Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.     

Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.     

Change of insulin-like growth factor gene expression in Chinese hamster ovary cells cultured in serum-free media

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.     

Glycosylation defects alter insulin but not insulin-like growth factor I binding to Chinese hamster ovary cells

Insulin binding to two Chinese hamster ovary cell lines with well-defined defects in their glycosylation pathway has been characterized and compared to insulin-like growth factor I (IGF-I) binding in the same cell lines. Insulin competition curves indicate that B4-2-1 cells, which transfer co-translationally to proteins an endoglycosidase H insensitive, truncated lipid-linked oligosaccharide, bind insulin with higher than normal affinity. Lec 1 cells, which fail to process oligosaccharide side chains to complex types, bind with a reduced affinity. The potencies of chicken and guinea pig insulins are appropriate for an insulin receptor in the control (WTB) and both mutant cell lines, whereas rat IGF-II is 3 times more potent than expected in the Lec 1 cells and human IGF-I is less potent than anticipated. Insulin bound to Lec 1 cells dissociates more quickly upon dilution than does insulin bound to either WTB or B4-2-1 cells. The Lec 1 insulin receptor is insensitive to pH change, whereas the other lines show the usual optimum of 8. 125I-IGF-I binds well to all three cell lines and is equally pH-sensitive in all three. Serum from a patient with circulating autoantibodies to the insulin receptor competes for insulin but not IGF-I binding, whereas alpha IR3, a monoclonal antibody directed toward the human IGF-I receptor inhibits IGF-I but not insulin binding. Cross-linking of either 125I-insulin or 125I-IGF-I reveals a typical alpha-subunit in the WTB and B4-2-1 cells but a band with faster mobility in the Lec 1 cells. Insulin (10(-8) M) stimulates autophosphorylation of a beta-subunit in all three lines, but again the Lec 1 subunit demonstrates an anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data demonstrate the differential effect of glycosylation on two closely related receptor molecules.     

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.     

Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells

Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR⁺ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Recombinant expression of human transforming growth factor-β isoforms in Chinese hamster ovary cells

Transforming growth factor-βs (TGF-βs) are multi functional growth modulators implicated in several physiological processes which include embryogenesis, inflammation, immune-suppression, wound healing, carcinogenesis and cellular differentiation. For clinical use, recombinant expression of TGF-βs is the only practical source because of very low yields from natural sources. Here, we report the recombinant expression of human TGF-βl and TGF-β2 in a mammalian expression system using a high expression eukaryotic vector driven by a cytomegalovirus promoter. Expression levels are as high as 0.97 μg/ml of TGF-βl and 0.24 μg/ml of TGF-β2 in conditioned media, sufficient for purification without the need for amplification of the gene using methotrexate.     

rhVEGF165在中国仓鼠卵巢细胞中的稳定表达、纯化及其生物学活性

利用哺乳动物细胞表达系统,稳定表达和纯化高生物学活性的人重组血管内皮生长因子 (VEGF165) 蛋白。将VEGF165克隆于表达载体pCDNA4.0,与T-GS载体共同转染CHO-S (中国仓鼠卵巢细胞) 细胞,MSX (Methionine sulphoximine) 加压筛选高表达细胞株,5 L发酵罐培养,细胞培养上清液通过三步纯化得到rhVEGF165蛋白,通过Western blotting、Biacore和人脐静脉内皮细胞增殖实验等对表达蛋白的特异性、亲和力及生物学活性等进行检测。所建立的细胞     

X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining

Six CHO mutants have previously been described as being sensitive to ionizing radiation and bleomycin treatment, with little or no cross sensitivity to UV-radiation (Jeggo and Kemp, 1983). Their ability to rejoin single- and double-strand breaks has been examined here. Using two techniques, gradient sedimentation and alkaline elution, no difference could be observed between wild-type and mutant strains in the initial number of single-strand breaks induced, the rate of rejoining, or the final level of single-strand breaks rejoined. Thus, a major inability to rejoin single-strand breaks is not the basis for sensitivity in these mutants. In contrast, all 6 mutants showed a decreased ability to rejoin the double-strand breaks induced by gamma-irradiation as measured by neutral elution. Rejoining of half of the breaks occurred in 37 min in wild-type cells and reached a maximum level of 72% after 2 h. All the mutants showed a decreased rate of rejoining, and the final level was 17% of that observed in the wild-type in the most defective mutant, and ranged from 35 to 69% in the other 5 mutants. These are the first mammalian cell mutants to be described with a defect in double-strand break rejoining.     

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10⁶ cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced inducible mGlu1α receptor expression in Chinese hamster ovary cells

Inducible expression of the group-I metabotropic glutamate receptor (mGlu1alpha) in Chinese hamster ovary cells allows for the study of receptor density dependent effects. However, expression levels attainable with this system are lower than those reported for various brain regions and achieved by conventional (constitutive) transfection. Thus, direct comparison of mGlu1alpha receptor-mediated responses in this inducible expression system with those for receptors expressed heterologously or in vivo is compounded. We show here that inducible expression can be selectively augmented by butyrate pretreatment to levels approaching those reported for cerebral tissue. Enhanced mGlu1alpha receptor protein levels, agonist-induced inositol phosphate accumulation, as well as single-cell inositol 1,4,5-trisphosphate production and intracellular Ca(2+) mobilization occurred following co-induction with butyrate. In contrast, endogenous purinoceptor function was unaffected. Importantly, the ability to titrate receptor expression by varying isopropyl beta-thiogalactoside concentration was retained. Sodium butyrate thus offers a simple and convenient method to enhance inducible gene expression to levels found in vivo.     

Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. (c) 1996 John Wiley & Sons, Inc.     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.