Cytotechnology

, Volume 15, Issue 1–3, pp 117–128

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

  • Sylvain Mercille
  • Bernard Massie
Article

Abstract

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.

Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.

Key words

Animal cell culture anoxia apoptosis cell death hybridoma hypoxia 

Abbreviations

VNA

Viable non-apoptotic cells

VA

Viable apoptotic cells

NVNA

Nonviable non-apoptotic or necrotic cells

NVA

Nonviable apoptotic cells

CF

Chromatin-free cells (late nonviable apoptotic cells)

AO

Acridine orange

EB

Ethidium Bromide

MAb

Monoclocnal antibody

D.O.

Dissolved oxygen

qMAb

Specific MAb production rate (mg. (109 cells)−1.day−1)

μ

Specific growth rate (h−1)

Xv

Viable cell number (105 cells.mL−1)

Xt

Total cell number (105 cells.mL−1)

Ylac/glc

Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Al-Rubeai M, Mills D and Emery AN (1990) Electron microscopy of hybridoma cells with regard to monoclonal antibody production. Cytotechnology 4: 13–28.Google Scholar
  2. Allen J, Winterford C, Axelsen RA and Gobe GC (1992) Effects of hypoxia on morphological and biochemical characteristics of renal epithelial cell and tubule cultures. Ren. Fail. 14: 453–460.Google Scholar
  3. Barraf CE and Bowen ID (1986) Kinetic studies on a murine sarcoma and an analysis of apoptosis. Br. J. Cancer 54: 989–998.Google Scholar
  4. Cai JW, Henderson BW, Shen JW and Subjeck JR (1993) Induction of glucose regulated proteins during growth of a murine tumor. J. Cell. Physiol. 154: 229–237.Google Scholar
  5. Carlos RQ, Seidler FJ and Slotkin TA (1991) Fetal dexamethasone exposure sensitizes neonatal rat brain to hypoxia effects on protein and DNA synthesis. Dev. Brain Res. 64: 161–166.Google Scholar
  6. Cattaneo MV, Luong JHT and Mercille S (1992) Monitoring glutamine in mammalian cell cultures using an amperometric biosensor. Biosensors and Biolectronics 7: 329–334.Google Scholar
  7. Chauret N, Coté J, Archambault J and André G (1992) High-performance gel-permeation chromatographic analysis of IgM produced by hybridoma culture. J. Chromatography 594: 179–185.Google Scholar
  8. Collins RJ, Harmon BV, Gobé GC and Kerr JFR (1992) Internucleosomal DNA cleavage should not be used as the sole criterion for identifying apoptosis. Int. J. Radiat. Biol. 61: 451–453.Google Scholar
  9. Cotter TG, Lennon SV, Glynn JG and Martin SJ (1990) Cell death via apoptosis and its relationship to growth, development and differentiation of both tumor and normal cells. Anticancer Res. 10: 1153–1160.Google Scholar
  10. Dragunow M, Young D, Hughes P, MacGibbon G, Lawlor P, Singleton K, Sirimanne E, Beilharz E and Gluckman P (1993) Is c-Jun involved in nerve cell death following status epilepticus and hypoxic-ischaemic brain injury? Brain Res. Mol. Brain Res. 4: 347–352.Google Scholar
  11. Duke RC and Cohen JJ (1992) Morphological and biochemical assays of apoptosis. In: Janssen K (ed.) Current protocols in immunology. (pp 3.17.1–3.17.16) Wiley, New York.Google Scholar
  12. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Lard H, Brooks M, Waters CM, Penn LK and Hancock DC (1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell 69: 119–1128.Google Scholar
  13. Franek F, Vomastek T and Dolnikova J (1992) Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology 9: 117–123.Google Scholar
  14. Fukuda K, Kojiro M and Chiu JF (1993) Demonstration of extensive chromatin cleavage in transplantated Morris hepatoma 7777 tissue: apoptosis or necrosis? Am. J. Pathol. 142: 935–946.Google Scholar
  15. Harrington EA, Fanidi A and Evan GI (1994) Oncogenes and cell death. Current opinion in Genetics and development 4: 120–129.Google Scholar
  16. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL and Korsmeyer SJ (1993) BCl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241–251.Google Scholar
  17. Koli K and Keski-Oja J (1992) Cellular senescence. Annals of Medicine. 24: 313–318.Google Scholar
  18. Lanks KW (1983) Metabolic regulation of heat shock protein levels. Proc. Natl. Acad. Sci. 80: 5325.Google Scholar
  19. Lanks KW (1986) Modulators of the eukariotic heat shock response. Exp. Cell. Res. 165: 1.Google Scholar
  20. Lee AS (1992) Mammalian stress response: induction of the glucose-regulated protein family. Current opinion in cell biology 4: 267–273.Google Scholar
  21. Lin AA and Miller WM (1987) CHO cell responses to low oxygen: Regulation of oxygen consumption and sensitization to oxidative stress. Biotechnol. Bioeng. 40: 505–516.Google Scholar
  22. Marcu KB, Bossone SA and Patel AJ (1992) Myc function and regulation. Annual Rev. Biochem. 61: 809–860.Google Scholar
  23. Martel M, Bazin R, Verrette S and Lemieux R (1988) Characterization of higher avidity monoclonal antibodies produced by murine B-Cell hybridoma variants selected for increased antigen binding of membrane Ig. J. Immunol. 141: 1624–1626.Google Scholar
  24. Martin SJ, Lennon SV, Bonham AM and Cotter TG (1990) Induction of apoptosis (programmed cell death) in leukemic HL-60 cells by inhibition of RNA or protein synthesis. J. Immunol. 145: 1859–1862.Google Scholar
  25. McDonald HR and Lees RK (1990) Programmed death of autoreactive thymocytes. Nature 343: 623–644.Google Scholar
  26. McKeehan WL (2986) Glutaminolysis in animal cells. In: Morgan MJ (ed.) Carbohydrate metabolism in cultured cells. Chapt. 4. Plenum Press, New York.Google Scholar
  27. Mercille S, Johnson M, Lemieux R and Massie B (1994) Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I. Biotechnol. Bioeng. 43: 833–846.Google Scholar
  28. Mercille S and Massie B (1994) Induction of apoptosis in nutrient-deprived cultures of hybridoma cells. Biotechnol. and Bioeng. 44: 1140–1154.Google Scholar
  29. Miller WM, Wilke CR and Blanch HW (1987) Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture. J. Cell. Physiol. 132: 524–530.Google Scholar
  30. Mishell BB, Shiigi SM, Henry C, Chan EL, North J, Gallily R, Slomich M, Miller K, Marbrook J, Parks D and Good AH (1980) Preparation of mouse cell suspensions. In: Mishell BB and Shiigi SM (eds.), Selected Methods in cellular immunology, pp 21–22 WH Freeman, New York.Google Scholar
  31. Mosser DD and Massie B (1994) Genetically engineering mammalian cell lines for increased viability and productivity. Biotech. Adv. 12: 253–277.Google Scholar
  32. Murphy BJ, Laderoute KR, Short SM and Sutherland RM (1991) The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells. Br. J. Cancer 64: 69–73.Google Scholar
  33. Ozturk SS and Palsson BO (1990) Effects of dissolved oxygen on hybridoma growth, metabolism and antibody production in continuous culture. Biotechnol. Prog. 6: 437–446.Google Scholar
  34. Perreault J and Lemieux R (1994) Essential role of optimal protein synthesis in preventing the apoptotic death of cultured B cell hybridomas. Cytotechnology 13: 99–105.Google Scholar
  35. Perreault J and Lemieux R (1993) Rapid apoptotic cell death of B-cell hybridomas in absence of gene expression. J. Cell Physiology 156: 286–293.Google Scholar
  36. Sciandra JJ, Subjeck JR and Hughes CS (1984) Induction of glucose-regulated proteins during anaerobic exposure and of heat-shock proteins after reoxygenetation. Proc. Nat. Acad. Sci. USA 81: 4843.Google Scholar
  37. Sheng-Ong GLC, Keath EJ, Piccoli SP and Cole MD (1982) Novel myc oncogene RNA from abortive immunoglobulin-gene recombination in mouse plasmacytomas. Cell 31: 443–452.Google Scholar
  38. Shi Y, Ryu DDY and Park SH (1993) Monoclonal antibody productivity and the metabolic pattern of perfusion cultures under varying oxygen tensions. Biotech. Bioeng. 42: 430–439.Google Scholar
  39. Shi Y, Sahai BM and Green DR (1989) Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature 339: 625–626.Google Scholar
  40. Singh RP, Al-Rubeai M, Gregory CD and Emery AN (1994) Cell death by necrosis and apoptosis during the culture of commercially important cell lines. In: Spier RE (ed.), Animal Cell Technology: Products of Today, Prospects for Tomorrow. Butterworth-Heinemann, Oxford, England.Google Scholar
  41. Smith CA, Williams GT, Kingston R, Jenkinson EJ and Owen JJ (1989) Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337: 181–184.Google Scholar
  42. Smith CG and Greenfield PF (1992) Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum Pluronic F68, and albumin supplements. Biotechnol. Bioeng. 40: 1045–1055.Google Scholar
  43. Stoler DL, Anderson GR, Russo CA, Spina AM and Beerman TA (1992) Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells. Cancer Research 52: 4372–4378.Google Scholar
  44. Sulik KK, Cook CS and Webster WS (1988) Teratogens and cranial malformations: relationships to cell death. Development 103: 213–231.Google Scholar
  45. Tedesco JL (1987) Analysis of glucose and lactic acid in cell culture media by ion moderated partitioning high performance liquid chromatography. Biotechniques 5: 46–51.Google Scholar
  46. Thömmes J, Gatgens J, Biselli M, Runstadler PW and Wandrey C (1993) The influence of dissolved oxygen tension on the metabolic activity of anim immobilized hybridoma population. Cytotechnology 13: 29–39.Google Scholar
  47. Tomei LD (1991) Apoptosis: a program for death or survival? In: Apoptosis: The molecular bais of cell death, edited by LD Tomei and FO Cope (Cold Spring Harbor Laboratory Press, New York) pp 279–316.Google Scholar
  48. Tozer GM and Griffith JR (1992) The contribution made by cell death and oxygenation to31P MRS observations of tumour energy metabolism. NMR in Biomedicine 5: 279–289.Google Scholar
  49. Veeraragavan K, Colpitts T and Gibbs BF (1990) Purification and characterization of two distinct lipases from Geotrichum Candidum. Biochem. Biophys. Acta 1044: 26–33.Google Scholar
  50. Vomastek T and Franek F (1993) Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures. Immunol. Lett. 35: 19–24.Google Scholar
  51. Wilson DF, Erecinska M, Drown C and Silver IA (1979) Arch Biochem Biophys 195: 485–493.Google Scholar
  52. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556.Google Scholar
  53. Zielke HR, Zielke CL and Ozand PT (1984) Glutamine: a major energy source for cultured mammalian cells. Fed. Proc. 43: 121–125.Google Scholar
  54. Zimmerman LH, Levine RA and Farber HW (1991) Hypoxia induces a specific set of stress proteins in cultured endothelial cells. J. Clin. Invest. 87: 908–914.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Sylvain Mercille
    • 1
  • Bernard Massie
    • 2
  1. 1.Biomira Inc.MontréalCanada
  2. 2.Groupe d'Ingénierie des cellules animales, Institut de recherche en BiotechnologieConseil National de Recherches du CanadaMontréalCanada

Personalised recommendations