Skip to main content
Cytotechnology logoLink to Cytotechnology
. 1999 Sep;31(1-2):143–151. doi: 10.1023/A:1008080407581

Co-expression of bcl-2 and bag-1, apoptosis suppressing genes, prolonged viable culture period of hybridoma and enhanced antibody production

Satoshi Terada, Tomoaki Komatsu, Tetsuo Fujita, Ain Terakawa, Teruyuki Nagamune, Shinichi Takayama, John C Reed, Eiji Suzuki
PMCID: PMC3449778  PMID: 19003134

Abstract

Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures.

Keywords: antibody productivity, apoptosis, BAG-1, Bcl-2, cell survival, hybridoma

Full Text

The Full Text of this article is available as a PDF (112.3 KB).

References

  1. Duke RC, Cohen JJ. Morphological and biochemical assays of apoptosis. In: Janssen K, editor. Current protocols in immunology. New York: Wiley; 1992. pp. 3.17.1–3.17.16. [Google Scholar]
  2. Duval D, Demangel C, Geahel I, Blondeau K, Marcaded A. Comparison of various methods for monitoring hybridoma cell proliferation. J Immunol Meth. 1990;134:177–185. doi: 10.1016/0022-1759(90)90379-A. [DOI] [PubMed] [Google Scholar]
  3. Itoh Y, Ueda H, Tsujimoto Y, 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]
  4. Makishima F, Terada S, Mikami T, Suzuki E. Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivuty. Cytotechnology. 1992;10:15–23. doi: 10.1007/BF00376096. [DOI] [PubMed] [Google Scholar]
  5. Pettersson M, Jernberg-Wiklund H, Larsson LG, Sundstrom C, Givol I, Tsujimoto Y, Nilsson K. Expression of the bcl-2 gene in human miltiple myeloma cell lines and normal plasma cells. Blood. 1992;79:495–502. [PubMed] [Google Scholar]
  6. Perreault J, Lemieux R. Essential role of optimal protein synthesis in preventing the apoptotic death of cultured B cell hybridomas. Cytotechnology. 1994;13:99–105. doi: 10.1007/BF00749936. [DOI] [PubMed] [Google Scholar]
  7. Simpson NH, Milner AE, Al-Rubeai M. Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. Biotechnol Bioeng. 1997;54:1–16. doi: 10.1002/(SICI)1097-0290(19970405)54:1<1::AID-BIT1>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  8. Singh RP, Emery AN, Al-Rubeai M. Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2. Biotechnol Bioeng. 1996;52:166–175. doi: 10.1002/(SICI)1097-0290(19961005)52:1<166::AID-BIT17>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  9. Suzuki E, Ollis DF. Enhanced antibody production at slowed growth rates: experimental demonstration and a simple structure model. Biotechnol Prog. 1990;6:231–236. doi: 10.1021/bp00003a013. [DOI] [PubMed] [Google Scholar]
  10. Takahashi K, Terada S, Ueda H, Makishima F, Suzuki E. Growth rate suppression of cultured mammalian cells enhances protein productivity. Cytotechnology. 1994;15:54–64. doi: 10.1007/BF00762379. [DOI] [PubMed] [Google Scholar]
  11. Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, Reed JC. Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. Cell. 1995;80:279–284. doi: 10.1016/0092-8674(95)90410-7. [DOI] [PubMed] [Google Scholar]
  12. Terada S, Itoh Y, Ueda H, Suzuki E. Characterization and Fed-batch Culture of Hybridoma Overexpressing Apoptosis Suppressing Gene bcl-2. Cytotechnology. 1997;24:135–141. doi: 10.1023/A:1007922104344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Terada S, Fukuoka K, Fujita T, Komatsu T, Takayama S, Reed JC, Suzuki E. Anti-apoptotic genes, bag-1 and bcl-2, enabled hybridoma cells to survive under treatment for arresting cell cycle. Cytotechnology. 1997;25:17–23. doi: 10.1023/A:1007954103572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Terada S, Itoh Y, Suzuki E. Establishing an apoptosis-resistant hybridoma cell line for usage in serum starvation culture. Kagaku Kogaku Ronbunshu. 1998;24:161–164. [Google Scholar]
  15. Tomei LD. Apoptosis: a program for death or survival? In: Tomei LD, Cope FO, editors. Apoptosis: the molecular basis of cell death. Cold Spring, NY: Cold Spring Harbor Laboratory Press; 1991. pp. 279–316. [Google Scholar]
  16. Tsujimoto Y. Stress-resistance conferred by high level of bcl-2a protein in human B lymphoblastoid cell. Oncogene. 1989;4:1331–1336. [PubMed] [Google Scholar]
  17. Vomastek T, Franek F. Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures. Immunol Lett. 1993;35:19–24. doi: 10.1016/0165-2478(93)90142-O. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES