Effects of Antioxidants on Cell Viability and hGM-CSF Production by Transgenic Nicotiana tabacum Suspension Cultures

형질전환된 Nucotiana tabacum 현탁세포배양에서 항산화제가 세포생존도 및 hGM-CSF 생산에 미치는 영향

  • Kim Yong Hoon (Department of Biological Engineering, Inha University) ;
  • Lee Sang Yoon (Department of Biological Engineering, Inha University) ;
  • Kim Dong Il (Department of Biological Engineering, Inha University)
  • 김용훈 (인하대학교 공과대학 생명화학공학부) ;
  • 이상윤 (인하대학교 공과대학 생명화학공학부) ;
  • 김동일 (인하대학교 공과대학 생명화학공학부)
  • Published : 2004.10.01

Abstract

Production of therapeutic proteins by transgenic plant cell suspension cultures is an attractive system alternative to the other expression system. However, plant cell cultures have shown low expression level of foreign proteins and decreased cell viability by the changes of culture conditions. Therefore, it is necessary to enhance cell viability during the culture period. In this study, a quantitative analysis technique was designed to measure relative cell viability for plant suspension cells which have cell wall and aggregates. It was found that the programmed cell death of plant cells by apoptosis was essentially linked with the apoptotic pathway of animal cells. Therefore, effects of nicotinamide, 3-aminobenzamide and antioxidants on cell viability and apoptosis were examined in transgenic Nicotiana tabacum cells producing hGM-CSF. With those additives, cell viability could be maintained and apoptosis could be redued. In the result, the extracellular production of hGM-CSF could be enhanced 2.5 fold. It was also found that the supplementation of glutathione and ascorbic acid suppressed both the cold stress-induced decrease in cell viability and the increase of total genomic DNA fragmentation.

Keywords

References

  1. Solomon, M., B. Belenghi, M. Delledonne, E. Menachem, and A. Levine (1999), The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants, Plant Cell 11, 431-443
  2. Steward, N., R. Martin, J. M. Engasser, J. L. Goergen (1999), A new methodology for plant cell viability assessment using intracellular esterase activity, Plant Cell Rep. 19, 171-176
  3. Lindahl, T., M. S. Satoh, G. G. Poirier, and A. Klungland (1995), Post-translational modification of poly (ADP-ribose) polymerase induced by DNA strand breaks, Trends Biochem. Sci. 20, 405-411
  4. Yang, J., L. K. Klaidman, M. L. Chang, S. Kem, T. Sugawara, P. Chan, and J. D. Adams (2002), Nicotinamide therapy protects against both necrosis and apoptosis in a stroke model, Pharm. Biochem. Behavior 73, 901-910
  5. Tian, R. H., G. Y. Zhang, C. H. Yan, and Y. R. Dai (2000), Involvement of poly (ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock -induced apoptosis in tobacco suspension cells, FEBS Lett. 474, 11-15
  6. Amor, Y., E. Babiychuk, D. Inze, and A. Levine (1998), The involvement of poly (ADP-ribose) polymerase in the oxidative stress responses in plants, FEBS Lett. 440, 1-7
  7. Korsmeyer, S. J., X. M. Yin, Z. N. Oltvai, D. J. Veis-Novack, and G. P. Linette (1995), Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family, Biochim. Biophys. Acta 1271, 63-66
  8. Kruman, I. I. and M. P. Mattson (1999), Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis, J. Neurochem. 72, 529-540
  9. McConkey, D. J., P. Hartzell, P. Nicotera, and S. Orrenius (1989), Calcium activated DNA fragmentation kills immature thymocytes, FASEB J. 3, 1843-1849
  10. Grub, S., W. E. Trommer, and A. Wolf (2002), Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZIMM125)-induced apoptosis in rat hepatocytes, Biochem. Pharmacol. 64, 1725-1736
  11. Orrenius, S., D. J. McConkey, G. Bellomo, and P. Nicotera (1989), Role of $Ca^{2+}$ in toxic cell killing, Trends Pharmacol. Sci. 10, 281-285
  12. Carpaneto, A., A. M. Cantu, and F. Gambale (1999), Redox agents regulate ion channel activity in vacuoles from higher plant cells, FEBS. Lett. 442, 129-132
  13. Kohler, C., S. Orrenius, and B. Zhivotovsky (2002), Evaluation of caspase activity in apoptotic cells, J. Immunol. Methods 265, 97-110
  14. Greenberg, J. T. (1996), Programmed cell death: A way of life for plants, Proc. Natl. Acad. Sci. USA 93, 12094-12097
  15. McCabe, P. F., and C. J. Leaver (2000), Programmed cell death in cell cultures, Plant Mol. Biol. 44, 359-368
  16. De Jong, A. J., E. T. Yakimova, V. M. Kapchina, and E. J. Woltering (2002), A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells, Planta 214, 537-545
  17. Koukalova, B., A. Kovarik, J. Fajkus, and J. Siroky (1997), Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress, FEBS Lett. 414, 289-292
  18. Korthout, H., G. Berecki, W. Bruin, B. V. Duijn, and M. Wang (2000), The presence and subcellular localization of caspase 3-like proteinases in plant cells, FEBS Lett. 475, 139-144
  19. Solomon, M., B. Belenghi, M. Delledonne, E. Menachem, and A. Levine (1999), The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants, Plant Cell 11, 431-443
  20. Gotoh, T., Y. Miyazaki, W. Sato, K. I. Kikuchi, and W. E. Bentley (2001), Proteolytic activity and recombinant protein production in virus-infected sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors, J. Biosci. Bioeng. 92, 248-255
  21. Haendeler, J., A. M. Zeiher, and S. Dimmeler (1996), Vitamin C and E prevent lipopolysaccharide-induced apoptosis in human endothelial cells by modulation of Bcl-2 and Bax, Eur. J. Pharmacol. 317, 407-411