• Journal of Pharmaceutical Investigation
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• v.33 no.4
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• pp.267-272
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• 2003

Herpes simplex virus (HSV) thymidine kinase (TK) has been widely used for suicidal gene therapy in combination with nucleoside analogs such as ganciclovir (GCV). The use of HSV-TK is limited due to the side effect of GCV at high concentrations. Previous studies showed that stable transfectants of mutant HSV-TK with enhanced affinity to GCV were killed at lower GCV concentrations. In this study, we tested whether mutant HSV-TK can provide enhanced suicidal effect when transiently transfected with Epstein-Barr virus (EBV)-based plasmid. EBV-based plasmid which contains OriP and EBNA-1 sequence is well known for a stable episomal maintenance in human cells. Optimal transfection condition was assessed for SNU-638 gastric cancer cell line using polyetylnimine (PEI). Maximum transfection efficiency was achieved when DNA:PEI was 1:3 (w/v). Cytotoxicities of mutant and wild type HSV-TK were compared before and after partially selecting transfected cells. The cells were sensitive to $100\;{\mu}g/ml$ hygromycin. Following GCV treatment, more cells were killed after hygromycin selection than before selection. The mutant HSV-TK showed enhanced cytotoxicity compared with the wild type HSV-TK. Our results suggest that the EBV-based plasmid encoding mutant HSV-TK may be useful to treat the diseases caused by uncontrolled cell proliferation such as cancer and rheumatoid arthritis.

• Journal of Life Science
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• v.29 no.10
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• pp.1159-1163
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• 2019

Although there is a growing interest in male menopause, a phenomenon associated with male hormone depletion, current kits using antibodies to quantify male hormones are expensive. In this study, we constructed an in vitro system for verifying the activity or concentration of male steroid hormones using a transcriptional activity test. A reporter plasmid, pGL2-Neo-ARE-AdE1BTATA, which reacts to testosterone, was constructed. In this plasmid, the ARE-AdE1bTATA sequences can be bounded by the testosterone - androgen receptor complex to express luciferase as a reporter. Then, a stable transfection was performed on the human prostate cancer cell line, LNcap-LN3. The constructed LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA testosterone compound detection (TCD) system showed quantitatively proportional luciferase activities to concentrations of $10^{-13}$ to $10^{-8}M$ of standard testosterone. The established in vitro TCD system will contribute to the development of materials for health/functional foods and drugs as it will be possible to search en masse for testosterone-like or testosterone-inhibiting substances derived from natural materials.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.66-69
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• 2001

Previously we developed a CHO cell line (CHO-OTC1-A19) expressing the first two enzymes of urea cycle. This cell line showed higher ammonia removal activity and faster growth rate than the vector controlled CHO cells (CHO-neo-5). The purpose of this study was to develop a cell line with higher ammonia removal activity than the cell line developed previously. To accomplish this, we constructed stable CHO cell lines expressing the first three, the first four, or all five enzymes of urea cycle by the stable transfection method. We finally selected CHO-AL-19 cell line expressing the first three, the first four enzymes of the cycle with higher ammonia activity than CHO-OTC1-A19 and CHO-n대-5 cell lines: 40% and 15% higher than those of CHO-neo-5 and CHO-OTC1-A19 cell lines 72 hour after culture started, respectively. It also showed 44% and 10% higher cell viability than CHO-neo-5 and CHO- OTC1-A19 cell lines at higher cell density. In addition, CHO-AL-19 cells showed 45%-60% and about 20% lower ammonia concentration per cell than those of CHO-neo-% and CHO-OTC1-A19 cell lines, respectively. These results indicate that CHO-AL-19 could be used in the production of human therapeutic proteins with higher efficiency.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.799-804
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• 2008

Retrovirus tropism can be restricted by host cell factors such as Fv1, TRIM5${\alpha}$, and LvI that inhibit infection by targeting the incoming viral capsid. The Fv1 gene inhibits murine leukemia virus infection in mice, but the precise mechanism of Fv1-mediated restriction is poorly understood. Our previous studies had demonstrated that Fv1-mediated viral tropism can be determined within the capsid protein at position 114. To study the interaction between Fv1 and CA, we introduced amino acid substitution and deletion at this site in the N-tropic AKV capsid gene. The mutated two-LTR proviral DNAs were introduced into SC-1 cells by transfection. After transfection, cell supernatants collected from transfected cells were tested for host range susceptibility. The result indicated that substitution of amino acids did not alter tropism, but the deletion of 114His produced a virus with unusual tropism. The novel phenotype produced here failed to replicate in Fv1-expressing cells. This mutant virus showing such an extreme restriction pattern would be useful for studying the mechanism of Fv1-mediated restriction.

• Molecules and Cells
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• v.25 no.1
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• pp.105-111
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• 2008

Radiotherapy is an important treatment for many malignant tumors, but there are recent reports that radiation may increase the malignancy of cancer cells by stimulating expression of type IV collagenases. In this study, we examined changes in matrix metalloproteinase (MMP) inhibitors, such as the tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and RECK, in response to irradiation in Panc-1 pancreatic cancer cells. Irradiation increased RECK protein levels but not mRNA levels, whereas no significant changes were found in TIMP-1 and TIMP-2. The enhanced RECK protein levels were associated with an increase in MMP inhibitory activity. However, irradiation slightly but reproducibly increased the invasiveness of the Panc-1 cells. Like irradiation, treatment of Panc-1 cells with transforming growth factor $(TGF)-{\beta}1$ led to a 2-fold increase in RECK protein levels. Transient transfection with Smad3 also increased RECK protein levels, but transfection with Smad7 markedly reduced them. Stable expression of Smad7 and treatment with SB431542, an inhibitor of $TGF-{\beta}$ receptor I kinase, abolished $TGF-{\beta}1$- and radiation-mediated effects on RECK. Furthermore, irradiation increased levels of phosphorylated Smad3. We conclude that radiation post-transciptionally enhances RECK protein levels in Panc-1 cells, at least in part, via $TGF-{\beta}$ signaling, and that irradiation increases Panc-1 invasiveness via a mechanism that may not be linked to MMP-2 activity.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.98-98
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• 2003

Direct gene transfer to mammalian tissues has significant potential for gene therapy and transgenesis. Liposome-mediated in vivo transfection has begun to gain attention as an alternative to viral vectors, and may also be a good mode of transfection in gene transfer. Interestingly, polymerized cationic liposomes are reported to be very stable in the bloods and efficient for in vivo gene transfer. To examine a possible gene delivery in vivo, we investigated the efficacy and safety of the liposome-mediated gene transfer using vein injection in chick or mouse as model animals. The number of injected pGFP-LacZ using either a commercial or home-made liposomes was 8 and 19 at 16 and 7 day of hatch, respectively. One of injected chick of each experiments was analyzed and the rest is being bred. In mouse, 4/22 showed expression of pGFP-LacZ but 8/22 showed no expression and the remaining animals are also being bred. After injection of liposome/pGFP-LacZ complex into wing vein of 7 or 16 day-old chick, pGFP-LacZ was detected in various tissues isolated from not only young chick but also old chick were turned out to possess. exogenous DNA. Transcripts and proteins of the transgene were also detected by RT-PCR or histochemical analysis, respectively. These results suggest that injected DNA were inserted to genome and produced mRNA and proteins in various tissues and may give an important tools for effective gene delivery in gene therapy or transgenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2837-2840
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• 2012

We investigated whether IFN-${\beta}$ inhibits the growth of human malignant glioma and induces glioma cell apoptosis using the human IFN-${\beta}$ gene transfected into glioma cells. A eukaryonic expression vector ($pSV2IFN{\beta}$) for IFN-${\beta}$ was transfected into the glioma cell line SHG44 using liposome transfection. Stable transfection and IFN-${\beta}$ expression were confirmed using an enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was also assessed by Hoechst staining and electron microscopy. In vivo experiments were used to establish a SHG44 glioma model in nude mice. Liposomes containing the human IFN-${\beta}$ gene were injected into the SHG44 glioma of nude mice to observe glioma growth and calculate tumor size. Fas expression was evaluated using immunohistochemistry. The IFN-${\beta}$ gene was successfully transfected and expressed in the SHG44 glioma cells in vitro. A significant difference in the number of apoptotic cells was observed between transfected and non-transfected cells. Glioma growth in nude mice was inhibited in vivo, with significant induction of apoptosis. Fas expression was also elevated. The IFN-${\beta}$ gene induces apoptosis in glioma cells, possibly through upregulation of Fas. The IFN-${\beta}$ gene modulation in the Fas pathway and apoptosis in glioma cells may be important for the treatment of gliomas.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.217-224
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• 2003

Previously, we developed a CHO cell line (CHO-OTC1-Al9) that expresses the first two enzymes in the urea cycle and exhibits a higher ammonia-removing ability and faster growth rate than a vector-controlled CHO cell line (CHO-neo-5). The current study was undertaken to develop a cell line with an ammonia-removing ability higher than the cell line developed previously. To accomplish this, CHO cell lines expressing the first three, first four, or all five enzymes of the urea cycle were constructed using a stable transfection method. Finally, the CHO-AS-16, CHO-AL-19, and CHO-Arg-11 cell lines expressing the first three, first four, and all five enzymes of the urea cycle, respectively, were selected and found to exhibit higher ammonia-removing ability than the CHO-OTC1-Al9 cell line. Among the three selected cell lines, CHO-AL-19 showed the highest ammonia-removing ability and highest cell viability at a higher cell density, with 40% and 15% lower ammonia concentration in the, culture media than that of CHO-neo-5 and CHO-OTC1-A19 cell lines, respectively. CHO-AL-19 also showed 44% and 10% higher cell viability than the CHO-neo-5 and CHO-OTC-Al9 cell lines, at a higher cell density, respectively. The ammonia concentrations in the culture media were expressed as the ammonia concentration/cell, and the CHO-AL-19 cells revealed 45-60% and 20% lower ammonia concentration/cell than the CHO-neo-5 and CHO-OTC1-Al9 cells, respectively.

• Molecules and Cells
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• v.38 no.4
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• pp.349-355
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• 2015

EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 co-precipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinase-inactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptor-like protein acts as a biochemical linker between the Eph receptor and caspase-8.

• Bulletin of the Korean Chemical Society
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• v.33 no.11
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.