• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.105-108
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• 2003

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g/L YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 ${\mu}g/mL$, which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced $q_{hTPO}$ and increased culture longevity. In addition YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.220-226
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• 2005

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcar-bamoylase (OTC). When supplements were added to the medium at $33\;^{\circ}C$, the growth of IBE and CO5 cells increased by $27\%\;and;26\%$, respectively and the maximum yield of EPO was increased by $40\%$ in both cell lines. The absolute EPO concentration in the CO5 cells was always $55{\sim}60\%$ higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at $33\;^{\circ}C$ until their growth rates approached those at $37\;^{\circ}C$, the growth rates of both IBE and CO5 cells increased by $54\%$ and their maximum EPO levels increased by up to $73\%\;and\;56\%$, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

• Environmental Mutagens and Carcinogens
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• v.24 no.3
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• pp.113-120
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• 2004

Chromium compounds are known human and animal carcinogens. In this study, the effects of sodium chromate on apoptosis and cell cycle were investigated in order to unveil the elements of early cellular responses to the metal. Using Chinese hamster ovary cells(CHO-K1-BH4), we found taht chromium (VI) treatment induced apoptosis in these cells, as signified by nuclear fragmentation, DNA laddering on agarose gel electrophoresis, and an increased proportionof cells with hypodiploid DNA. Preceding these changes, chromium (VI) treatment increased caspase 3 pritease activity and also increased expression of p53 protein, while the level of bcl2 protein was not changed. Coincubation with caspase inhibitor, Z-DEVD-FMK, inhibited chromium-induced apoptosis. In the flow cytometric analysis using propidium iodide fluorescence, an increase of cell population in G2/M phase was shown in cells exposed to at least 160 $\mu\textrm{m}$ of sodium chromate for 72h, form 9.8% for 0$\mu\textrm{m}$ chromium (VI) to 26.4% for 320$\mu\textrm{m}$ chromium(VI). Taken together, these findings suggest that chromium(VI)-induced apoptosis is accompanied by G2/M cell cycle arrest, and that p53-mediated pathway may be involved in positive regulation of G2/M arrest and a concurred apoptosis in CHO cells.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1281-1287
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• 2017

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.

• Environmental Mutagens and Carcinogens
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• v.16 no.2
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• pp.83-87
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• 1996

This study was performed by the sister chromatid exchanges (SCEs) to investigate the effects of Aphidicolin (APC) or 2, 4-dinitrophenoi (DNP) on cross-adaptive response to ultraviolet radiation (UV) or ethyl methanesulfonate (EMS) in Chinese hamster ovary (CHO) cells. The pretreatment with 1 J/m$^2$ UV decreased the yield of SCEs induced by subsequent treatment with 8 mM EMS in CHO cells. And the treatment with 10 $\mu$g/ml APC or 50 $\mu$M DNP during incubation after pretreatment with 1 J/m$^2$ UV increased the yield of SCEs induced by 8 mM EMS. The pretreatment with 2 mM EMS decreased the yield of SCEs induced by subsequent treatment with 5 J/m$^2$ UV. The treatment with 10 $\mu$g/ml APC during incubation after 2 mM EMS increased the yield of SCEs induced by 5 J/m$^2$ UV. These results suggest that APC and DNP inhibit cross-adaptive response to pretreatment with UV and subsequent treatment with EMS, and also cross-adaptive response to pretreatment with EMS and subsequent treatment with UV is inhibited by APC in CHO cells.

• Environmental Mutagens and Carcinogens
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• v.15 no.2
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• pp.94-99
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• 1995

The adaptive response and cross-adaptive response to sister chromatid exchanges (SCEs) and DNA single-strand breaks (SSBs) in Chinese hamster ovary (CHO)-K$_1$ cells treated with ultraviolet radiation (UV), ethyl methanesuffonate (EMS), or bleomycin (BLM) were investigated. Two assays were used in this study; SCEs and alkaline elution. The pretreatment with low conditioning dose of 2 mM EMS or 1 J/m$^2$ UV decreased the yield of SCEs induced by subsequent treatment with 8 mM EMS, 5 J/m$^2$ UV or 5 $\mu$g/ml BLM. And the pretreatment with low conditioning dose of 1 $\mu$g/ml BLM decreased the yield of SCEs induced by subsequent treatment with 5 $\mu$g/ml BLM or 5 J/m$^2$ UV. The rejoining of DNA SSBs in cells subsequently treated with 2 J/m$^2$ UV, 50 mM EMS or 400 $\mu$g/ml BLM is higher than that only treated with 2 J/m$^2$ UV, 50 mM EMS or 400 $\mu$g/ml BLM. These results suggest that there are the adaptive response and cross-adaptive response to SCEs, and is the adaptive response to the rejoining of DNA SSBs in CHO cells.

• Environmental Mutagens and Carcinogens
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• v.9 no.1
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• pp.13-22
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• 1989

The effect of novobiocin (NOV), and inhibitor of topoisomerase II, on ethyl methanesulfonate (EMS)-or bleomycin (BLM)-induced DNA repair synthesis was examined during the cell cycle of Chinese hamster ovary (CHO)-K1 cells. Three assays were employed in this study: cell survival, alkaline elution and unscheduled DNA synthesis. EMS was effective at killing CHO cells in G1 phase, wheras BLM preferentially killed cells in G2 and S phases. EMS induced the much more amount of DNA damage in G1 phase, while BLM induced in G2 phase than the other phases. The both of pre- and post-treatment with BOV inhibitied EMS- or BLM-induced DNA repair synthesis in G1 and G2 phases, and pretreatment with NOV inhibited more effectively than the post-treated group. These results suggested that CHO cells exhibited a differential sensitivity to cell lethality and DNA damage in relation to cell cycle according to used chemical agents, and that DNA topoisomerase II participated in an initial stage of DNA repair.

• Korean journal of applied entomology
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• v.43 no.2
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• pp.155-162
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• 2004

Serotonin receptor binds to serotonin (5-HT) and activates effector proteins such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase or ion channel through G protein on the cell membrane, resulting in various physiological responses like diuresis, memory and development. To examine the comparative expression of the 5-HT$\_$7/ receptor of Aedes aegypti, the Aedes 5-HT$\_$7/ receptor gene was transfected into Drosophila Schneider2 (S2) cells and mammalian Chinese hamster ovary (CHO)-Kl cells. The expression of the Aedes 5-HT$\_$7/ receptor gene in selected cell lines, Tr-CHO and Tr-S2, was confirmed with reverse transcription-PCR, Western blot and immunocytochemistry. Compared with the induced intracellular cAMP level of Tr-S2 cell line to 5-HT, the induced cAMP in the Tr-CHO cell line was over 9 times higher and was dose-dependent. These results suggest that the functionality of Aedes 5-HT$\_$7/ receptor is much more effective in mammalian CHO-K 1 cells and that the Tr-CHO cell line expressing Aedes 5-HT$\_$7/ receptor can be used for synthetic agonist or antagonist candidate screening.

• Molecules and Cells
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• v.25 no.4
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• pp.504-509
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• 2008

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.459-462
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• 2001

Angiopoietin-2 (Ang2) is a naturally occurring antagonist for angiopoietin-l (Angl) and its Tie2 receptor during vasculogenesis, Although angiopoietins have been expressed in several mammalian cell lines, their expression levels are low. Recombinant Chinese hamster ovary (CHO) cell lines expressing a high level of human Ang2 or its analog, human $Ang2_{443}$, with an amino-terminal FLAG-tag were constructed by transfecting the expression vectors into dhfr-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate level. Secreted Ang2 or human $Ang2_{443}$ were purified from the cultured medium using an anti-FLAG- agarose affinity chromatography, The purified Ang2 and $Ang2_{443}$ migrated on SOS-PAGE as a broad band, characteristic of glycosylated protein. Their biological activity in vitro was demonstrated in a serum deprivation-induced apoptosis assay. Ang2 at high concentration, like AngI, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor.