Abstract
Most cell lines that are used for the production of recombinant proteins proliferate spontaneously at a high rate. In many types of cultivation systems these cells still keep growing after having reached the desired cell density. Further proliferation in batch cultures leads to cell death as a consequence of nutrient and oxygen depletion as well as to accumulation of lactate and toxic products. Consequently, in many technical processes, the surplus of cells is removed.
We have established cell lines in which proliferation is controlled by a physiological regulator, IRF-1. IRF-1 (Interferon Regulatory Factor 1) is a transcriptional activator and acts as a tumor suppressor. Constitutive overexpression of recombinant IRF-1 leads to inhibition of cell growth. The extent of this growth arrest depends on the intracellular concentration of active IRF-1. To allow IRF-1 expression in various mammalian cells a system for conditional IRF-1 activation has been established. A fusion protein composed of IRF-1 and the hormone binding domain of the human estrogen receptor, was used. This system allows to control gradually the growth of several mammalian cell lines by adjusting the intracellular concentration of active IRF-1 via estradiol in the medium. We have evaluated BHK-21 cells with respect to IRF-1 mediated cell growth inhibition and expression of two secreted proteins. Whereas the productivity of proliferation inhibited cells with respect to constitutively transcribed IgG genes is reduced, productivity of another secreted protein which is controlled by an IRF-1 inducible promoter is strongly enhanced under these conditions.
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Bebbington CR, Renner G, Thomson S, King D, Abrams D and Yarranton G (1992) High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10: 169–175.
Berger J, Hauber R, Hauber R, Geiger R and Cullen BR (1988) Secreted alkaline phosphatase: a powerfull new quantitative indicator of gene expression in eukaryotic cells. Gene 66: 1–10.
Colbère-Garapin F, Horidniceau F, Khourilsky P and Garapin AC (1981) A new dominant selective marker for higher eukaryotic cells. J Mol Biol 150: 1–13.
Dirks W, Mielke C, Karreman S, Haase B, Wirth M, Lindenmaier W and Hauser H (1994a) Applications of expression vectors containing bicistronic transcription units in mammalian cells. In: Cell culture in pharmaceutical research. Fusenig NE and Graf H, (eds) Springer, Heidelberg, 239–266.
Dirks W, Schaper F and Hauser H (1994b) A new hybrid promoter directs transcription at identical start points in mammalian cells and in vitro. Gene, 149: 389–390.
Dirks W, Schaper F, Kirchhoff S, Morelle C and Hauser H (1994c) A multifunctional vector family for gene expression in mammalian cells. Gene, 149: 387–388.
Fujita T, Kimura Y, Miyamoto M, Barsoumian EL and Taniguchi T (1989) Induction of endogenous IFN-alpha and IFN-beta genes by a regulatory transcription factor, IRF-1. Nature (London) 337: 270–272.
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.
Gross G and Hauser H (1995) Gene analysis by heterologous expression. J Biotechnology, 41: 91–110.
Kamijo R, Harada H, Matsuyama T, Bosland M, Gerecitano J, Shapiro D, Le J, Koh SI, Kimura T, Green SJ, Mak TW, Taniguchi T and Vilcek J (1994) Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 262: 1612–1615.
Kirchhoff S, Koromilas A, Schaper F, Grashoff M, Sonenberg N and Hauser H (1995a) IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR., Oncogene, 11: 439–445.
Kirchhoff S, Schaper F and Hauser H (1993) Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes. Nucleic Acids Res 21: 2881–2889.
Kirchhoff S, Köster M, Wirth M, Schaper F, Gossen M, Bujard H and Hauser H (1995b) Identification of mammalian cell clones exhibiting highly regulated expressionn from inducible promoters. TIG, 11: 219–220.
Köster M, Kirchhoff S, Schaper F and Hauser H (1995) Proliferation control of mammalian cells by the tumor suppressor IRF-1. Cytotechnology, 18: 67–75.
Matsuyama T, Kimura T, Kitagawa M, Pfeffer K, Kwakami T, Watanabe N, Kündig TM, Amakawa R, Kishihara K, Wakeham A, Potter J, Furlonger CL, Narendan A, Suzuki H, Ohashi PS, Paige CJ, Taniguchi T and Mak TW (1993) Target disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75: 83–97.
Pine R, Decker T, Kessler DS, Levy DE and Darnell Jr JE (1990) Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcription activator of either. Mol Cell Biol 10: 2448–2457.
Pine R (1992) Constitutive expression of an ISGF'/IRF1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection. J Virol 66: 4470–4478.
Racher AJ, Moreira JL, Alves PM, Wirth M, Weidle UH, Hauser H Carrondo MJT and Griffiths JB (1994) Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics. Appl Microbiol Biotechnol 40: 851–856.
Reich NC and Darnell Jr JE (1989) Differential binding of interferon-induced factors to an oligonucleotide that mediates transcriptional activation. Nucleic Acids Res 17: 3415–3424.
Reis LFL, Harada H, Wolchok JD, Taniguchi T and Vilcek J (1992) Critical role of a common transcription factor, IRF-1 in the regulation of IFN-β and IFN-inducible genes. EMBO J 11: 185–193.
Reis LFL, Ruffner H, Stark G, Aguet M and Weissmann C (1994) Mice devoid of IRF-1 show normal expression of type I IFN genes. EMBO J 13: 4798–4806.
Ruffner H, Reis LFL, Näf D and Weissmann C (1993) Induction of type I interferon genes and interferon-inducible genes in embryonal stem cells devoid of interferon regulatory factor 1. Proc Natl Acad Sci USA 90: 11503–11507.
Sambrook J, Frisch EF and Maniatis T (1989) Molecular Cloning: A laboratory manual. Cold Spring Harbor Press (New York).
Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, Matsuyama T, Mak TW, Taki S and Taniguchi T (1995) An IRF-1 dependent pathway of DNA-damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 376: 596–599.
Tanaka N, Oishi T, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T, Lamphier MC, Aizawa S, Mak T and Taniguchi T (1994) Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1 Cell 77: 829–839.
Vara J, Portela A, Ortin J and Jimenez A (1986) Expression cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res 14: 4617–4624.
Willman CL, Sever CE, Pallacvivini MG, Harada H, Tanaka N, Slovak ML, Yamamoto H, Harada K, Meeker TC. ListAF and TaniguchiT (1993) Deletion of IRF-1, mapping to chromosome 5q31.1, in human leukemia and preleukemic myelodysplasia. Science 259: 968–971.
Wirth M and Hauser H (1993) Genetic engineering of animal cells, In: Biotechnology Vol 2 Pühler A (ed.) VCH, Weinheim 663–744.
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Kirchhoff, S., Kröger, A., Cruz, H. et al. Regulation of cell growth by IRF-1 in BHK-21 cells. Cytotechnology 22, 147–156 (1996). https://doi.org/10.1007/BF00353934
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DOI: https://doi.org/10.1007/BF00353934