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Jaffaraghaei M, Ghafouri H, Vaziri B, Taheri M, Talebkhan Y, Heravi M, Parand M. Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies. PLoS One 2024; 19:e0300702. [PMID: 38696377 PMCID: PMC11065310 DOI: 10.1371/journal.pone.0300702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Accepted: 03/04/2024] [Indexed: 05/04/2024] Open
Abstract
The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.
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Affiliation(s)
- Morteza Jaffaraghaei
- Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran
| | - Hossein Ghafouri
- Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran
| | - Behrouz Vaziri
- Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Maryam Taheri
- Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran
| | - Yeganeh Talebkhan
- Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Mansooreh Heravi
- Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Mohammad Parand
- Department of Research and Development, PersisgenPar, Tehran, Iran
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In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line. Pharmaceutics 2022; 14:pharmaceutics14102013. [PMID: 36297448 PMCID: PMC9609024 DOI: 10.3390/pharmaceutics14102013] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 09/07/2022] [Accepted: 09/14/2022] [Indexed: 11/18/2022] Open
Abstract
Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and β-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.
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Malm M, Kuo CC, Barzadd MM, Mebrahtu A, Wistbacka N, Razavi R, Volk AL, Lundqvist M, Kotol D, Tegel H, Hober S, Edfors F, Gräslund T, Chotteau V, Field R, Varley PG, Roth RG, Lewis NE, Hatton D, Rockberg J. Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins. Metab Eng 2022; 72:171-187. [PMID: 35301123 PMCID: PMC9189052 DOI: 10.1016/j.ymben.2022.03.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 02/09/2022] [Accepted: 03/10/2022] [Indexed: 10/31/2022]
Abstract
Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
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Affiliation(s)
- Magdalena Malm
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Chih-Chung Kuo
- Departments of Pediatrics and Bioengineering, University of California, San Diego, La Jolla, CA, 92093, USA; The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, CA, 92093, USA
| | - Mona Moradi Barzadd
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Aman Mebrahtu
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Num Wistbacka
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Ronia Razavi
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Anna-Luisa Volk
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Magnus Lundqvist
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - David Kotol
- Science for Life Laboratory, KTH - Royal Institute of Technology, Solna, 171 65, Sweden
| | - Hanna Tegel
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Sophia Hober
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Fredrik Edfors
- Science for Life Laboratory, KTH - Royal Institute of Technology, Solna, 171 65, Sweden
| | - Torbjörn Gräslund
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden
| | - Veronique Chotteau
- Dept. of Industrial Biotechnology, KTH - Royal Institute of Technology, Stockholm, SE-10691, Sweden
| | - Ray Field
- Cell Culture and Fermentation Sciences, BioPharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | - Paul G Varley
- Cell Culture and Fermentation Sciences, BioPharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | - Robert G Roth
- Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Nathan E Lewis
- Departments of Pediatrics and Bioengineering, University of California, San Diego, La Jolla, CA, 92093, USA; The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, CA, 92093, USA.
| | - Diane Hatton
- Cell Culture and Fermentation Sciences, BioPharmaceutical Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | - Johan Rockberg
- Dept. of Protein Science, KTH - Royal Institute of Technology, Stockholm, SE-106 91, Sweden.
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Patil AA, Bhor SA, Rhee WJ. Cell death in culture: Molecular mechanisms, detections, and inhibition strategies. J IND ENG CHEM 2020. [DOI: 10.1016/j.jiec.2020.08.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Formas‐Oliveira AS, Basílio JS, Rodrigues AF, Coroadinha AS. Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production. Biotechnol J 2020; 15:e1900562. [DOI: 10.1002/biot.201900562] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 05/22/2020] [Indexed: 12/18/2022]
Affiliation(s)
- Ana S. Formas‐Oliveira
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - João S. Basílio
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - Ana F. Rodrigues
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - Ana S. Coroadinha
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
- The Discoveries centre for Regenerative and Precision Medicine Nova University Lisbon Oeiras Campus, Av. da República 2780‐157 Oeiras Portugal
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Towards development of plasmacytoma cells-based expression systems utilizing alphavirus vectors: An NS0-VEE model. J Virol Methods 2019; 274:113734. [PMID: 31525396 DOI: 10.1016/j.jviromet.2019.113734] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2019] [Revised: 07/19/2019] [Accepted: 09/12/2019] [Indexed: 11/24/2022]
Abstract
Plasmacytoma (myeloma) cells have a large protein expression capacity, although their industrial use is confined to stable expression systems. Vectors derived from genomes of viruses from the genus Alphavirus allow obtaining of high yields of target proteins but their use is limited to transient expression. Little information has been published to date on attempts to combine the myeloma cells as hosts with alphaviruses as expression vectors. A plasmid construct which allows rescue of a model alphavirus Venezuelan equine encephalitis virus (VEE) upon transfection of a cell culture was created. Mutations in the capsid and nsP2 genes allow for less cytopathogenic propagation of the virus. A cDNA-copy of the genome was placed in a plasmid under the control of the CMV promoter for virus rescue following DNA transfection. Parameters for the virus rescue by electroporating of the infectious clone in murine myeloma cells (NS0) were optimized. The highest FFU counts (1.2 × 105 FFU per 10 ug DNA) were produced with 2 pulses (voltage 250 V, capacitance 960 u F) and the best electroporation buffer was selected from eight buffers. Self-sustained VEE infection was established in NS0 cultures with high titers (8 × 108 FFU/ml) of the virus, despite a fraction of infected cells dying during 5-days observation. Further development of the NS0-VEE expression system may require addressing of apoptosis induced by VEE.
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Grilo AL, Mantalaris A. Apoptosis: A mammalian cell bioprocessing perspective. Biotechnol Adv 2019; 37:459-475. [PMID: 30797096 DOI: 10.1016/j.biotechadv.2019.02.012] [Citation(s) in RCA: 118] [Impact Index Per Article: 19.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Revised: 02/08/2019] [Accepted: 02/19/2019] [Indexed: 02/07/2023]
Abstract
Apoptosis is a form of programmed and controlled cell death that accounts for the majority of cellular death in bioprocesses. Cell death affects culture longevity and product quality; it is instigated by several stresses experienced by the cells within a bioreactor. Understanding the factors that cause apoptosis as well as developing strategies that can protect cells is crucial for robust bioprocess development. This review aims to a) address apoptosis from a bioprocess perspective; b) describe the significant apoptotic mechanisms linking them to the most relevant stresses encountered in bioreactors; c) discuss the design of operating conditions in order to avoid cell death; d) focus on industrially relevant cell lines; and e) present anti-apoptosis strategies including cell engineering and model-based optimization of bioprocesses. In addition, the importance of apoptosis in quality-by-design bioprocess development from clone screening to production scale are highlighted.
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Affiliation(s)
- Antonio L Grilo
- Biological Systems Engineering Laboratory, Centre for Process Systems Engineering, Department of Chemical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.
| | - Athanasios Mantalaris
- Biological Systems Engineering Laboratory, Centre for Process Systems Engineering, Department of Chemical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.
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Gulis G, Simi KCR, de Toledo RR, Maranhao AQ, Brigido MM. Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein. BMC Biotechnol 2014; 14:26. [PMID: 24725707 PMCID: PMC3995513 DOI: 10.1186/1472-6750-14-26] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2013] [Accepted: 04/08/2014] [Indexed: 01/23/2023] Open
Abstract
Background The optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability. Results In this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction. Conclusions Our data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production.
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Affiliation(s)
- Galina Gulis
- Institute of Biological Sciences, Department of Cell Biology, University of Brasilia, Campus Universitário Darcy Ribeiro, Brasília, DF 70910-900, Brazil.
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Vestrheim AC, Moen A, Egge-Jacobsen W, Bratlie DB, Michaelsen TE. Different glycosylation pattern of human IgG1 and IgG3 antibodies isolated from transiently as well as permanently transfected cell lines. Scand J Immunol 2013; 77:419-28. [PMID: 23488770 DOI: 10.1111/sji.12046] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2012] [Accepted: 03/07/2013] [Indexed: 12/17/2022]
Abstract
The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.
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Affiliation(s)
- A C Vestrheim
- Department of Bacteriology & Immunology, Norwegian Institute of Public Health, 0403 Oslo, Norway.
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A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates. Immunobiology 2011; 216:1239-47. [DOI: 10.1016/j.imbio.2011.07.004] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2010] [Accepted: 07/03/2011] [Indexed: 11/19/2022]
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11
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Meleady P, Doolan P, Henry M, Barron N, Keenan J, O'Sullivan F, Clarke C, Gammell P, Melville MW, Leonard M, Clynes M. Sustained productivity in recombinant Chinese hamster ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype. BMC Biotechnol 2011; 11:78. [PMID: 21781345 PMCID: PMC3170212 DOI: 10.1186/1472-6750-11-78] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2011] [Accepted: 07/24/2011] [Indexed: 11/25/2022] Open
Abstract
Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.
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Affiliation(s)
- Paula Meleady
- National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland.
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Meng X, Xu X, Hu J, Jin F, Hu Q, Sun Q, Yu X, Ren S. Toxicity and differential protein analysis following destruxin A treatment of Spodoptera litura (Lepidoptera: Noctuidae) SL-1 cells. Toxicon 2011; 58:327-35. [PMID: 21718714 DOI: 10.1016/j.toxicon.2011.06.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2011] [Revised: 06/03/2011] [Accepted: 06/06/2011] [Indexed: 10/18/2022]
Abstract
The cytotoxicity of a destruxin A (DA) treatment of Spodoptera litura SL-1 cells was investigated. An MTT assay showed that DA was highly toxic to SL-1 cells in a concentration- and time-dependent manner. The IC(50) values of DA, after 24 h and 48 h of treatment, were 17.86 μg/mL and 7.80 μg/mL, respectively. Under inverted phase contrast microscopy (IPCM), it was found that prolonged treatment with DA could induce cell rounding, cellular membrane shrinking, formation of apoptotic bodies, vacuole appearance and cytoplasm leak out. Apoptosis induced by DA was further confirmed by fluorescence microscopy (FM) and flow cytometry (FCM) studies. SL-1 cells entered early apoptosis following a treatment with 2.5 μg/mL DA and entered late apoptosis following a treatment with increasing concentrations of DA. Furthermore, two-dimensional gel electrophoresis (2-DE) analysis was used to identify 22 proteins which were differentially expressed (≥2-fold difference) between control cells and DA-treated cells, and the expression level of these proteins was significantly different between the treated and untreated cells. Our results suggest that these differentially expressed proteins may help explain the diverse biological effects caused by the destruxin A treatment of cells; additionally, some of the identified proteins may have roles in SL-1 cellular proliferation and apoptosis.
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Affiliation(s)
- Xiang Meng
- Engineering Research Center of Biological Control, Ministry of Education, South China Agricultural University, Guangzhou 510642, China
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Park JH, Park HH, Park TH. Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems. KOREAN J CHEM ENG 2010. [DOI: 10.1007/s11814-010-0278-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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Yang X, He H, Yang W, Song T, Guo C, Zheng X, Liu Q. Effects of HSP70 antisense oligonucleotide on the proliferation and apoptosis of human hepatocellular carcinoma cells. ACTA ACUST UNITED AC 2010; 30:337-43. [PMID: 20556578 DOI: 10.1007/s11596-010-0353-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2009] [Indexed: 12/19/2022]
Abstract
The study investigated the effects of heat shock protein 70 (HSP70) antisense oligonucleotide (ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line (SMMC-7721 cells) in vitro. HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of Sofast transfection reagent. Inhibition rate of SMMC-7721 cells was determined by using MTT method. Apoptosis rate and cell cycle distribution were measured by flow cytometry. Immunocytochemistry staining was used to observe the expression of HSP70, Bcl-2 and Bax. The results showed that HSP70 ASODN at various concentrations could significantly inhibit the growth of SMMC-7721 cells, and the inhibition effect peaked 48 h after transfection with 400-nmol/L HSP70 ASODN. Cytometric analysis showed the apoptotic rate was increased in a dose- and time-dependent manner in the HSP70 ASODN-treated cells. The percentage of cells in the G2/M and S phases was significantly decreased and that in the G0/G1 phase increased as the HSP70 ASODN concentration was elevated and the exposure time prolonged. Immunocytochemistry showed that treatment of SMMC-7721 cells with HSP70 ASODN resulted in decreased expressions of HSP70 and Bcl-2 proteins, and an increased expression of Bax protein. It was concluded that the HSP70 ASODN can inhibit the growth of the SMMC-7721 cells and increase cell apoptosis by down-regulating the expression of HSP70. HSP70 ASODN holds promise for the treatment of hepatocellular carcinoma.
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Affiliation(s)
- Xue Yang
- Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710061, China.
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Krampe B, Al-Rubeai M. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. Cytotechnology 2010; 62:175-88. [PMID: 20502964 DOI: 10.1007/s10616-010-9274-0] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2010] [Accepted: 04/12/2010] [Indexed: 12/15/2022] Open
Abstract
Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.
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Affiliation(s)
- Britta Krampe
- School of Chemical and Bioprocess Engineering, and Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Republic of Ireland
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Engineering mammalian cells in bioprocessing - current achievements and future perspectives. Biotechnol Appl Biochem 2010; 55:175-89. [PMID: 20392202 DOI: 10.1042/ba20090363] [Citation(s) in RCA: 92] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Over the past 20 years, we have seen significant improvements in product titres from 50 mg/l to 5-10 g/l, a more than 100-fold increase. The main methods that have been employed to achieve this increase in product titre have been through the manipulation of culture media and process control strategies, such as the optimization of fed-batch processes. An alternative means to increase productivity has been through the engineering of host cells by altering cellular processes. Recombinant DNA technology has been used to over-express or suppress specific genes to endow particular phenotypes. Cellular processes that have been altered in host cells include metabolism, cell cycle, protein secretion and apoptosis. Cell engineering has also been employed to improve post-translational modifications such as glycosylation. In this article, an overview of the main cell engineering strategies previously employed and the impact of these strategies are presented. Many of these strategies focus on engineering cell lines with more efficient carbon metabolism towards reducing waste metabolites, achieving a biphasic production system by engineering cell cycle control, increasing protein secretion by targeting specific endoplasmic reticulum stress chaperones, delaying cell death by targeting anti-apoptosis genes, and engineering glycosylation by enhancing recombinant protein sialylation and antibody glycosylation. Future perspectives for host cell engineering, and possible areas of research, are also discussed in this review.
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Kaviarasan S, Ramamurthy N, Gunasekaran P, Varalakshmi E, Anuradha CV. Induction of alcohol-metabolizing enzymes and heat shock protein expression by ethanol and modulation by fenugreek seed polyphenols in Chang liver cells. Toxicol Mech Methods 2009; 19:116-22. [PMID: 19778255 DOI: 10.1080/15376510802305039] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The present study investigated the effect of fenugreek seed polyphenolic extract (FPEt) on ethanol-induced protein expression in Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in the presence of FPEt for 24 h. Cells were harvested and assessed for expression of alcohol metabolizing enzymes-alcohol dehydrogenase (ADH(2) isoform), aldehyde dehydrogenase (ALDH(2) isoform), cytochrome P450 (CYP2E1), the electron transport component (cytochrome-c), and the heat shock proteins. The expression of ADH(2), ALDH(2), and CYP2E1 were upregulated, whereas the expression of cytochrome-c was downregulated in the ethanol-treated cells. The expression of cellular heat shock proteins-HSP70, HSC70, HSC92, and mitochondrial protein mtHSP70 were induced in ethanol-treated Chang liver cells. FPEt modulated the protein expression changes induced by ethanol and had no effect when incubated with normal Chang liver cells. FPEt might exert cytoprotective action on ethanol-induced liver cell damage, possibly by enhancing cellular redox status.
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Affiliation(s)
- S Kaviarasan
- Department of Biochemistry, Annamalai University, Annamalai, Tamil Nadu, India. kavi
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18
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Lee YY, Wong KT, Tan J, Toh PC, Mao Y, Brusic V, Yap MG. Overexpression of heat shock proteins (HSPs) in CHO cells for extended culture viability and improved recombinant protein production. J Biotechnol 2009; 143:34-43. [DOI: 10.1016/j.jbiotec.2009.05.013] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2009] [Revised: 05/19/2009] [Accepted: 05/30/2009] [Indexed: 10/20/2022]
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19
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Jain E, Kumar A. Upstream processes in antibody production: Evaluation of critical parameters. Biotechnol Adv 2008; 26:46-72. [DOI: 10.1016/j.biotechadv.2007.09.004] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2007] [Accepted: 09/04/2007] [Indexed: 10/22/2022]
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20
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Ishaque A, Thrift J, Murphy JE, Konstantinov K. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol Bioeng 2007; 97:144-55. [PMID: 17054114 DOI: 10.1002/bit.21201] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.
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Affiliation(s)
- Adiba Ishaque
- Research and Development, Process Sciences, Bayer Healthcare, Biological Products Division, 800 Dwight Way, Berkeley, California 94701, USA
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21
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Kuystermans D, Krampe B, Swiderek H, Al-Rubeai M. Using cell engineering and omic tools for the improvement of cell culture processes. Cytotechnology 2007; 53:3-22. [PMID: 19003186 DOI: 10.1007/s10616-007-9055-6] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2006] [Accepted: 01/25/2007] [Indexed: 12/26/2022] Open
Abstract
Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the 'omic' technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.
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Affiliation(s)
- Darrin Kuystermans
- School of Chemical and Bioprocess Engineering and Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
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22
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Fernandes C, Parrilha GL, Lessa JA, Santiago LJ, Kanashiro MM, Boniolo FS, Bortoluzzi AJ, Vugman NV, Herbst MH, Horn A. Synthesis, crystal structure, nuclease and in vitro antitumor activities of a new mononuclear copper(II) complex containing a tripodal N3O ligand. Inorganica Chim Acta 2006. [DOI: 10.1016/j.ica.2006.04.007] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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23
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Arden N, Betenbaugh MJ. Regulating apoptosis in mammalian cell cultures. Cytotechnology 2006; 50:77-92. [PMID: 19003072 DOI: 10.1007/s10616-006-9008-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2006] [Accepted: 03/31/2006] [Indexed: 12/19/2022] Open
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.
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Affiliation(s)
- Nilou Arden
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA
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24
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Seth G, Hossler P, Yee JC, Hu WS. Engineering cells for cell culture bioprocessing--physiological fundamentals. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2006; 101:119-64. [PMID: 16989260 DOI: 10.1007/10_017] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.
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Affiliation(s)
- Gargi Seth
- Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, MN 55455-0132, USA
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25
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Andersen DC, Reilly DE. Production technologies for monoclonal antibodies and their fragments. Curr Opin Biotechnol 2005; 15:456-62. [PMID: 15464378 DOI: 10.1016/j.copbio.2004.08.002] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
In recent years, monoclonal antibodies have emerged as an increasingly important class of human therapeutics. A variety of forms of antibodies, including fragments such as Fabs, Fab'2s and single-chain Fvs, are also being evaluated for a range of different purposes. A variety of expression systems and improvements within these systems have been developed to address these growing and diverse needs.
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Affiliation(s)
- Dana C Andersen
- Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080, USA.
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26
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Dinnis DM, James DC. Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol Bioeng 2005; 91:180-9. [PMID: 15880827 DOI: 10.1002/bit.20499] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
In this review we consider how cell specific recombinant monoclonal antibody (Mab) production by engineered mammalian cells can be improved. Whilst it is generally recognized that Mab production is limited post-transcriptionally at folding and assembly reactions, genetic engineering strategies based on overexpression of individual chaperones or foldases in mammalian cells have not reliably increased cell specific Mab production. Given that recent studies have established that chaperones and foldases themselves exist in a large multiprotein complex, which may coordinate the sequential processing of Mabs, we propose that global expansion of all components of the secretory pathway will likely be necessary to generically improve recombinant Mab production by mammalian cells. In this context, what can be learnt from nature? Important recent studies have delineated some of the main cellular pathways involved in the differentiation of B-cells into nature's own high level Mab producers, plasma cells. This is achieved by a dramatic re-programming of cellular function where the coordinated expansion of metabolic and secretory machinery precedes Ig production, then is maintained by induction of a key intracellular signaling pathway, the unfolded protein response (UPR). Here we review genetic engineering strategies to increase cell specific production rate and discuss whether manipulation of intracellular signaling systems such as the UPR will provide a novel means to engineer mammalian cells for high level recombinant Mab production.
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Affiliation(s)
- Diane M Dinnis
- School of Engineering, University of Queensland, St. Lucia, QLD 4072, Australia
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27
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Komarova EY, Afanasyeva EA, Bulatova MM, Cheetham ME, Margulis BA, Guzhova IV. Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone hsp70. Cell Stress Chaperones 2005; 9:265-75. [PMID: 15544164 PMCID: PMC1065285 DOI: 10.1379/csc-27r1.1] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.
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Affiliation(s)
- Elena Yu Komarova
- Institute of Cytology RAS, Tikhoretsky pr. 4, St Petersburg 194064, Russia
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28
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Seth G, Philp RJ, Denoya CD, McGrath K, Stutzman-Engwall KJ, Yap M, Hu WS. Large-scale gene expression analysis of cholesterol dependence in NS0 cells. Biotechnol Bioeng 2005; 90:552-67. [PMID: 15830340 DOI: 10.1002/bit.20429] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r). Large-scale transcriptional analyses were done using the Affymetrix GeneChip array, MG-U74Av2. The transcripts expressed differentially across the two cell lines were identified. Additionally, proteomic tools were employed to analyze cell lysates from these two cell lines. Cellular proteins from both NS0 and NS0_r were subjected to 2D gel electrophoresis. MALDI-TOF mass spectrometry was performed to determine the identity of the differentially expressed spots. We examined the expression level of mouse genes directly involved in cholesterol biosynthesis, lipid metabolism, and central energy metabolism. Most of these genes were downregulated in the revertant cell type, NS0_r, compared to NS0. Overall, a large number of genes are expressed differentially, indicating that the reversal of cholesterol dependency has a profound effect on cell physiology. It is probable that a single gene mutation, activation, or inactivation is responsible for cholesterol auxotrophy. However, the wide-ranging changes in gene expression point to the distinct possibility of a regulatory event affecting the reversibility of auxotrophy, either directly or indirectly.
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Affiliation(s)
- Gargi Seth
- Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Ave. SE, Minneapolis, Minnesota 55455-0132, USA
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29
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Wu YF, Cao MF, Gao YP, Chen F, Wang T, Zumbika EP, Qian KX. Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells. World J Gastroenterol 2004; 10:2944-8. [PMID: 15378770 PMCID: PMC4576249 DOI: 10.3748/wjg.v10.i20.2944] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B.
METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis.
RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change.
CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.
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Affiliation(s)
- Yi-Feng Wu
- College of Life Sciences, Zhejiang University, Hangzhou 310027, Zhejiang Province, China
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Arden N, Betenbaugh MJ. Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 2004; 22:174-80. [PMID: 15038922 DOI: 10.1016/j.tibtech.2004.02.004] [Citation(s) in RCA: 164] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Mammalian cell culture is widely used to produce valuable biotherapeutics including monoclonal antibodies, vaccines and growth factors. Industrial cell lines such as Chinese hamster ovary (CHO), mouse myeloma (NS0), baby hamster kidney (BHK) and human embryonic kidney (HEK)-293 retain many molecular components of the apoptosis cascade. Consequently, these cells often undergo programmed cell death upon exposure to stresses encountered in bioreactors. The implementation of strategies to control apoptosis and enhance culture productivities represents a major goal of biotechnologists. Fortunately, previous research has uncovered many intracellular proteins involved in activating and inhibiting apoptosis. Here, we summarize three apoptotic pathways and discuss different environmental and genetic methodologies implemented to limit cell death for biotechnology applications.
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Affiliation(s)
- Nilou Arden
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
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