• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.32-35
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• 1996

Amoving aeration-membrane (MAM) bioreactor was employed for the production of 2$\mu$g/mL of tissue type Plasminogen Activator (tPA)in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating condition, shear stress was as low as 0.65 dynes/$\textrm{cm}^2$ at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2$\mu$g tPA/mL while maintaining a high cell denisty of 1.0$\times$107 viable cell/mL.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.566-571
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• 1997

To gain further insight into how estrogens modulate cell function, the effects of estrogen on cell proliferation were studied inhuman breast cancer cells. We examined the effects of estrogen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Ten nM estradiol markedly stimulated the proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor $1.15{\pm}0.03 pmole/mg protein)$(over that of control. In T47D cells that contained low levels of estrogen receptor $0.23{\pm}0.05 pmole/mg protein)$, Ten nM estrogen slightly stimulated the proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by estrogen. These results showed their sensitivity to growth stimulation by estrogen correlated well with their estrogen receptor content. Also we examined the effect of estrogen on cellular progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. Ten nM estradiol showed maximal stimulation of progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. It is not clear whether these stimulations of progesterone receptor and plasminogen activator activity by estrogen are related to the estrogen stimulation of cell proliferation of MCF-7 cells. Studies with estrogen in human breast cancer cells in culture indicate that sensitivity to growth stimulation by estrogen correlates well with estrogen receptor contents.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1116-1122
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• 2013

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.522-525
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• 1988

A method to produce tissue type Plasminogen Activator (tPA) from normal human fibroblast is developed by cultivating cells in serum free media containing heparin as an inducer. Optimal dose of this inducer was 30$\mu$g/m$\ell$. The composition of serum free medium was also defined to fit to the industrial scale cultivation. 1.42 ug of tPA per 10$^5$ viable cells per ml was produced. 1.1 gram of tPA can be produced every day from this cell line under normal perfusion chemostat operations assuming that same productivity is maintained when the process is sealed up. This method could reduce pro-duction costs and simplify purification processes by using serum free medium. Tissue type PA produced from this cell line has high ability of dissolving clots, based upon fibrin lysis test showing 50mm$^2$ of clearing zones in agarose gel plate. These results were reproducible and in good agreement with results of ELISA assay. tPA from normal human cells will be safer than that from melanoma and recombinant cells in human clinical trials.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.572-578
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• 1997

To gain further insight into how antiestrogens modulate cell function, the effects of antiestrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of trans-tamoxifen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Trans-tamoxifen $(1{\mu}M)$ markedly inhibited the estrogen stimulated proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor $(1.15{\pm}0.03 pmole/mg protein)$ over that of control. In T47D cells that contained low levels of estrogen receptor $(0.23{\pm}0.05 pmole/mg protein)$, trans-tamoxifen $(1{\mu}M)$ showed minimal inhibition of estrogen stimulated cell proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by trans-tamoxifen treatment. These results showed their sensitivity to growth inhibition by antiestrogen conrrelated well with their estrogen receptor content. Also we examined the effect of antiestrogen on cellular progestrone receptor level as well as plasminogen activator activity in MCF-7 cells. Trans-tamoxifen $(1{\mu}M)$ showed maximal inhibition of estrogen stimulated progestrone receptor level as well as plasminogen activator activity in MCF-7 cells that were stimulated by estrogen. It is not clear whether these inhibitions of progestrone receptor and plasminogen activator activity by estrogen are related to the antiestrogen inhibition of cell proliferation of MCF-7 cells. From the results of this study, it is clearly demonstrated that trans-tamoxifen is an antiestrogen in MCF-7 human breast cancer cells. Our data suggest that the biological effectiveness of trans-tamoxifen appear to result from its affinity of interaction with the estrogen receptor.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.43-43
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• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.37-42
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• 2013

Receptor activator of NF-${\kappa}B$ ligand (RANKL) is an essential cytokine for osteoclast differentiation, activation and survival. T lymphocytes such as $T_{17}$ cells, a subset of T helper cells that produce IL-17, play an important role in rheumatoid arthritic bone resorption by producing inflammatory cytokines and RANKL. It has not yet been clearly elucidated how T cell activation induces RANKL expression. T cell receptor activation induces the activation of nuclear factor of activated T cell (NFAT) and expression of its target genes. In this study, we examined the role of NFAT in T cell activation-induced RANKL expression. EL-4, a murine T lymphocytic cell line, was used. When T cell activation was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin, RANKL expression increased in a time-dependent manner. In the presence of cyclosporin, an inhibitor of NFAT activation, this PMA/ionomycin-induced RANKL expression was blocked. Overexpression of either NFATc1 or NFATc3 induced RANKL expression. Chromatin immunoprecipitation results demonstrated that PMA/ionomycin treatment induced the binding of NFATc1 and NFATc3 to the mouse RANKL gene promoter. These results suggest that NFATc1 and NFATc3 mediates T cell receptor activation-induced RANKL expression in T lymphocytes.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.261-264
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• 2002

CHO (Chinese hamster ovary) cells were transfected with plasmids containing both cis-acting HRE (hypoxia response element) and CMV-promoter that controls tissue-type plasminogen activator (t-PA). CHO cells with HRE produced 16.2 fold higher t-PA concentration than CHO cells without HRE. It was noted that hypoxia strongly induced CHO cell apoptosis. which resulted in decrease of cell viability and protein production. In this study. by introducing Bcl-2, anti-apoptotic gene, we tried to recover cell viability and increase the protein production. When batch culture of both control cells without transfection of Bcl-2 and cells transfected with Bcl-2 were performed in the absence of CoCl ι hypoxia mimic condition. the cells with Bcl-2 were effected specific cell growth rates, maximum cell density. Immunoblotting assay showed Bcl-2 was recombinant with HRE dependent t- P A expression cassette, and their expression level was depended on hypoxia. By introducing Bcl-2, both cell viability and maximum cell density could be increased.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.75-82
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• 2005

This study were examined whether plasminogen activators (PAs) are produced by porcine fresh or frozen-thawed cumulus-oocytes complexes (COCs) and cumulus cell free-oocytes. In fresh or frozen-thawed COCs and oocytes for 0 hour cultured, no activity of PAs was detected. However, at 24 hours of culture urokinase-type plasminogen activator (uPA) was detected in COCs and denuded oocytes. In the frozen-thawed COCs and cumulus cell free-oocytes cultured for 24 hours, no PAs were observed. After COCs were cultured for 48 hours, tissue-type plasminogen activator (tPA) and tPA-PAI were observed in COCs only. In the frozen-thawed COCs and cumulus cell free-oocytes cultured for 48 hours, no PAs were observed. These results suggest that uPA, tPA and tPA-PAI are produced by porcine COCs, but only uPA by oocytes during maturation for 24 hours. Only tPA, and tPA-PAI are produced by COCs cultured for 48 hours, and no PAs are produced by denuded-oocytes cultured for 48 hours. In all of the frozen-thawed groups, no PAs are observed by COCs and denuded-oocytes.

• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.107-113
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• 2013

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.