear.
Einfluss von Karotten- und Tomatensaft-Konsum auf Coloncarcinogenese-relevante Faecesmarker beim Menschen
(2006) Schnäbele, Kerstin; Briviba, Karlis
Colorectal cancer is one of the most common tumor diseases in the world. Most of the colorectal tumors are sporadic and develop somatically in epithelial cells. Nutritional factors can markedly affect tumor development. A high intake of fruits and vegetables is often associated with a reduced risk of colorectal cancer. Protective effects of fruits and vegetables are attributed to ingredients, such as fibers, vitamins, and secondary plant products (e.g. carotenoids), which have potential anticarcinogenic properties. The aim of this study was to investigate, by means of a human intervention trial with carrot and tomato juice consumption, whether a diet rich in carotenoids, especially high in beta-carotene and lycopene, can modify processes relevant to colon carcinogenesis in the gastrointestinal lumen. Therefore, several faecal markers had to be established and used in this study. In the randomized crossover trial, 22 healthy male subjects on a low-carotenoid diet consumed 330ml of carrot or tomato juice daily for a period of two weeks. The two juice intervention periods were preceded by two-week depletion phases. At the end of each study period the stool of twelve volunteers was collected over a 48-hour period. This stool was used to produce some preparations such as non-filtered and sterile-filtered faecal water, as well as faecal lipid extracts, in order to use them in cell culture systems. Spectral photometric and flow cytometric methods were used to determine the effects of the above-mentioned preparations on colon adenocarcinoma cells (HT-29), as well as to determine the activities of the bacterial enzymes beta- glucosidase and beta-glucuronidase in faecal water. HPLC methods were used to measure the concentrations of several bile acids in faecal water, as well as to determine the concentrations of carote-noids and malondialdehyde (MDA) in faecal samples. The concentrations of the major short chain fatty acids (SCFA) were measured via gas chromatography. Consumption of carrot juice led to a marked increase of beta-carotene and alpha-carotene in faeces and in non-filtered faecal water, as did lycopene after consumption of tomato juice. In the succeeding depletion phases, the contents of those carotenoids in faeces and faecal water returned to their initial values. Changes in faecal MDA concentrations by carrot and tomato juice interventions could not be observed. Faecal water showed high, dose-dependent cytotoxic effects on HT-29 cells. Those effects were, however, not markedly changed by carrot and tomato juice consumption. Neither bile acid concentrations nor the bile acid profile in faecal water changed after carrot and tomato juice consumption. Bacterial activities of beta-glucosidase and beta-glucuronidase also did not change. While tomato juice consumption did not significantly affect the pH value of faecal water, this value was, however, decreased by carrot juice consumption. Although faecal water concentrations of acetate and butyrate contributed to the decrease in faecal water pH values, SCFA were probably not responsible for the observed pH changes after carrot juice consumption. SCFA concentrations in faecal water and SCFA proportions did not change significantly. Neither bile and SCFA concentrations, nor the activities of tested bacterial enzymes, had any influence on the cytotoxic effects of sterile-filtered faecal water. These cytotoxic effects, however, decreased with increasing proportions of the primary bile acids cholic and chenodesoxycholic acid, independent of the study phases. As determined by multiple regression analysis, the most probable leading factors for the growth inhibitory effects of faecal water are the faecal MDA content and bacterial beta-glucosidase activity. Further studies should investigate whether the parameters mentioned directly influence cytotoxic and antiproliferative effects of faecal water or if those parameters are indirect markers for the activity of individual microflora. Carrot and tomato juice consumption strongly increased the cytotoxic effects of faecal lipid extracts in HT-29 cells, likely caused by the induction of apoptosis. Which mechanisms account for these effects and the consequences of these effects in the in vivo situation should be investigated in further studies. This work shows that two-week interventions with carotenoid-rich juices lead only to minor changes in luminal processes relevant to colon carcinogenesis in young healthy volunteers on an energy- and macronutrient-balanced diet. Lacking effects on 1) the toxic and antiproliferative properties of faecal water, 2) lipid peroxidation in faeces, 3) the bile and SCFA concentrations in faecal water, and 4) bacterial enzyme activities indicate that related physiological effects can not be influenced by a diet rich in carotenoids under the just described conditions. Other anticarcinogenic mechanisms seem to be of greater importance.
Mechanismen der Mastzellaktivierung durch gram-negative Bakterien und Bakterienprodukte aus der Darmflora.
(2006) Krämer, Sigrid; Bischoff, Stephan C.
The role of mast cells (MC) as effector cells in IgE dependent processes like the type 1 allergy has been known for a long time. During the decade, it has been shown that MC are also involved in other pathophysiological processes such as mucosal polyposis, rheumatoid arthritis, inflammatory bowl disease, tissue fibrosis, and atherosclerosis. Furthermore, MC play an important role in the regulation of host defense against microbes, tissue remodeling processes, and neuro-immunology-interaction. The first aim of the present study was to clarify the question whether human intestinal MC express toll-like receptors (TLR), which recognize conserved bacterial and viral components, and can MC be activated through TLR-ligands. The second major focus of the present study was to investigate if the stimulation of human intestinal MC with different E. coli and Shigella strains, respectively, results in an activation of MC and to identify the underlying mechanism(s). Accordingly, human intestinal MC were isolated from surgery tissue with a mechanical and enzymatical protocol. The purity of the MC cultures used in all experiments was between 98 and 100% which was achieved by positive selection (MACS). We could show, that human intestinal MC express mRNA for TLR 1, 2, 3, 4, 5, 6, 8, and 9. However, neither the stimulation with LPS (lipopolysaccharide, TLR 4 ligand), LTA (lipoteichoic acid, TLR 2 ligand), Zymosan (TLR 2 ligand), poly I:C (polyinosinic-polycytidylic acid, TLR 3 ligand), R848 (TLR 7/8 ligand), CpG (C poly G oligo-desoxy-nucleotide, TLR 9 ligand) and non CpG, respectively resulted in a release of histamine, leucotriens, TNF-alpha, or IL-8. Furthermore, mRNA expression levels of TNF-alpha and IL-8 were not induced by any of the treatments. Similar results where found when human intestinal MC were stimulated with E. coli (O101:H-) isolated from human faeces or the probiotic strain E. coli Nissle 1917. Even after stimulation with pathogenic bacteria strains such as the invasive S. flexneri M90T and the fimbriated E. coli, respectively, no induction of any of the parameters mentioned above was found. However, E. coli strains activate the intracellular signal molecule and transcription factors ERK1/2, c-Fos, and AP1, but this activation failed to induce a complete immune answer. In contrast, the hemolytic E. coli stains ATCC 25922 and ATCC 35218 provoked strong activation of intestinal MC. Using the isogenic hemolysin negative E. coli mutants and the hemolysin positive transformants of the probiotic E. coli Nissle 1917 it was shown, that human intestinal MC are sensitive target cells for E. coli alpha hemolysin. Stimulation of MC with sublytic concentration of hemolysin resulted in an induction of TNF-alpha, IL-3, IL-5, IL-6, IL-8 mRNA expression, the release of histamine as well as leucotrien. This activation was found to be regulated by calcium dependent signal cascades. Inhibition of intracellular signal molecules showed that the activation depends on L-typ calcium channels, calcineurin, NFAT and NFkappaB. Prolonged infection with hemolytic E. coli strains resulted in lysis of intestinal MC indicating a biphasic activation of hemolysin.
Die kleine GTPase RagA und der potentielle Pankreastumormarker TCTP, zwei Bindungspartner des Kerntransport-Proteins RanBP3
(2006) Schleicher Lilia; Biesalski, Hans-Konrad
RanBP3 is a component of the Crm1-mediated protein export from the nucleus. It favors the binding of export substrate to the RanGTP-Crm1 complex in the nucleoplasm. Upon translocation through the nuclear pore into the cytoplasm, the export complex is dissociated by RanBP1 and RanGAP. It was the objective of this thesis to advance the understanding of RanBP3 function by identifying novel binding partners. Using Yeast-Two-Hybrid Screening, two new interactors of nuclear protein RanBP3 were found, the small GTPase RagA and the translationally controlled tumour protein (TCTP). As a minimal requirement a short C-terminal part of RagA (residues 275-313) binds to the N-terminal region of RanBP3. Full length recombinant RagA was expressed. To substantiate the two-hybrid interactions, the binding of RagA to RanBP3 was verified by in vitro binding assays using recombinant proteins. RagA competes with exportin Crm1 for binding to RanBP3. Likewise, the nucleotide exchange factor for Ran, RCC1, binds to the RanBP3-Ran complex and inhibits RagA binding. Expression of GFP-fusion proteins established the cellular localisation of RagA in living and fixed cells. It is found predominantly in and at the nucleus. A model of RagA-import and -export is suggested. Residues 100-172 in the C-terminal part of TCTP form the binding site for its interaction with RanBP3. Using Northern Blot analysis, two mRNAs of TCTP were found in all human tissues examined. The TCTP sequence was completed using an ovarian cDNA library and the TCTP was cloned and expressed. By means of quantitative PCR it was shown that TCTP belongs to the small group of very abundant mRNA molecules in the cell. The TCTP-RanBP3 interaction was verified by in vitro binding assays using recombinant proteins. A polyclonal antibody to TCTP was generated. By using a GFP-TCTP fusion protein and anti-TCTP antibody the cellular localisation of TCTP was analysed. In Cos7-cells TCTP is found in and around the nucleus. In human cell lines most of the TCTP is localised at the nuclear membrane. Using specific anti-TCTP antibody it was shown that the TCTP level is increased in pancreatic tumours. A differential diagnosis of pancreatitis and pancreas tumour is feasible. TCTP has the potential to become a pancreatic tumour marker or a prognostic factor for ductal type adenocarcinomas of the exocrine pancreas.
Einfluss kurzkettiger Fettsäuren und mikrobieller Fermentationsprodukte neuartiger Oligosaccharide auf Cytotoxizität, Proliferation und Apoptose von humanen Coloncarcinom-Zelllinien
(2006) Roser, Silvia; Rechkemmer, Gerhard
Colon cancer is the second most common cancer in Germany. The role of dietary fibre in the prevention of colon cancer is still controversial: Promising results from in vitro and animal studies are contradictory to inconsistent results from epidemiological stu-dies. Functional carbohydrates as constituents of prebiotic food can modify the colonic microflora for the benefit of short chain fatty acid (SCFA)-producing microbial strains. The SCFA-concentrations should also be increased in the distal part of the colon where most colon carcinomas are developing. SCFA are considered to be preventive against colon cancer. For this study, three different new functional oligosaccharides (OS, made of Isomaltulose and resistant starch) were produced from the Südzucker company and fermented in vitro with human feces of healthy test subjects. The resulting fermentation supernatants (FS) were tested in a cell culture system, using colon carcinoma cell lines of various degrees of differentiation (HT29, HT29 Clone 19A, T84). Cytotoxicity, proliferation, the induction of apoptosis, influences on the cell cycle and electrophysiological parameters were measured. Spectral photometric and flow cytometric methods were performed, as well as measurements in vertical diffusion chambers (Ussing chambers). The parallel testing of SCFA-mixtures with the same SCFA-concentrations as in the FS was included, as well as the testing of a FS ?Control? which was produced without OS-fermentation. Several independent fermentations revealed reproducible results regarding the SCFA-concentrations of the FS. After OS-fermentation, the ratio of the three major SCFA in the FS, acetate, propionate, and butyrate, was similar to that observed in vivo. The FS and SCFA-mixtures tested had a cytotoxic effect on all cell lines at the con-centration of 50 %. A dose dependent decrease in cell proliferation could be found, as well as the induction of apoptosis at a concentration of 50 %. Parallel testing of the analogous SCFA-mixtures showed that cytotoxic and proliferation inhibiting effects of the FS could be primarily attributed to their SCFA-content. This could not be confirmed for apoptosis induction: the SCFA-mixtures were mostly able to induce a higher apoptosis rate than the FS. Similarly, the effects of FS and SCFA-mixtures on the cell cycle were different: The SCFA-mixtures showed more potent inhibition of DNA-synthesis than the analogous FS, which generally led to an arrest in the G2-phase of the cell cycle. Neither FS nor SCFA-mixtures had an impact on transepithelial resistance or short circuit current of differentiated cell monolayers in Ussing chambers. The difference in the fermentation patterns of the various FS and the SCFA-concentrations of the SCFA-mixtures was not great enough to achieve significantly different results in the test systems used. Also, the various differentiation grades of the cell lines showed inconsistent results after treatment with FS and their SCFA-mixtures, so that no correlation could be found between degree of differentiation and test compound action. This study shows that the in vitro fermentation of OS with human feces results in reproducible SCFA-patterns in the FS, similar to the in vivo situation. For the screening of FS and their SCFA-mixtures, respectively, a spectrum of methods was established for the incubation with colon carcinoma cell lines of various differentiation states and of all stages of growth (exponential, subconfluent, confluent, fully differentiated monolayer). Indeed, the effects measured after incubation with FS could only in part been ascribed to their SCFA content. Other FS components than SCFA that play a role, especially regarding to their apoptosis inhibiting and cell cycle influencing effects, remain to be identified. Also, this study allows no conclusions to be drawn, which of the fermented OS is more promising in it?s beneficial influence on colon cancer preventing factors, e.g. the induction of apoptosis, than the other. Future studies should investigate FS with greater differences in their SCFA-concentrations. The same OS which were used for the in vitro fermentation, should also be tested in animal studies and human intervention studies to elucidate their fermentation patterns in vivo.
Transepitheliale Stimulation humaner Leukozyten durch Bakterien und ihre Oberflächenbestandteile
(2007) Bäuerlein, Annette; Parlesak, Alexandr
Background: The intestinal mucosa plays an important role in the discrimination of immune response between pathogenic and non-pathogenic bacteria as well as in mediating the systemic immunity. To adress the question whether probiotic, commensal, pathogenic germs and bacteria of food origin as well as their membrane components modify the immune response of the intestinal mucosa, we co-cultivated enterocyte-like CaCo-2 cells with human blood leucocytes in transwell cultures. Of further interest was the sequence of enteroxyte-leukocyte activation. Methods: PBMC (Peripheral blood mononuclear cells) were stimulated transepithelially in CaCo-2/PBMC co-cultures and directly challenged with probiotic, commensal, enteropathogenic and food-originating bacteria as well as with membrane components of grampositive and gramnegative bacteria. The expression of inflammatory cytokines (TNF-α, IL-8, IL-6, IL-10 and IFN-γ) was studied by enzyme linked immunosorbant assay (ELISA). The ratio of IL-8/18S mRNA was detected using quantitative reverse transcription polymerase chain reaction (qRT PCR). The permeation of endotoxin was quantified via Limulus amobocyte (LAL) assay and the integrity of the CaC-2 cell monolayer was detected via fluorescein-dextran and transepithelial electric resistance (TEER). Results: Grampositive bacteria did not activate immunocompetent cells in leukocyte-enterocyte co-cultures whereas a stimulation with the gramnegative probiotic E. coli Nissle resulted in higher expressions of TNF-α, IL-8, IL-6, IL-10 and IFN-γ than stimulation with the enteropathogenic E. coli (EPEC). The feature ?probiotic? results not necessarily in an enhanced production of inflammatory cytokines. Differences in epithelial permeability were not necessarily associated with an enhanced activation of immunocompetent cells. There was no activation of immunocompetent cells after direct or transepithelial stimulation with lipoteichoic acid (LTA) of E. faecalis. In contrast, endotoxin depending on its structure was a very potent (E. coli K12, E. coli Nissle, S. Typhimurium) or a moderately potent (B. vulgatus, B. vulgatus MPK) inducer of an inflammatory cytokine response. A neutralisation of endotoxin permeating into the basolateral compartment with Polymyxin B and Colisin resulted in a nearly total inhibition of inflammatory response. Furthermore, directly stimulated PBMC with comparable amounts of the permeating endotoxin in CaCo-2/PBMC co-cultures showed the same activation status as transepithelially stimulated cells. The probiotic, nonstimulating bacteria (Lb. rhamnosus GG, B. vulgatus, B. bifidum) were not able to reduce the E. coli K12 induced TNF-α, IL-8, IL-6, IL-10 and IFN-γ production. A co-stimulation of LPS from E. coli K12 (1 µg/ml) with non-stimulating endotoxins (B. vulgatus and B. vulgatus MPK) 100 µg/ml tended to reduce cytokine expression. Conclusion: These results show that the attribute ?probiotic?does not result in an obligatory activation of immunocompetent cells. The ability to stimulate immunocompetent cells is preferentially dependent on the presence of endotoxin, regarding the structure which is responsible for the stimulating capacity and is not mediated by enterocytes in first line. Activation of the basolaterally located lymphocytes occurs via permeating endotoxin. The permeability of the intestinal epithelial layer is only relevant when permeating endotoxin is able to stimulate immunocompetent cells to due its structural features.
Probiotic bacteria enhance the antibacterial barrier of enterocytes: insights into their mechanism of action
(2007) Schlee, Miriam; Bode, Christiane
In the healthy intestine there is a stable balance of luminal bacteria and host factors to prevent infections or inflammatory bowel diseases (IBD). A complex network of environmental, genetic, and immunoregulatory factors may precipitate the onset of ulcerative colitis (UC) and Crohn's disease (CD), the primary manifestations of inflammatory bowel disease (IBD). It is currently believed that IBD results from an aberrant immune response of the intestinal mucosa towards the normal commensal bacterial flora. Alternatively, a primary defect in the mucosal barrier might permit bacterial invasion and trigger inflammation. In our research group the hypothesis was proposed that the defective barrier in Crohn´s disease may be due to a lack of defensins which form a chemical barrier against luminal bacteria. A major gut defensin is the human beta defensin-2 (hBD-2) which is an inducible antimicrobial peptide synthesized and secreted by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. In recent studies, Fellermann et al demonstrated that the defective expression of hBD-2 which was measured in the gut mucosa of patients with Crohn´s disease was due to a reduced copy number of the hBD-2 gene. In patients with ulcerative colitis beta-defensin expression is low in the colon during remission, but readily inducible during inflammation. Probiotic bacteria might act beneficially in the human gut by inducing the expression of defensins and thereby reinforcing the mucosal barrier. Recently, our group has been the first to describe hBD-2 induction by the probiotic strain E. coli Nissle (Mutaflor®) which is an effective treatment for ulcerative colitis during remission. The aim of the present work was to determine the underlying molecular mechanisms. We determined a time- and dose-dependent expression pattern of hBD-2 in Caco-2 cells upon stimulation with IL-1 beta;, E. coli Nissle culture supernatant and diverse other probiotic strains. We further investigated the transcriptional regulation of hBD-2 expression mediated by probiotics. The hBD-2 promoter contains several elements known to be involved in transcriptional upregulation such as the NF-kappa B element, which is believed to be one of the main regulators of the hBD-2 gene expression. However, for certain signals, the expression of the hBD-2 gene has been reported to depend on the activation of a second transcription factor, such as AP-1. Most importantly, E. coli Nissle was shown to shed or secrete factors, contained in the bacterial supernatant, which were sufficient to trigger activation of NF-kappa B and AP-1 and to induce hBD-2. Our results indicated further that the supernatant-induced activation of the MAP kinase pathways ERK½, JNK, and p38 may be directly responsible for the probiotic supernatant-induced activation of the transcription factors AP-1 and NF-kappa B and subsequent synthesis of hBD-2. A further aim of the present study was to identify and isolate the bacterial components which are responsible for E. coli Nissle mediated hBD-2 induction. As E. coli Nissle culture supernatant was found to be a more potent stimulant than the bacterial pellet, we investigated the characteristics of the unknown soluble or shed molecules in the bacterial culture media. The first analysis revealed the factor as a heat resistant and proteinase sensitive molecule. Both, E. coli Nissle specific lipopolysaccharide (LPS) and bacterial DNA, which might contain immunostimulatory DNA motifs, failed to trigger hBD-2 expression. Based on the knowledge of the surface composition several E. coli Nissle deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin, the flagella filament protein, specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Next, we isolated flagellins from different bacteria strains (Salmonella enterica serovar Enteritidis, E. coli ATCC 25922, E. coli Nissle and the uropathogenic E. coli strain CFT073 Delta hly, whose genome structure resembles closely that of E. coli Nissle). In the Western blot anti-H1 flagellin displayed immunoreactivity against the different types of flagellins, due to the highly conserved central region of the flagellin filament structure. Incubation of Caco-2 cells with isolated E. coli Nissle flagellin (molecular size 60.81 kDa) induced hBD-2 promoter activation in a dose-dependent manner. The induction of hBD-2 expression by flagellin was confirmed with a positive control (Salmonella flagellin). Interestingly, the serotype-identical CFT073 Delta hly flagellin expressed only moderate hBD-2 inducing ability compared to E. coli Nissle flagellin. Thus, differences in extracellular matrix e.g. the glycosilation degree might underlie the differentially modulated hBD-2 response of Caco-2 cells by the two flagellins. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle. In conclusion, flagellin of E. coli Nissle provides reinforcement of mucosal antimicrobial function, apparently without inducing inflammation. This might explain the beneficial effects of E. coli Nissle on remission maintenance in ulcerative colitis. In patients with Crohn´s disease there is evidence against a therapeutic effect of probiotics and this may be explained by a defective defensin system. Future investigations about strain-specific beneficial functions might contribute to the therapeutic application of science-based probiotic products.
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.
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.
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.
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.
Nuclear activation of proteasome in oxidative stress and aging
(2009) Catalgol, Betul; Grune, Tilman
Poly(ADP-ribosyl)ation reactions are of interest in recent years and they take place in DNA repair in different processes especially following oxidative nuclear damage. Proteasomal reactions also take place in repair following oxidative nuclear damage with the degradation of oxidized histones. Antitumor chemotherapy is generally believed to act via the oxidation of nuclear material in the tumor cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the nucleus. Tumor cells are supposed to have a higher nuclear proteasome activity than do nonmalignant cells. Poly(ADP-ribosyl)ation reactions take place in the tumor cells as a consequence of chemotherapy. Such a reaction might occur with the 20S proteasome ?which is known to increase the activity- and also with histones ?which is firstly shown to decrease the degradation in this study. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increased accompanied by an increase in PARP-1 mRNA expression. In the recovery of the level of protein carbonyls, single strand breaks and 8-OHdG, proteasome and PARP-1 were shown to play a role together. This was tested with inhibitor treatments. The proteasomal activation following poly(ADP-ribosyl)ation of proteasome and the decrease in poly(ADP-ribosyl)ation of histones and increase in the proteasomal degradation of histones following H2O2 treatment confirmed our hypothesis. The second part of the thesis shows the changes in PARP-1 and proteasome in different aged fibroblasts with population doublings 19, 36, and 56. The nuclear protective mechanisms were shown to be effected during the senescence process. PARP-1 protein amount decreased whereas there was no change in proteasome amount. PARP activation following H2O2 treatment increased only in young and middle aged cells. In the nuclear extracts of young and old cells, poly(ADP-ribosyl)ation potentials were tested with NAD+ addition into the reaction. In addition to that active proteasome and PARP enzymes were added into the reaction and proteasome activity was measured. With active PARP, proteasome activity was increased both in young and old cells whereas there was no increase in old cells without PARP addition. These results show that proteasome activation is mainly limited by PARP activity. Taken together all results demonstrate the importance of PARP mediated proteasome activation in the repair of oxidatively damaged chromatin.
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.
Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907
(2010) Fritsche, Louise; Schleicher, Erwin D.
Insulin receptor substrate (IRS) proteins are major transducers of the insulin and IGF-1 signal into the PI-3 kinase/PKB and the MAP kinase pathway. In addition to tyrosine phosphoryla-tion, a large number of serine/threonine phosphorylation sites enable the IRS proteins to inte-grate different extra- and intracellular stimuli resulting in positive and negative modulation of the insulin and IGF-1 signal. Chronic hyperphosphorylation of serine/threonine sites of IRS-1 is involved in the development of insulin resistance. IRS-2 is of great importance for β-cell survival and for the regulation of hepatic metabolism. The study of serine/threonine phos-phorylations is required to understand the physiological and pathophysiological regulation of this important mediator of insulin signaling. In this thesis two novel IRS-2 serine phosphoryla-tion sites have been identified and characterized (mouse amino acid numbering): Ser 675, which is located in the kinase regulatory loop binding (KRLB) domain unique to IRS-2 and Ser 907, which is adjacent to the Grb2 binding site Tyr 911. Using phospho-site specific antibod-ies both sites were demonstrated to be phosphorylated upon insulin, phorbol ester and ani-somycin treatment in Fao rat hepatoma cells. The phosphorylation was also detected in pri-mary human hepatocytes and in liver tissue of insulin treated or refed mice. The insulin-induced phosphorylation of Ser 907 was mediated by the MAP kinase ERK1/2. Simulation of a permanent phosphorylation of this site in BHK cells expressing IRS-2 Glu 907 led to a slight decrease of IRS-2 tyrosine phosphorylation with no apparent effect on insulin downstream signaling. The insulin-induced association of IRS-2 with Grb2 in HEK293 cells was abrogated by mutation of the adjacent Tyr 911 to Phe, but not influenced by mutation of Ser 907 to Ala. Of note, the activation of MAP kinase signaling was not impaired in HEK293 cells expressing IRS-2 Phe 911 and not regulated by the expression level of IRS-2 wildtype, but completely dependent on IR expression, indicating the importance of an alternative, IRS-2-Grb2-independent pathway for the activation of MAP kinase signaling in these cells. The insulin-induced phosphorylation of Ser 675 was dependent on mTOR, but not on the downstream kinase p70 S6K1. Prevention of this phosphorylation in BHK cells or HEK293 cells expressing IRS-2 Ala 675 had no effect on proximal or distal insulin signal transduction. But compared with IRS-2 wildtype, the mutated IRS-2 protein Ala 675 showed increased half life in cycloheximide-treated HEK293 cells. Thus, phosphorylation of Ser 675 could have a similar function as its homologous site Ser 632 in IRS-1 and could be involved in the regula-tion of mTOR-dependent IRS-2 proteasom-mediated protein degradation.
Glucocorticoid-induziertes Wachstum von Tumorzellen : systematische Quantifizierung, Signalmechanismen und Inhibition
(2010) Gündisch, Sibylle; Jeremias, Irmela
Glucocorticoids (GCs) like Dexamethasone (Dex) are widely used in cancer patients, as cytotoxic drugs in hematopoetic tumors or adjuvants in solid tumors to reduce severe side effects. Nevertheless, GCs are accused to reduce anti-cancer treatment efficiency. Due to preliminary works in our research group the suspicion arose that GCs are able to induce proliferation of tumor cells. The present work provides the first systematic quantification of the proproliferative effects of GCs on tumor cells. Enhanced tumor cell growth was validated by repetitive microscopy, impedance analysis, investigation of DNA synthesis rate, enzymatic activity as well as absolute cell number. It could be proven that 6 out of 10 cell lines from solid tumors showed enhanced proliferation after stimulation with Dex, whereas this phenotype was not limited to one tumour entity or a common origin. In vivo, Dex significantly promoted tumor cell growth in a preclinical mouse model with a lung carcinoma cell line. Furthermore the effect of GCs was detected on 139 primary, patient-derived acute childhood leukemia cells. In 15% GCs were able to increase the in vitro survival of the tumor cells and one sample showed even GC-induced proliferation. Accordingly the anti-apoptotic and pro-proliferative effects of GCs could be proven not only on established solid tumor cell lines but also on primary hematopoetic tumor cells. Knockdown studies in cells of solid tumors showed that GC-induced proliferation was mediated by the glucocorticoid receptor and was further transmitted by the proteinkinases Akt and p38-MAPK. GC-induced proliferation could be prevented by induction of apoptosis which was caused either by clinically applicable substances, as for example Vincristine, or by inducible expression of the pro-apoptotic molecule Caspase-3. To sum up, the present work identified GC-induced proliferation of tumor cells as a new, tumor cell directed side effect of GCs. Of direct translational relevance, our data argue towards a restricted use of GCs during anti-cancer therapy as well as the need for preclinical and clinical studies which demonstrate a more effective and safer application of GCs during anti-cancer therapy.
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.
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.
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.

Browse

Browsing Institut für Ernährungswissenschaften by Issue Date