• Molecules and Cells
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• v.41 no.5
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• pp.436-443
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• 2018

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

• Journal of Radiation Protection and Research
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• v.34 no.2
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• pp.49-54
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• 2009

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with $^{137}Cs$ $\gamma$-rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and $H_2O_2$-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and $H_2O_2$-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

• Journal of Life Science
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• v.24 no.8
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• pp.837-842
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• 2014

The purpose of this study was to investigate the effect of Brunfelsia grandiflora ethanol extract (BGEE) on the induction of autophagy via regulation of SIRT1 expression and p53 activation in a human lung fibroblast cell line, IMR 90. BGEE at a concentration of $5{\mu}g/ml$ or more exhibited a cytotoxic effect on IMR 90 cells. For the first time, this study showed that BGEE induces autophagy in normal human lung fibroblasts. BGEE also increased the expression level of beclin-1 at $2.5{\mu}g/ml$ or less and Atg7 at $5{\mu}g/ml$, both of which are known to be involved in the induction of autophagy. In addition, BGEE modulated the expression of other proteins related to autophagy in normal human lung fibroblasts. The expression levels of p53 and p-p53, an active form of p53, were decreased in the presence of BGEE at a noncytotoxic concentration. In contrast, the expression level of SIRT1 was increased in human lung fibroblasts treated with BGEE at a noncytotoxic concentration. Moreover, SA-${\beta}$-Gal staining, an aging marker, was reduced in the normal human lung fibroblasts treated with BGEE. These findings suggest that BGEE promotes the induction of autophagy and antiaging through the modulation of p53 and SIRT1 in human lung fibroblasts.

• Molecules and Cells
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• v.37 no.8
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• pp.620-627
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• 2014

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) downregulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the ${\alpha}$ subunit of CK2 ($CK2{\alpha}$) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the $CK2{\alpha}3^{\prime}$-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for $CK2{\alpha}$ downregulation. The four miRNAs increased senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) staining, p53 and $p21^{Cip1/WAF1}$ expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. $CK2{\alpha}$ overexpression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through $CK2{\alpha}$ downregulation-dependent ROS generation.

• Journal of electromagnetic engineering and science
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• v.15 no.3
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• pp.134-141
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• 2015

The effect of a 60 Hz time-varying electric field was studied using a facing-electrode device (FED) and a coplanar-electrode device (CED) for further investigation of the genotoxicity of 60 Hz time-varying magnetic field (MF) from preceding research. Neither a single 30-minute exposure to the CED or to the FED had any obvious biological effects such as DNA double strand break (DSB) and apoptosis in cancerous SCC25, and HeLa cells, normal primary fibroblast IMR90 cells, while exposures of 60 Hz time-varying MF led to DNA damage with induced electric fields much smaller than those used in this experiment. Nor did repetitive exposures of three days or a continuous exposure of up to 144 hours with the CED induce any DNA damage or apoptosis in either HeLa or IMR90 cells. These results imply that the solitary electric field produced by time-varying MF is not a major cause of DSBs or apoptosis in cancer or normal cells.

• Journal of Radiation Protection and Research
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• v.36 no.3
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• pp.119-126
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• 2011

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and $H_2O_2$-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and $H_2O_2$-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-${\beta}$-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

• Journal of Life Science
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• v.19 no.4
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• pp.436-441
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• 2009

B23/nucleophosmin, a nucleolar protein, translocates into the nucleus from the nucleolus when cells are damaged by extracellular stresses. Recently, it was shown that such translocation of B23/nucleophosmin in normal fibroblasts under stress conditions increases both the stability and activation of the p53 protein by disrupting its interaction with MDM2. Senescent cells have a single large nucleolus and a diminished capacity to induce p53 stability upon exposure to various DNA damaging agents. To investigate the role of B23/nucleophosmin in p53 stability in senescent cells, we established a senescence model system by expressing the ras oncogene in IMR90 cells. The stability of p53 was reduced in these cells in response to nucleolar stress, although the level of B23/nucleophosmin protein was not changed. In addition, p53 did not accumulate in the nucleus and B23/nucleophosmin did not translocate into the nucleoplasm. The binding affinity of B23/nucleophosmin with p53 was reduced in senescent cells, whereas the interaction between MDM2 and p53 was stable. Taken together, the stability of p53 in ras-induced senescent cells may be influenced by the ability of B23/nucleophosmin to interact with p53 in response to nucleolar stress.

• Korean Journal of Pharmacognosy
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• v.38 no.4
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• pp.308-311
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• 2007

Inhibition of acetylcholinesterase and amyloid beta(1-42) peptide is good drug targets for Alzheimer's disease therapeutics. Among the twenty enthusiasm reducing herbals, the 70% methanol extracts (1 mg/ml) of Moutan Radicis Cortex and Forsythiae Fructus showed 91.5% and 85.3% about acethylcholinesterase inhibition, respectively. The extracts (1 mg/ml) of Coptidis Rhizoma and Paeoniae Radix Rubra showed more than 85% inhibition rate against amyloid beta (1-42) peptide aggregation. The neuroprotective effect of the extracts (1 mg/ml) of Moutan Radicis Cortex, Forsythiae Fructus and Paeoniae Radix Rubra showed 90.0%, 87.4% and 85.1% to compare with amyloid beta (1-42) peptide treated cells (IMR-32), respectively. Three herbs, Moutan Radicis Cortex, Forsythiae Fructus and Paeoniae Radix Rubra are promising candidates from natural products for development of Alzheimer's disease therapeutics.

• Bulletin of the Korean Chemical Society
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• v.32 no.1
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• pp.53-58
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• 2011

Cysteine and histidine-capped water-dispersible ZnS:Mn nanocrystals (ZnS:Mn-Cys and ZnS:Mn-His) were synthesized and their effects on E. coli and human cells were investigated. Particle sizes of these nanocrystals were found from HR-TEM images to be 3.5 nm and 4.0 nm, respectively. Their solution photoluminescence spectra showed identical broad emission peaks at 580 nm. ZnS:Mn-His significantly suppressed the growth of E. coli at $100{\mu}g/mL$ and 1 mg/mL concentrations, something not observed with ZnS:Mn-Cys. Consistent with this, greater inhibition of cell proliferation and viability were observed in HEK293 and IMR90 cells in ZnS:Mn-His at $100{\mu}g/mL$ and 1 mg/mL concentrations.

• Journal of Life Science
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• v.23 no.4
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• pp.501-509
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• 2013

Formaldehyde (FA) is widely used in industries, and it is an indoor and outdoor pollutant. Exposure to FA may cause inflammation and respiratory oxidative stress. Studies have demonstrated that FA can cause cancer in animal models. During the regeneration process of injured starfish (Asterina pectinifera), several changes have been observed in the expression of cytokines. In particular, higher TGF-${\beta}1$ expression has been detected in arm cut starfish extract after eight days. The current study was designed to elucidate the in-vitro and the in-vivo pharmacological effects of starfish extract on FA exposure. We investigated the protective effects of intact starfish extract and arm cut starfish extract on an IMR-90 cell line and on mouse lung injury in response to FA exposure. In the presence of FA, inhalation of the arm cut starfish extract was associated with more promising cell proliferation, TNF-${\alpha}$, NF-${\kappa}B$ decrement, and $I{\kappa}-B{\alpha}$ increment. In the experimental group, the pulmonary structure of the arm cut starfish extract-treated group in the presence of FA exposure was similar to the control group, whereas the FA exposure group showed damage to the pulmonary structure. Moreover, the arm cut starfish extracts was more effective than the intact starfish extracts in terms of the expression of TNF-${\alpha}$, NF-${\kappa}B$, $I{\kappa}-B{\alpha}$, and surfactant protein A. The results obtained in this study demonstrate that arm cut starfish extracts are more effective in protecting pulmonary structure and function against FA exposure than intact starfish extracts.