中国仓鼠卵巢细胞表观遗传调控研究进展

中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞因其具有可悬浮培养及进行蛋白质糖基化等翻译后修饰等优势,在生物制药重组蛋白生产方面具有不可替代的重要作用。但转基因沉默、表观遗传修饰等影响基因表达调控,造成CHO细胞表达稳定性降低而导致重组蛋白产量下降。本文对CHO细胞中表观遗传修饰包括DNA甲基化、组蛋白修饰和miRNA的作用研究,以及对基因表达调控的影响进行了综述。     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells

Cytotechnology - Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production...     

Control of starvation-induced apoptosis in Chinese hamster ovary cell cultures

The application of the unscented Kalman filter to control starvation-induced programmed cell death-apoptosis-in Chinese hamster ovary cells was investigated. Neural network-based sensitivity analysis identified glutamine and asparagine as two major amino acids that play a key role in the suppression of apoptosis. Dynamic equations that accounted for the dependence of apoptotic cells on the concentrations of viable cells, glutamine, and asparagine were derived. These state equations were highly nonlinear and included nine state variables. An oxygen mass balance was written in the liquid phase. It served as the output equation for the unscented Kalman filter. Using the oxygen uptake rate as the observer, it was possible to estimate the states. A model predictive controller was then implemented once the apoptotic cells in the bioreactor approached a concentration of 1.5 x 10(4) cells/mL, taking into account the operating range of the flow cytometer and measurement error. The manipulated variables were the flow rates of glucose, glutamine, and asparagine. Simulation results showed that the controller was able to keep the apoptotic cells at a concentration of 1.5 x 10(4) cells/mL.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF‐1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT‐PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.     

Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture

Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.Abbreviations ANOVA analysis of variance - CHO Chinese hamster ovary cells - CFA colony-forming ability - dhfr gene for dihydrofolate reductase - DHAP dihydroxyacetone phosphate - FBS fetal bovine serum - G-3-P glyceraldehyde-3-phosphate - GloI glyoxalase I - GloII glyoxalase II - GSH reduced glutathione - HPLC high-performance liquid chromatography - IMDM Iscove's modified Dulbecco's medium - MTX methotrexate - 2-MQ 2-methylquinoxaline - 5-MQ 5-methylquinoxaline - MEM minimal essential medium - P_i inorganic phosphate - PCA perchloric acid - o-PD o-phenylenediamine     

RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity

RNA interference (RNAi) has been recently applied to improve the yield and quality of recombinant proteins produced in Chinese hamster ovary (CHO) cells, the most commonly used mammalian cell line for production of complex biopharmaceuticals. Proteomic profiling of CHO cells undergoing gene amplification identified cofilin, a key regulatory protein of actin cytoskeletal dynamics, as a cellular target for genetic engineering studies. Transient reduction of cofilin by small interfering RNA (siRNA) enhanced specific productivity in recombinant CHO cells by up to 80%. CHO cell lines expressing cofilin-specific short hairpin RNA (shRNA) vectors showed up to a 65% increase in specific productivity. These results suggest that modulation of cofilin, and its regulatory pathways, may be a new approach to enhance recombinant protein productivity in CHO cells.     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.     

Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

批次和流加培养中国仓鼠卵巢工程细胞的细胞周期相关基因转录谱分析

以表达人重组尿激酶原中国仓鼠卵巢 (CHO) 工程细胞系11G-S为研究对象,运用基因芯片技术比较了CHO工程细胞在批次及流加培养不同生长阶段基因表达水平的差异,在此基础上采用Genmapp软件,同时结合已知的细胞周期信号通路图,着重分析了批次及流加培养CHO工程细胞的细胞周期调控基因转录谱差异。在基因芯片涉及的19 191个目标基因中,批次和流加培养不同生长阶段CHO工程细胞的下调表达的基因数量多于上调表达基因数目;两种培养模式下的基因差异表达有着明显的不同,尤其是在细胞生长的衰退期,流加培养CHO工程细胞中下调表达的基因数量明显多于批次培养。有关调控细胞周期关键基因的转录谱分析表明,CHO工程细胞主要是通过下调表达CDKs、Cyclin及CKI家族中的Cdk6、Cdk2、Cdc2a、Ccne1、Ccne2基因及上调表达Smad4基因,来达到调控细胞增殖及维持自身活力的目的。     

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.     

Rapid serum-free/suspension adaptation: Medium development using a definitive screening design for Chinese hamster ovary cells

The biopharmaceutical industry prefers to culture the mammalian cells in suspension with a serum-free media (SFM) due to improved productivity and process consistency. However, mammalian cells preferentially grow as adherent cells in a complete medium (CM) containing serum. Therefore, cells require adaptation from adherence in CM to suspension culture in SFM. This work proposes an adaptation method that includes media supplementation during the adaption of Chinese hamster ovary cells. As a result, the adaptation was accelerated compared to the traditional repetitive subculturing. Ca²⁺/Mg²⁺ supplementation significantly reduced the doubling time compared to the adaptation without supplementation during the adaptation of adherent cells from 100% CM to 75% CM (p < 0.05). Furthermore, a definitive screening design (DSD) was applied to select essential nutrients during the adaptation from 10% CM to 0% CM. The main effects of Ca²⁺ and Dulbecco's modified essential medium (DMEM) were found significant to both viable cell density and viability at harvest. Additionally, the interaction term between Ca²⁺ and DMEM was found significant, which highlights the ability of DSD to capture interaction terms. Eventually, the media supplementation method resulted in adaptation SFM in 27 days, compared to the previously reported 66 days. Additionally, the membrane surface integrin expression was found significantly decreased when adherent cells were adapted to suspension. Moreover, the Ca²⁺/Mg²⁺ supplementation correlated with faster integrin recovery after trypsinization. However, faster integrin recovery did not contribute to the accelerated cell growth when subculturing from 100% CM to 75% CM.