Abstract
Hybridoma 2E3-O cells were transfected with bcl-2 alone or with bcl-2 and bag-1 in combination. The bcl-2/bag-1 transfectant survived maintaining viability above 75% for almost 5 days when the cells were treated with excess (30 mM) thymidine for arresting cell cycle, whereas the mock transfectant survived for only 2 days, and the bcl-2 alone transfectant lived for 4 days. Owing to this extended viable culture period, the bcl-2/bag-1 transfectant produced twofold amount of antibody in comparison with the mock transfectant in non-proliferating state prepared by the excess thymidine treatment. When their proliferation was arrested by serum limitation, the bcl-2/bag-1 transfectant and the bcl-2 alone transfectant survived for 3 days maintaining viability above 75% while the mock transfectant survived only 1 day. The bcl-2/bag-1 transfectans produced the antibody at the rate three times as high as the bcl-2 alone transfectant and the mock transfectant in non-proliferating state established by serum limitation. Such genetic engineering of hybridoma cells for improving survival in the non-proliferating state will be useful for using nutrients in culture medium efficiently to produce antibody, since nutrients could be diverted from cell proliferation to antibody production in such non-proliferating viable cell culture.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Al-Reubeai M, Rookes and Emery An (1989) Flow cytometric studies during synchronous and asynchronous suspension cultures of hybridoma cells. In: Spier RE, Griffiths HB, Stephenne J and Crooy PJ (eds.). Advances in animal cell biology and technology for bioprocesses (pp. 241–245) Butterworths, Kent.
Bardelli A, Longati P, Albero D, Goruppi S, Schneider C, Ponzetto C and Comoglio PM (1996) HGF receptor associates with the antiapoptotic protein BAG-1 and presents cell death. EMBO Journal 15: 6205–6212.
Bueall DN and Fahey JL (1969) Limited periods of gene expression in immunoglobulin-synthesizing cells. Science 164: 1524–1525.
Byars N and Kisdon C (1970) Programmed synthesis and export of immunoglobulin by synchronized myeloma cells. Nature 226: 648–650.
Engvall E (1980) Enzyme immunoassay ELISA and EMIT. In: Van Vunakis H and Langone JJ (ed.) Methods in Enzymology. Vol. 70 (pp. 419–439) Acedemic Press, New York.
Garatun-Tjeldstø O, Pryme IF, Weltman JK and Dowben RM (1971) Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells. J. Cell Biol. 68: 232–239.
Itoh Y, Udea H and Suzuki E (1995) Overexpression of bcl-2, apoptosis suppressing gene: prolonged viable culture period of hydridoma and enhanced antibody production. Biotechnol. Bioeng. 48: 118–122.
Johnson RT, Downes CS and Meyn RE (1993) The synchronization of mammalian cells. In: Fantes P and Brooks R (ed.) The cell cycle a practical approach. (pp. 1–22) IRL Press, Oxford.
Karasuyama H, Kudo A and Melchers F (1990) The proteins encoded by the VpreB and λ 5 pre-B cell-specific genes can associate with each other and with m heavy chain. J. Exp. Med. 172: 969–972.
Makishima F, Terada S, Mikami T and Susuki E (1992) Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity. Cytotechnology 10: 15–23.
Mercille S and Massie B (1994) Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells. Biotechnol. Bioeng. 44: 1140–1154.
Miyashita T and Reed JC (1992) bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cell to cell death and DNA fragmentation induced by glucocorticoids and multiple chemitherapeutic drugs. Cancer Research 52: 5407–5411.
Nunez G, London L, Hockenberry D, Alexander M, McKearn JP and Korshmeyer H (1990) Deregulated bcl-2 gene expression selectively prolongs survival of growth factor-deprived haempoietic cell lines. J. Immunol. 144: 3692–3610.
Patterson M, Jernberg-Wiklund H, Larsson LG, Sundstrom C, Givol I, Tsujimoto Y and Nilsson K (1992) Exression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells. Blood 79: 495–501.
Pryme IF (1974) The stimulation of light chain immunoglobulin synthesis in vitro by 0.5 M salt wash of MPC-11 membrane-bound bound polysomes isolated in the G1 and G2 phases of the cell cycle. Biochem. Biophys. Res. Commun. 61: 838–844.
Ramirez OT and Mutharasan R (1990) Cell cycle-and growth phase-dependent variations in size distribution, antibody productivity, and oxyhen demand in hybridoma cultures. Biotechnol. Bioeng. 36: 839–848.
Reed JC (1994) Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 124: 1–6.
Singh RP, Al-Rubeai M, Gregory CD and Emery AN (1994) Cell death in bioreactors: a role for apoptosis. Biotechnol. Bioeng. 44: 720–726.
Stein GS and Borun TE (1972) The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S3 cells J. Cell Biol. 52: 292–307.
Suzuki E and Ollis DF (1990) Enhanced antibody production at slowed growth rates: experimental demonstration and a simple structure model. Biotechnol. Prog. 6: 231–236.
Suzuki E, Takahashi K and Ollis DF (1992) A simple structured model predicted positively-, negatively-, or non-growth associated antibody productionn rate depending on culture condition. In: Murakami H, Shirahata S and Tachibana H (ed.) Animal cell technology:basic and applied aspects, Vol. 4, (pp. 15–21) Kluwer Academic Publisher, Dordrecht.
Takahashi K, Terada S, Ueda H, Makishima F and Suzuki E (1994) Growth rate suppression of cultured mammalian cells enhances protein productivity. Cytotechnology 15: 54–64.
Takahashi M, Yagi Y, Moore GE and Pressman D (1969) Immunoglobulin production in synchronized cultures of human hematopoietic cell lines. J. Immin. 103, 834–843.
Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA and Reed JC (1995) Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. cell 80: 279–284.
Takayama S, Bimston DN, Matsuzawa S and Reed JC (1997) BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO in press.
Terashima T and Tolmach IJ (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cell. Exp. Cell Res. 30: 344–362.
Tsujimoto Y (1989) Stress-resistance conferred by high level of Bcl-2a protein in human B lymphoblastoid cell. Oncogene 4: 1331–1336.
Vaux DL, Cory and Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalise pre-B cells. Nature 335: 440–442.
Wang H-G, Takayama S, Rapp UR and Reed JC (1996) Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl. Acad. Sci. USA. 93: 7063–7068.
Zetterburg A and Larson O (1985) Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 82: 5365–53699.
Rights and permissions
About this article
Cite this article
Terada, S., Fukuoka, K., Fujita, T. et al. Anti-apoptotic genes, bag-1 and bcl-2, enabled hybridoma cells to survive under treatment for arresting cell cycle. Cytotechnology 25, 17–23 (1997). https://doi.org/10.1023/A:1007954103572
Issue Date:
DOI: https://doi.org/10.1023/A:1007954103572