<i>Ras</i>Amplification in BHK-21 Cells Produces a Host Cell Line for Further Rapid Establishment of Recombinant Protein Hyper-producing Cell Lines

Teruya, Kiichiro; Yano, Takahiro; Shirahata, Sanetaka; Watanabe, Junko; Osada, Kyoichi; Ohashi, Hideya; Tachibana, Hirofumi; Kim, Eun‐Ho; Murakami, Hiroki

doi:10.1271/bbb.59.341

Cited by 18 publications

(9 citation statements)

References 3 publications

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“…After 48 h, 10 μL of WST‐1 (Roche, Indianapolis, IN, USA) was added to each well and cell proliferation was analyzed according to the manufacturer's protocol. The WST‐1 colorimetric assay is designed for spectrophotometric quantification of cell proliferation based on cellular enzymatic activity for cleavage of tetrazolium salts 31, 32. The Benchmark Plus™ Bio‐Rad microplate reader was used for the measurement of absorbance of samples at 450 nm (reference wavelength 690 nm).…”

Section: Methodsmentioning

confidence: 99%

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Yan

Jovanović

et al. 2010

Proteomics

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Transforming growth factor β (TGF-β) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-β type II receptor (TβRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TβRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TβRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TβRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-β signaling pathway in fibroblasts because systemic inhibition of TGF-β signaling pathways is being considered as a potential cancer therapy.

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Section: Methodsmentioning

confidence: 99%

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Yan

Jovanović

et al. 2010

Proteomics

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“…This effect disappeared after an activityfree continued incubation for a further 2 days. This radiation-induced rise in cell viability might be explained by the phenomenon of 'hormesis', first described by Feinendegen and colleagues [24][25][26][27][28][29]. On the other hand, an early cellular response to radiation damage (by DNA repair process or cellular changes in early apoptosis) might increase the overall activity of mitochondrial dehydrogenases and therefore only simulate an increase in the number of viable cells.…”

Section: Discussionmentioning

confidence: 99%

“…Upon incubation with viable cells, the tetrazolium ring of WST (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) is cleaved by mitochondrial dehydrogenases to yield a purple formazan product [23][24][25]. Briefly, aliquots of the WST-1 solution were added to each well for substrate reaction either immediately or 2 days after irradiation.…”

Section: Cell Viability Assaymentioning

confidence: 99%

Cell death induced by 131I in a differentiated thyroid carcinoma cell line in vitro: Necrosis or apoptosis?

Marx

Moka

Schomäcker

et al. 2006

Nuclear Medicine Communications

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The data suggest that the nature of the cytotoxic effect of I and whether it leads to apoptotic or necrotic cell death is dose-dependent. High I doses seem to produce mainly necrotic phenomena, whereas at low I activity concentrations apoptotic phenomena prevail. The predominance of delayed apoptosis could explain why radioiodine therapy at lower doses is often linked to delayed onset and possible continuation of thyroid volume reduction over some months and even up to a year.

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“…We previously developed a promoter-activated production (PAP) system to enhance recombinant protein productivity in cultured cells by activating specific promoters via transcriptional activators such as oncogenes (Yano et al 1994;Teruya et al 1995;Shirahata et al 1995). This PAP system was shown to be very effective in CHO cells, in which the peak productivity reached 13.6 lg 10 À6 cells day À1 (Katakura et al 1999).…”

Section: Introductionmentioning

confidence: 99%

An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

et al. 2005

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The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 lg 10 À6 cells day À1 , one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-jB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-jB activation, was examined. Suppression of NF-jB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.Abbreviations: CHO -Chinese hamster ovary; dhfr -dihydrofolate reductase; DMEM -Dulbecco's modified Eagle's medium; ECL -enhanced chemiluminescence; ELISA -enzyme-linked immunosorbent assay; EMSA -electrophoretic mobility shift assay;

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RasAmplification in BHK-21 Cells Produces a Host Cell Line for Further Rapid Establishment of Recombinant Protein Hyper-producing Cell Lines

Cited by 18 publications

References 3 publications

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Cell death induced by 131I in a differentiated thyroid carcinoma cell line in vitro: Necrosis or apoptosis?

An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

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