• BMB Reports
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• v.39 no.6
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• pp.730-736
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• 2006

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced $G_1$ arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of $\beta$-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces $G_1$ arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of $\beta$-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.170-170
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• 2002

College of Pharmacy, Ewha womans University, Seoul, 120-750, Korea 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent halogenated aromatic hydrocarbon congener that induces expression of several genes including CYP1A1. Exposure to TCDD results in many toxic actions such as carcinogenesis, hepatotoxicity, immune suppression, and reproductive and developmental toxicity. Dramatic differences in dioxin toxicity have been observed between the sexes of some animal species, suggesting hormonal modulation of dioxin action. Many studies have been reported and propose several mechanisms of anti-estrogenic effects of TCDD. In contrast, the effect of estrogen on the regulation of CYP1A1 are not clear at present. There are several reports showing conflicting results. It seems that induction/inhibition of CYP1A1 may be dependent on cell-type and concentration. The purpose of this study was to investigate the regulation of TCDD-induced CYP1A1 gene expression by estradiol and its metabolites. We examined whether estradiol and its metabolites altered TCDD-mediated induction of CYP1A1 enzyme activity. 17 ${\beta}$ estradiol and 16 ${\alpha}$ estriol at non cytotoxic concentrations caused a significant concentration dependent decline of TCDD-induced EROD activity To determine whether reduced EROD activity reflected altered CYP1A1 mRNA expression, we measured CYP1A1 mRNA level by RT-PCR. And to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level, we also peformed transient transfection with an AhR responsive reporter plasmid containing the 5' flanking region of the human CYP1A1 gene to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level.

• Journal of Pharmaceutical Investigation
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• v.40 no.6
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• pp.357-362
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• 2010

To enhance therapeutic effects of insulin-sensitizing adipokine, ADN gene and potent agonists, rosiglitazone for the $PPAR{\gamma}$, cationic liposomes as non-viral vectors were formulated. The particle size and zeta potential of drug loaded and unloaded cationic liposomes were investigated. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. In vitro transfection was investigated in HepG2, HeLa, and HEK293 cells by mRNA expression of ADN. Encapsulation efficacy of rosiglitazone-loaded liposomes was determined by UV detection. Particle sizes of cationic liposomes were in the range of 110-170 nm and those of rosiglitazone-loaded cationic liposomes were in the range of 130-180 nm, respectively. Gel retardation of complexes indicated that the complex was formed at weight ratios of cationic lipid to plasmid DNA higher than 20:1. Both complexes protected plasmid DNA from DNase either drug free or drug loading. Encapsulation efficiency of rosiglitazone-loaded emulsion was increased by drug dose. The mRNA expression levels of ADN were dose-dependently increased in cells transfected with plasmid DNA. Therefore, cationic liposomes could be potential co-delivery system for drug and gene.

• Journal of Environmental Health Sciences
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• v.30 no.3
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• pp.230-238
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• 2004

The adverse health effects of a number of environment pollutions are related to the formation of free radicals. Induction of antioxidant defensive system in the response to an oxidative attack is an essential element of the cell to survive. CYP2E1 is easily induced by organic solvents and induces continuous formation of reactive oxygen species (ROS). ${\gamma}$-Glutamyltranspeptidase (${\gamma}$GT) plays an important role in glutathione metabolism and xenobiotic detoxification. To evaluate the characteristic of oxidative stress which induces GGT expression and to understand human antioxidant defensive response against oxidative stress induced by CYP2E1, we studied regulation of ${\gamma}$GT enzyme expression in response to various oxidative stresses in human HepG2 cells. The ${\gamma}$GT activity was not modified after exposure of acute oxidative stress inducing agents (ferric nitrilotriacetate, cumene hydroperoxide, ADP-Fe, O-tetradecanoylphorbol-13-acetate, tumor necrosis factor-alpha). To induce continuous exposure of cells to ROS, HepG2 cells were transfected by human CYP2E1 gene transiently. The CYP2E1 activity was verified with chlorzoxazone hydroxylation. Transfection of CYP2E1 showed continuous 60% increase in intracellular ROS and 240 % increase in microsomal ROS. CYP2E1 overexpressing cells showed increased ${\gamma}$GT activity (2.5-fold). The observed enhancement of ${\gamma}$GT activity correlated with a significant increase of ${\gamma}$GT mRNA (2.1-fold). Treatment with antioxidant strongly prevented the increase in ${\gamma}$GT activity. The CYP2E1 overexpression did not modify toxicity index and increased glutathione levels. These results show that continuous exposure of cells to ROS produced by CYP2E1 up-regulates ${\gamma}$GT; This may be one of the adaptive antioxidant responses of cells to oxidative insult. Present study also suggests that the induction of ${\gamma}$GT could be used as a marker of oxidative stress induced by exposure to organic solvents.

• Biomolecules & Therapeutics
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• v.25 no.2
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• pp.202-212
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• 2017

Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however, the dose-dependent cardiotoxicity associated with DOX significantly limits its clinical application. In the present study, we investigated whether Rb1 could prevent DOX-induced apoptosis in H9C2 cells via aryl hydrocarbon receptor (AhR). H9C2 cells were treated with various concentrations ($-{\mu}M$) of Rb1. AhR, CYP1A protein and mRNA expression were quantified with Western blot and real-time PCR analyses. We also evaluated the expression levels of caspase-3 to assess the anti-apoptotic effects of Rb1. Our results showed that Rb1 attenuated DOX-induced cardiomyocytes injury and apoptosis and reduced caspase-3 and caspase-8, but not caspase-9 activity in DOX-treated H9C2 cells. Meanwhile, pre-treatment with Rb1 decreased the expression of caspase-3 and PARP in the protein levels, with no effects on cytochrome c, Bax, and Bcl-2 in DOX-stimulated cells. Rb1 markedly decreased the CYP1A1 and CYP1A2 expression induced by DOX. Furthermore, transfection with AhR siRNA or pre-treatment with AhR antagonist CH-223191 significantly inhibited the ability of Rb1 to decrease the induction of CYP1A, as well as caspase-3 protein levels following stimulation with DOX. In conclusion, these findings indicate that AhR plays an important role in the protection of Ginsenoside Rb1 against DOX-triggered apoptosis of H9C2 cells.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.140-147
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• 2010

Japanese encephalitis virus (JEV), a member of the mosquito-borne flaviviruses, causes epidemics of viral encephalitis in the Southeastern Asia. JEV is a small enveloped virus with a positive-sense RNA genome; the infectious virion consists of three structural proteins, namely capsid, membrane (M; a mature form of its prM precursor), and envelope proteins. Here, we investigated a role of the charged residues found at the N-terminus of the JEV M protein in virus production. Using an infectious JEV cDNA, we generated two mutant cDNAs, Mm1 and Mm2, by charged-to-alanine substitution for $E^9$ and $K^{15}K^{16}E^{17}$ residues of the M protein, respectively. By transfection of wild-type or each of the two mutant RNAs transcribed from the corresponding cDNAs, we found that Mm2, but not Mm1, had a ~3-log decrease in virus production, even though a comparable amount of all three structural proteins were produced in transfected cells. Interestingly, the prM protein expressed in Mm2 RNA-transfected cells was not recognized by the polyclonal antiserum raised against the N-terminal 44 amino acids of the wild type M protein, but reacted to the antiserum raised against the corresponding region of the mutant Mm2. Our results indicate that three charged residues ($K^{15}K^{16}E^{17}$) in JEV M protein play a role in virus production. Two polyclonal antisera specifically recognizing the wild-type or Mm2 version of the M protein would provide a useful reagent for the functional study of this protein in the virus life cycle.

• Journal of Life Science
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• v.31 no.9
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• pp.806-817
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• 2021

Increasing evidence suggests that depression is associated with impairments in neural plasticity. Sirtuin 1 plays an important role in neural plasticity, and the activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling is known to improve neural plasticity. In this study, we aimed to determine whether sirtuin 1 affects dendrite outgrowth and spine formation through mTORC1 signaling. Resveratrol (sirtuin 1 activator; 1 and 10 μM) and sirtinol (sirtuin 1 inhibitor; 1 and 10 μM) were treated in primary cortical culture with and without dexamethasone (500 μM). Levels of sirtuin 1, phospho-extracellular signal regulated protein kinase 1/2 (ERK1/2), phospho-mTORC1, and phospho-p70 ribosomal protein S6 kinase (p70S6K) were evaluated using Western blot analysis. Dendritic outgrowth and spine density were assessed using immunostaining. Resveratrol significantly increased levels of sirtuin 1 expression and phosphorylation of ERK1/2 (a downstream target of sirtuin 1), mTORC1, and p70S6K (a downstream target of mTORC1) in a concentration-dependent manner under dexamethasone conditions. Resveratrol also significantly increased dendritic outgrowth and spine density. Conversely, sirtinol significantly decreased levels of sirtuin 1 expression and phosphorylation of ERK1/2, mTORC1, and p70S6K in a concentration-dependent manner under normal conditions. Moreover, sirtinol significantly decreased dendritic outgrowth and spine density. Consistent with the results of sirtinol, sirtuin 1 knockdown using sirtuin 1 siRNA transfection significantly decreased dendritic outgrowth and spine density as well as phosphorylation levels of ERK1/2 and mTORC1. These data suggest that sirtuin 1 enhances dendritic outgrowth and spine density by activating mTORC1 signaling.

• Journal of Life Science
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• v.15 no.1 s.68
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• pp.38-44
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• 2005

Infection with hepatitis B virus (HBV) is a major health problem worldwide. Although a tremendous amount has been known about HBV, there have been obstacles in the study of HBV due to the narrow host range of HBV limited to humans and primates. In the present study, we investigated the susceptibility to HBV infection of mouse hepatoma cell line, Hepa-1c1c7. In addition, based on that human hepatocytes infected by HBV increase the expression of the pro-inflammatory cytokine TNF-a, the inducibility of TNF-a expression by HBV in the cells was determined. HBV surface antigen (HBsAg) secretion was measured by the microparticle enzyme immunoassay and steady state mRNA expression was analyzed by quantitative competitive RT-PCR. Transient transfection of Hepa-1c1c7 cells with HBV expression vector resulted in a dose-dependent induction of TNF-a expression. Infection of Hepa-1c1c7 cells with the serum of HBV carrier also increased TNF-a mRNA expression. Both in the transfected and infected cells, HBV mRNA was expressed and significant HBsAg secretion was detected. There was no significant variation in $\beta-actin$ mRNA expression by HBV. These results demonstrate that HBV is infectious to Hepa-lc1c7 in vitro and the viral infection induces TNF-a expression, which suggests that Hepa-lc1c7, a mouse hepatoma cell line, may be a possible model system for analysis of various molecular aspects of HBV infection.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.153-158
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• 2008

Tissue-specific and temporal regulation of milk protein gene expression is advantageous when creating transgenic animal that produces foreign protein into milk. Gene expression, i.e. protein production, is regulated not only by promoter strength but also mRNA stability. Especially, poly A tail length by polyadenylation affects in vivo and in vitro mRNA stability and translation efficiency of the target gene. In the present study, nucleotide sequence of 3'-UTR was analyzed to evaluate the effects of mRNA stability on the target gene expression. Based on the poly A signal of 3' -untranslated region (UTR), nucleotide sequences of putative cytoplasmic polyadenylation elements (CPEs) and downstream elements (DSEs: U-rich, G-rich, GU-rich) were analyzed and used to construct 15 luciferase reporter vectors. Each vector was transfected to HC11 and porcine mammary gland cell (PMGC) and measured for dual luciferase expression levels after 48 hours of incubation. Luciferase expression was significantly higher in construct #6 (with CPE 2, 3 and DSE 1 of exon 9) and #11 (with CPE 2, 3 and DSE 1, 2 and 3 of exon 9) than construct #1 in the PMGC. These results suggest that expression of target genes in PMGC may be effectively expressed by using the construct #6 and #11 on production of transgenic pig.

• BMB Reports
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• v.46 no.10
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• pp.501-506
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• 2013

Oligodendrocyte precursor cells (OPCs) are most susceptible to oxidative stress in the brain. However, the cause of differences in susceptibility to oxidative stress between OPCs and mature oligodendrocytes (mOLs) remains unclear. Recently, we identified in vivo that ${\alpha}B$-crystallin (aBC) is expressed in mOLs but not in OPCs. Therefore, we examined in the present study whether aBC expression could affect cell survival under oxidative stress induced by hydrogen peroxide using primary cultures of OPCs and mOLs from neonatal rat brains. Expression of aBC was greater in mOLs than in OPCs, and the survival rate of mOLs was significantly higher than that of OPCs under oxidative stress. Suppression of aBC by siRNA transfection resulted in a decrease in the survival rate of mOLs under oxidative stress. These data suggest that higher susceptibility of OPCs than mOLs to oxidative stress is due, at least in part, to low levels of aBC expression.