UV-light-induced mutations in synchronous CHO cells

Asynchronous and synchronous CHO cells were irradiated with germicidal UV light to determine the fluence response curve for cell killing, and the induction of resistance to 6-thioguanine, ouabain, and diphtheria toxin. For asynchronous populations the data show a sigmoidal response for induced reproductive death, as has been seen by other, with a D₀ of 6 J/m² and an extrapolation number of 2.5. The induction of mutations appears to be a linear function for all three mutagenic markers up to a dose of 17 J/m².Reproductive death induced in the synchronous populations is a function of the time at which exposure occurs in the cell cycle, with late G₁ and early S being the sensitive stages. The induction of resistance to 6TG, ouabain, and diphtheria toxin (DT) all seem to depend on the time of exposure in the cell cycle. As in the case of UV-induced reproductive death, the more sensitive periods for mutation induction appear also to be the G₁ and early S period of the cell cycle, with the largest cyclic variation occurring for induced DT resistance.A comparison of the results reported here for the UV exposure with exposures of synchronous CHO cells to X-rays and ethylnitrosourea suggests that there are different age-specific responses to mutation induction for each agent, and that there are often different age responses for different mutagenic end- points with the same mutagen.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.     

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin,Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of ¹²⁵I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Effects of retinoids and phorbol esters on the sensitivity of different cell lines to the polypeptide toxins modeccin, abrin, ricin and diphtheria toxin.

The effects of retinoic acid and 12-O-tetradecanoylphorbol 13-acetate on the sensitivities of a number of cell lines to the toxins modeccin, abrin, ricin and diphtheria toxin were studied. Retinoic acid and some other retinoids were found to protect a number of the cell lines against the toxins. HeLa cells that were protected bound much more retinoic acid than L-cells that were not protected. The tumour promoter 12-O-tetradecanoylphorbol 13-acetate was found to increase the sensitivity of cells to abrin, ricin and modeccin in the absence as well as in the presence of retinoic acid. Neither retinoic acid nor 12-O-tetradecanoylphorbol 13-acetate affected the extent of binding and pinocytotic uptake of toxins by the cells. Apparently retinoic acid and 12-O-tetradecanoylphorbol 13-acetate interfere with the entry of the toxins through the cell membrane.     

Towards dynamic metabolic flux analysis in CHO cell cultures

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.     

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.     

Reduced permeability in CHO cells as a mechanism of resistance to colchicine

Colchicine resistant (CH^R) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [³H] colchicine uptake by whole cells and binding of [³H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CH^R lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.     

Optimized signal peptides for the development of high expressing CHO cell lines

Recombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large‐scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed‐batch experiments with stably transfected cell pools, in which cell‐specific productivities up to 90 pg cell^?1 day^?1 and product concentrations up to 4 g L^?1 could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non‐antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product‐independent manner. Biotechnol. Bioeng. 2013; 110: 1164–1173. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A

22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle- specific coats.     

Characterization of site-specific recombination mediated by Cre recombinase during the development of erythropoietin producing CHO cell lines

The establishment of erythropoietin (EPO) producing Chinese hamster ovary (CHO) cell lines was conducted using Cre-mediated cassette exchange. The characterization of site-specific recombination mediated by Cre-recombinase during the cell line development was also performed. A total of six parental clones, which had various green fluorescence levels ranging from high to low and containing a single copy of insertion vector (pEGFP-m2), were screened. The EPO targeting vector (pIC-m2-EPO) was targeted into the 6 parental clones by Cre-mediated cassette exchange. Correctly targeted clones were obtained from 4 out of 6 parental clones with 0∼15% of targeting efficiencies. Moreover, there was a positive relationship (R² = 0.87) between fluorescence levels of the parental clones before Cre-mediated cassette exchange and specific EPO productivities (q _EPO ) of the correctly targeted clones after Cre-mediated cassette exchange. Therefore, it was verified that the chromosomal loci’s characteristic gene expression level was not modified even after cassette exchange mediated by Cre recombinase during the development of EPO producing CHO cell lines. This finding implies that the reproducible development of CHO cell lines largely producing a desired protein is expected to be achieved by Cre-mediated cassette exchange.     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell.

By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55IU and HmAb rCH8 had 1.09IU/100 micro g of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 micro g IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94IU/100 micro g IgG.     

APRT缺陷型CHO细胞系的建立及其重组蛋白表达能力评价北大核心CSCD

中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞,腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸,是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因,验证获得的APRT缺陷型CHO细胞系的生物学特性;构建两种真核表达载体:对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP),分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池;重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度,并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明,CHO细胞aprt基因成功敲除;获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明,与野生型CHO细胞相比,转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%;特别是长期传代培养时,转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05);构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。     

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCA^r and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCA^r mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCA^r mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway.     

Studies on the MgSO4-induced cytoplasmic uptake of proteins by cells in culture

When healthy HeLa cells were exposed to an intracellular acting cytocidal protein extracted from vaccinia virus-infected cells, or non-toxic diphtheria A fragment, there was no detectable drop in cell viability. However, when the cells were treated with vaccinia cytocidal protein, low non-toxic doses of whole diphtheria toxin, or high doses of non-toxic diphtheria A fragment in the presence of 0.85 M MgSO₄ for 15 min, to induce intra-cytoplasmic uptake, 25–30% of the cells died.This partial killing effect was shown to be due to a heterogeneous cytoplasmic uptake of the cytotoxic protein; induced protein uptake occurred in only 25–30% of the cell population. It was not found possible to correlate this cell sub-population with a genetically determined cell sub-population, cells in a specific stage of the active cell division cycle or, inactive G0-like cells. Electron microscopic studies of cells after 0.85 M MgSO₄ treatment showed increased vacuolation and uptake of horse-radish peroxidase into such newly formed vacuoles was demonstrated. Light microscope studies indicated that this induced vacuolation was also not homogeneous within the population, which led to the possibility that excessive induced vacuolation and eventual cytoplasmic entry of macromolecules were linked phenomena. This hypothesis was supported by studies on rabbit kidney (RK), and baby hamster kidney (BHK) cells, which showed similar degrees of vacuole inducibility and toxin susceptibilities as HeLa cells, and by studies on HEp-2 cells, Chang conjunctival cells (W-K derivative), and McCoy cells, which showed no MgSO₄-induced vacuolation and were totally resistant to vaccinia cytocidal protein and diphtheria toxin A fragment in the presence of hypertonic MgSO₄. Evidence was obtained which indicated that excessive vacuolation is also linked with aberrant vacuolar fusion events within the cell after treatment with hypertonic MgSO₄.