Advertisement

Cytotechnology

, Volume 67, Issue 2, pp 237–254 | Cite as

Dynamics of unfolded protein response in recombinant CHO cells

  • Kamal Prashad
  • Sarika MehraEmail author
Original Research

Abstract

Genes in the protein secretion pathway have been targeted to increase productivity of monoclonal antibodies in Chinese hamster ovary cells. The results have been highly variable depending on the cell type and the relative amount of recombinant and target proteins. This paper presents a comprehensive study encompassing major components of the protein processing pathway in the endoplasmic reticulum (ER) to elucidate its role in recombinant cells. mRNA profiles of all major ER chaperones and unfolded protein response (UPR) pathway genes are measured at a series of time points in a high-producing cell line under the dynamic environment of a batch culture. An initial increase in IgG heavy chain mRNA levels correlates with an increase in productivity. We observe a parallel increase in the expression levels of majority of chaperones. The chaperone levels continue to increase until the end of the batch culture. In contrast, calreticulin and ERO1-l alpha, two of the lowest expressed genes exhibit transient time profiles, with peak induction on day 3. In response to increased ER stress, both the GCN2/PKR-like ER kinase and inositol-requiring enzyme-1alpha (Ire1α) signalling branch of the UPR are upregulated. Interestingly, spliced X-Box binding protein 1 (XBP1s) transcription factor from Ire1α pathway is detected from the beginning of the batch culture. Comparison with the expression levels in a low producer, show much lower induction at the end of the exponential growth phase. Thus, the unfolded protein response strongly correlates with the magnitude and timing of stress in the course of the batch culture.

Keywords

Productivity Recombinant CHO cells Unfolded protein response (UPR) pathway ER stress Gene expression 

Notes

Acknowledgments

This work was partially supported by a grant from Department of Biotechnology, Government of India. We would like to thank Dr. Miranda Yap and Dr. Niki Wong, Bioprocessing Technology Institute, Singapore for providing the CHO cell lines. We would also like to thank Prof. Wei Shou Hu of University of Minnesota for useful discussions and comments on the manuscript.

Supplementary material

10616_2013_9678_MOESM1_ESM.tif (1.1 mb)
Estimate of relative abundance of various genes profiled in this study in HP cell line. Time profiles of ΔCT are plotted for a) IgG HC and LC b) Chaperones c) PERK pathway genes and d) Ire1α pathway genes. ΔCT is defined as , Positive values imply abundance greater than β-actin whereas negative values imply lower abundance. Note that β-actin is a moderately abundant gene (TIFF 1084 kb)

References

  1. Barnes LM, Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol Bioeng 96:337–348CrossRefGoogle Scholar
  2. Borth N, Mattanovich D, Kunert R, Katinger H (2005) Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line. Biotechnol Prog 21:106–111CrossRefGoogle Scholar
  3. Brush MH, Shenolikar S (2008) Control of cellular GADD34 Levels by the 26S proteasome. Mol Cell Biol 28:6989–7000CrossRefGoogle Scholar
  4. Chen XL, Kunsch C (2004) Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des 10:879–891CrossRefGoogle Scholar
  5. Chen K, Liu Q, Xie L, Sharp PA, Wang DIC (2001) Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production. Biotechnol Bioeng 72:55–61CrossRefGoogle Scholar
  6. Chung JY, Lim SW, Hong YJ, Hwang SO, Lee GM (2004) Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant chinese hamster ovary cells. Biotechnol Bioeng 85:539–546CrossRefGoogle Scholar
  7. Chusainow J, Yang YS, Yeo JHM, Toh PC, Asvadi P, Wong NSC, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196CrossRefGoogle Scholar
  8. Clark RA, Li S-L, Pearson DW, Leidal KG, Clark JR, Denning GM, Reddick R, Krause KH, Valente AJ (2002) Regulation of calreticulin expression during induction of differentiation in human myeloid cells: evidence for remodeling of the endoplasmic reticulum. J Biol Chem 277:32369–32378Google Scholar
  9. Cudna RE, Dickson AJ (2006) Engineering responsiveness to cell culture stresses: growth arrest and DNA damage gene 153 (GADD153) and the unfolded protein response (UPR) in NS0 myeloma cells. Biotechnol Bioeng 94:514–521CrossRefGoogle Scholar
  10. Cullinan SB, Zhang D, Hannink M, Arvisais E, Kaufman RJ, Diehl JA (2003) Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol Cell Biol 23:7198–7209CrossRefGoogle Scholar
  11. Davis R, Schooley K, Rasmussen B, Thomas J, Reddy P (2000) Effect of PDI overexpression on recombinant protein secretion in CHO cells. Biotechnol Prog 16:736–743CrossRefGoogle Scholar
  12. Dietmair S, Hodson MP, Quek L-E, Timmins NE, Gray P, Nielsen LK (2012) A multi-omics analysis of recombinant protein production in Hek293 cells. PLoS one 7:e43394CrossRefGoogle Scholar
  13. Doolan P, Melville M, Gammell P, Sinacore M, Meleady P, McCarthy K, Francullo L, Leonard M, Charlebois T, Clynes M (2008) Transcriptional profiling of gene expression changes in a PACE-transfected CHO DUKX cell line secreting high levels of rhBMP-2. Mol Biotechnol 39:187–199Google Scholar
  14. Dorai H, Csirke B, Scallon B, Ganguly S (2006) Correlation of heavy and light chain mRNA copy numbers to antibody productivity in mouse myeloma production cell lines. Hybridoma 25:1–9CrossRefGoogle Scholar
  15. Downham MR, Farrell WE, Jenkins HA (1996) Endoplasmic reticulum protein expression in recombinant NS0 myelomas grown in batch culture. Biotechnol Bioeng 51:691–696CrossRefGoogle Scholar
  16. Du Z, Treiber D, McCoy RE, Miller AK, Han M, He F, Domnitz S, Heath C, Reddy P (2013) Non-invasive UPR monitoring system and its applications in CHO production cultures. Biotechnol Bioeng 110:2184–2194Google Scholar
  17. DuRose JB, Tam AB, Niwa M (2006) Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress. Mol Biol Cell 17:3095–3107CrossRefGoogle Scholar
  18. Elanchezhian R, Palsamy P, Madson CJ, Mulhern ML, Lynch DW, Troia AM, Usukura J, Shinohara T (2012) Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells. Cell Death Dis 3:e301Google Scholar
  19. Eletto D, Maganty A, Dersh D, Makarewich C, Biswas C, Paton JC, Paton AW, Doroudgar S, Glembotski CC, Argon Y (2012) Limitation of individual folding resources in the ER leads to outcomes distinct from the unfolded protein response. J Cell Sci 12:4865–4875Google Scholar
  20. Figueroa B, Ailor E, Osborne D, Hardwick JM, Reff M, Betenbaugh MJ (2007) Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19 K and aven in the production of a monoclonal antibody with Chinese hamster ovary cells. Biotechnol Bioeng 97:877–892CrossRefGoogle Scholar
  21. Gass JN, Jiang H-Y, Wek RC, Brewer JW (2008) The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells. Mol Immunol 45:1035–1043CrossRefGoogle Scholar
  22. Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000a) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6:1099–1108CrossRefGoogle Scholar
  23. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D (2000b) Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5:897–904CrossRefGoogle Scholar
  24. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11:619–633Google Scholar
  25. Heal R, McGivan J (1998) Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells. Biochem J 329:389–394Google Scholar
  26. Hendrick V, Winnepenninckx P, Abdelkafi C, Vandeputte O, Cherlet M, Marique T, Renemann G, Loa A, Kretzmer G, Werenne J (2001) Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis. Cytotechnology 36:71–83Google Scholar
  27. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102Google Scholar
  28. Ifandi V, Al-Rubeai M (2005) Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2. Biotechnol Prog 21:671–677CrossRefGoogle Scholar
  29. Jayapal K, Wlaschin K, Hu W-S, and Yap M (2007) Recombinant protein therapeutics from Cho cells—20 years and counting. CHO Consortium, SBE Special Edition, pp 40–47Google Scholar
  30. Jiang Z, Huang Y, Sharfstein ST (2006) Regulation of recombinant monoclonal antibody production in chinese hamster ovary cells: a comparative study of gene copy number, mRNA level, and protein expression. Biotechnol Prog 22:313–318CrossRefGoogle Scholar
  31. Kantardjieff A, Jacob NM, Yee JC, Epstein E, Kok Y-J, Philp R, Betenbaugh M, Hu WS (2010) Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment. J Biotechnol 145:143–159CrossRefGoogle Scholar
  32. Kaspar JW, Jaiswal AK (2010) An autoregulatory loop between Nrf2 and Cul3-Rbx1 controls their cellular abundance. J Biol Chem 285:21349–21358CrossRefGoogle Scholar
  33. Kaufmann H, Mazur X, Fussenegger M, Bailey JE (1999) Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol Bioeng 63:573–582CrossRefGoogle Scholar
  34. Kim NS, Lee GM (2002) Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3. Biotechnol Bioeng 78:217–228CrossRefGoogle Scholar
  35. Kim JY, Kim Y-G, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93:917–930CrossRefGoogle Scholar
  36. Kober L, Zehe C, Bode J (2012) Development of a novel ER stress based selection system for the isolation of highly productive clones. Biotechnol Bioeng 109:2599–2611CrossRefGoogle Scholar
  37. Koumenis C, Naczki C, Koritzinsky M, Rastani S, Diehl A, Sonenberg N, Koromilas A, Wouters BG (2002) Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2α. Mol Cell Biol 22:7405–7416Google Scholar
  38. Ku SCY, Ng DTW, Yap MGS, Chao S-H (2008) Effects of overexpression of X-box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells. Biotechnol Bioeng 99:155–164CrossRefGoogle Scholar
  39. Ku SCY, Toh PC, Lee YY, Chusainow J, Yap MGS, Chao S-H (2010) Regulation of XBP-1 signaling during transient and stable recombinant protein production in CHO cells. Biotechnol Prog 26:517–526Google Scholar
  40. Lambert N, Merten O-W (1997) Effect of serum-free and serum-containing medium on cellular levels of ER-based proteins in various mouse hybridoma cell lines. Biotechnol Bioeng 54:165–180CrossRefGoogle Scholar
  41. Lee A-H, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23:7448–7459CrossRefGoogle Scholar
  42. Lengwehasatit I, Dickson AJ (2002) Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells. Biotechnol Bioeng 80:719–730CrossRefGoogle Scholar
  43. Li J, Zhang C, Jostock T, Dübel S (2007) Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site. Protein Eng Des Sel 20:491–496CrossRefGoogle Scholar
  44. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  45. Meents H, Enenkel B, Eppenberger HM, Werner RG, Fussenegger M (2002) Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-xL on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng 80:706–716CrossRefGoogle Scholar
  46. Merquiol E, Uzi D, Mueller T, Goldenberg D, Nahmias Y, Xavier RJ, Tirosh B, Shibolet O (2011) HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response. PLoS one 6:e24660Google Scholar
  47. Mohan C, Park SH, Chung JY, Lee GM (2007) Effect of doxycycline-regulated protein disulfide isomerase expression on the specific productivity of recombinant CHO cells: thrombopoietin and antibody. Biotechnol Bioeng 98:611–615CrossRefGoogle Scholar
  48. Murphy TC, Woods NR, Dickson AJ (2001) Expression of the transcription factor GADD153 is an indicator of apoptosis for recombinant chinese hamster ovary (CHO) cells. Biotechnol Bioeng 75:621–629CrossRefGoogle Scholar
  49. Nissom PM, Sanny A, Kok Y, Hiang Y, Chuah S, Shing T, Lee YY, Wong KT, Hu WS, Sim MY, Philp R (2006) Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells. Mol Biotechnol 34:125–140Google Scholar
  50. Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2α. J Cell Biol 153:1011–1022CrossRefGoogle Scholar
  51. Ohya T, Hayashi T, Kiyama E, Nishii H, Miki H, Kobayashi K, Honda K, Omasa T, Ohtake H (2008) Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression. Biotechnol Bioeng 100:317–324Google Scholar
  52. Pahl HL, Baeuerle PA (1997) The ER-overload response: activation of NF-κB. Trends Biochem Sci 22:63–67CrossRefGoogle Scholar
  53. Pena J, Harris E (2011) Dengue virus modulates the unfolded protein response in a time-dependent manner. J Biol Chem 286:14226–14236CrossRefGoogle Scholar
  54. Rutkowski DT, Arnold SM, Miller CN, Wu J, Li J, Gunnison KM, Mori K, Sadighi, Akha AA, Raden D, Kaufman RJ (2006) Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS Biol 4:e374Google Scholar
  55. Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES (1989) NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J 8(13):4251–4258Google Scholar
  56. Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog 21:122–133CrossRefGoogle Scholar
  57. Schröder M (2006) The unfolded protein response. Mol Biotechnol 34:279–290CrossRefGoogle Scholar
  58. Schröder M (2008) Endoplasmic reticulum stress responses. Cell Mol Life Sci 65:862–894CrossRefGoogle Scholar
  59. Schröder M, Kaufman RJ (2005) The mammalian unfolded protein response. Annu Rev Biochem 74:739–789CrossRefGoogle Scholar
  60. Seth G, Hossler P, Yee J, Hu W-S (2006) Engineering Cells for cell culture bioprocessing—physiological fundamentals. Adv Biochem Eng Biotechnol 101:119–164Google Scholar
  61. Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ, Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol Bioeng 88:474–488Google Scholar
  62. Stansfield SH, Allen EE, Dinnis DM, Racher AJ, Birch JR, James DC (2007) Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture. Biotechnol Bioeng 97:410–424CrossRefGoogle Scholar
  63. Stępkowski TM, Kruszewski MK (2011) Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis. Free Radic Biol Med 50:1186–1195CrossRefGoogle Scholar
  64. Teruya K, Daimon Y, Dong X-Y, Katakura Y, Miura T, Ichikawa A, Fujiki T, Yamashita M, Mori T, Ohashi H, Shirahata S (2005) An approach to further enhance the cellular productivity of exogenous protein hyper-producing Chinese hamster ovary (CHO) cells. Cytotechnology 47:29–36Google Scholar
  65. Tigges M, Fussenegger M (2006) Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells. Metab Eng 8:264–272CrossRefGoogle Scholar
  66. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258CrossRefGoogle Scholar
  67. Underhill MF, Birch JR, Smales CM, Naylor LH (2005) eIF2α phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells. Biotechnol Bioeng 89:805–814CrossRefGoogle Scholar
  68. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D (2000) Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287:664–666Google Scholar
  69. Watanabe S, Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1. Biotechnol Bioeng 77:1–7CrossRefGoogle Scholar
  70. Yoon SK, Hong JK, Choo SH, Song JY, Park HW, Lee GM (2006a) Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production. J Biotechnol 122:463–472CrossRefGoogle Scholar
  71. Yoon SK, Kim SH, Song JY, Lee GM (2006b) Biphasic culture strategy for enhancing volumetric erythropoietin productivity of Chinese hamster ovary cells. Enzyme Microb Technol 39:362–365CrossRefGoogle Scholar
  72. Yurochko AD, Mayo MW, Poma EE, Baldwin AS, Huang ES (1997) Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kappaB promoters. J Virol 71:4638–4648Google Scholar
  73. Zhang P, McGrath BC, Reinert J, Olsen DS, Lei L, Gill S, Wek SA, Vattem KM, Wek RC, Kimball SR, Jefferson LS, Cavener DR (2002) The GCN2 eIF2α kinase is required for adaptation to amino acid deprivation in mice. Mol Cell Biol 22:6681–6688Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Department of Chemical EngineeringIndian Institute of Technology BombayPowai, MumbaiIndia

Personalised recommendations