Skip to main content

Advertisement

Log in

Regulating apoptosis in mammalian cell cultures

  • Original Paper
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams JM (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev 17:2481–2495

    Article  CAS  Google Scholar 

  • Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326

    Article  CAS  Google Scholar 

  • Adams JM, Cory S (2001) Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26:61–66

    Article  CAS  Google Scholar 

  • Al-Rubeai M, Singh RP (1998) Apoptosis in cell culture. Curr Opin Biotechnol 9:152–156

    Article  CAS  Google Scholar 

  • Al-Rubeai M, Mills D, Emery AN (1990) Electron microscopy of hybridoma cells with special regard to monoclonal antibody production. Cytotechnology 4:13–28

    Article  CAS  Google Scholar 

  • Arden N, Betenbaugh MJ (2004). Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 22:174–180

    Article  CAS  Google Scholar 

  • Ashkenazi A (2002) Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2:420–430

    Article  CAS  Google Scholar 

  • Bhonde MR, Hanski ML, Budczies J, Cao M, Gillissen B, Moorthy D, Simonetta F, Scherubl H, Truss M, Hagemeier C et al (2006) DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: The lack of this suppression in p53mut␣cells contributes to apoptosis. J Biol Chem 281:8675–8685

    Article  CAS  Google Scholar 

  • Boatright KM, Salvesen GS (2003) Mechanisms of caspase activation. Curr Opin Cell Biol 15:725–731

    Article  CAS  Google Scholar 

  • Boya P, Roumier T, Andreau K, Gonzalez-Polo RA, Zamzami N, Castedo M, Kroemer G (2003) Mitochondrion-targeted apoptosis regulators of viral origin. Biochem Biophys Res Commun 304:575–581

    Article  CAS  Google Scholar 

  • Breckenridge DG, Germain M, Mathai JP, Nguyen M, Shore GC (2003) Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 22:8608–8618

    Article  CAS  Google Scholar 

  • Charbonneau JR, Furtak T, Lefebvre J, Gauthier ER (2003) Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures. Biotechnol Bioeng 81:279–290

    Article  CAS  Google Scholar 

  • Cheng EH, Levine B, Boise LH, Thompson CB, Hardwick JM (1996) Bax-independent inhibition of apoptosis by Bcl-XL. Nature 379:554–556

    Article  CAS  Google Scholar 

  • Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8:705–711

    Article  CAS  Google Scholar 

  • Chiang GG, Sisk WP (2005a) Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells. Biotechnol Bioeng 91:779–792

    Article  CAS  Google Scholar 

  • Chiang GG, Sisk WP (2005b) Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells. Biotechnol Bioeng. Jun 28

  • Chipuk JE, Green DR (2006) Dissecting p53-dependent apoptosis. Cell Death Differ

  • Cho Y, Gorina S, Jeffrey PD, Pavletich NP (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265:346–355

    Article  CAS  Google Scholar 

  • Chung JD, Sinskey AJ, Stephanopoulos G (1998) Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line. Biotechnol Bioeng 57:164–171

    Article  CAS  Google Scholar 

  • Cohen JJ (1993) Apoptosis. Immunol Today 14:126–130

    Article  CAS  Google Scholar 

  • Cory S, Huang DC, Adams JM (2003) The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22:8590–8607

    Article  CAS  Google Scholar 

  • Cudna RE, Dickson AJ (2003) Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 81:56–65

    Article  CAS  Google Scholar 

  • Cummings MC, Winterford CM, Walker NI (1997) Apoptosis. Am J Surg Pathol 21:88–101

    Article  CAS  Google Scholar 

  • deZengotita VM, Schmelzer AE, Miller WM (2002) Characterization of hybridoma cell responses to elevated pCO(2) and osmolality: intracellular pH, cell size, apoptosis, and metabolism. Biotechnol Bioeng 77:369–380

    Article  CAS  Google Scholar 

  • Ferri KF, Kroemer G (2001) Organelle-specific initiation␣of cell death pathways. Nat Cell Biol 3:E255–E263

    Article  CAS  Google Scholar 

  • Figueroa B Jr, Sauerwald TM, Mastrangelo AJ, Hardwick JM, Betenbaugh MJ (2001) Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults. Biotechnol Bioeng 73:211–222

    Article  CAS  Google Scholar 

  • Figueroa B Jr, Sauerwald TM, Oyler GA, Hardwick JM, Betenbaugh MJ (2003) A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures. Metab Eng 5:230–245

    Article  CAS  Google Scholar 

  • Follstad BD, Wang DI, Stephanopoulos G (2002) Mitochondrial membrane potential selects hybridomas yielding high viability in fed-batch cultures. Biotechnol Prog 18:1–5

    Article  CAS  Google Scholar 

  • Franek F, Dolnikova J (1991) Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: effect of nutrient concentration. Cytotechnology 7:33–38

    Article  CAS  Google Scholar 

  • Franek F, Sramkova K (1996) Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids. Immunol Lett 52:139–144

    Article  CAS  Google Scholar 

  • Goswami J, Sinskey AJ, Steller H, Stephanopoulos GN, Wang DI (1999) Apoptosis in batch cultures of Chinese hamster ovary cells. Biotechnol Bioeng 62:632–640

    Article  CAS  Google Scholar 

  • Gueven N, Becherel OJ, Birrell G, Chen P, DelSal G, Carney JP, Grattan-Smith P, Lavin MF (2006) Defective p53 response and apoptosis associated with an ataxia-telangiectasia-like phenotype. Cancer Res 66:2907–2912

    Article  CAS  Google Scholar 

  • Guo B, Zhai D, Cabezas E, Welsh K, Nouraini S, Satterthwait AC, Reed JC (2003) Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature 423:456–461

    Article  CAS  Google Scholar 

  • Huo JX, Metz SA, Li GD (2004) p53-independent induction of p21(waf1/cip1) contributes to the activation of caspases in GTP-depletion-induced apoptosis of insulin-secreting cells. Cell Death Differ 11:99–109

    Article  CAS  Google Scholar 

  • Jendrossek V, Muller I, Eibl H, Belka C (2003) Intracellular mediators of erucylphosphocholine-induced apoptosis. Oncogene 22:2621–2631

    Article  CAS  Google Scholar 

  • Jung D, Cote S, Drouin M, Simard C, Lemieux R (2002) Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures. Biotechnol Bioeng 79:180–187

    Article  CAS  Google Scholar 

  • Kaufman RJ (2002) Orchestrating the unfolded protein response in health and disease. J Clin Invest 110:1389–1398

    CAS  Google Scholar 

  • Kaufman RJ, Scheuner D, Schroder M, Shen X, Lee K, Liu CY, Arnold SM (2002) The unfolded protein response in nutrient sensing and differentiation. Nat Rev Mol Cell Biol 3:411–421

    Article  CAS  Google Scholar 

  • Kelekar A, Chang BS, Harlan JE, Fesik SW, Thompson CB (1997) Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 17:7040–7046

    CAS  Google Scholar 

  • Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257

    CAS  Google Scholar 

  • Kim NS, Lee GM (2002a) Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3. Biotechnol Bioeng 78:217–228

    Article  CAS  Google Scholar 

  • Kim NS, Lee GM (2002b). Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression. J Biotechnol 95:237–248

    Article  CAS  Google Scholar 

  • Kischkel FC, Lawrence DA, Tinel A, LeBlanc H, Virmani A, Schow P, Gazdar A, Blenis J, Arnott D, Ashkenazi A (2001) Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J Biol Chem 276:46639–46646

    Article  CAS  Google Scholar 

  • Komiyama T, Ray CA, Pickup DJ, Howard AD, Thornberry NA, Peterson EP, Salvesen G (1994) Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition. J Biol Chem 269:19331–19337

    CAS  Google Scholar 

  • Lasunskaia EB, Fridlianskaia AA, Darieva ZA, da Silva MS, Kanashiro MM, Margulis BA (2003) Transfection of NS0 myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis. Biotechnol Bioeng 81:496–504

    Article  CAS  Google Scholar 

  • Lasunskaia EB, Fridlianskaia II, Darieva ZA, Da Silva MSR, Kanashiro MM, Margulis BA (2005) Transfection of NS0 myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis. Biotechnol Bioeng 81:496–504

    Google Scholar 

  • LeBlanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10:66–75

    Article  CAS  Google Scholar 

  • Lee SK, Lee GM (2003) Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line. Biotechnol Bioeng 82:872–876

    Article  CAS  Google Scholar 

  • Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K et al (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6:1389–1399

    Article  CAS  Google Scholar 

  • Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157

    Article  CAS  Google Scholar 

  • Mastrangelo AJ, Betenbaugh MJ (1998) Overcoming apoptosis: new methods for improving protein-expression systems. Trends Biotechnol 16:88–95

    Article  CAS  Google Scholar 

  • Mastrangelo AJ, Hardwick JM, Betenbaugh MJ (1996) BCl-2 apoptosis and extends recombinant protein production cells infected with Sindbis viral vectors. Cytotechnology 22:169–178

    Article  CAS  Google Scholar 

  • Mastrangelo AJ, Hardwick JM, Bex F, Betenbaugh MJ (2000a) Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors. Biotechnol Bioeng 67:544–554

    Article  CAS  Google Scholar 

  • Mastrangelo AJ, Hardwick JM, Zou S, Betenbaugh MJ (2000b) Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults. Biotechnol Bioeng 67:555–564

    Article  CAS  Google Scholar 

  • Mazur X, Fussenegger M, Renner WA, Bailey JE (1998) Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27. Biotechnol Prog 14:705–713

    Article  CAS  Google Scholar 

  • Meents H, Enenkel B, Eppenberger HM, Werner RG, Fussenegger M (2002) Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng 80:706–716

    Article  CAS  Google Scholar 

  • Mercille S, Massie B (1994a). Induction of apoptosis in nutrient deprived cultures of hybridoma and myeloma cells. Biotechnol Bioeng 44:1140–1154

    Article  CAS  Google Scholar 

  • Mercille S, Massie B (1994b). Induction of apoptosis in oxygen-deprived cultures of hybridoma cells. Cytotechnology 15:117–128

    Article  CAS  Google Scholar 

  • Mercille S, Jolicoeur P, Gervais C, Paquette D, Mosser DD, Massie B (1999) Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19 K adenoviral gene. Biotechnol Bioeng 63:516–528

    Article  CAS  Google Scholar 

  • Nicholls CD, McLure KG, Shields MA, Lee PW (2002). Biogenesis of p53 involves cotranslational dimerization of monomers and posttranslational dimerization of dimers. Implications on the dominant negative effect. J Biol Chem 277:12937–12945

    Article  CAS  Google Scholar 

  • Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288:1053–1058

    Article  CAS  Google Scholar 

  • Patil C, Walter P (2001) Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr Opin Cell Biol 13:349–355

    Article  CAS  Google Scholar 

  • Perani A (1998) VAriable functions of bcl-2 in mediating strss-induced apoptosis in hybridoma cells. Cytotechnology 28:177–188

    Article  CAS  Google Scholar 

  • Raff M (1998) Cell suicide for beginners. Nature 396(6707):119–122

    Article  CAS  Google Scholar 

  • Rao RV, Poksay KS, Castro-Obregon S, Schilling B, Row RH, del Rio G, Gibson BW, Ellerby HM, Bredesen DE (2004) Molecular components of a cell death pathway activated by endoplasmic reticulum stress. J Biol Chem 279:177–187

    Article  CAS  Google Scholar 

  • Ron D (2002) Translational control in the endoplasmic reticulum stress response. J Clin Invest 110:1383–1388

    CAS  Google Scholar 

  • Sak A, Wurm R, Elo B, Grehl S, Pottgen C, Stuben G, Sinn B, Wolf G, Budach V, Stuschke M (2003) Increased radiation-induced apoptosis and altered cell cycle progression of human lung cancer cell lines by antisense oligodeoxynucleotides targeting p53 and p21(WAF1/CIP1). Cancer Gene Ther 10:926–934

    Article  CAS  Google Scholar 

  • Sanfeliu A, Stephanopoulos G (1999) Effect of glutamine limitation on the death of attached Chinese hamster ovary cells. Biotechnol Bioeng 64:46–53

    Article  CAS  Google Scholar 

  • Sauerwald TM, Betenbaugh MJ (2002) Apoptosis in biotechnology: its role in mammalian cell culture and methods of inhibition. BioProcessing 1:61–68

    Google Scholar 

  • Sauerwald TM, Betenbaugh MJ, Oyler GA (2002) Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants. Biotechnol Bioeng 77:704–716

    Article  CAS  Google Scholar 

  • Sauerwald TM, Oyler GA, Betenbaugh MJ (2003) Study of caspase inhibitors for limiting death in mammalian cell culture. Biotechnol Bioeng 81:329–340

    Article  CAS  Google Scholar 

  • Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300:135–139

    Article  CAS  Google Scholar 

  • Simpson NH, Singh RP, Emery AN, Al-Rubeai M (1999)␣Bcl-2 over-expression reduces growth rate and␣prolongs G1 phase in continuous chemostat cultures of hybridoma cells. Biotechnol Bioeng 64:174–186

    Article  CAS  Google Scholar 

  • Singh RP, al-Rubeai M (1998) Apoptosis and bioprocess technology. Adv Biochem Eng Biotechnol 62:167–184

    CAS  Google Scholar 

  • Singh RP, al-Rubeai M, Gregory CD, Emery AN (1994) Cell death in bioreactors: a role for apoptosis. Biotechnol Bioeng 44:720–726

    Article  CAS  Google Scholar 

  • Singh RP, Finka G, Emery AN, al-Rubeai M (1997) Apoptosis and its control in cell cultures systems. Cytotechnology 23:87–93

    Article  Google Scholar 

  • Sitailo LA, Tibudan SS, Denning MF (2002) Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes. J Biol Chem 277:19346–19352

    Article  CAS  Google Scholar 

  • Strasser A, O’Connor L, Dixit VM (2000) Apoptosis signaling. Annu Rev Biochem 69:217–245

    Article  CAS  Google Scholar 

  • Tey BT, Al-Rubeai M (2004) Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity. Apoptosis 9:843–852

    Article  CAS  Google Scholar 

  • Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000a) Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures. J Biotechnol 79:147–159

    Article  CAS  Google Scholar 

  • Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000b) Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng 68:31–43

    Article  CAS  Google Scholar 

  • Vives J, Juanola S, Cairo JJ, Prats E, Cornudella L, Godia F (2003) Protective effect of viral homologues of bcl-2 on hybridoma cells under apoptosis-inducing conditions. Biotechnol Prog 19:84–89

    Article  CAS  Google Scholar 

  • Weinberg RL, Veprintsev DB, Fersht AR (2004) Cooperative binding of tetrameric p53 to DNA. J Mol Biol 341:1145–1159

    Article  CAS  Google Scholar 

  • White C, Li C, Yang J, Petrenko NB, Madesh M, Thompson CB, Foskett JK (2005) The endoplasmic␣reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R. Nat Cell Biol 7:1021–1028

    Article  CAS  Google Scholar 

  • Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80:285–291

    Article  CAS  Google Scholar 

  • Zha J, Weiler S, Oh KJ, Wei MC, Korsmeyer SJ (2000) Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290:1761–1765

    Article  CAS  Google Scholar 

  • Zhang H (2006) p53 plays a central role in UVA and UVB induced cell damage and apoptosis in melanoma cells. Cancer Lett

  • Zhang X, Li J, Sejas DP, Pang Q (2005) The ATM/p53/p21 pathway influences cell fate decision between apoptosis and senescence in reoxygenated hematopoietic progenitor cells. J Biol Chem 280:19635–19640

    Article  CAS  Google Scholar 

  • Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB (2003) Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol 162:59–69

    Article  CAS  Google Scholar 

  • Zong WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB (2001) BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev 15:1481–1486

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M. J. Betenbaugh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arden, N., Betenbaugh, M. Regulating apoptosis in mammalian cell cultures. Cytotechnology 50, 77–92 (2006). https://doi.org/10.1007/s10616-006-9008-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-006-9008-5

Keywords

Navigation