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Analysis of Oxygen Sensing and Its Application for Cell Technology

Research Project

Project/Area Number 09660085
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

NAGAO Masaya  KYOTO UNIVERSITY, GRADUATE SCHOOL OF BIOSTUDIES, ASSOSIATE PROFESSOR, 生命科学研究科, 助教授 (10237498)

Co-Investigator(Kenkyū-buntansha) MASUDA Seiji  KYOTO UNIVERSITY, GRADUATE SCHOOL OF BIOSTUDIES, ASSOSIATE PROFESSOR, 生命科学研究科, 助教授 (20260614)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsoxygen sensor / hypoxia / erythropoietin / transcription / cell technology / 酵素センサー / 低酵素
Research Abstract

Erythropoietin (Epo) is a glycoprotein, and its sugar chains are important for its in vivo biological activity. So, this year we examined the biological activities of recombinant Epo (rEpo) produced by cultured Chinese hamster ovary (CHO) cells in normoxia or hypoxia and analyzed the structure of its sugar chains. For this purpose we used the promoter of lactate dehydrogenase A gene, which is active in CHO cells and its vicinal hypoxia-response enhancer (HRE) stimulates the promoteractivity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing rEPO under control of this promoter/HRE. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells in 21% and 2% oxygen. Further more, forced expression of hypoxia-inducible factor-1α (HIF-1α) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even under low oxygen concentration.
HIF-1 is involved in hypoxia response. AKT kinase thought to be involved in the signal transudation of hypoxia response. So we co-transfected a constitutive active AKT kinase expression plasmid and a luciferase reporter plasmid under control of LDH A promoter/HRE into Cos1 cells. Expression of constitutive active AKT kinase alone could not stimulate the expression of the reporter in normoxic condition, but it could potentiate the expression of the reporter in hypoxia. It was reported that dominant-negative AKT could depress the hypoxic induction. These results suggest that AKT kinase is necessary but not sufficient for hypoxic induction.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Masuda,S.: "In vitro neuroprotective action of recombinant rat eryhropoitein produced by astrocyte cell lines and comparetive studies with erythropoietin produced by Chinese hamster ovary cells"Cytotechnology. 29. 207-213 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda,S.: "Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production"Int.J.Hematol.. 70. 1-6 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda,S.: "A new biologicl strategy for high productivity of recombinant proteins in aminal cells by the use of hypoxia-response enhancer"Biotechnol. Bioeng.. 67・2. 157-164 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda,S.: "Animal Cell Technology : Challenges for the 21st Century"Kluwer Academic Publishers, Netherland. 5/452 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda, S.: "In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells"Cytotechnology. 29. 207-213 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda, S.: "Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production"Int. J.Hematol.. 70. 1-6 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda, S.: "A new biological strategy for high productivity of recombinant proteins in aminal cells by the use of hypoxia-response enhancer"Biotechnol. Bioeng.. 67(2). 157-164 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda, S.: "Erythropoietin Protects Neurons from Ischemic Damage"Animal Cell Technology : Challenges for the 21st Century, Kluwer Acade* Publishers Netherland. 249-253 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda S.: "In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells"Cytotechnology. 29. 207-213 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masuda S.: "Erythropoietic,neurotrophic,and angiogenic functions of erythropoietin and regulation of erythropoietin production"Int.J.Hematol.. 70. 1-6 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masuda S.: "A new biological strategy for high productivity of recombinant proteins in aminal cells by the use of hypoxia-response enhancer"Biotechnol.Bioeng.. 67・2. 157-164 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masuda,S: "Animal Cell Technology:Challenges for the 21st Century"Kluwer Academic Publishers,Netherland. 5/452 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Sakanaka,M.: "In vivo evidence that erythropoietin protects neurons from ischemic damage" Proc.Natl.Acad.Sci.USA. 95・8. 4635-4640 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Yasuda,Y.: "Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis" J.Biol.Chem.273・39. 25381-25387 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] P.J.Ratcliffe et al.: "Oxygen regulated gene expression:Erythropoietin as a model system" Kidney Int.51. 514-526 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] C.W.Pugh et al.: "Activation of Hypoxia-inducible Factor-1:Definition of Regulatory Domains within the αSubunit" J.Biol.Chem.272. 11205-11214 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] S-K.Moon et al.: "Erythropoietin enhancer stimulates production of a recombinant protein by Chinese hamster ovary(CHO)cells under hypoxic condition" Cytotechnology. 25. 79-88 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Shirai et al.: "Effect of hypoxia duration on the oxygen-dependent production of a recombinant protein,β-galactosidase by an animal cell line F6D2,with a hypoxia-inducible enhancer" Cytotechnology. 25. 71-77 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] T.Kambe et al.: "Embryonal Carcinama P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner" Blood. 91. 1-12 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 永尾雅哉: "酸素による遺伝子発現の制御現象の解明とその動物細胞工学への応用に関する研究" 日本農芸化学会誌. 71. 1253-1264 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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