Apoptosis

, Volume 7, Issue 3, pp 231–239

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture

  • A. Ishaque
  • M. Al-Rubeai
Article

Abstract

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.

apoptosis bcl-2 cell culture hybridoma nutrients vitamins 

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References

  1. 1.
    Kruse PS, Patterson MK. Tissue Culture-Methods and Applications. New York: Academic press, 1973.Google Scholar
  2. 2.
    Pollack R, ed. Readings in Mammalian Cell Culture. Cold Springs Harbour, New York: Cold Spring Harbour Laboratory, 1975.Google Scholar
  3. 3.
    Reitzer LJ, Wice BM, Kenell D Evidence that glutamine not sugar is the main energy source for cultured HeLa cells. J Biol Chem 1979; 254: 2669–2676.Google Scholar
  4. 4.
    Dalili M, Sayels GD, Ollis D Glutamine limited batch hybridoma growth and antibody production: Experiment and model. Biotechnol Bioeng 1990; 36: 74–82.Google Scholar
  5. 5.
    Franek F Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures. Biotechnol Bioeng 1995; 45: 86–90.Google Scholar
  6. 6.
    Al-Rubeai M, Mills D, Emery AN Electron microscopy of hybridoma cells with special regard to monoclonal antibody production. Cytotechnol 1990; 4: 13.Google Scholar
  7. 7.
    Franek F, Dolnikova J Nucleosomes occurring in protein-free hybridoma cell cultures. Evidence for programmed cell death. FEBS Lett 1991; 248: 285–287.Google Scholar
  8. 8.
    Mercille S, Massie B Induction of apoptosis in oxygen-deprived cultures of hybridoma cells. Cytotechnology 1994; 15: 117–128.Google Scholar
  9. 9.
    Singh RP, Al-Rubeai M, Gregory CD, Emery AN Cell death in bioreactors: A role for apoptosis. Biotechnol Bioeng 1994; 44: 720–726.Google Scholar
  10. 10.
    Kerr JFR, Wyllie AH, Currie R Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257.Google Scholar
  11. 11.
    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.Google Scholar
  12. 12.
    Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M In hybridoma cultures deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng 1998; 59: 90–98.Google Scholar
  13. 13.
    Ishaque A, Al-Rubeai M Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture. Apoptosis 1999; 4: 1–21.Google Scholar
  14. 14.
    Waymouth C Simple nutrient solutions for animal cells. Internat. Rev Cytol 1954; 3: 1–68.Google Scholar
  15. 15.
    Shapiro D, Schrier BK Cell cultures of fetal rat brain: Growth and marker enzyme development. Exp Cell Res 1972; 89: 139–142.Google Scholar
  16. 16.
    Ishaque A, Al-Rubeai M Use of intracellular pH and annexin V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J Immunological Methods 1998; 221: 43–57.Google Scholar
  17. 17.
    Bettger WJ, Ham RG The nutrient requirements of cultured mammalian cells. Adv Nutr Res 1982; 4: 249–286.Google Scholar
  18. 18.
    Kurano S, Kurano N, Leist C, Fiechter A Utilization and stability of vitamins in serum-containing and serum-free medium in CHO cell culture. Cytotechnol 1990; 4: 243–250.Google Scholar
  19. 19.
    Del Buono BJ, Williamson PL, Schlegel RA Alterations in plasma membrane lipid organisation during lymphocyte differentiation. J Cell Pysiol 1986; 126: 379–388.Google Scholar
  20. 20.
    Ormerod MG, Sun XM, Snowden RT, Davies R, Fearnhead H, Cohen GM Increased membrane permeability of apoptotic thymocytes: A flow cytometric study. Cytometry 1993; 14: 595–602.Google Scholar
  21. 21.
    Horwitt MK, Witting LA Riboflavin. IX. Biochemical Systems. In: Sebrell WH Jr, Harris RS, eds. The Vitamins. New York: Academic Press, 1972: 53–70, Vol. V, Ch. 14.Google Scholar
  22. 22.
    Rieger D Relationships between energy metabolism and development of early mammalian embryos. Theriogenology 1992; 37: 75–93.Google Scholar
  23. 23.
    Lane M, Gardner DK Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum Reprod 1998; 13: 991–997.Google Scholar
  24. 24.
    Zamzami N, Marchetti P, Castedo M, et al Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett 1996; 384: 53–57.Google Scholar
  25. 25.
    Kroemer G The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 1997; 3: 614–620.Google Scholar
  26. 26.
    Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G Subcellular and submitochondrial mode of action of bcl-2 like oncoproteins. Oncogene 1998; 16: 2265–2282.Google Scholar
  27. 27.
    Vairo G, Inmes KM, Adams JM Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival. Oncogene 1996; 13: 1511–1519.Google Scholar
  28. 28.
    Borner C Diminished cell proliferation associated with the death protective activity of bcl-2. J Biol Chem 1996; 271: 12695–12698.Google Scholar
  29. 29.
    Simpson NH, Singh RP, Emery AN, Al-Rubeai M Bcl-2 overexpression reduces the growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells. Biotechnol Bioeng 1999; 64: 174–186.Google Scholar
  30. 30.
    Shimizu S, Eguchi Y, Kosaka H, Kamiike W, Matsuda H, Tsujimoto Y Prevention of hypoxia induced cell death by bcl-2 and bcl-xL Nature 1995; 374: 811–813.Google Scholar
  31. 31.
    Shimizu S, Eguchi Y, Kamiike W, et al Retardation of chemical hypoxia-induced necrotic cell death by bcl-2 and ICE inhibitors possible involvement of common mediators in apoptotic and necrotic signal transduction. Oncogene 1996; 12: 2045–2050.Google Scholar
  32. 32.
    Gardner DK Embryo development and culture techniques. In: Clark J, ed. Animal Breeding: Technology for the 21st Century. UK: Harwood Academic, 1998: 446.Google Scholar
  33. 33.
    Fischer AB, Hess C, Neubauer T, Eikmann T Testing of chelating agents and vitamins against lead toxicity using mammalian cell cultures. Analyst 1998; 123: 155–158.Google Scholar
  34. 34.
    Gardner DK, Lane M Alleviation of the '2-cell block' and development to the blastocyst of CFI mouse embryos: Role of amino acids, EDTA and physical parameters. Hum Reprod 1996; 11: 2703–2712.Google Scholar
  35. 35.
    Tsai FC, Gardner DK Nicotinamide, a component of complex culture media inhibits mouse embryo development in vitro and reduces subsequent development after transfer. Fertil Steril 1994; 61: 2376–2382.Google Scholar
  36. 36.
    Yan Q, Briehl M, Crowley CL, Payne CM, Bernstein H, Bernstein C The NAD+precursors, nicotinic acid and nicotinamide upregulate glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenasemRNAin Jurkat cells. Biochem Biophys Res Commun 1999; 255: 133–136.Google Scholar
  37. 37.
    Klaidman LK, Mukherjee SK, Hutchin TP, Adams JD Nicotinamide as a precursor for NAD+ prevents apoptosis in the mouse brain induced by tertiary-butylhydroperoxide. Neurosci Lett 1996; 206: 5–8.Google Scholar
  38. 38.
    Satoh MS, Lindahl T Enzymatic repair of oxidative DNA damage. Cancer Res 1994; 54: 1899s-1901s.Google Scholar
  39. 39.
    Petronini PG, Urbani S, Alferi R, Borghetti AF, Guidotti GG Cell susceptibility to apoptosis by glutamine deprivation and rescue: Survival and apoptotic death in cultured lymphomaleukemia cell line. J Cell Physiol 1996; 169: 175–185.Google Scholar
  40. 40.
    Singh RP, Al-Rubeai M. In: M Al-Rubeai, ed. Apoptosis and Bioprocess Technology Advances in Biochemical Engineering, 1998: 168–184.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • A. Ishaque
    • 1
  • M. Al-Rubeai
    • 1
  1. 1.Animal Cell Technology Group, Department of Chemical EngineeringThe University of BirminghamBirminghamUK

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