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  • Research Article
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Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells

Abstract

The eukaryotic cell cycle is regulated by a complex network of many proteins. Effective reprogramming of this complex regulatory apparatus to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production, can require coordinated manipulation of multiple genes. Previous efforts to establish inducible cell-cycle arrest of Chinese hamster ovary (CHO) cells by regulated expression of the cyclin-dependent kinase inhibitor (GDI) p21 failed. By tetracy-cline-regulated coexpression of p21 and the differentiation factor CCAAT/enhancer-binding protein α (which both stabilizes and induces p21), we have achieved effective cell-cycle arrest. Production of a model heterologous protein (secreted alkaline phosphatase; SEAP) has been increased 10–15 times, on a per cell basis, relative to an isogenic control cell line. Because activation of apoptosis response is a possible complication in a proliferation-arrested culture, the survival gene bcl-xL was coexpressed with another GDI, p27, found to enable CHO cell-cycle arrest predominantly in G1 phase. CHO cells stably transfected with a tricistronic construct containing the genes for these proteins and for SEAP showed 30-fold higher SEAP expression than controls.

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References

  1. Holmberg, N., Lilius, G., Bailey, J.E., and Bülow, L. 1997. Transgenic tabacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolic production. Nature Biotechnology 15: 244–247.

    Article  CAS  Google Scholar 

  2. Renner, W.A., Lee, K.H., Hatzimanikatis, V., Bailey, J.E., and Eppenberger, H.M. 1995. Recombinant cyclin E expression activates proliferation and obviates surface attachment of Chinese hamster ovary (CHO) cells in protein-free medium. Biotechnology and Bioengineering 47: 476–482.

    Article  CAS  Google Scholar 

  3. McDaniel, R., Ebert-KhosIa, S., Hopwood, D.A., and Khosia, C. 1993. Engineered biosynthesis of novel polyketides. Science 262: 1546–1550.

    Article  CAS  Google Scholar 

  4. McDaniel, R., Ebert-KhosIa, S., Hopwood, D.A., and Khosia, C. 1995. Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375: 549–554.

    Article  CAS  Google Scholar 

  5. Alvarez, M.A., Fu, H., Khosla, C., Hopwood, D.A., and Bailey, J.E. 1996. Engineered biosynthesis of novel polyketides: properties of the whiE aro-matase/cyclase. Nature Biotechnology 14: 335–338.

    Article  CAS  Google Scholar 

  6. Graña, X. and Reddy, E.P. 1995. Cell cycle control in mammalian cells: role of cyclins, cyclin-dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11: 211–219.

    PubMed  Google Scholar 

  7. EI-Diery, W.W., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M. et al. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817–825.

    Article  Google Scholar 

  8. Polyak, K., Lee, M.-L., Erdjument-Bromage, H., Koff, A., Roberts, J.M., Tempst, P., and Massagué, J. 1994. Cloning of p27Kip1, a cyclin-dependent kinase I inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78: 59–66.

    Article  CAS  Google Scholar 

  9. Toyoshima, H. and Hunter, T. 1994. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78: 67–74.

    Article  CAS  Google Scholar 

  10. Harper, J.W., Adami, N., Wei, K., Keyomarsi, K., and Elledge, S.J. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75: 805–816.

    Article  CAS  Google Scholar 

  11. Xiong, Y., Hannon, G.J., Zhang, D., Casso, R., Kobayashi, R., and Beach, D. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366: 701–704.

    Article  CAS  Google Scholar 

  12. Gartel, A.L., Serfas, M.S., and Tyner, A.L. 1996. p21-negative regulator of the cell cycle. Proceedings of Experimental and Biological Medicine 213: 138–149.

    Article  CAS  Google Scholar 

  13. Timchenko, N.A., Wilde, M., Nakanishi, M., Smith, J.R. and Darlington, G.J. 1996. CCAAT/enhancer-binding protein a (C/EBPa) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev. 10: 804–815.

    Article  CAS  Google Scholar 

  14. Hartwell, L.H. and Kastan, M.B. 1994. Cell cycle control and cancer. Science 266: 1821–1828.

    Article  CAS  Google Scholar 

  15. Bertelsen, A.H., Beaudry, G.A., Stoller, T.J., Trotta, P.P., and Sherman, M.I. 1995. Tumor suppressor genes: prospects for cancer therapies. Bio/Technology 13: 127–131.

    CAS  PubMed  Google Scholar 

  16. Chen, J., Willingham, T., Shuford, M., and Nisen, P.D. 1996. Tumor suppression and inhibition of aneuploid cell accumulation in human brain tumor cells by extopic overexpression of the cyclin-dependent kinase inhibitor p27-KIP1. J. Clin. Invest. 97: 1983–1988.

    Article  CAS  Google Scholar 

  17. Clayman, G.L., Liu, T.J., Overholt, S.M., Mobley, S.R., Wang, M., Janot, F., and Goepfert, H. . 1996. Gene therapy for head and neck cancer: Comparing the tumor suppressor gene p53 and a cell-cycle regulator WAF1/CIP1. Arch. Otolaryngol. Head Neck Surg. 122: 489–493.

    Article  CAS  Google Scholar 

  18. Pennisi, E. 1996. Will a twist of viral fate lead to a new cancer treatment? Science 274: 342–343.

    Article  CAS  Google Scholar 

  19. Yang, Z., Perkins, N.D., Ohno, T., Nabel, E.G., and Nabel, G.J. 1995. The p21 cyclin-dependent kinase-inhibitor suppresses tumorigenicity in vivo. Nat. Med. 1: 1052–1056.

    Article  CAS  Google Scholar 

  20. Fussenegger, M., Mazur, X., and Bailey, J.E. 1997. A novel cytostatic process enhances the productivity of Chinese hamster ovary cell. Biotechnology and Bioengineering 55: 927–939.

    Article  CAS  Google Scholar 

  21. Fussenegger, M., Mazur, X., Renner, W.A., and Bailey, J.E., 1998. Productivity of growth-arrested Chinese hamster ovary (CHO) cells expressing cyclin-dependent kinase inhibitor gene p27. Biotechnology and Bioengineering. In press.

  22. Rowan, S., Ludwig, R.L., Haupt, Y., Bates, S., Lu, X., Oren, M., and Vousden, K.H. 1996. Specific loss of apoptotic but not cell-cycle arrest function in a human tumor derived p53 mutant. EMBO J. 15: 827–838.

    Article  CAS  Google Scholar 

  23. Itoh, Y., Ueda, H., and Suzuki, E., 1995. overexpression of bcl-2, apoptosis suppressing gene: prolonged viable culture period of hybridoma and enhanced antibody production. Biotechnology and Bioengineering 48: 118–122.

    Article  CAS  Google Scholar 

  24. Singh, R.P., Emery, A.M., and AI-Rubeai, M. 1996. Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2. Biotechnology and Bioengineering. 52: 166–175.

    Article  CAS  Google Scholar 

  25. Huang, D.C., Cory, S., and Strasser, A. 1997. Bcl2, Bcl-xL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene 14: 405–414.

    Article  CAS  Google Scholar 

  26. Fussenegger, M., Mazur, X., and Bailey, J.E. 1998. pTRIDENT, a novel vector family for tricistronic gene expression in mammalian cells. Biotechnology and Bioengineering 57: 1–10.

    Article  CAS  Google Scholar 

  27. Minn, A.J., Boise, L.H., and Thompson, C.B. 1996. Expression of bcl-xL and loss of p53 can cooperate to overcome a cell cycle checkpoint induced by mitotic spindle damage. Genes Dev. 10: 2621–2631.

    Article  CAS  Google Scholar 

  28. EI-Diery, W.S., Harper, J.W., O'Connor, P.M., Velculescu, V.E., Canman, C.E., Jackman, J. et al. 1994. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 54: 1169–1174.

    Google Scholar 

  29. Polyak, K., Kato, J.-Y., Solomon, M.J., Sherr, C.J., Massagué, J., Roberts, J.M., and Koff, A. 1994. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-β and contact inhibition to cell-cycle arrest. Genes Dev. 8: 9–22.

    Article  CAS  Google Scholar 

  30. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Qren, M. 1991. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352: 345–347.

    Article  CAS  Google Scholar 

  31. Ko, L.J. and Prives, C. 1996. p53: Puzzle and paradigm. Genes Dev. 10: 1054–1072.

    Article  CAS  Google Scholar 

  32. Gossen, M., and Bujard, H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad, Sci. USA 89: 5547–5551.

    Article  CAS  Google Scholar 

  33. Zang, M., Trautmann, H., Gandor, C., Messi, F., Asselbergs, F., Leist, C. et al. 1995. Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium. Bio/Technology 13: 389–392.

    CAS  PubMed  Google Scholar 

  34. Berger, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B.R. 1988. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66: 1–10.

    Article  CAS  Google Scholar 

  35. Behrens, T.W., Jagadeesh, J., Scherle, P., Dearns, G.M., Yewdell, J.W., and Staudt, L.M. 1994. Jaw1, a lymphoid-restricted membrane protein localized to the endoplasmic reticulum. J. Immunol. 153: 682–690.

    CAS  PubMed  Google Scholar 

  36. Tsujimoto, Y. and Croce, C.M. 1986. Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. USA. 83: 5214–5218.

    Article  CAS  Google Scholar 

  37. Boise, L.H., Gonzalez-Garcia, M., Postema, C.E., Ding, L., Lindsten, T., Turka, L.A. et al. 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74: 597–608.

    Article  CAS  Google Scholar 

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Fussenegger, M., Schlatter, S., Dätwyler, D. et al. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol 16, 468–472 (1998). https://doi.org/10.1038/nbt0598-468

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