Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture
Cite this article as: Ishaque, A. & Al-Rubeai, M. Apoptosis (2002) 7: 231. doi:10.1023/A:1015343616059 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 References
Kruse PS, Patterson MK.
Tissue Culture-Methods and Applications
. New York: Academic press, 1973.
Pollack R, ed.
Readings in Mammalian Cell Culture
. Cold Springs Harbour, New York: Cold Spring Harbour Laboratory, 1975.
Reitzer LJ, Wice BM, Kenell D Evidence that glutamine not sugar is the main energy source for cultured HeLa cells.
J Biol Chem
Dalili M, Sayels GD, Ollis D Glutamine limited batch hybridoma growth and antibody production: Experiment and model.
Franek F Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures.
Al-Rubeai M, Mills D, Emery AN Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.
Franek F, Dolnikova J Nucleosomes occurring in protein-free hybridoma cell cultures. Evidence for programmed cell death.
Mercille S, Massie B Induction of apoptosis in oxygen-deprived cultures of hybridoma cells.
Singh RP, Al-Rubeai M, Gregory CD, Emery AN Cell death in bioreactors: A role for apoptosis.
Kerr JFR, Wyllie AH, Currie R Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics.
Br J Cancer
Simpson NH, Milner AE, Al-Rubeai M Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions.
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.
Ishaque A, Al-Rubeai M Role of Ca
ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture.
Waymouth C Simple nutrient solutions for animal cells. Internat.
Shapiro D, Schrier BK Cell cultures of fetal rat brain: Growth and marker enzyme development.
Exp Cell Res
Ishaque A, Al-Rubeai M Use of intracellular pH and annexin V flow cytometric assays to monitor apoptosis and its suppression by
over-expression in hybridoma cell culture.
J Immunological Methods
Bettger WJ, Ham RG The nutrient requirements of cultured mammalian cells.
Adv Nutr Res
Kurano S, Kurano N, Leist C, Fiechter A Utilization and stability of vitamins in serum-containing and serum-free medium in CHO cell culture.
Del Buono BJ, Williamson PL, Schlegel RA Alterations in plasma membrane lipid organisation during lymphocyte differentiation.
J Cell Pysiol
Ormerod MG, Sun XM, Snowden RT, Davies R, Fearnhead H, Cohen GM Increased membrane permeability of apoptotic thymocytes: A flow cytometric study.
Horwitt MK, Witting LA
Riboflavin. IX. Biochemical Systems
. In: Sebrell WH Jr, Harris RS, eds.
. New York: Academic Press, 1972: 53–70, Vol. V, Ch. 14.
Rieger D Relationships between energy metabolism and development of early mammalian embryos.
Lane M, Gardner DK Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts.
Zamzami N, Marchetti P, Castedo M,
Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis.
Kroemer G The proto-oncogene Bcl-2 and its role in regulating apoptosis.
Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G Subcellular and submitochondrial mode of action of bcl-2 like oncoproteins.
Vairo G, Inmes KM, Adams JM Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival.
Borner C Diminished cell proliferation associated with the death protective activity of bcl-2.
J Biol Chem
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.
Shimizu S, Eguchi Y, Kosaka H, Kamiike W, Matsuda H, Tsujimoto Y Prevention of hypoxia induced cell death by bcl-2 and bcl-xL
Shimizu S, Eguchi Y, Kamiike W,
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.
Gardner DK Embryo development and culture techniques. In: Clark J, ed.
Animal Breeding: Technology for the 21st Century
. UK: Harwood Academic, 1998: 446.
Fischer AB, Hess C, Neubauer T, Eikmann T Testing of chelating agents and vitamins against lead toxicity using mammalian cell cultures.
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.
Tsai FC, Gardner DK Nicotinamide, a component of complex culture media inhibits mouse embryo development
and reduces subsequent development after transfer.
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
Klaidman LK, Mukherjee SK, Hutchin TP, Adams JD Nicotinamide as a precursor for NAD
prevents apoptosis in the mouse brain induced by tertiary-butylhydroperoxide.
Satoh MS, Lindahl T Enzymatic repair of oxidative DNA damage.
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
Singh RP, Al-Rubeai M. In: M Al-Rubeai, ed.
Apoptosis and Bioprocess Technology Advances in Biochemical Engineering, 1998: 168–184. Copyright information
© Kluwer Academic Publishers 2002