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. 2001 Jan;35(1):3–8. doi: 10.1023/A:1008192221674

Growth and secretion of erythropoietin of Chinese hamster ovary cells coexpressing epidermal growth factor receptor and erythropoietin genes: Design of cells for cell culture matrix

Guoping Chen 1, Yoshihiro Ito 2,✉, Seiji Masuda 4, Ryuzo Sasaki 4
PMCID: PMC3466621  PMID: 19003275

Abstract

Chinese hamster ovary (CHO) cells were transfected withboth genes encoding erythropoietin (Epo) and epidermal growthfactor receptor (EGFR). The transfection of the Epo gene wasconfirmed by an enzyme-linked immunoassay. Overexpression ofEGFR was confirmed by Western blotting of EGFR. Thetransfected CHO cells were cultured in serum-free medium inthe presence of soluble epidermal growth factor (EGF) orimmobilized EGF. The CHO cells overexpressing EGFR grew in thepresence of less EGF than the cells not overexpressing EGFR.In addition, the growth of EGFR-overexpressing CHO cells wasenhanced in the presence of immobilized EGF more efficientlythan in the presence of soluble EGF. The amount of Eposecreted from the cells increased linearly with the increaseof growth rate. Consequently, culture of CHO cellscoexpressing Epo and EGFR on EGF-immobilized matrix was themost efficient for Epo production.

Keywords: epidermal growth factor, epidermal growth factor receptor, erythropoietin, immobilization, transfection

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References

  1. Bosenberg MW, Massague J. Juxtacrine cell signaling molecules. Curr Opin Cell Biol. 1993;5:822–838. doi: 10.1016/0955-0674(93)90032-l. [DOI] [PubMed] [Google Scholar]
  2. Chen G, Ito Y, Imanishi Y. Photo-immobilization of epidermal growth factor enhances its mitogenic effect by artificial juxtacrine signaling. Biochim Biophys Acta. 1997;1358:200–208. doi: 10.1016/s0167-4889(97)00065-7. [DOI] [PubMed] [Google Scholar]
  3. Felgner PL, Holm M. Cationic liposome-mediated transfection. Focus. 1989;11:21–25. [PubMed] [Google Scholar]
  4. Fiore PPD, Pierce JH, Fleming TP, Hazan R, Ullrich A, King CR, Schlessinger J, Aaronson SA. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell. 1987;51:1063–1070. doi: 10.1016/0092-8674(87)90592-7. [DOI] [PubMed] [Google Scholar]
  5. Glover DM. Gene Cloning. New York: Chapman and Hall; 1984. [Google Scholar]
  6. Goto M, Akai K, Murakami A, Hashimoto C, Tsuda E, Ueda M, Kawanishi G, Takahashi N, Ishimoto A, Chiba H, Sasaki R. Production of recombinant human erythropoietin in mammalian cells: host-cell dependency of the biological activity of the cloned glycoprotein. Bio/Technology. 1988;6:67–71. [Google Scholar]
  7. Goto M, Murakami A, Akai K, Kawanishi G, Ueda M, Chiba H, Sasaki R. Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants. Blood. 1989;74:1415–1423. [PubMed] [Google Scholar]
  8. Hofmann C, Goldfine ID, Whittaker J. The metabolic and mitogenic effects of both insulin and insulin-like growth factor are enhanced by transfection of insulin receptors into NIH3T3 fibroblast. J Biol Chem. 1989;264:8606–8611. [PubMed] [Google Scholar]
  9. Ishiyama M, Shiga M, Sasamoto K, Mizoguchi M, He P. A new sulfonated tetrazolium salt that produces a highly watersoluble formazan dye. Chem Pharm Bull. 1993;41:1118–1122. [Google Scholar]
  10. Ishiyama M, Tominaga H, Shiga M, Sasamoto K., Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull. 1996;19:1518–1520. doi: 10.1248/bpb.19.1518. [DOI] [PubMed] [Google Scholar]
  11. Ito Y, Chen G, Imanishi Y. Artificial juxtacrine stimulation for tissue engineering. J Biomat Sci. 1998;9:879–888. doi: 10.1163/156856298x00217. [DOI] [PubMed] [Google Scholar]
  12. Ito Y, Liu SQ, Imanishi Y. Enhancement of cell growth by growth-factor-immobilized polymer films. Biomaterials. 1991;12:449–453. doi: 10.1016/0142-9612(91)90141-v. [DOI] [PubMed] [Google Scholar]
  13. Ito Y, Ueda H, Suzuki E. Overexpression of bcl-2, apoptosis suppressing gene: prolonged viable culture period of hybridoma and enhanced antibody production. Biotechnol Bioeng. 1995;48:118–122. doi: 10.1002/bit.260480205. [DOI] [PubMed] [Google Scholar]
  14. Ito Y, Zheng J, Imanishi Y, Yonezawa K, Kasuga M. Protein-free cell culture on an artificial substrate with covalently immobilized insulin. Proc Natl Acad Sci USA. 1996;93:3598–3601. doi: 10.1073/pnas.93.8.3598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krantz SB. Erythropoietin. Blood. 1991;77:419–434. [PubMed] [Google Scholar]
  16. Massague J, Pandiella A. Membrane-anchored growth factors. Annu Rev Biochem. 1993;62:15–541. doi: 10.1146/annurev.bi.62.070193.002503. [DOI] [PubMed] [Google Scholar]
  17. Moon SK, Takeuchi S, Kambe T, Tsuchiya T, Masuda S, Nagao M, Sasaki R. Erythropoietin enhancer stimulates production of a recombinant protein by Chinese hamster ovary (CHO) cells under hypoxic condition. Cytotechnology. 1997;25:79–88. doi: 10.1023/A:1007963800362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Reddy CC, Wells A, Lauffenburger DA. Proliferative response of fibroblasts expressing internalization-deficient epidermal growth factor (EGF) receptors is altered via differential EGF depletion effect. Biotechnol Prog. 1994;10:377–384. doi: 10.1021/bp00028a006. [DOI] [PubMed] [Google Scholar]
  19. Tajima Y, Huang EJ, Vosseller K, Ono M, MAS, Besmer P. Role of dimerization of the membrane-associated growth factor kit ligand in juxtacrine signaling: The Sl17H mutation affects dimerization and stability - phenotypes in hematopoiesis. J Exp Med. 1998;187:1451–1461. doi: 10.1084/jem.187.9.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tanaka M, Itai T, Adachi M, Nagata S. Dowunregulation of Fas ligand by shedding. Nat Med. 1998;4:1–36. doi: 10.1038/nm0198-031. [DOI] [PubMed] [Google Scholar]

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