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  • 301.
    Kowanetz, Katarzyna
    et al.
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Szymkiewicz, Iwona
    Haglund, Kaisa
    Kowanetz, Marcin
    Husnjak, Koraljka
    Taylor, Jon D.
    Soubeyran, Philippe
    Engstrom, Ulla
    Ladbury, John E.
    Dikic, Ivan
    Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors.2003In: J Biol Chem, Vol. 278, no 41, p. 39735-46Article in journal (Refereed)
  • 302.
    Kowanetz, Katarzyna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Szymkiewicz, Iwona
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Haglund, Kaisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kowanetz, Marcin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Husnjak, Koraljka
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Taylor, Jonathan D
    Soubeyran, Philippe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ladbury, John E
    Dikic, Ivan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 41, p. 39735-39746Article in journal (Refereed)
    Abstract [en]

    CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.

  • 303.
    Kowanetz, Katarzyna
    et al.
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Terzic, Janos
    Dikic, Ivan
    Dab2 links CIN85 with clathrin-mediated receptor internalization.2003In: FEBS Lett, Vol. 554, no 1-2, p. 81-7Article in journal (Refereed)
  • 304.
    Kowanetz, Marcin
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Novel Regulators of the TGF-β Signaling Pathway2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.

    The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, Id2 and Id3. Interestingly, we found that TGF-β-induced repression of Ids is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of Ids and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.

    Furthermore, we identified a serine/threonine kinase, SNF1LK, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling.

    List of papers
    1. TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition
    Open this publication in new window or tab >>TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition
    Show others...
    2005 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 16, no 4, p. 1987-2002Article in journal (Refereed) Published
    Abstract [en]

    Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-beta/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-beta superfamily establishes that TGF-beta but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-beta-induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-beta target genes with ligand-specific responses. Using a TGF-beta type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-beta1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, alpha-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-beta and predict functional links to the control of cell proliferation and EMT.

    Place, publisher, year, edition, pages
    The American Society for Cell Biology, 2005
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-93311 (URN)10.1091/mbc.E04-08-0658 (DOI)15689496 (PubMedID)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2018-06-04Bibliographically approved
    2. Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein
    Open this publication in new window or tab >>Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein
    Show others...
    2004 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 10, p. 4241-4254Article in journal (Refereed) Published
    Abstract [en]

    Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.

    Place, publisher, year, edition, pages
    American Society for Microbiology, 2004
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-93312 (URN)10.1128/MCB.24.10.4241-4254.2004 (DOI)15121845 (PubMedID)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2017-12-14Bibliographically approved
    3.
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  • 305.
    Kowanetz, Marcin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lönn, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Vanlandewijck, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Kowanetz, Katarzyna
    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.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    TGFβ induces SIK to negatively regulate type I receptor kinase signaling2008In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 182, no 4, p. 655-662Article in journal (Refereed)
    Abstract [en]

    Signal transduction by transforming growth factor beta (TGFbeta) coordinates physiological responses in diverse cell types. TGFbeta signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFbeta signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFbeta/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFbeta. We thus identify in SIK a negative regulator that controls TGFbeta receptor turnover and physiological signaling.

  • 306.
    Kowanetz, Marcin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Valcourt, Ulrich
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bergström, Rosita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology.
    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, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein2004In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 24, no 10, p. 4241-4254Article in journal (Refereed)
    Abstract [en]

    Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.

  • 307.
    Kozlova, Inna
    et al.
    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.
    Ruusala, Aino
    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.
    Voytyuk, Oleksandr
    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.
    Skandalis, Spyros S
    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.
    Heldin, Paraskevi
    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.
    IQGAP1 regulates hyaluronan-mediated fibroblast motility and proliferation2012In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 24, no 9, p. 1856-1862Article in journal (Refereed)
    Abstract [en]

    IQGAP1, an essential scaffolding protein, forms a complex with the hyaluronan receptor CD44. In this study, we have examined the importance of IQGAP1 for hyaluronan-mediated fibroblast migration and proliferation. Hyaluronan induced formation of F-actin fibers and focal adhesions, which was dependent on IQGAP1. IQGAP1 was required for hyaluronan- but not for platelet-derived growth factor (PDGF)-BB-induced cell migration, and was required for both hyaluronan- and PDGF-BB-mediated fibroblast proliferation, but not for proliferation induced by 10% fetal bovine serum. Depletion of IQGAP1 suppressed hyaluronan-induced activation of Rac1 and enhanced the activation of RhoA. Taken together, these findings indicate important roles for IQGAP1 in hyaluronan-stimulated migration and proliferation of fibroblasts.

  • 308.
    Krampert, Monika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Chirasani, Sridhar Reddy
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Wachs, Frank-Peter
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Aigner, Robert
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Bogdahn, Ulrich
    Department of Neurology, University of Regensburg, Regensburg, Germany.
    Yingling, Jonathan M.
    Eli Lilly, Lilly Research Laboratories, Cancer Research, Indianapolis, Indiana, USA.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Aigner, Ludwig
    Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
    Heuchel, Rainer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Smad7 Regulates the Adult Neural Stem/Progenitor Cell Pool in a Transforming Growth Factor β- and Bone Morphogenetic Protein-Independent Manner2010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 14, p. 3685-3694Article in journal (Refereed)
    Abstract [en]

    Members of the transforming growth factor beta (TGF-beta) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-beta and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-beta and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-beta and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-beta and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/progenitor cell proliferation in a TGF-beta- and BMP-independent manner.

  • 309.
    Krampert, Monika
    et al.
    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.
    Heuchel, Rainer L.
    Dept. of Clinical Science, Intervention and Technology, Karolinska University Hospital, Huddinge, Sweden.
    A gain-of-function mutation in the PDGFR-beta alters thekinetics of injury response in liver and skin2008In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 88, no 11, p. 1204-1214Article in journal (Refereed)
    Abstract [en]

    Platelet-derived growth factor (PDGF) isoforms stimulate cell proliferation, migration and survival. We recently generated mice carrying a gain-of-function mutation within the activation loop of PDGF beta-receptor (PDGFR-beta D849N). Embryonic fibroblasts derived from these mice show elevated basal phosphorylation and altered kinetics for ligand-induced activation of PDGFR-beta, as well as enhanced proliferation and migration. To investigate the effect of this mutation in vivo, we used carbon tetrachloride-induced liver injury as a model system. We observed a higher basal activation of mutant PDGFR-beta in unchallenged livers; however, the difference in activation upon carbon tetrachloride stimulation was lower than expected, an effect that might be explained by a delayed response of the mutated receptor toward reactive oxygen species. Mutant mice showed enhanced proliferation of nonparenchymal liver cells and activation of hepatic stellate cells, leading to a small increase in early fibrosis formation. Another mouse strain lacking the binding site for phosphatidylinositol-3' kinase in PDGFR-beta showed the reverse phenotype. These results suggest an important role for PDGFR-beta signaling in the early injury-response. We confirmed this hypothesis with a second injury model, cutaneous wound healing, where we observed earlier proliferation and formation of granulation tissue in D849N-mutant mice.

     

  • 310. Kubinski, Konrad
    et al.
    Zielinski, Rafal
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mazur, Elzbieta
    Szyszka, Ryszard
    Yeast Elf1 factor is phosphorylated and interacts with protein kinase CK22006In: Journal of Biochemistry and Molecular Biology, ISSN 1225-8687, E-ISSN 0219-1024, Vol. 39, no 3, p. 311-318Article in journal (Refereed)
    Abstract [en]

    One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2alpha' (K(m) 0.38 microM) and holoenzyme (K(m) 0.13 microM). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Coimmunoprecypitation experiments show that Elf1 interacts with catalytic (alpha and alpha') as well as regulatory (beta and beta') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.

  • 311.
    Kurisaki, Akira
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kose, Shingo
    Yoneda, Yoshihiro
    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, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner2001In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 12, no 4, p. 1079-1091Article in journal (Refereed)
    Abstract [en]

    Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

  • 312.
    Kurisaki, Akira
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kurisaki, Keiko
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kowanetz, Marcin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Sugino, Hiromu
    Yoneda, Yoshihiro
    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, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    The mechanism of nuclear export of smad3 involves exportin 4 and Ran2006In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 26, no 4, p. 1318-1332Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.

  • 313.
    Kurisaki, Keiko
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kurisaki, Akira
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Valcourt, Ulrich
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Terentiev, Alexei A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Pardali, Katerina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    ten Dijke, Peter
    Heldin, Carl-Henrik
    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.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation2003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 13, p. 4494-4510Article in journal (Refereed)
    Abstract [en]

    Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.

  • 314.
    Kurtovic, Sanela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Grehn, Leif
    Medicago AB, Danmark Berga, Uppsala SE-75598, Sweden.
    Karlsson, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Glutathione transferase activity with a novel substrate mimics the activation of the prodrug azathioprine2008In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 375, no 2, p. 339-344Article in journal (Refereed)
    Abstract [en]

    Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.

  • 315.
    Kłosowska-Wardega, Agnieszka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hasumi, Yoko
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Burmakin, Mikhail
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Åhgren, Aive
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Stuhr, Linda
    Department of Biomedicine, University of Bergen, Norway.
    Moen, Ingrid
    Department of Biomedicine, University of Bergen, Norway.
    Reed, Rolf K.
    Department of Biomedicine, University of Bergen, Norway.
    Rubin, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hellberg, Carina
    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.
    Combined anti-angiogenic therapy targeting PDGF and VEGF receptors lowers the interstitial fluid pressure in a murine experimental carcinoma2009In: PloS one, ISSN 1932-6203, Vol. 4, no 12, p. e8149-Article in journal (Refereed)
    Abstract [en]

    Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

  • 316.
    Kłosowska-Wardega, Agnieszka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hasumi, Yoko
    Department of Dermatology, Faculty of Medicine, University of Yamanashi, Shimokato, Tamaho, Nakakoma, Yamanashi, Japan.
    Åhgren, Aive
    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.
    Hellberg, Carina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Combination therapy using imatinib and vatalanib improves the therapeutic efficiency of paclitaxel towards a mouse melanoma tumor2011In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 21, no 1, p. 57-65Article in journal (Refereed)
    Abstract [en]

    Melanomas respond poorly to chemotherapy. In this study, we investigated the sensitization of B16 mouse melanoma tumors to paclitaxel by a combination of two tyrosine kinase inhibitors: vatalanib, targeting vascular endothelial growth factor receptors, and imatinib, an inhibitor targeting for example, Abl/BCR-ABL, the platelet-derived growth factor receptor, and stem cell factor receptor c-Kit. C57Bl6/J mice carrying B16/PDGF-BB mouse melanoma tumors were treated daily with vatalanib (25 mg/kg), imatinib (100 mg/kg), or a combination of these drugs. Paclitaxel was given subcutaneously twice during the study. The effects of the drugs on tumor cell proliferation in vitro were determined by counting cells. B16/PDGF-BB mouse melanoma tumors were not sensitive to paclitaxel at doses of either 5 or 20 mg/kg. However, the tumor growth was significantly reduced by 58%, in response to paclitaxel (5 mg/kg) when administered with daily doses of both vatalanib and imatinib. Paclitaxel only inhibited the in-vitro growth of B16/PDGF-BB tumor cells when given in combination with imatinib. Imatinib presumably targets c-Kit, as the cells do not express platelet-derived growth factor receptor and as another c-Abl inhibitor was without effect. This was supported by data from three c-Kit-expressing human melanoma cell lines showing varying sensitization to paclitaxel by the kinase inhibitors. In addition, small interfering RNA knockdown of c-Kit sensitized the cells to paclitaxel. These data show that combination of two tyrosine kinase inhibitors, imatinib and vatalanib, increases the effects of paclitaxel on B16/PDGF-BB tumors, thus suggesting a novel strategy for the treatment of melanomas expressing c-Kit.

  • 317.
    Kłosowska-Wardęga, Agnieszka
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Combination Therapies Targeting PDGF and VEGF Signaling Pathways in Solid Tumors2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are independently involved in several cancer-associated mechanisms including autocrine stimulation of cancer cells, stimulation of tumor angiogenesis and regulation of interstitial fluid pressure (IFP). The scope of this thesis was to investigate the combinatory effect of anti-VEGF and anti-PDGF treatment on tumor angiogenesis and tumor IFP.

    Angiogenesis is a process of formation of blood vessels. Based on the tumors dependency on the blood vessels to supply them with oxygen and nutrients, several anti-angiogenic therapies have been tried and shown to have beneficial anti-tumor effects. More recently, anti-angiogenic treatment appeared to transiently “normalize” disorganized tumor vasculature and therefore to improve the uptake of cytotoxic agents.

    In the first study, treatment was performed on two tumor models that differ only with regard to the degree of maturation of the vasculature, reflected by different number of pericytes that are the target for anti-PDGF treatment in these tumors. The aim was to study the role of pericyte coverage in protecting endothelial cells from anti-VEGF therapies. In the pericyte-rich tumor model the combination treatment gave a more efficient anti-angiogenic effect. Interestingly, it was only a subset of pericytes that was sensitive for the treatment.

    In the second paper, the effect of anti-VEGF and anti-PDGF treatment on tumor IFP was measured. IFP is elevated in most solid tumors, which is linked to poor prognosis and higher recurrence rate. Additionally, this serves as a problem in ant-cancer therapies since it makes the uptake of cytotoxic agents inefficient. PDGF is known to actively regulate the IFP by regulating the contractile activity of fibroblasts, while VEGF regulates IFP primarily by affecting vessel leakiness. In the current study, combination of anti-VEGF and anti-PDGF therapies was shown to have an additive effect. However, the timing of administration of inhibitors appeared to be crucial. It was only short, but not long term combination treatment that further reduced IFP as compared to monotherapies. Surprisingly, the additive effect on IFP did not translate into an increased efficiency of chemotherapy when comparing combination treatment with monotherapies.

    The last paper is a follow up of the first study, where it was shown that combination of anti-VEGF and anti-PDGF treatment affect the tumor vasculature. Here we investigated if the anti-angiogenic effect improves treatment efficiency of a cytotoxic agent. There was a significant effect of the combination of anti-VEGF and anti-PDGF on Taxol treatment efficiency in this Taxol resistant tumor model. However, the mechanism for the treatment effect and the relative contribution of the targeted vasculature in the outcome of the therapy remains to be determined, since tumor cells were also sensitized for Taxol in vitro.

    In summary, we have shown that targeting of PDGF and VEGF signaling pathways simultaneously affect both vasculature and IFP to a higher extent than monotherapies.

    List of papers
    1. Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling
    Open this publication in new window or tab >>Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling
    Show others...
    2007 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 121, no 12, p. 2606-2614Article in journal (Refereed) Published
    Abstract [en]

    The aim of our study was to further explore the use of anti-angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet-derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF-BB (B16/PDGF-BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size-matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF-BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF-BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of alpha-smooth muscle actin and PDGFRbeta-positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF-BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.

    Keywords
    Pericytes, anti-angiogenic therapy, PDGF, VEGF, tumor
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-119798 (URN)10.1002/ijc.22999 (DOI)000251109000004 ()17691110 (PubMedID)
    Available from: 2007-11-06 Created: 2007-11-06 Last updated: 2017-12-12Bibliographically approved
    2. Combined anti-angiogenic therapy targeting PDGF and VEGF receptors lowers the interstitial fluid pressure in a murine experimental carcinoma
    Open this publication in new window or tab >>Combined anti-angiogenic therapy targeting PDGF and VEGF receptors lowers the interstitial fluid pressure in a murine experimental carcinoma
    Show others...
    2009 (English)In: PloS one, ISSN 1932-6203, Vol. 4, no 12, p. e8149-Article in journal (Refereed) Published
    Abstract [en]

    Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112888 (URN)10.1371/journal.pone.0008149 (DOI)000272829200003 ()19997591 (PubMedID)
    Available from: 2010-01-21 Created: 2010-01-21 Last updated: 2017-11-10Bibliographically approved
    3. Combination therapy using imatinib and vatalanib improves the therapeutic efficiency of paclitaxel towards a mouse melanoma tumor
    Open this publication in new window or tab >>Combination therapy using imatinib and vatalanib improves the therapeutic efficiency of paclitaxel towards a mouse melanoma tumor
    Show others...
    2011 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 21, no 1, p. 57-65Article in journal (Refereed) Published
    Abstract [en]

    Melanomas respond poorly to chemotherapy. In this study, we investigated the sensitization of B16 mouse melanoma tumors to paclitaxel by a combination of two tyrosine kinase inhibitors: vatalanib, targeting vascular endothelial growth factor receptors, and imatinib, an inhibitor targeting for example, Abl/BCR-ABL, the platelet-derived growth factor receptor, and stem cell factor receptor c-Kit. C57Bl6/J mice carrying B16/PDGF-BB mouse melanoma tumors were treated daily with vatalanib (25 mg/kg), imatinib (100 mg/kg), or a combination of these drugs. Paclitaxel was given subcutaneously twice during the study. The effects of the drugs on tumor cell proliferation in vitro were determined by counting cells. B16/PDGF-BB mouse melanoma tumors were not sensitive to paclitaxel at doses of either 5 or 20 mg/kg. However, the tumor growth was significantly reduced by 58%, in response to paclitaxel (5 mg/kg) when administered with daily doses of both vatalanib and imatinib. Paclitaxel only inhibited the in-vitro growth of B16/PDGF-BB tumor cells when given in combination with imatinib. Imatinib presumably targets c-Kit, as the cells do not express platelet-derived growth factor receptor and as another c-Abl inhibitor was without effect. This was supported by data from three c-Kit-expressing human melanoma cell lines showing varying sensitization to paclitaxel by the kinase inhibitors. In addition, small interfering RNA knockdown of c-Kit sensitized the cells to paclitaxel. These data show that combination of two tyrosine kinase inhibitors, imatinib and vatalanib, increases the effects of paclitaxel on B16/PDGF-BB tumors, thus suggesting a novel strategy for the treatment of melanomas expressing c-Kit.

    Place, publisher, year, edition, pages
    Wolters Kluwer Health | Lippincott Williams & Wilkins, 2011
    Keywords
    combination therapy, inhibitor, mouse melanoma, paclitaxel, receptor tyrosine kinase
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-119825 (URN)10.1097/CMR.0b013e32833faf4d (DOI)000285709200006 ()20975605 (PubMedID)
    Available from: 2010-03-02 Created: 2010-03-02 Last updated: 2018-01-12Bibliographically approved
  • 318. Labropoulou, V. T.
    et al.
    Theocharis, A. D.
    Symeonidis, A.
    Skandalis, Spyros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Karamanos, N. K.
    Kalofonos, H. P.
    Pathophysiology and Pharmacological Targeting of Tumor-Induced Bone Disease: Current Status and Emerging Therapeutic Interventions2011In: Current Medicinal Chemistry, ISSN 0929-8673, E-ISSN 1875-533X, Vol. 18, no 11, p. 1584-1598Article, review/survey (Refereed)
    Abstract [en]

    Bone disease is a common complication of metastatic solid tumors but also of primary hematological malignancies such as multiple myeloma. Our understanding of the molecular mechanisms underlying the development of bone disease by solid tumors and multiple myeloma has been significantly improved. A complex inter-dependence exists between bone disease and malignant cell growth, creating a vicious cycle of extensive bone destruction and tumor progression. Although myeloma and solid tumors share a number of common molecular pathogenetic mechanisms, they involve distinct pathophysiological pathways, resulting in osteoclastic bone resorption and inhibition of bone formation. In this review, we analyze the molecular mechanisms, involved in tumor-induced bone disease and discuss the current therapeutic approaches and the most recent clinical developments of emerging targeted therapies.

  • 319.
    Larsson, Annika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Linda
    Westermark, Gunilla T.
    Sletten, Knut
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Tjernberg, Lars O.
    Näslund, Jan
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Unwinding fibril formation of medin, the peptide of the most common form of human amyloid.2007In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 361, no 4, p. 822-828Article in journal (Refereed)
    Abstract [en]

    Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.

  • 320.
    Lehmann, Laura C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hewitt, Graeme
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Aibara, Shintaro
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden..
    Leitner, Alexander
    Swiss Fed Inst Technol, Inst Mol Syst Biol, Dept Biol, CH-8093 Zurich, Switzerland..
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Maslen, Sarah L.
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    Maturi, Varun
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Skehel, J. Mark
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    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.
    Boulton, Simon J.
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC12017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 5, p. 847-859.e7Article in journal (Refereed)
    Abstract [en]

    Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying-mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.

  • 321.
    Lennartsson, Johan
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Stem Cell Factor Induced Signal Transduction2002Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Stem Cell Factor (SCF) can function as a survival factor, a mitogen or a chemoattractant depending on cell type. Binding of SCF to c-Kit induces dimerization and subsequent autophosphorylation of the receptor. This thesis describes the intracellular signal transduction elicited by c-Kit.

    In the search for signal transduction molecules binding to activated c-Kit, we identified the adaptor proteins Grb2 and Grb7 as interacting partners. Grb2 could associate to Tyr-703 in the kinase insert as well as to Tyr-936 in the C-terminal tail of c-Kit. However, Grb7 could only bind to Tyr-936.

    Tyr-568 in c-Kit is essential for SCF induced association and activation of Src family kinases (SFK). A mutated receptor that could not activate SFK (Y568F or Y568/570F) showed reduced Shc phosphorylation, Ras GTP loading, Erk activation and induction of c-fos. However, activation of SFK is not essential for the mitogenic response as measured by DNA synthesis.

    Tyr-900 in c-Kit was identified as a SFK dependent phosphorylation site. The adaptor protein Crk and the p85 subunit of PI3’-kinase could associate with phosphorylated Tyr-900. In addition, we could demonstrate a constitutive complex between Crk and p85, suggesting indirect binding of Crk to Tyr-900 via p85. Mutation of Tyr-900 (Y900F) led to a reduced phosphorylation of Crk-II, loss of the second wave of Erk phosphorylation and a reduced mitogenic response. In addition the mutated receptor showed an increased ligand-induced degradation as compared to the wild-type receptor.

    There exist two splice forms of c-Kit that differ in the presence or absence of four amino acids in the extracellular juxtamembrane region. These splice forms bind SCF with similar affinity but display striking differences in signaling characteristics, e.g. in phosphorylation kinetics, ligand-induced c-Kit degradation and activation of Erks. However, other pathways are activated similarly by both splice forms, such as the Ser/Thr kinase Akt which lies downstream of PI3’-kinase. In this study we show that differential phosphorylation of the various tyrosine residues occurs. Interestingly, Tyr-568 is more efficiently phosphorylated in the shorter form leading to stronger binding of SFK, whereas the PI3’-kinase binding site showed a similar degree of phosphorylation consistent with the data on Akt activation.

  • 322.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Burovic, Fatima
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Witek, Barbara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Jurek, Aleksandra
    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.
    Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis2010In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 22, no 6, p. 955-960Article in journal (Refereed)
    Abstract [en]

    Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor beta (PDGFRbeta). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.

  • 323.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ma, Haisha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wardega, Piotr
    Pelka, Karin
    Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellberg, Carina
    School of Biosciences, University of Birmingham, Birmingham, UK.
    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.
    The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 22, p. 15736-15744Article in journal (Refereed)
    Abstract [en]

    Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF β-receptor (PDGFRβ) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRβ kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.

  • 324.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Experimental Clinical Chemistry, Wallenberg Laboratory, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden.
    Stem Cell Factor Receptor/c-Kit: From Basic Science to Clinical Implications.2012In: Physiological Reviews, ISSN 0031-9333, E-ISSN 1522-1210, Vol. 92, no 4, p. 1619-1649Article, review/survey (Refereed)
    Abstract [en]

    Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Activation of c-Kit leads to its autophosphorylation and initiation of signal transduction. Signaling proteins are recruited to activated c-Kit by certain interaction domains (e.g., SH2 and PTB) that specifically bind to phosphorylated tyrosine residues in the intracellular region of c-Kit. Activation of c-Kit signaling has been found to mediate cell survival, migration, and proliferation depending on the cell type. Signaling from c-Kit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and some aspects of the nervous system. Deregulated c-Kit kinase activity has been found in a number of pathological conditions, including cancer and allergy. The observation that gain-of-function mutations in c-Kit can promote tumor formation and progression has stimulated the development of therapeutics agents targeting this receptor, e.g., the clinically used inhibitor imatinib mesylate. Also other clinically used multiselective kinase inhibitors, for instance, sorafenib and sunitinib, have c-Kit included in their range of targets. Furthermore, loss-of-function mutations in c-Kit have been observed and shown to give rise to a condition called piebaldism. This review provides a summary of our current knowledge regarding structural and functional aspects of c-Kit signaling both under normal and pathological conditions, as well as advances in the development of low-molecular-weight molecules inhibiting c-Kit function.

  • 325.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Experimental Clinical Chemistry, Dept. of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden.
    The stem cell factor receptor/c-Kit as a drug target in cancer2006In: Current Cancer Drug Targets, ISSN 1568-0096, E-ISSN 1873-5576, Vol. 6, no 1, p. 65-75Article in journal (Refereed)
    Abstract [en]

    Tyrosine phosphorylation has a key role in intracellular signaling. Inappropriate proliferation and survival cues in tumor cells often occur as a consequence of unregulated tyrosine kinase activity. Much of the current development of anti-cancer therapies tries to target causative proteins in a specific manner to minimize side-effects. One attractive group of target proteins is the kinases. c-Kit is a receptor tyrosine kinase that normally controls the function of primitive hematopoietic cells, melanocytes and germ cells. It has become clear that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loops or mutational activation. This makes c-Kit an excellent target for cancer therapies in these tumors. In this review we will highlight the current knowledge on the signal transduction molecules and pathways activated by c-Kit under normal conditions and in cancer cells, and the role of aberrant c-Kit signaling in cancer progression. Recent advances in the development of specific inhibitors interfering with these signal transduction pathways will be discussed.

  • 326.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Voytyuk, Olexandr
    Heiss, Elke
    Sundberg, Christina
    Sun, Jianmin
    Rönnstrand, Lars
    C-Kit signal transduction and involvement in cancer2005In: Cancer Therapy, Vol. 3, no A, p. 5-28Article in journal (Refereed)
  • 327.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wardega, Piotr
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    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.
    Alix Facilitates the Interaction between c-Cbl and Platelet-derived Growth Factor beta-Receptor and Thereby Modulates Receptor Down-regulation2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 51, p. 39152-39158Article in journal (Refereed)
    Abstract [en]

    Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) β-receptor (PDGFRβ) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRβ removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRβ. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRβ. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRβ. Our data suggest that Alix inhibits down-regulation of PDGFRβ by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.

  • 328.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wardega, Piotr
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Ma, Haisha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pelka, Karin
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellberg, Carina
    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.
    The Fer tyrosine kinase is necessary for platelet-derived growth factor-BB-induced Stat3 phosphorylation and colony formation in soft agarManuscript (preprint) (Other academic)
    Abstract [en]

    Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF b-receptor (PDGFRb) through multiple autophosphorylation sites, i.e. Tyr579, Tyr581, Tyr740 and Tyr1021. Using low molecular weight inhibitors we found that PDGF-BB-induced Fer activation is dependent on PDGFRb kinase activity, but not on the enzymatic activity of Src or Jak kinases. To elucidate the function of Fer downstream of PDGFRb, we downregulated the expression using siRNA; under conditions with reduced Fer expression, PDGF-BB was unable to induce phosphorylation of Stat3, whereas Stat5, Erk1/2 and Akt were unaffected. In addition, PDGFRb autophosphorylation was partially dependent on Fer expression. On a functional level, we could demonstrate that expression of Fer is dispensable for PDGF-BB-induced proliferation and migration. However, Fer was found to be essential for colony formation in soft agar, consistent with its critical role in Stat3 activation, which has frequently been implicated in cell transformation.

  • 329.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 288, no 1, p. 110-118Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.

  • 330. Lexander, Helena
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Palmberg, Carina
    Auer, Gert
    Hellström, Magnus
    Franzén, Bo
    Jörnvall, Hans
    Egevad, Lars
    Evaluation of two sample preparation methods for prostate proteome analysis2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 13, p. 3918-3925Article in journal (Refereed)
    Abstract [en]

    For laboratory techniques that require well-preserved proteins, such as 2-DE, fresh tissue must be harvested and processed as fast as possible to avoid proteolytic degradation. We describe a modified method for harvesting tissue from radical prostatectomy specimens for proteome analysis and compare it with the standard technique. Cells were scraped from cut surfaces of 11 prostate specimens. A fraction of the material was smeared on a glass slide and Giemsa stained for morphological control. The sample was collected in a medium with protease inhibitors, and the protein material was prepared for 2-DE. Filtering and Percoll centrifugation were omitted. Sample locations were noted on a specimen map. From the same area, a tissue block was harvested for comparison. The block was processed with the conventional technique including mechanical disintegration, filtering and Percoll centrifugation. Quality measures of 2-DE were similar with both methods. With the scrape sampling technique, control smears showed abundant epithelial cells and a cleaner background and processing was faster than with tissue block sampling. For proteomic analysis, the scrape sample technique has several advantages over the tissue block method.

  • 331. Lexander, Helena
    et al.
    Palmberg, Carina
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Auer, Gert
    Hellström, Magnus
    Franzén, Bo
    Jörnvall, Hans
    Egevad, Lars
    Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 15, p. 4370-4380Article in journal (Refereed)
    Abstract [en]

    The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.

  • 332.
    Li, Li
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Da, Jiping
    Landström, Marené
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ulmsten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Fu, Xin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Antiproliferative activity and toxicity of 2-Methoxyestradiol in cervical cancer xenograft mice2005In: International Journal of Gynecological Cancer, ISSN 1048-891X, E-ISSN 1525-1438, Vol. 15, no 2, p. 301-307Article in journal (Refereed)
    Abstract [en]

    2-methoxyestradiol (2-ME) is considered to be an effective anticancer compound for many types of tumors. We have previously demonstrated that 2-ME inhibits the growth of human cervical cancer HeLaS3 cells in vitro. In this study, we investigated the antitumoral effects of 2-ME on human cervical carcinoma in severe combined immune deficient (SCID) mice. The potential side effects of 2-ME on the SCID mice were also investigated. SCID mice were injected with HeLaS3 cells (3 x 10(6) to 4 x 10(6)/mouse) and a 15-day administration of 2-ME followed after a 1-week cell implantation. Tumor weight, volume, body weight, and blood chemistry were determined. Tumor tissues were examined with an antibody against the proliferative cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Liver, spleen, kidney, heart, and lung were screened by pathologic examinations. 2-ME (75 mg/kg p.o.) inhibited growth of human cervical carcinoma by 34% (P < 0.05) as compared with control. Necrosis was found in both 2-ME-treated and untreated tumor tissues, but the necrotic area was larger in 2-ME-treated mice. A low expression of proliferative cell nuclear antigen and an increased number of apoptotic cells were found in 2-ME-treated tumor sections as compared to those in controls. No significant difference was detected in blood chemistry. In addition, the liver showed hyperplastic Kupffer cells, hydropic swelling of hepatocytes, and liquefactive necrosis. The spleen showed an increased number of megakaryocytes and apoptotic cells after 2-ME treatment. Thus, 2-ME has an antitumor effect on human cervical carcinoma, and it is toxic to liver and spleen in this mouse model.

  • 333.
    Li, Lingli
    Uppsala University, Units outside the University, Ludwig Institute for Cancer Research.
    Effect of Hyaluronan-activation of CD44 on Cell Signaling and Tumorigenesis2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance.

    Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine CXCL1/GRO1 gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates HAS2 transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of HAS2 gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan.

    In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.

    List of papers
    1. Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner
    Open this publication in new window or tab >>Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner