TY - JOUR A1 - Kiener, Mirjam A1 - Chen, Lanpeng A1 - Krebs, Markus A1 - Grosjean, Joȅl A1 - Klima, Irena A1 - Kalogirou, Charis A1 - Riedmiller, Hubertus A1 - Kneitz, Burkhard A1 - Thalmann, George N. A1 - Snaar-Jagalska, Ewa A1 - Spahn, Martin A1 - Kruithof-de Julio, Marianna A1 - Zoni, Eugenio T1 - miR-221-5p regulates proliferation and migration in human prostate cancer cells and reduces tumor growth in vivo JF - BMC Cancer N2 - Background Despite latest advances in prostate cancer (PCa) therapy, PCa remains the third-leading cause of cancer-related death in European men. Dysregulation of microRNAs (miRNAs), small non-coding RNA molecules with gene expression regulatory function, has been reported in all types of epithelial and haematological cancers. In particular, miR-221-5p alterations have been reported in PCa. Methods miRNA expression data was retrieved from a comprehensive publicly available dataset of 218 PCa patients (GSE21036) and miR-221-5p expression levels were analysed. The functional role of miR-221-5p was characterised in androgen- dependent and androgen- independent PCa cell line models (C4–2 and PC-3M-Pro4 cells) by miR-221-5p overexpression and knock-down experiments. The metastatic potential of highly aggressive PC-3M-Pro4 cells overexpressing miR-221-5p was determined by studying extravasation in a zebrafish model. Finally, the effect of miR-221-5p overexpression on the growth of PC-3M-Pro4luc2 cells in vivo was studied by orthotopic implantation in male Balb/cByJ nude mice and assessment of tumor growth. Results Analysis of microRNA expression dataset for human primary and metastatic PCa samples and control normal adjacent benign prostate revealed miR-221-5p to be significantly downregulated in PCa compared to normal prostate tissue and in metastasis compared to primary PCa. Our in vitro data suggest that miR-221-5p overexpression reduced PCa cell proliferation and colony formation. Furthermore, miR-221-5p overexpression dramatically reduced migration of PCa cells, which was associated with differential expression of selected EMT markers. The functional changes of miR-221-5p overexpression were reversible by the loss of miR-221-5p levels, indicating that the tumor suppressive effects were specific to miR-221-5p. Additionally, miR-221-5p overexpression significantly reduced PC-3M-Pro4 cell extravasation and metastasis formation in a zebrafish model and decreased tumor burden in an orthotopic mouse model of PCa. Conclusions Together these data strongly support a tumor suppressive role of miR-221-5p in the context of PCa and its potential as therapeutic target. KW - prostate cancer KW - miR-221-5p KW - proliferation KW - migration KW - tumor suppressor miRNA Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325762 VL - 19 ER - TY - JOUR A1 - Koderer, Corinna A1 - Schmitz, Werner A1 - Wünsch, Anna Chiara A1 - Balint, Julia A1 - El-Mesery, Mohamed A1 - Volland, Julian Manuel A1 - Hartmann, Stefan A1 - Linz, Christian A1 - Kübler, Alexander Christian A1 - Seher, Axel T1 - Low energy status under methionine restriction is essentially independent of proliferation or cell contact inhibition JF - Cells N2 - Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy. KW - methionine restriction KW - caloric restriction KW - mass spectrometry KW - LC/MS KW - liquid chromatography/mass spectrometry KW - metabolomics KW - L929 KW - amino acid KW - proliferation KW - contact inhibition Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262329 SN - 2073-4409 VL - 11 IS - 3 ER - TY - JOUR A1 - Dapergola, Eleni A1 - Menegazzi, Pamela A1 - Raabe, Thomas A1 - Hovhanyan, Anna T1 - Light Stimuli and Circadian Clock Affect Neural Development in Drosophila melanogaster JF - Frontiers in Cell and Developmental Biology N2 - Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. Here we demonstrate that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, we show that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, we define that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain. KW - neuroblast growth KW - proliferation KW - circadian clock KW - light stimuli Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231049 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Gabbert, Lydia A1 - Dilling, Christina A1 - Meybohm, Patrick A1 - Burek, Malgorzata T1 - Deletion of Protocadherin Gamma C3 Induces Phenotypic and Functional Changes in Brain Microvascular Endothelial Cells In Vitro JF - Frontiers in Pharmacology N2 - Inflammation of the central nervous system (CNS) is associated with diseases such as multiple sclerosis, stroke and neurodegenerative diseases. Compromised integrity of the blood-brain barrier (BBB) and increased migration of immune cells into the CNS are the main characteristics of brain inflammation. Clustered protocadherins (Pcdhs) belong to a large family of cadherin-related molecules. Pcdhs are highly expressed in the CNS in neurons, astrocytes, pericytes and epithelial cells of the choroid plexus and, as we have recently demonstrated, in brain microvascular endothelial cells (BMECs). Knockout of a member of the Pcdh subfamily, PcdhgC3, resulted in significant changes in the barrier integrity of BMECs. Here we characterized the endothelial PcdhgC3 knockout (KO) cells using paracellular permeability measurements, proliferation assay, wound healing assay, inhibition of signaling pathways, oxygen/glucose deprivation (OGD) and a pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) treatment. PcdhgC3 KO showed an increased paracellular permeability, a faster proliferation rate, an altered expression of efflux pumps, transporters, cellular receptors, signaling and inflammatory molecules. Serum starvation led to significantly higher phosphorylation of extracellular signal-regulated kinases (Erk) in KO cells, while no changes in phosphorylated Akt kinase levels were found. PcdhgC3 KO cells migrated faster in the wound healing assay and this migration was significantly inhibited by respective inhibitors of the MAPK-, β-catenin/Wnt-, mTOR- signaling pathways (SL327, XAV939, or Torin 2). PcdhgC3 KO cells responded stronger to OGD and TNFα by significantly higher induction of interleukin 6 mRNA than wild type cells. These results suggest that PcdhgC3 is involved in the regulation of major signaling pathways and the inflammatory response of BMECs. KW - blood-brain barrier KW - protocadherin gamma C3 KW - inflammation KW - oxygen/glucose deprivation KW - stroke KW - tumor necrosis factor-α KW - proliferation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219828 SN - 1663-9812 VL - 11 ER - TY - JOUR A1 - Sun, Ping A1 - Ortega, Gabriela A1 - Tan, Yan A1 - Hua, Qian A1 - Riederer, Peter F. A1 - Deckert, Jürgen A1 - Schmitt-Böhrer, Angelika G. T1 - Streptozotocin impairs proliferation and differentiation of adult hippocampal neural stem cells in vitro-correlation with alterations in the expression of proteins associated with the insulin system JF - Frontiers in Aging Neuroscience N2 - Rats intracerebroventricularily (icv) treated with streptozotocin (STZ), shown to generate an insulin resistant brain state, were used as an animal model for the sporadic form of Alzheimer's disease (sAD). Previously, we showed in an in vivo study that 3 months after STZ icv treatment hippocampal adult neurogenesis (AN) is impaired. In the present study, we examined the effects of STZ on isolated adult hippocampal neural stem cells (NSCs) using an in vitro approach. We revealed that 2.5 mM STZ inhibits the proliferation of NSCs as indicated by reduced number and size of neurospheres as well as by less BrdU-immunoreactive NSCs. Double immunofluorescence stainings of NSCs already being triggered to start with their differentiation showed that STZ primarily impairs the generation of new neurons, but not of astrocytes. For revealing mechanisms possibly involved in mediating STZ effects we analyzed expression levels of insulin/glucose system-related molecules such as the glucose transporter (GLUT) 1 and 3, the insulin receptor (IR) and the insulin-like growth factor (IGF) 1 receptor. Applying quantitative Real time-PCR (qRT-PCR) and immunofluorescence stainings we showed that STZ exerts its strongest effects on GLUT3 expression, as GLUT3 mRNA levels were found to be reduced in NSCs, and less GLUT3-immunoreactive NSCs as well as differentiating cells were detected after STZ treatment. These findings suggest that cultured NSCs are a good model for developing new strategies to treat nerve cell loss in AD and other degenerative disorders. KW - Alzheimer’s disease KW - streptozotocin KW - proliferation KW - neural stem cells KW - insulin-like growth factor 1 receptor KW - insulin receptor KW - glucose transporter KW - differentiation Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176741 VL - 10 IS - 145 ER - TY - JOUR A1 - Otto, Wolfgang A1 - Rubenwolf, Peter C. A1 - Burger, Maximilian A1 - Fritsche, Hans-Martin A1 - Rößler, Wolfgang A1 - May, Matthias A1 - Hartmann, Arndt A1 - Hofstädter, Ferdinand A1 - Wieland, Wolf F. A1 - Denzinger, Stefan T1 - Loss of aquaporin 3 protein expression constitutes an independent prognostic factor for progression-free survival: an immunohistochemical study on stage pT1 urothelial bladder cancer JF - BMC Cancer N2 - Background: Treatment of patients with stage pT1 urothelial bladder cancer (UBC) continues to be a challenge due to its unpredictable clinical course. Reliable molecular markers that help to determine appropriate individual treatment are still lacking. Loss of aquaporin (AQP) 3 protein expression has previously been shown in muscle-invasive UBC. The aim of the present study was to investigate the prognostic value of AQP3 protein expression with regard to the prognosis of stage pT1 UBC. Method: AQP 3 protein expression was investigated by immunohistochemistry in specimens of 87 stage T1 UBC patients, who were diagnosed by transurethral resection of the bladder (TURB) and subsequent second resection at a high-volume urological centre between 2002 and 2009. Patients underwent adjuvant instillation therapy with Bacillus Calmette-Guerin (BCG). Loss of AQP3 protein expression was defined as complete absence of the protein within the whole tumour. Expression status was correlated retrospectively with clinicopathological and follow-up data (median: 31 months). Multivariate Cox regression analysis was used to assess the value of AQP3 tumour expression with regard to recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS). RFS, PFS and CSS were calculated by Kaplan-Meier analysis and Log rank test. Results: 59% of patients were shown to exhibit AQP3-positive tumours, whereas 41% of tumours did not express the marker. Loss of AQP3 protein expression was associated with a statistically significantly worse PFS (20% vs. 72%, p=0.020). This finding was confirmed by multivariate Cox regression analysis (HR 7.58, CI 1.29 - 44.68; p=0.025). Conclusions: Loss of AQP3 protein expression in pT1 UBC appears to play a key role in disease progression and is associated with worse PFS. Considering its potential prognostic value, assessment of AQP3 protein expression could be used to help stratify the behavior of patients with pT1 UBC. KW - urothelial bladder carcinoma KW - progression KW - transitional cell carcinoma KW - bacillus calmette guerin KW - water channels KW - follow up KW - in vitro KW - recurrence KW - growth KW - T1 KW - tumor KW - proliferation KW - stage pT1 KW - aquaporin 3 protein KW - immunohistochemistry Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135679 VL - 12 IS - 459 ER - TY - JOUR A1 - Schmidt, Marianne A1 - Skaf, Josef A1 - Gavril, Georgiana A1 - Polednik, Christine A1 - Roller, Jeanette A1 - Kessler, Michael A1 - Holzgrabe, Ulrike T1 - The influence of Osmunda regalis root extract on head and neck cancer cell proliferation, invasion and gene expression JF - BMC Complementary and Alternative Medicine N2 - Background: According to only a handful of historical sources, Osmunda regalis, the royal fern, has been used already in the middle age as an anti-cancer remedy. To examine this ancient cancer cure, an ethanolic extract of the roots was prepared and analysed in vitro on its effectiveness against head and neck cancer cell lines. Methods: Proliferation inhibition was measured with the MTT assay. Invasion inhibition was tested in a spheroid-based 3-D migration assay on different extracellular matrix surfaces. Corresponding changes in gene expression were analysed by qRT-PCR array. Induction of apoptosis was measured by fluorescence activated cell sorting (FACS) with the Annexin V binding method. The plant extract was analysed by preliminary phytochemical tests, liquid chromatography/mass spectroscopy (LC-MS) and thin layer chromatography (TLC). Anti-angiogenetic activity was determined by the tube formation assay. Results: O. regalis extract revealed a growth inhibiting effect on the head and neck carcinoma cell lines HLaC78 and FaDu. The toxic effect seems to be partially modulated by p-glycoprotein, as the MDR-1 expressing HLaC79-Tax cells were less sensitive. O. regalis extract inhibited the invasion of cell lines on diverse extracellular matrix substrates significantly. Especially the dispersion of the highly motile cell line HlaC78 on laminin was almost completely abrogated. Motility inhibition on laminin was accompanied by differential gene regulation of a variety of genes involved in cell adhesion and metastasis. Furthermore, O. regalis extract triggered apoptosis in HNSCC cell lines and inhibited tube formation of endothelial cells. Preliminary phytochemical analysis proved the presence of tannins, glycosides, steroids and saponins. Liquid chromatography/mass spectroscopy (LC-MS) revealed a major peak of an unknown substance with a molecular mass of 864.15 Da, comprising about 50% of the total extract. Thin layer chromatography identified ferulic acid to be present in the extract. Conclusion: The presented results justify the use of royal fern extracts as an anti-cancer remedy in history and imply a further analysis of ingredients. KW - head and neck carcinoma KW - invasion KW - plant extract KW - proliferation KW - HNSCC KW - metastasis KW - gene expression KW - Osmunda regalis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158704 VL - 17 IS - 518 ER - TY - JOUR A1 - Muturi, Harrison T. A1 - Dreesen, Janine D. A1 - Nilewski, Elena A1 - Jastrow, Holger A1 - Giebel, Bernd A1 - Ergun, Suleyman A1 - Singer, Berhard B. T1 - Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior JF - PLOS ONE N2 - Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1 000 nm in diameter), which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter), derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM)-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis. KW - carcinoembryonic anitgen family KW - biliary glycoprotein CD66A KW - adhesion molecule-1 KW - epithelial cells KW - membrane vesicles KW - growth-factor KW - cancer KW - expression KW - proliferation Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128373 SN - 1932-6203 VL - 8 IS - 9 ER - TY - JOUR A1 - Sanz-Moreno, Adrian A1 - Fuhrmann, David A1 - Wolf, Elmar A1 - von Eyss, Björn A1 - Eilers, Martin A1 - Elsässer, Hans-Peter T1 - Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation JF - PLOS ONE N2 - Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15(Ink4)) or Cd-kn1a (p21(Cip1)). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1 Delta POZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1 Delta POZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype. KW - C-MYC KW - transcription factor MIZ-1 KW - breast-cancer cells KW - gene expression KW - epithelial cells KW - prolactin KW - transgenic mice KW - growth KW - differentiation KW - proliferation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117286 VL - 9 IS - 2 ER - TY - JOUR A1 - Liedert, Astrid A1 - Röntgen, Viktoria A1 - Schinke, Thorsten A1 - Benisch, Peggy A1 - Ebert, Regina A1 - Jakob, Franz A1 - Klein-Hitpass, Ludger A1 - Lennerz, Jochen K. A1 - Amling, Michael A1 - Ignatius, Anita T1 - Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice JF - PLOS ONE N2 - The canonical Wnt/beta-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5(-/-)) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5(-/-) mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5(-/-) mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active beta-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis. KW - autosomal-dominant osteopetrosis KW - receptor related protein KW - high-bone-mass KW - WNT pathway KW - in-vitro KW - cells KW - gene KW - proliferation KW - osteoclasts KW - mutations Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115782 SN - 1932-6203 VL - 9 IS - 7 ER - TY - JOUR A1 - Bensaad, Karim A1 - Favaro, Elena A1 - Lewis, Caroline A. A1 - Peck, Barrie A1 - Lord, Simon A1 - Collins, Jennifer M. A1 - Pinnick, Katherine E. A1 - Wigfield, Simon A1 - Buffa, Francesca M. A1 - Li, Ji-Liang A1 - Zhang, Qifeng A1 - Wakelam, Michael J. O. A1 - Karpe, Fredrik A1 - Schulze, Almut A1 - Harris, Adrian L. T1 - Fatty Acid Uptake and Lipid Storage Induced by HIF-1 alpha Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation JF - Cell Reports N2 - An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O-2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via beta-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo. KW - inducible factor-I KW - binding protein KW - triglyceride accumulation KW - cancer cell KW - complex-III KW - beta-oxidation KW - metabolism KW - lipogenesis KW - proliferation KW - resistance Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115162 SN - 2211-1247 VL - 9 IS - 1 ER - TY - JOUR A1 - Rath, Subha N. A1 - Brandl, Andreas A1 - Hiller, Daniel A1 - Hoppe, Alexander A1 - Gbureck, Uwe A1 - Horch, Raymund E. A1 - Boccaccini, Aldo R. A1 - Kneser, Ulrich T1 - Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells JF - PLOS ONE N2 - Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. KW - arteriovenous loop KW - calcium-phosphate KW - iron release KW - bone KW - angiogenesis KW - expression KW - differentation KW - proliferation KW - osteoblasts KW - growth Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114339 SN - 1932-6203 VL - 9 IS - 12 ER - TY - THES A1 - Hovhanyan, Anna T1 - Functional analyses of Mushroom body miniature (Mbm) in growth and proliferation of neural progenitor cells in the central brain of Drosophila melanogaster T1 - Funktionelle Analyse des Mushroom body minature (Mbm) in das Wachstum und die Proliferation von neuronalen Vorläuferzellen im zentralen Gehirn von Drosophila melanogaster N2 - Zellwachstum und Zellteilung stellen zwei miteinander verknüpfte Prozesse dar, die dennoch grundsätzlich voneinander zu unterscheiden sind. Die Wiederaufnahme der Proliferation von neuralen Vorläuferzellen (Neuroblasten) im Zentralhirn von Drosophila nach der spät-embryonalen Ruhephase erfordert zunächst Zellwachstum. Der Erhalt der regulären Zellgröße ist eine wichtige Voraussetzung für die kontinuierliche Proliferation der Neuroblasten über die gesamte larvale Entwicklungsphase. Neben extrinsischen Ernährungssignalen ist für das Zellwachstum eine kontinuierliche Versorgung mit funktionellen Ribosomen notwendig, damit die Proteinsynthese aufrechterhalten werden kann. Mutationen im mushroom body miniature (mbm) Gen wurden über einen genetischen Screen nach strukturellen Gehirnmutanten identifiziert. Der Schwerpunkt dieser Arbeit lag in der funktionellen Charakterisierung des Mbm Proteins als neues nukleoläres Protein und damit seiner möglichen Beteiligung in der Ribosomenbiogenese. Der Vergleich der relativen Expressionslevel von Mbm und anderen nuklearen Proteinen in verschiedenen Zelltypen zeigte eine verstärkte Expression von Mbm in der fibrillären Komponente des Nukleolus von Neuroblasten. Diese Beobachtung legte die Vermutung nahe, dass in Neuroblasten neben generell benötigten Faktoren der Ribosomenbiogenese auch Zelltyp-spezifische Faktoren existieren. Mutationen in mbm verursachen Proliferationsdefekte von Neuroblasten, wirken sich jedoch nicht auf deren Zellpolarität, die Orientierung der mitotischen Spindel oder die Asymmetrie der Zellteilung aus. Stattdessen wurde eine Reduktion der Zellgröße beobachtet, was im Einklang mit einer Beeinträchtigung der Ribosomenbiogenese steht. Insbesondere führt der Verlust der Mbm Funktion zu einer Retention der kleinen ribosomalen Untereinheit im Nukleolus, was eine verminderte Proteinsynthese zur Folge hat. Interessanterweise wurden Störungen der Ribosomenbiogenese nur in den Neuroblasten beobachtet. Zudem ist Mbm offensichtlich nicht erforderlich, um Wachstum oder die Proliferation von Zellen der Flügelimginalscheibe und S2-Zellen zu steuern, was wiederum dafür spricht, dass Mbm eine Neuroblasten-spezifische Funktion erfüllt. Darüber hinaus wurden die transkriptionelle Regulation des mbm-Gens und die funktionelle Bedeutung von posttranslationalen Modifikationen analysiert. Mbm Transkription wird von dMyc reguliert. Ein gemeinsames Merkmal von dMyc Zielgenen ist das Vorhandensein einer konservierten „E-Box“-Sequenz in deren Promotorregionen. In der Umgebung der mbm-Transkriptionsstartstelle befinden sich zwei „E-Box“-Motive. Mit Hilfe von Genreporteranalysen konnte nachgewiesen werden, dass nur eine von ihnen die dMyc-abhängige Transkription vermittelt. Die dMyc-abhängige Expression von Mbm konnte auch in Neuroblasten verifiziert werden. Auf posttranslationaler Ebene wird Mbm durch die Proteinkinase CK2 phosphoryliert. In der C-terminalen Hälfte des Mbm Proteins wurden in zwei Clustern mit einer Abfolge von sauren Aminosäuren sechs Serin- und Threoninreste als CK2- Phosphorylierungsstellen identifiziert. Eine Mutationsanalyse dieser Stellen bestätigte deren Bedeutung für die Mbm Funktion in vivo. Weiterhin ergaben sich Evidenzen, dass die Mbm-Lokalisierung durch die CK2-vermittelte Phosphorylierung gesteuert wird. Obwohl die genaue molekulare Funktion von Mbm in der Ribosomenbiogenese noch im Unklaren ist, unterstreichen die Ergebnisse dieser Studie die besondere Rolle von Mbm in der Ribosomenbiogenese von Neuroblasten um Zellwachstum und Proliferation zu regulieren. N2 - Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis. Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm. Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites. Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation. Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation. KW - Taufliege KW - Mbm KW - Neuroblast KW - cell growth KW - proliferation KW - ribosome biogenesis KW - CK2 KW - Myc KW - Vorläuferzellen KW - Drosophila melanogaster Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-91303 ER -