View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UEF Electronic Publications KUOPION YLIOPISTON JULKAISUJA G. - A.I. VIRTANEN -INSTITUUTTI 56 KUOPIO UNIVERSITY PUBLICATIONS G. A.I. VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 56 MERVI T. HYVÖNEN Regulation of Spermidine/Spermine N1-Acetyltransferase and its Involvement in Cellular Proliferation and Development of Acute Pancreatitis Doctoral dissertation To be presented by permission of the Faculty of Medicine of the University of Kuopio for public examination in Tietoteknia Auditorium, Tietoteknia building, University of Kuopio, on Friday 14th December 2007, at 12 noon Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Kuopio JOKA KUOPIO 2007 Distributor: Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 17 163 430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html Series Editors: Research Director Olli Gröhn, Ph.D. Department of Neurobiology A.I. Virtanen Institute for Molecular Sciences Research Director Michael Courtney, Ph.D. Department of Neurobiology A.I. Virtanen Institute for Molecular Sciences Author’s address: Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 17 162 092 Fax +358 17 163 025 E-mail: [email protected] Supervisors: Professor Leena Alhonen, Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Kuopio Professor Juhani Jänne, M.D., Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Kuopio Reviewers: Professor Seppo Lapinjoki, Ph.D. Department of Pharmaceutical Chemistry University of Kuopio Docent Pauli Seppänen, Ph.D. Savonia-polytechnic Kuopio Opponent: Professor Eero Vuorio, M.D., Ph.D. University of Turku ISBN 978-951-27-0615-0 ISBN 978-951-27-0437-8 (PDF) ISSN 1458-7335 Kopijyvä Kuopio 2007 Finland Hyvönen, Mervi T. Regulation of spermidine/spermine 1N­acetyltransferase and its involvement in cellular proliferation and development of acute pancreatitis. Kuopio University Publications G. – A. I. Virtanen Institute for Molecular Sciences 56. 2007. 79 p. ISBN 978•951•27­0615­0 ISBN 978•951•27­0437­8 (PDF) ISSN 1458­7335 ABSTRACT The naturally occurring organic polycations, the polyamines spermidine, spermine and their precursor putrescine, are essential for cellular proliferation and differentiation. Their intracellular level is maintained by strictly regulated metabolic pathways. In order to investigate the functions and metabolism of polyamines, several genetically manipulated rodent lines have been generated. Activation of polyamine catabolism in transgenic rats overexpressing spermidine/spermine1­ N acetyltransferase (SSAT) under the control of heavy metal­inducible metallothionein I (MT) promoter results in a rapid depletion of spermidine and spermine and leads to the development of severe acute pancreatitis. Previous studies have shown that prophylactic administration of a stable spermidine analog, a­methylspermidine (MeSpd), can prevent the development of zinc­induced acute pancreatitis and restore the delayed liver regeneration after partial hepatectomy of MT­SSAT transgenic rats. In this work, the role of polyamines in other experimental models of pancreatitis, the pathogenesis of polyamine depletion­induced pancreatitis and the therapeutic potential of methylpolyamines were investigated. Activation of polyamine catabolism and depletion of higher polyamines were evident in both cerulein and L­arginine experimental models of pancreatitis, and also in two human pancreatic specimens obtained from patients with acute pancreatitis. Early pathogenesis in MT­ SSAT transgenic rats involved the activation of cathepsin B and trypsinogen, whereas prior administration of MeSpd inhibited the activation of both proteases. Importantly, the therapeutic administration of MeSpd ora,w­bismethylspermine (Me2Spm) could dramatically protect MT­ SSAT transgenic rats from pancreatitis­associated mortality. The ability of stereoisomers of methylpolyamines to protect pancreatic integrity and support cellular growth was also tested. Although only (S,S)­Me2Spm was metabolized to MeSpd, both (R,R)• and (S,S)­enantiomers were equally effective in preventing the development of pancreatitis and restoring liver regeneration in MT­SSAT transgenic rats.In vitro, all stereoisomers of both MeSpd and Me2Spm effectively rescued cells from acute cytostasis caused by inhibition of polyamine biosynthesis with a­difluoromethylornithine (DFMO). However, only (S)­MeSpd was able to support growth after prolonged exposure to DFMO. 2D­immunoblot analysis of eukaryotic translation initiation factor 5A (eIF5A) indicated that only (S)­MeSpd could serve as a precursor of hypusine, a unique aminoacid derivative essential for the synthesis of functional eIF5A. The physiological relevance of alternative splicing of SSAT pre­mRNA was also investigated. The alternative splice variant was targeted to a protein synthesis­dependent degradation pathway known as nonsense­mediated mRNA decay. Furthermore, the intracellular polyamine level regulated the balance of the two splice variants: polyamine supplementation favored the generation of the productive variant, subsequently resulting in decreased polyamine levels, whereas polyamine depletion favored the production of the alternative, unproductive variant. Thus, polyamine­regulated unproductive splicing and translation represents a novel posttranscriptional regulation mechanism of SSAT. In conclusion, these findings emphasize the importance of polyamine homeostasis for cellular proliferation and for the integrity and normal function of the liver and the exocrine pancreas, and open up possibilities for novel therapeutic approaches. National Library of Medicine classification: QT 120, QU 61, QU 450, QY 58, WI 702, WI 805 Medical Subject Headings: Acetyltransferases; Animals, Genetically Modified; Disease Models, Animal; Homeostasis; Liver/metabolism; Liver Regeneration; Pancreas/metabolism; Pancreatitis/therapy; Polyamines/metabolism; Putrescine; Rats; Spermidine; Spermidine/analogs & derivatives; Spermine; Spermine/analogs & derivatives; Trypsinogen If it happens, it must be possible. (Unnamed law) ACKNOWLEDGEMENTS This thesis work was carried out in the Department of Biotechnology and Molecular Medicine, at A. I. Virtanen Institute, University of Kuopio, during the years 2002­2007. I wish to express my deepest gratitude to my supervisors, Professors Leena Alhonen, Ph.D., and Juhani Jänne, M.D., Ph.D., for introducing me to the interesting field of polyamines and giving me the opportunity to work in their research group using state­of­the­art biotechnical equipment. Your belief in me has been encouraging during all these years. I am grateful to Docent Pauli Seppänen and Professor Seppo Lapinjoki, the official reviewers of this thesis, for offering valuable advice and constructive criticism. I also thank Ewen MacDonald, Ph.D., for linguistic revision of this thesis. Many thanks to Riitta Sinervirta for invaluable help with the animal work and all general issues during these years. Heartfelt thanks to our post doctoral fellows Tuomo Keinänen, Ph.D., and Anne Uimari, Ph.D., for their inexhaustive guidance, advice, understanding and all the joyful moments and stimulating discussions. Thank also to Sami Heikkinen, Ph.D., Aki Järvinen, Ph.D., and Marko Pietilä, Ph.D. Extra thanks to Suvikki Loimas, Ph.D., for encouragement and guidance at the beginning of my scientific career. I thank Marc Cerrada­Gimenez, M.Sc. and Mari Merentie, M.Sc., for significant contribution to this thesis work. I also thank our present and former group members. I thank the technical staff for the invaluable help in all practical issues during these years. Thanks to Marita Heikkinen, Sisko Juutinen, Anne Karppinen, Arja Korhonen, Tuula Reponen and Anu Heikkinen for excellent technical assistance and for numerous invigorating conversations in the lab and coffee room. I am also grateful to Eeva Hakala, Riitta Keinänen, Ph.D., Helena Pernu, Pekka Alakuijala and Jouko Mäkäräinen for keeping all practical and technical things running smoothly. I warmly thank our collaborators Ale Närvänen, Ph.D., Karl­Heinz Herzig, M.D., Ph.D., Professor Isto Nordback, M.D., Ph.D. I wish to give extra­special thanks to our "chemistry guys" Professor Jouko Vepsäläinen, Ph.D., Alex R. Khomutov, Ph.D. and Nikolay Grigorenko, Ph.D. for work­ related and non­work­related issues. Thanks also to Jarmo Wahlfors, Ph.D., Tiina Wahlfors, Ph.D., and Riikka Pellinen, Ph.D. for collaboration. Special thanks to Tero Hongisto for enjoyable conversations and help with my current studies. Beyond scientific world, I am most grateful to my parents Eila and Veikko for encouragement, understanding and support. I thank Marika for sharing all the joys and sorrows with me. Once more, special thanks to my soul­mate and husband Tuomo for all the support and TLC during these years. Your belief, support and love have made this possible. And Tolstoi & flock, my specials, I love you all! Special thanks to my close friends and "second family": Impi, Pia, Asko, Riitta, Mikko, Maija, Ripi and Anni, as well as Vigrate and Spint Wind, thank you for fun & relaxing time together and giving me the much­needed distraction from the "World of Science"