Differential expression of microRNAs in recombinant Chinese hamster ovary cells treated with sodium butyrate using digital RNA counting.

Sodium butyrate (NaBu) is an efficient supplement for increasing recombinant protein production in Chinese hamster ovary (CHO) cell culture. To elucidate the effects of NaBu on miRNA expression profile in recombinant CHO (rCHO) cells, differentially expressed miRNAs in NaBu-treated rCHO cells were assessed by NanoString nCounter analysis. This result showed that eight mature mouse miRNAs (let-7b, let-7d, miR-15b, miR-25, miR-27a, miR-99a, miR-125a-5p, and miR-125b-5p) were differentially expressed. Furthermore, quantitative real-time RT-PCR analysis of eight mature CHO miRNAs, annotated using a miRBase database, confirmed the transcriptomic findings. Among the potential corresponding target mRNAs for the selected mature miRNAs, seven cell growth-related target genes (e2f2, akt2, mtor, bcl-2, bim, p38α, and bmf) and five N-glycosylation-related target genes (neu1, b4galt3, gale, man1b1 and mgat4a) were selected by considering the effectiveness of NaBu on rCHO cell culture. The altered expression patterns of the 12 target mRNAs were inversely correlated with those of the selected mature miRNAs. Altogether, NanoString nCounter analysis may be useful for identifying differentially expressed miRNAs in rCHO cells.

Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells.

Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition.

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (ß-tubulin and ß-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of ß-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.

CHO cells in biotechnology for production of recombinant proteins: current state and further potential.

Recombinant Chinese hamster ovary cells (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Recent advances in cell culture technology for rCHO cells have achieved significant improvement in protein production leading to titer of more than 10 g/L to meet the huge demand from market needs. This achievement is associated with progression in the establishment of high and stable producer and the optimization of culture process including media development. In this review article, we focus on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells. The approaches that manipulate various DNA elements for gene targeting by site-specific integration and cis-acting elements to augment and stabilize gene expression are reviewed here. The genetic modulation strategy by "direct" cell engineering with growth-promoting and/or productivity-enhancing factors and omics-based approaches involved in transcriptomics, proteomics, and metabolomics to pursue cell engineering are also presented.

Effect of constitutively active Ras overexpression on cell growth in recombinant Chinese hamster ovary cells.

Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)-producing rCHO cell line with regulated CA-Ras overexpression (EPO-off-CA-Ras) was established using the Tet-off system. The CA-Ras expression level in EPO-off-CA-Ras cells was tightly regulated by doxycycline addition. Although CA-Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA-Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA-Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates.

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (µ, SFM#S1) or the highest specific antibody productivity (q(Ab,) SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells.

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahistidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by two-dimensional gel electrophoresis (2DE) and identified by MALDI-TOF MS/MS. A total of 27 protein spots including glucose-regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells.

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture.

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310-610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase-3, caspase-7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3-II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310-610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality.

Effect of XIAP overexpression on sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells producing erythropoietin.

Previously, overexpression of X-linked inhibitor of apoptosis (XIAP), which is known to inhibit activities of caspase-3, -7, and -9 in CHO-K1 cells offered protection against Sindbis virus-induced apoptosis. In this study, the potential role of XIAP overexpression in recombinant CHO (rCHO) cells treated with sodium butyrate (NaBu), which can increase the specific productivity, was investigated by establishing erythropoietin (EPO)-producing rCHO cells with regulated XIAP overexpression (EPO-off-XIAP). The XIAP overexpression in EPO-off-XIAP was tightly regulated by doxycycline. The XIAP overexpression could simultaneously reduce the activation of caspase-3, -7, and -9 induced by NaBu addition. However, XIAP overexpression could not inhibit NaBu-induced apoptosis, as evidenced by DNA fragmentation. In addition, it also did not help the maintenance of the mitochondrial membrane potential in the presence of NaBu, suggesting that the release of mitochondrial proteins might induce caspase-independent apoptosis. As a result, XIAP overexpression did not affect cell growth and EPO production significantly. Taken together, XIAP overexpression, which was reported to inhibit Sindbis virus-induced apoptosis, could not inhibit the NaBu-induced apoptosis in rCHO cells.

Inhibition of Japanese encephalitis virus replication by peptide nucleic acids targeting cis-acting elements on the plus- and minus-strands of viral RNA.

Japanese encephalitis virus (JEV) is a major cause of acute viral encephalitis in humans. The single-stranded, plus-sense viral genome, which is used for translation and minus-strand RNA synthesis, and its complementary minus-strand viral RNA contain various sequences and RNA secondary structures conserved in flaviviruses, providing potential targets for antisense agents. Here, we investigated the antiviral effects of peptide nucleic acids (PNAs) targeting cis-acting signals at the 5'-untranslated region (UTR), 3'-UTR, and genome cyclization motifs on the plus-strand RNA, as well as the 95-nucleotide 3'-end of the minus-strand RNA, which serves as a template for plus-strand RNA synthesis by the viral RNA-dependent RNA polymerase (RdRp). Among the tested cell-penetrating peptide (CPP)-PNA conjugates, a 17-mer PNA conjugate targeting the top of the 3'-UTR loop structure was most effective in suppressing virus proliferation. In vitro RdRp assays and electrophoretic mobility shift assays using a functional recombinant JEV RdRp showed that the 3'-terminal region-targeting PNAs could inhibit RNA synthesis by competing with viral RdRp for binding to a selected cis-acting element at the 3'-end of plus- and minus-strand viral RNAs. Collectively, our results suggest that CPP-PNA conjugates can suppress JEV proliferation by blocking RNA-protein or RNA-RNA interactions essential for productive viral infection.

Effect of Bcl-xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-deprived condition.

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl-x(L) overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x(L) overexpression (EPO-off-Bcl-x(L)) was established using the Tet-off system. The expression level of Bcl-x(L) in EPO-off-Bcl-x(L) cells was tightly regulated by doxycycline in a dose-dependent manner. Bcl-x(L) overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl-x(L) overexpression suppressed apoptosis by inhibiting the activation of caspase-3 and -7. Simultaneously, Bcl-x(L) overexpression also delayed autophagy, characterized by LC3-II accumulation. Immunoprecipitation analysis with a Flag-tagged Bcl-x(L) revealed that Bcl-x(L) interacts with Bax and Bak, essential mediators of caspase-dependent apoptosis, as well as with Beclin-1, an essential mediator of autophagy, and may inhibit their pro-cell death function. Taken together, it was found that Bcl-x(L) overexpression inhibits both apoptosis and autophagy in rCHO cell culture.