Browsing by Subject "Glucocorticoids"

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    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.
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    Der Glucocorticoidrezeptor des Schweins: Herstellung und Charakterisierung eines polyklonalen Antiserums, sowie Studien zur Verteilung des GCR im Intestinaltrakt von Ebern und Kastraten
    (2002) Gutscher, Monika; Claus, Rolf
    Glucocorticoids are well known to be essential for many physiological and developmental processes. Such functions include their effects on carbohydrate and protein metabolism and their regulatory influences on the immune system. In cell regulation they play a dose-dependent key role for differentiation and apoptosis. In rapidly renewing tissues the stringent control of these mechanisms is central to the maintenance of tissue homeostasis. ln the gastrointestinal tract both the adaption to changing nutrients and the presentation with a vast array of different types of antigens, including potential pathogens and harmless dietary antigens requires a granular regulation of cell proliferation, differentiation and cell death. In the pig, the differences in the turn-over rate for instance between skeletal muscle and the gut tissue could be attributed to different GCR concentrations respectively. This explains the tissue specific sensitivty on circulating corticoids. Thus studies on GCR distribution contributes to the clarification of the role of glucocorticoids in the regulation of these mechanisms in the intestinal tract. In the pig, so far receptor detection has been performed by radio ligand binding assays, which only measures steroid unoccupied non-activated receptors in the cytoplasm. Selective GCR antibodies react with both occupied and unoccupied GCR. In addition, antibodies enable celltype specific detection of the GCR in complex tissues by immunocytochemistry. The aim of this investigation was the production of porcine GCR-specific polyclonal antibodies by detailed analysis of the cDNA sequence of the GCR and the recombinant expression of a suitable antigen fragment. A fragment with 2.1 kb of the GCR cDNA (gcr2.1) was sequenced. Based on Blast sequence analysis a GCR antigen fragment for recombinant expression was selected from the modulatory region (GCRmr) and cloned in a T7-expression system as a His-tag fusion protein. After affinity chromatographic und preparative purification The anti-pGCR-antibodies bind the pGCRmr antigen with high affinity, as well as the denatured receptor in western blot analysis. In additon, immunoprecipitation assays demonstrated that cytosolic GCR is recognized regardless of whether it is unoccuppied or occuppied with dexamethasone. Thus, the antiserum is able to bind the native GCR both in its inactivated form as a multiprotein complex in association with HSP90 and in its activated form with shed HSP 90. Our investigations with immunoprecipitation assays support the applicability of the anti-pGCR antiserum in immunohistochemistry. The characterized antibodies were implemented in immunohistochemy for studies of distribution and localization of the GCR in the small bowl and colon of boars and barrows. The intracellular distribution of the GCR was examined by western blot assays. Immunohistochemical studies showed an increased number of immunostained GCR in the colon compared with the small intestine, as has been shown earlier with ligand-binding assays. 32,9 % and 14,5 % of the cells of the lamina propria were GCR immunoreactive in the small intestine of barrows and boars. In the colon 49,3 % and 43,3 % showed immunostaining. Epithelial cells showed a reversed pattern compared to the lamina propria in both groups. The number of GCR immunoreactive cells in barrows and boars decreased from 9,6 % and 9 % in the small intestine to 5,4 and 5,6 % in the colon, respectively. Comparison of both groups ? barrows and boars - revealed significant differences in the number of GCR immunoreactive cells in the lamina propria of the small bowl. Boars showed a decreased GCR expression of 10 % in the duodenum and 30 % in the jejunum. The number of GCR immuostained colonic cells amounts to 36,9 % in the colon ascendens and 49,2% in the colon descendens of boars and 47,5 % and 51 % in barrows. Studies of the subcellular localization by western blot analysis of cytosolplasmic and nuclear proteins demonstrated that in both groups in the ileum a higher amount of GCR was translocated into the nucleus. In the colon the number of cytoplasmic GCR was higher. The different subcellular GCR distribution in the two segments of the intestine can be explained by the increased expression of 11â-hydroxysteroid dehydrogenase 2 in the colon. 11â-HSD 2 inactivates cortisol and thus inhibits receptor activation and thereby translocation to the nucleus.
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    Steuerungsmechanismen der Spermatogenese beim Eber: Auswirkungen einer GnRH-Immunisierung und anschließender Estradiolinfusion
    (2005) Wagner, Anna; Claus, Rolf
    Whereas the regulation of spermatogenesis is well known in various species, specific differences, however, exist for the boar, which require species-specific investigation. High concentrations of estrogens are synthesized in the boar testis. It is known, that they play a synergistic role for male behaviour and function of accessory sex glands. Additionally, estrogens in the ejaculate reach the female genital tract and contribute to sperm transport and ovulation in the sow. A possible involvement of estrogens in spermatogenesis, however, was not investigated so far. Similarly, high concentrations of glucocorticoids occur in the tubuli of mature boars. Their regulatory function in spermatogenesis so far is unknown. For clarification, boars were actively immunized against GnRH. GnRH stimulates gonadotropin-release from the pituitary. The evaluation of hormone concentrations in blood plasma after GnRH immunization showed unexpectedly, that the gonadotropin FSH is not influenced, whereas LH- and in consequence steroid- (androgen and estrogen) concentrations were abolished. This offers the possiblity to characterize the selecitve function of estrogens in the presence of FSH. Three groups were evaluated: intact boars (n=5), immunized boars (n=5), 17ß-estradiol infused, immunized boars (n=6). All animals were fitted with indwelling cephalic vein catheters on both sides. One of them was reserved for infusion, the other one for uncontaminated blood sampling. All animals were infused with physiological saline over 7 weeks. For the 17ßEstradiol infused, immunized boars, a defined concentration of estradiol was added to the solution. Blood samples were collected over the whole 7-week period for LH, FSH, testosterone and estradiol. Blood samples were later analysed by established radioimmunological procedures. At the end of the 7-week period, all animals were killed. Tissue samples were fixed for histological examination. Morphological criteria included e.g. tubular diameter, seminiferous epithelial height and number of Leydig cells. Imunocytochemical evaluation was performed for mitosis (Ki-67), apoptosis (TUNEL) and the glucocorticoid receptor. The number of germ cell in the tubuli was reduced by 60% in immunized boars compared to the intact controls. These changes were explained by a more pronounced expression of the glucocorticoid receptor in spermatogonia, followed by the appearance of apoptosis in spermatogonia and primary spermatocytes. In consequence these findings demonstrate for the first time an involvment of glucocorticoids in the quantitative regulation of spermatogenesis. They counteract tubular mitosis by reducing the number of germ cells by apoptosis. In the 17ßestradiol infused group of immunized boars, the infusion led to the restoration of physiological estradiol concentrations (230 pg/ml) in peripheral blood plasma. As expected, androgen concentrations were minimal, so that the specific role of estrogens for spermatogenesis could be clarified. Data from the histological evaluation showed, that the expression of the estrogen receptor alpha occured in spermatogonia, selectively in the mitotic active stage of the seminiferous epithelial cycle leading to a 41-50% rise of germ cell numbers compared to immunized boars. The apoptotic rate in the tubuli of the estradiol infused boars however was not changed compared to immunized boars, so that physiological sperm numbers as they are characteristic for intact boars, were not reached. This study shows for the first time that both glucocorticoids and estrogens play an essential role for spermatogenesis in the boar. They are involved in the regulation of sperm yield by influencing the mitosis-apoptosis equilibrium in the tubuli of boars.