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  • 1.
    Aase, Karin
    et al.
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ernkvist, Mira
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ebarasi, Lwaki
    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
    Jakobsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Majumdar, Arindam
    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
    Yi, Chunling
    Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
    Birot, Olivier
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Ming, Yue
    Department of Clinical Neuroscience, Section of Ophthalmology and Vision, Karolinska Institutet, St Erik’s Hospital, SE-11284 Stockholm, Sweden.
    Kvanta, Anders
    Department of Clinical Neuroscience, Section of Ophthalmology and Vision, Karolinska Institutet, St Erik’s Hospital, SE-11284 Stockholm, Sweden.
    Edholm, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kissil, Joseph
    Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Shimono, Akihiko
    Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Chuou-ku, Kobe 650-0047, Japan.
    Holmgren, Lars
    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden.
    Angiomotin regulates endothelial cell migration during embryonic angiogenesis2007In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 21, no 16, p. 2055-2068Article in journal (Refereed)
    Abstract [en]

    The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.

  • 2. Abramczyk, Olga
    et al.
    Zien, Piotr
    Zielinski, Rafał
    Pilecki, Marek
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Szyszka, Ryszard
    The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD12003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 307, no 1, p. 31-40Article in journal (Refereed)
    Abstract [en]

    The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.

  • 3.
    Afrakhte, Mozhgan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Morén, Anita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Jossan, Surinder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Itoh, Susumu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Sampath, Kuber
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Nils-Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    ten Dijke, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Induction of inhibitory Smad6 and Smad7 mRNA by TGF-beta family members1998In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 249, no 2, p. 505-11Article in journal (Refereed)
    Abstract [en]

    Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.

  • 4.
    Agnarsdóttir, Margrét
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sooman, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Bolander, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Rexhepaj, Elton
    UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland.
    Bergqvist, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gallagher, William
    UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ekman, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Uhlen, Mathias
    Department of Proteomics, School of Biotechnology, AlbaNova University Center, KTH-Royal Institute of Technology, Stockholm, Sweden.
    Hedstrand, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    SOX10 expression in superficial spreading and nodular malignant melanomas2010In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 20, no 6, p. 468-478Article in journal (Refereed)
    Abstract [en]

    SOX10 is a transcription factor expressed in nerve cells and melanocytes. The aim of this study was to investigate the protein expression pattern of SOX10 in malignant melanoma tumors and to analyze whether the results correlated with clinical parameters and the proliferation marker Ki-67. Furthermore, proliferation and migration were analyzed in three different cell lines employing SOX10 small interfering RNA-mediated silencing. Expression patterns were determined in 106 primary tumors and 39 metastases in addition to 16 normal skin samples and six benign nevi employing immunohistochemistry and tissue microarrays. The immunohistochemical staining was evaluated manually and with an automated algorithm. SOX10 was strongly expressed in the benign tissues, but for the malignant tumors superficial spreading melanomas stained stronger than nodular malignant melanomas (P=0.008). The staining intensity was also inversely correlated with T-stage (Spearman's ρ=-0.261, P=0.008). Overall survival and time to recurrence were significantly correlated with SOX10 intensity, but not in multivariate analysis including T-stage. With the automated algorithm there was an inverse correlation between the SOX10 staining intensity and the proliferation marker, Ki-67 (ρ=-0.173, P=0.02) and a significant difference in the intensity signal between the benign tissues, the primary tumors and the metastases where the metastases stained the weakest (P≤0.001). SOX10 downregulation resulted in variable effects on proliferation and migration rates in the melanoma cell lines. In conclusion, the SOX10 intensity level differed depending on the tissue studied and SOX10 might have a role in survival. No conclusion regarding the role of SOX10 for in-vitro proliferation and migration could be drawn.

  • 5. Agüero, Fernán
    et al.
    Noé, Griselda
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Repetto, Yolanda
    Zaha, Arnaldo
    Cazzulo, Juan José
    Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus2004In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 137, no 2, p. 207-214Article in journal (Refereed)
    Abstract [en]

    Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.

  • 6. All-Ericsson, Charlotta
    et al.
    Girnita, Leonard
    Müller-Brunotte, Anja
    Brodin, Bertha
    Seregard, Stefan
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Larsson, Olle
    c-Kit-dependent growth of uveal melanoma cells: a potential therapeutic target?2004In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 45, no 7, p. 2075-82Article in journal (Refereed)
    Abstract [en]

    PURPOSE: This study was conducted to investigate the expression and functional impact of the proto-oncogene c-kit in uveal melanoma. METHODS: Based on immunohistochemical (IHC) study of paraffin-embedded specimens from 134 uveal melanomas and Western blot analysis on eight fresh-frozen samples the expression of c-kit in uveal melanoma was studied. Furthermore, the phosphorylation of c-kit and the impact of the tyrosine kinase inhibitor STI571 was examined in the three uveal melanoma cell lines OCM-1, OCM-3, and 92-1. RESULTS: Eighty-four of 134 paraffin-embedded samples and six of eight fresh-frozen samples expressed c-kit. c-Kit was strongly expressed and tyrosine phosphorylated in cultured uveal melanoma cells compared with cutaneous melanoma cells. Moreover, in contrast to cutaneous melanoma cell lines c-kit maintained a high phosphorylation level in serum-depleted uveal melanoma cells. No activation-related mutations in exon 11 of the KIT gene were found. On the contrary, expression of the stem cell growth factor (c-kit ligand) was detected in all three uveal melanoma cell lines, suggesting the presence of autocrine (paracrine) stimulation pathways. Treatment of uveal melanoma cell lines with STI571, which blocks c-kit autophosphorylation, resulted in cell death. The IC(50) of the inhibitory effects on c-kit phosphorylation and cell proliferation was of equal size and less than 2.5 microM. CONCLUSIONS: The results confirm that c-kit is vastly expressed in uveal melanoma, suggest that the c-kit molecular pathway may be important in uveal melanoma growth, and point to its use as a target for therapy with STI571.

  • 7. Alvarado-Kristensson, Maria
    et al.
    Melander, Fredrik
    Leandersson, Karin
    Rönnstrand, Lars
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Andersson, Tommy
    p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils2004In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 199, no 4, p. 449-458Article in journal (Refereed)
    Abstract [en]

    Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

  • 8.
    Amagasaki, Kenichi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kaneto, Hideaki
    Osaka University Graduate School of Medicine, Department of Internal Medicine and Therapeutics (A8), 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    c-Jun N-terminal kinase is necessary for platelet-derived growth factor-mediated chemotaxis in primary fibroblasts2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 31, p. 22173-22179Article in journal (Refereed)
    Abstract [en]

    c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

  • 9. Andersson, Torbjörn
    et al.
    Eriksson, Barbro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lindgren, PG
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Wilander, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Öberg, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Effects of Interferon on Tumor Tissue Content in Liver Metastases of Human Carcinoid Tumors1990In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, no 50, p. 3413-3415Article in journal (Refereed)
    Abstract [en]

    In 21 patients ultrasound-guided cutting biopsies, from carcinoid metastases of the liver, were taken before and after therapy with α-interferon. Each biopsy was examined under light microscopy and the amount of tumor tissue and connective tissue was quantified and then correlated to objective response to interferon therapy. A significant reduction of the amount of tumor tissue, in spite of unaltered metastatic size and a corresponding increase in connective tissue, was seen after interferon therapy. A more pronounced reduction of tumor tissue occurred after long-term interferon therapy. A positive correlation between objective therapy response and tumor tissue reduction was also present. Patients responding poorly, or not at all, to therapy did not show any significant decrease in tumor tissue.

    Since treatment with immune response modifiers is expected to increase in the near future, it is important to choose the right investigations for therapy monitoring, and since all patients in this investigation had unchanged tumor size on repeated radiological examinations, it is obvious that microscopic examination of core biopsies is a better method for evaluating effects of long-term therapy than tumor size measurement with radiological techniques. Further, the results may indicate that interferon exerts a cytotoxic effect on carcinoid tumor cells in vivo.

  • 10. Arase, Mayu
    et al.
    Horiguchi, Kana
    Ehata, Shogo
    Morikawa, Masato
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tsutsumi, Shuichi
    Aburatani, Hiroyuki
    Miyazono, Kohei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Koinuma, Daizo
    Transforming growth factor-beta-induced lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells2014In: Cancer Science, ISSN 1347-9032, E-ISSN 1349-7006, Vol. 105, no 8, p. 974-982Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor (TGF)-beta exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-beta is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-beta lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-beta in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-beta In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-beta type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-beta of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-beta-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-beta functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-beta signaling.

  • 11. Argyropoulos, C.
    et al.
    Chatziioannou, A. A.
    Nikiforidis, G.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kollias, G.
    Aidinis, V.
    Operational criteria for selecting a cDNA microarray data normalization algorithm2006In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 15, no 4, p. 983-996Article in journal (Refereed)
    Abstract [en]

    Microarray technology allows gene expression profiling at a global level. Many algorithms for the normalization of raw microarray data have been proposed, but no attempt has yet been made to propose operationally verifiable criteria for their comparative evaluation, which is necessary for the selection of the most appropriate method for a given dataset. This study develops a set of operational criteria for assessing the impact of various normalization algorithms in terms of accuracy (bias), precision (variance) and over-fitting (information reduction). The use of these criteria is illustrated by applying the three most widely used algorithms (global median normalization, spiked-in based normalization and lowess) on a specifically designed, multiply-controlled dataset.

  • 12.
    Arora, Deepika
    et al.
    Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
    Köthe, Susanne
    Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
    van den Eijnden, Monique
    Merck Serono, Geneva 1202, Switzerland.
    van Huijsduijnen, Rob Hooft
    Merck Serono, Geneva 1202, Switzerland.
    Heidel, Florian
    Department of Hematology/Oncology, Otto-von Guericke-University, Magdeburg, Germany.
    Fischer, Thomas
    Department of Hematology/Oncology, Otto-von Guericke-University, Magdeburg, Germany.
    Scholl, Sebastian
    Department of Hematology/Oncology, Clinic for Internal Medicine II, Jena University Hospital, Jena, Germany.
    Tölle, Benjamin
    Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
    Böhmer, Sylvia-Annette
    Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
    Lennartsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Isken, Fabienne
    Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
    Müller-Tidow, Carsten
    Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
    Böhmer, Frank-D
    Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
    Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression2012In: Cell Communication and Signaling, ISSN 1478-811X, E-ISSN 1478-811X, Vol. 10, no 1, p. 19-Article in journal (Refereed)
    Abstract [en]

    Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.

  • 13. Arsura, Marcello
    et al.
    Panta, Ganesh R.
    Bilyeu, Jennifer D.
    Cavin, Lakita G.
    Sovak, Mika A.
    Oliver, Aundrea A.
    Factor, Valentina
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mercurio, Frank
    Thorgeirsson, Snorri S.
    Sonenshein, Gail E.
    Transient activation of NF-kappaB through a TAK1/IKK kinase pathway by TGF-beta1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation.2003In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 22, no 3, p. 412-425Article in journal (Refereed)
    Abstract [en]

    NF-kappaB has been implicated in the regulation of apoptosis, a key mechanism of normal and malignant growth control. Previously, we demonstrated that inhibition of NF-kappaB activity by TGF-beta1 leads directly to induction of apoptosis of murine B-cell lymphomas and hepatocytes. Thus, we were surprised to determine that NF-kappaB is transiently activated in response to TGF-beta1 treatment. Here we elucidate the mechanism of TGF-beta1-mediated regulation of NF-kappaB and induction of apoptosis in epithelial cells. We report that TGF-beta1 activates IKK kinase, which mediates IkappaB-alpha phosphorylation. In turn, the activation of IKK following TGF-beta1 treatment is mediated by the TAK1 kinase. As a result of NF-kappaB activation, IkappaB-alpha mRNA and protein levels are increased leading to postrepression of NF-kappaB and induction of cell death. Inhibition of NF-kappaB following TGF-beta1 treatment increased AP-1 complex transcriptional activity through sustained c-Jun phosphorylation, thereby potentiating AP-1/SMADs-mediated cell killing. Furthermore, TGF-beta1-mediated upregulation of Smad7 appeared independent of NF-kappaB. In hepatocellular carcinomas of TGF-beta1 or TGF-alpha/c-myc transgenic mice, we observed constitutive activation of NF-kappaB that led to inhibition of JNK signaling. Overall, our data illustrate an autocrine mechanism based on the ability of IKK/NF-kappaB/IkappaB-alpha signaling to negatively regulate NF-kappaB levels thereby permitting TGF-beta1-induced apoptosis through AP-1 activity.

  • 14.
    Arvidsson, Ann-Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Rupp, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Nånberg, Eeva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Downward, Julian
    Signal Transduction Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wennström, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Schlessinger, Joseph
    Department of Pharmacology, New York University Medical Center, New York New York 10016, USA.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Tyr-716 in the platelet-derived growth factor beta-receptor kinase insertis involved in GRB2 binding and Ras activation1994In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 14, no 10, p. 6715-6726Article in journal (Refereed)
    Abstract [en]

    Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

  • 15.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Integration of signalling pathways regulated by small GTPases and calcium2004In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1742, no 1-3, p. 51-58Article, review/survey (Refereed)
    Abstract [en]

    The Ras superfamily of small GTPases constitutes a large group of structurally and functionally related proteins. They function as signalling switches in numerous signalling cascades in the cell. During the recent years, an increased awareness of a communication between signalling systems employing Ras-like GTPases and signalling systems employing calcium has emerged. For instance, the intensity of the activation of Ras-like GTPases is regulated by calcium-dependent mechanisms, acting on proteins that facilitate the activation or inactivation of the small GTPases. Other Ras-like GTPases have a direct influence on calcium signalling by regulating the activity of certain calcium channels. In addition, several small GTPases collaborate with calcium signalling in regulating cellular processes, such as cell adhesion, cell migration and exocytosis.

  • 16.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Roles of F-BAR/PCH Proteins in the Regulation of Membrane Dynamics and Actin Reorganization2009In: International Review of Cell and Molecular Biology, Vol. 272, p. 1-31Article, review/survey (Refereed)
    Abstract [en]

    The Pombe Cdc15 Homology (PCH) proteins have emerged in many species as important coordinators of signaling pathways that regulate actomyosin assembly and membrane dynamics. The hallmark of the PCH proteins is the presence of a Fes/ClP4 homology-Bin/Amphiphysin/Rvsp (F-BAR) domain; therefore they are commonly referred to as F-BAR proteins. The prototype F-BAR protein, Cdc15p of Schizosaccharomyces pombe, has a role in the formation of the contractile actomyosin ring during cytokinesis. Vertebrate F-BAR proteins have an established role in binding phospholipids and they participate in membrane deformations, for instance, during the internalization of transmembrane receptors. This way the F-BAR proteins will function as linkers between the actin polymerization apparatus and the machinery regulating membrane dynamics. Interestingly, some members of the F-BAR proteins are implicated in inflammatory or neurodegenerative disorders and the observations can be expected to have clinical implications for the treatment of the diseases.

  • 17.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The mammalian verprolin homologue WIRE participates in receptor-mediated endocytosis and regulation of the actin filament system by distinct mechanisms2004In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 298, no 2, p. 485-498Article in journal (Refereed)
    Abstract [en]

    The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor beta-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408-412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor beta-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells.

  • 18.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The verprolin family of proteins: Regulators of cell morphogenesis and endocytosis2005In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, no 24, p. 5253-5259Article, review/survey (Refereed)
    Abstract [en]

    The verprolin family of proteins, WIP, CR16 and WIRE/WICH, has emerged as critical regulators of cytoskeletal organisation in vertebrate cells. The founding father of the family, verprolin, was originally identified in budding yeast and later shown to be needed for actin polymerisation during polarised growth and during endocytosis. The vertebrate verprolins regulate actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. Interestingly, also the vertebrate verprolins have been implicated in endocytosis, demonstrating that most of the functional modules in this fascinating group of proteins have been conserved from yeast to man.

  • 19.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    The verprolins as regulators of actin dynamics.2006In: Actin-monomer-binding proteins., Austin, Texas: Landes Biosciences , 2006, p. 97-106Chapter in book (Refereed)
    Abstract [en]

    Verprolin is an actin-binding protein first identified in budding yeast Saccharomyces cerevisiae. The yeast verprolin is needed for actin polymerisation during polarised growth and during endocytosis. In vertebrate cells, three genes encoding Verprolin orthologues have been identified: WIP, CR16 and WIRE/WICH. The mammalian verprolins have been implicated in the regulation of actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. This review article will bring up to discussion the current understanding of the mechanisms underlying verprolin-dependent mobilisation of the actin filament system.

  • 20.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The WASP-binding protein WIRE has a role in the regulation of the actin filament system downstream of the platelet-derived growth factor receptor2002In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 279, no 1, p. 21-33Article in journal (Refereed)
    Abstract [en]

    Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WIP-related). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRbeta cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckbeta. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.

  • 21.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Richnau, Ninna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pombe Cdc15 homology proteins: regulators of membrane dynamics and the actin cytoskeleton2006In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 31, no 12, p. 670-679Article, review/survey (Refereed)
    Abstract [en]

    Pombe Cdc15 homology (PCH) proteins have emerged in many species as important coordinators of signalling pathways that regulate actomyosin assembly and membrane dynamics. For example, the prototype PCH protein, Cdc15p of Schizosaccharomyces pombe, has a role in assembly of the contractile ring, which is needed to separate dividing cells. Recently, mammalian PCH proteins have been found to bind phospholipids and to participate in membrane deformation. These findings suggest that PCH proteins are crucial linkers of membrane dynamics and actin polymerization, for example, during the internalization of transmembrane receptors. Intriguingly, some members of the PCH protein family are mutated in neurodegenerative and inflammatory diseases, which has implications for the identification of cures for such disorders.

  • 22. Aspenström, Pontus
    et al.
    Fransson, Åsa
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Saras, Jan
    Rho GTPases have diverse effects on the organization of the actin filament system2004In: Biochem J, Vol. 337, no Pt 2, p. 327-337Article in journal (Refereed)
  • 23.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rho GTPases have diverse effects on the organization of the actin filament system2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 377, no Pt 2, p. 327-337Article in journal (Refereed)
    Abstract [en]

    The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.

  • 24.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Richnau, Ninna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Johansson, Ann-Sofi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 12, p. 2180-2194Article in journal (Refereed)
    Abstract [en]

    Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.

  • 25.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ruusala, Aino
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pacholsky, Dirk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Taking Rho GTPases to the next level: the cellular functions of atypical Rho GTPases2007In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 17, p. 3673-3679Article, review/survey (Refereed)
    Abstract [en]

    The Rho GTPases are influential regulators of signalling pathways that control vital cellular processes such as cytoskeletal dynamics, gene transcription, cell cycle progression and cell transformation. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, but this protein family actually harbours 20 members. Recently, the less known Rho GTPases have received increased attention. Many of the less studied Rho GTPases have structural, as well as, functional features which makes it pertinent to classify them as atypical Rho GTPases. This review article will focus on the critical aspects of the atypical Rho GTPases, RhoH, Wrch-1, Chp and RhoBTB. These proteins are involved in a broad spectre of biological processes, such as cytoskeletal dynamics, T-cell signalling and protein ubiquitinylation. We will also discuss the roles of atypical Rho GTPases as oncogenes or tumour suppressors, as well as their potential involvement in human diseases.

  • 26.
    Aspenström-Fagerlund, Bitte
    et al.
    Toxicology Division, National Food Administration, P.O. Box 622, SE-75126 Uppsala, Sweden.
    Ring, Linda
    Toxicology Division, National Food Administration, P.O. Box 622, SE-75126 Uppsala, Sweden.
    Aspenström, Pontus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Tallkvist, Jonas
    Department of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Box 7028, SE-75007, Uppsala, Sweden.
    Ilbäck, Nils-Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Glynn, Anders W.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
    Oleic acid and docosahexaenoic acid cause an increase in the paracellular absorption of hydrophilic compounds in an experimental model of human absorptive enterocytes2007In: Toxicology, ISSN 0300-483X, E-ISSN 1879-3185, Vol. 237, no 1-3, p. 12-23Article in journal (Refereed)
    Abstract [en]

    Surface active compounds present in food possibly have the ability to enhance the absorption of water soluble toxic agents. Therefore, we investigated whether fatty acids such as oleic acid and docosahexaenoic acid (DHA), both commonly present in food, negatively affect the integrity of tight junctions (TJ) in the intestinal epithelium and thereby increase the absorption of poorly absorbed hydrophilic substances. Caco-2 cells, which are derived from human absorptive enterocytes, were grown on permeable filters for 20-25 days. Differentiated cell monolayers were apically exposed for 90min to mannitol in emulsions of oleic acid (5, 15 or 30mM) or DHA (5, 15 or 30mM) in an experimental medium with or without Ca(2+) and Mg(2+). Absorption of (14)C-mannitol increased and trans-epithelial electrical resistance (TEER) decreased in cell monolayers exposed to oleic acid and DHA, compared to controls. Cytotoxicity, measured as leakage of LDH, was higher in groups exposed to 30mM oleic acid and all concentrations of DHA. Morphology of the cell monolayers was studied by using fluorescence microscopy. Exposure of cell monolayers to 5mM DHA for 90min resulted in a profound alteration of the cell-cell contacts as detected by staining the cells for beta-catenin. Oleic acid (30mM) treatment also induced dissolution of the cell-cell contacts but the effect was not as pronounced as with DHA. Cell monolayers were also exposed for 180min to 250nM cadmium (Cd) in emulsions of oleic acid (5 or 30mM) or DHA (1 or 5mM), in an experimental medium with Ca(2+) and Mg(2+). Retention of Cd in Caco-2 cells was higher after exposure to 5mM oleic acid but lower after exposure to 30mM oleic acid and DHA. Absorption of Cd through the monolayers increased after DHA exposure but not after exposure to oleic acid. Our results indicate that fatty acids may compromise the integrity of the intestinal epithelium and that certain lipids in food may enhance the paracellular absorption of poorly absorbed hydrophilic substances.

  • 27.
    Avesson, Lotta
    et al.
    Department of Molecular Biology; Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Schumacher, Heiko T
    Merck Millipore AG; Zug, Switzerland.
    Fechter, Pierre
    Architecture et Réactivité de l‘ARN; Université de Strasbourg; CNRS; IBMC; Strasbourg, France.
    Romby, Pascale
    Architecture et Réactivité de l‘ARN; Université de Strasbourg; CNRS; IBMC; Strasbourg, France.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Söderbom, Fredrik
    Department of Molecular Biology; Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Abundant class of non-coding RNA regulates development in the social amoeba Dictyostelium discoideum2011In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 8, no 6, p. 1094-1104Article in journal (Refereed)
    Abstract [en]

    Non-coding (nc)RNAs are important players in most biological processes. Although small RNAs such as microRNAs and small interfering RNAs have emerged as exceptionally important regulators of gene expression, great numbers of larger ncRNAs have also been identified. Many of these are abundant and differentially expressed but their functions have in most cases not been elucidated. The social amoeba Dictyostelium discoideum contain the ncRNAs commonly found in eukaryotes. In addition, we previously reported the identification of two novel classes of 42-65 nt long stem-loop forming RNAs, Class I and Class II RNAs, with unknown function. In this study we have further characterized these abundant ncRNAs, which are down regulated during development. We have confirmed expression of 29 Class I RNAs and experimentally verified the formation of the computationally predicted short conserved stem structure. Furthermore, we have for the first time created knockout strains for several small ncRNA genes in D. discoideum and found that deletion of one of the Class I RNAs, DdR-21, results in aberrant development. In addition we have shown that this Class I RNA forms a complex with one or several proteins but do not appear to be associated with ribosomes or polysomes. In a pull down assay, several proteins interacting with DdR-21 were identified, one of these has two RNA recognition motifs (RRMs). The purified RRM containing protein was demonstrated to bind directly and specifically to DdR-21.

  • 28. Baan, Bart
    et al.
    Pardali, Evangelia
    ten Dijke, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    van Dam, Hans
    In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 9, p. 1982-1990Article in journal (Refereed)
    Abstract [en]

    Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.

  • 29.
    Ballagi, A E
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Odin, P
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Othberg-Cederström, A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Smits, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Duan, W M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lindvall, O
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Funa, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Platelet-derived growth factor receptor expression after neural grafting in a rat model of Parkinson's disease1994In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 3, no 6, p. 453-460Article in journal (Refereed)
    Abstract [en]

    Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13-E15 rat embryos possessed both PDGF alpha- and beta-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF alpha-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the alpha-receptor but the expression was unchanged following transplantation. The beta-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk beta-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.

  • 30.
    Baranowska-Kortylewicz, Janina
    et al.
    Departments of Radiation Oncology, University of Nebraska Medical Center, Omaha, Nebraska.
    Abe, Michio
    Departments of Radiation Oncology, University of Nebraska Medical Center, Omaha, Nebraska.
    Pietras, Kristian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kortylewicz, Zbigniew P
    Departments of Radiation Oncology, University of Nebraska Medical Center, Omaha, Nebraska.
    Kurizaki, Takashi
    Department of Surgery II, National Hospital Organization, Kumamoto University Medical School, Kumamoto, Japan.
    Nearman, Jessica
    Departments of Radiation Oncology, University of Nebraska Medical Center, Omaha, Nebraska.
    Paulsson, Janna
    Department of Pathology-Oncology, Karolinska Institute, Stockholm, Sweden.
    Mosley, R Lee
    Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska.
    Enke, Charles A
    Departments of Radiation Oncology, University of Nebraska Medical Center, Omaha, Nebraska.
    Östman, Arne
    Department of Pathology-Oncology, Karolinska Institute, Stockholm, Sweden.
    Effect of platelet-derived growth factor receptor-beta inhibition with STI571 on radioimmunotherapy2005In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 65, no 17, p. 7824-7831Article in journal (Refereed)
    Abstract [en]

    Whereas radioimmunotherapy of hematologic malignancies has evolved into a viable treatment option, the responses of solid tumors to radioimmunotherapy are discouraging. The likely cause of this problem is the interstitial hypertension inherent to all solid tumors. Remarkable improvements in tumor responses to radioimmunotherapy were discovered after the inclusion of STI571 in the therapy regimen. A combination of the tumor stroma-reactive STI571, a potent platelet-derived growth factor receptor-beta (PDGFr-beta) antagonist, and the tumor-seeking radiolabeled antibody B72.3 yielded long-lasting growth arrest of the human colorectal adenocarcinoma LS174T grown as s.c. xenografts in athymic mice. The interaction of STI571 with the stromal PDGFr-beta reduced tumor interstitial fluid pressure (P(IF)) by >50% and in so doing improved the uptake of B72.3. The attenuation of P(IF) also had a positive effect on the homogeneity of antibody distribution. These effects were dose-dependent and under optimized dosing conditions allowed for a 2.45 times increase in the tumor uptake of B72.3 as determined in the biodistribution studies. Single-photon emission computed tomography imaging studies substantiated these results and indicated that the homogeneity of the radioisotope distribution was also much improved when compared with the control mice. The increased uptake of radioimmunotherapy into the tumor resulted in >400% increase in the tumor absorbed radiation doses in STI571 + radioimmunotherapy-treated mice compared with PBS + radioimmunotherapy-treated mice. The improved antibody uptake in response to the attenuation of tumor P(IF) was identified as the primary reason for the growth arrest of the STI571 + radioimmunotherapy-treated tumors. Two related causes were also identified: (a) the improved homogeneity of monoclonal antibody distribution in tumor and (b) the increased tumor radiosensitivity resulting from the improved tumor oxygenation.

  • 31. Bardales, José R.
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Villamarí­n, J. Antonio
    CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis2007In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 461, no 1, p. 130-137Article in journal (Refereed)
    Abstract [en]

    Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.

  • 32.
    Bardales, José R.
    et al.
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Villamarín, J. Antonio
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 18, p. 4479-4489Article in journal (Refereed)
    Abstract [en]

    Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

  • 33. Barderi, P
    et al.
    Campetella, O
    Frasch, A C
    Santome, JA
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Cazzulo, JJ
    The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression1998In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 330, no 2, p. 951-958Article in journal (Refereed)
    Abstract [en]

    NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

  • 34. Bart, Genevieve
    et al.
    Vico, Nuria Ortega
    Hassinen, Antti
    Pujol, Francois M.
    Deen, Ashik Jawahar
    Ruusala, Aino
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Tammi, Raija H.
    Squire, Anthony
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kellokumpu, Sakari
    Tammi, Markku I.
    Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS32015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 18, p. 11479-11490Article in journal (Refereed)
    Abstract [en]

    In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

  • 35.
    Bellomo, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Caja, Laia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fabregat, Isabel
    Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet, and Department of Physiological Sciences, School of Medicine, University of Barcelona, ES-08908, Barcelona, Spain.
    Mikulits, Wolfgang
    Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, A-1090, Vienna, Austria.
    Kardassis, Dimitris
    Division of Basic Medical Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, GR-71003, Heraklion, Greece.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Snail mediates crosstalk between TGFβ and LXRα in hepatocellular carcinoma2018In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 25, no 5, p. 885-903Article in journal (Refereed)
    Abstract [en]

    Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor β (TGFβ) and liver X receptor α (LXRα) pathways. TGFβ is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFβ. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFβ and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFβ cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFβ.

  • 36.
    Bellomo, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Caja, Laia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Transforming growth factor beta as regulator of cancer stemness and metastasis2016In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 115, no 7, p. 761-769Article, review/survey (Refereed)
    Abstract [en]

    Key elements of cancer progression towards metastasis are the biological actions of cancer stem cells and stromal cells in the tumour microenvironment. Cross-communication between tumour and stromal cells is mediated by secreted cytokines, one of which, the transforming growth factor beta (TGF beta), regulates essentially every cell within the malignant tissue. In this article, we focus on the actions of TGF beta on cancer stem cells, cancer-associated fibroblasts and immune cells that assist the overall process of metastatic dissemination. We aim at illustrating intricate connections made by various cells in the tumour tissue and which depend on the action of TGF beta.

  • 37.
    Bellomo, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Gahman, T. C.
    Ludwig Canc Res, La Jolla, CA USA..
    Shiau, A. K.
    Ludwig Canc Res, La Jolla, CA USA..
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    TGF beta and the nuclear receptor LXR alpha crosstalk on lipid metabolism and epithelial to mesenchymal transition in hepatocellular carcinoma2016In: European Journal of Clinical Investigation, ISSN 0014-2972, E-ISSN 1365-2362, Vol. 46, p. 36-36Article in journal (Other academic)
  • 38.
    Bengoechea-Alonso, Maria T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    A phosphorylation cascade controls the degradation of active SREBP12009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 9, p. 5885-5895Article in journal (Refereed)
    Abstract [en]

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

  • 39.
    Bengoechea-Alonso, Maria T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Cdk1/cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis2006In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 5, no 15, p. 1708-1718Article in journal (Refereed)
    Abstract [en]

    Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We recently found that the mature forms of SREBP1a and SREBP1c are hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In addition, we found that mature SREBP1 was stabilized in a phosphorylation-dependent manner during mitosis. We have now mapped the major MPM-2 epitope to a serine residue, S439, in the C terminus of mature SREBP1. Using phosphorylation-specific antibodies, we demonstrate that endogenous SREBP1 is phosphorylated on S439 specifically during mitosis. Mature SREBP1 interacts with the Cdk1/cyclin B complex in mitotic cells and we demonstrate that Cdk1 phosphorylates S439, both in vitro and in vivo. Our results suggest that Cdk1-mediated phosphorylation of S439 stabilizes mature SREBP1 during mitosis, thereby preserving a critical pool of active transcription factors to support lipid synthesis. Taken together with our previous work, the current study suggests that SREBP1 may provide a link between lipid synthesis, proliferation and cell growth. This hypothesis was supported by our observation that siRNA-mediated inactivation of SREBP1 arrested cells in the G(1) phase of the cell cycle, thereby attenuating cell growth.

  • 40.
    Bengoechea-Alonso, Maria T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    SREBP in signal transduction: cholesterol metabolism and beyond2007In: Current Opinion in Cell Biology, ISSN 0955-0674, E-ISSN 1879-0410, Vol. 19, no 2, p. 215-222Article in journal (Refereed)
    Abstract [en]

    The last few years have seen important advances in defining the mechanisms that cells use to monitor changes in cholesterol levels and regulate lipid metabolism. This work has unraveled a feedback system that enables cholesterol and certain sterol intermediates to regulate the proteolysis and transport of specific membrane proteins. The sterol regulatory element-binding protein (SREBP) family of transcription factors is at the center of this feedback system. These membrane-embedded proteins are activated by ER-to-Golgi transport followed by limited proteolysis. In addition, both the activation of the SREBPs and the stability of the rate-limiting enzyme in cholesterol synthesis are regulated by the ubiquitin-proteasome system in a sterol-dependent manner. The ubiquitin-proteasome system also regulates the degradation of active SREBPs. Recent work also highlights the important role of this regulatory system in several organisms, ranging from yeast to humans. In addition, the SREBP pathway has been found to regulate a diverse set of cellular processes, including phagocytosis, cell cycle progression, oxygen sensing and survival in response to bacterial infection. These advances illustrate the wide-ranging roles that SREBPs and membrane biogenesis have in cell biology.

  • 41.
    Bengoechea-Alonso, Maria T
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hyperphosphorylation regulates the activity of SREBP1 during mitosis2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 33, p. 11681-11686Article in journal (Refereed)
    Abstract [en]

    The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G(2)/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G(2)/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G(2)/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G(2)/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.

  • 42. Ben-Saadon, Ronen
    et al.
    Fajerman, Ifat
    Ziv, Tamar
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Schwartz, Alan L
    Ciechanover, Aaron
    The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system: Direct evidence for ubiquitination at the N-terminal residue2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 40, p. 41414-41421Article in journal (Refereed)
    Abstract [en]

    Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.

  • 43. Berger, Karin
    et al.
    Sivars, Ulf
    Sörhede Winzell, Maria
    Johansson, Peter
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rippe, Catarina
    Erlanson-Albertsson, Charlotte
    Mitochondrial ATP synthase--a possible target protein in the regulation of energy metabolism in vitro and in vivo2002In: Nutritional neuroscience, ISSN 1028-415X, E-ISSN 1476-8305, Vol. 5, no 3, p. 201-210Article in journal (Refereed)
    Abstract [en]

    The increasing prevalence of obesity in the Western world has stimulated an intense search for mechanisms regulating food intake and energy balance. A number of appetite-regulating peptides have been identified, their receptors cloned and the intracellular events characterized. One possible energy-dissipating mechanism is the mitochondrial uncoupling of ATP-synthesis from respiratory chain oxidation through uncoupling proteins, whereby energy derived from food could be dissipated as heat, instead of stored as ATP. The exact role of the uncoupling proteins in energy balance is, however, uncertain. We show here that mitochondrial F1F0-ATP synthase itself is a target protein for an anorectic peptide, enterostatin, demonstrated both after affinity purification of rat brain membranes and through a direct physical interaction between enterostatin and purified F1-ATP synthase. In insulinoma cells (INS-1) enterostatin was found to target F1F0-ATP synthase, causing an inhibition of ATP production, an increased thermogenesis and increased oxygen consumption. The experiments suggest a role of mitochondrial F1F0-ATP synthase in the suppressed insulin secretion induced by enterostatin. It could be speculated that this targeting mechanism is involved in the decreased energy efficiency following enterostatin treatment in rat.

  • 44.
    Bergqvist, Ann-Sofi
    et al.
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Faculty of Veterinary Medicine andAnimal Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden.
    Yokoo, Masaki
    Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohuku University, Sendai, Japan.
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Frendin, Jan
    Division of Large Animal Surgery and Medicine, Section of Anaesthesiology and Intensive Care, Department of Clinical Sciences, Faculty of Veterinary Medicine andAnimal Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden.
    Sato, Eimei
    Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohuku University, Sendai, Japan.
    Rodríguez-Martínez, Heriberto
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Faculty of Veterinary Medicine andAnimal Science, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden.
    Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct2005In: Zygote (Cambridge. Print), ISSN 0967-1994, E-ISSN 1469-8730, Vol. 13, no 3, p. 207-218Article in journal (Refereed)
    Abstract [en]

    Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 microg, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 microg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.

  • 45.
    Bergsten, Erika
    et al.
    Ludwig Institute for Cancer Research, Stockholm Branch, PO Box 240, S-171 77 Stockholm, Sweden .
    Uutela, Marko
    Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, PO Box 21 (Haartmaninkatu 3), SF-00014 Helsinki, Finland .
    Li, Xuri
    Ludwig Institute for Cancer Research, Stockholm Branch, PO Box 240, S-171 77 Stockholm, Sweden .
    Pietras, Kristian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Alitalo, Kari
    Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, PO Box 21 (Haartmaninkatu 3), SF-00014 Helsinki, Finland .
    Eriksson, Ulf
    Ludwig Institute for Cancer Research, Stockholm Branch, PO Box 240, S-171 77 Stockholm, Sweden .
    PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor2001In: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 3, no 5, p. 512-516Article in journal (Refereed)
    Abstract [en]

    The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.

  • 46.
    Bergström, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yamashita, Taro
    Ando, Yukio
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 348, no 2, p. 532-539Article in journal (Refereed)
    Abstract [en]

    We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.

  • 47.
    Bergström, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gustavsson, Åsa
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Sletten, Knut
    Murphy, Charles
    Weiss, Deborah
    Solomon, Alan
    Olofsson, Bert-Ove
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Amyloid deposits in transthyretin derived amyloidosis: Cleaved transthyretin is associated with distinct amyloid morphology2005In: The Internet Journal of Pathology, ISSN 1528-8307, Vol. 206, no 2, p. 224-232Article in journal (Refereed)
    Abstract [en]

    The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild-type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub-endocardium, sub-epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly approximately 79-residue C-terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR-associated amyloidoses.

  • 48.
    Bergström, Rosita
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Savary, Katia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Morén, Anita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Guibert, Sylvain
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ohlsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Biology.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Transforming growth factor β promotes complexes between Smad proteins and the CCCTC-binding factor on the H19 imprinting control region chromatin2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 26, p. 19727-19737Article in journal (Refereed)
    Abstract [en]

    Whether signal transduction pathways regulate epigenetic states in response to environmental cues remains poorly understood. We demonstrate here that Smad3, signaling downstream of transforming growth factor beta, interacts with the zinc finger domain of CCCTC-binding factor (CTCF), a nuclear protein known to act as "the master weaver of the genome." This interaction occurs via the Mad homology 1 domain of Smad3. Although Smad2 and Smad4 fail to interact, an alternatively spliced form of Smad2 lacking exon 3 interacts with CTCF. CTCF does not perturb well established transforming growth factor beta gene responses. However, Smads and CTCF co-localize to the H19 imprinting control region (ICR), which emerges as an insulator in cis and regulator of transcription and replication in trans via direct CTCF binding to the ICR. Smad recruitment to the ICR requires intact CTCF binding to this locus. Smad2/3 binding to the ICR requires Smad4, which potentially provides stability to the complex. Because the CTCF-Smad complex is not essential for the chromatin insulator function of the H19 ICR, we propose that it can play a role in chromatin cross-talk organized by the H19 ICR.

  • 49.
    Bergström, Rosita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Whitehead, Joanne
    Kurukuti, Sreenivasulu
    Ohlsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    CTCF Regulates Asynchronous Replication of the Imprinted H19/Igf2 Domain2007In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 6, no 4, p. 450-454Article in journal (Refereed)
    Abstract [en]

    Asynchronous replication during S phase is a universal characteristic of genomically imprinted genes. Replication timing in imprinted domains is determined epigenetically, as it is parent of origin specific, and is seen in the absence of sequence divergence between the two alleles. At the imprinted H19/lgf2 domain, the methylated paternal allele replicates early while the CTCF-bound maternal allele replicates late during S phase. CTCF regulates the allele-specific epigenetic characteristics of this domain, including methylation, transcription and chromosome conformation. Here we show that maternal, but not paternal inheritance of a mutated H19 imprinting control region, lacking functional CTCF binding sites, underlies a late to early switch in replication timing of the maternal H19/ lgf2 domain.

  • 50.
    Bernert, Berit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Importance of Hyaluronan Metabolism and Signalling in Tumour Progression2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Hyaluronan, an unbranched glycosaminoglycan of the extracellular matrix, has an amazingly simple structure. Initially thought to fulfil only hydrating and space-filling functions in tissues, evidence generated during the past decades shows that hyaluronan is involved in intriguingly complex signalling events in health and disease. In cancer, increased hyaluronan levels have been correlated with poor patient survival.

    The research underlying this thesis sheds light on the interplay between hyaluronan, its producing and degrading enzymes as well as the triggered intracellular signalling in the metastatic cascade. Utilising breast cancer and normal mammary cells, paper I and II investigate the initial steps of tumour progression: proliferation, invasion and epithelial-mesenchymal transition. Hyaluronan synthase 2 plays a central role in all these processes. In paper III, the focus is shifted toward growth factor-induced hyaluronan production. Stimulation with PDGF-BB, which can be secreted by tumour cells, increased hyaluronan production via upregulation of HAS2 in fibroblast cultures. Finally, paper IV discusses the involvement of hyaluronidases and CD44 in angiogenesis and intravasation – events that are associated with advanced cancer stages.

    List of papers
    1. Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)
    Open this publication in new window or tab >>Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)
    2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 49, p. 42349-42359Article in journal (Refereed) Published
    Abstract [en]

    Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-168725 (URN)10.1074/jbc.M111.278598 (DOI)000298180900044 ()22016393 (PubMedID)
    Available from: 2012-02-15 Created: 2012-02-15 Last updated: 2017-12-07Bibliographically approved
    2. Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2
    Open this publication in new window or tab >>Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2
    Show others...
    2013 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 37, p. 4355-4365Article in journal (Refereed) Published
    Abstract [en]

    Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor β (TGFβ) is a well-studied inducer of EMT. We demonstrate that TGFβ potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFβ receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFβ-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFβ-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFβ-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFβ-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFβ-induced EMT.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-190691 (URN)10.1038/onc.2012.475 (DOI)000324404200004 ()23108409 (PubMedID)
    Available from: 2013-01-08 Created: 2013-01-08 Last updated: 2017-12-06Bibliographically approved
    3. Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB
    Open this publication in new window or tab >>Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB
    Show others...
    2007 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 404, p. 327-336Article in journal (Refereed) Published
    Abstract [en]

    The glycosaminoglycan hyaluronan is important in many tissue-repair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK 1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappa B (nuclear factor KB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta 1 (transforming growth factor-beta 1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Imponantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hennes-1, inhibited PDGF-BB-stimulated [H-3]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

    Keywords
    dermal fibroblast, growth factor, hyaluronan synthase (HAS), hyaluronidase, platelet-derived growth factor (PDGF), signal transduction
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-144329 (URN)10.1042/BJ20061757 (DOI)000247014700017 ()17324121 (PubMedID)
    Available from: 2011-01-31 Created: 2011-01-28 Last updated: 2017-12-11Bibliographically approved
    4. CD44 and Hyal2 affect capillary endothelial cell differentiation and breast cancer transmigration
    Open this publication in new window or tab >>CD44 and Hyal2 affect capillary endothelial cell differentiation and breast cancer transmigration
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-190704 (URN)
    Available from: 2013-01-08 Created: 2013-01-08 Last updated: 2013-04-29
1234567 1 - 50 of 674
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