• Animal cells and systems
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• 제1권3호
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• pp.521-526
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• 1997

We isolated a mouse nck cDNA from the thymus cDNA expression library. The cDNA encodes a 377 amino acid protein and displays 97% amino acid sequence identity to human oncogenic protein nck, which is composed almost exclusivelv of three src homology 3 (SH3) domains and one SH2 domain. The sequence analysis also showed that the isolated cDNA is the mouse counterpart of the human nck and different from the mouse grb4, which has been reported to be highly similar to the human nck and, therefore considered as a mouse nck, Northern blot analysis showed that the transcript of the gene was 1.8 kb and was highly expressed in the testis, thymus, and brain but moderately in the liver and lymph node. Western blot analysis showed that the size of the protein was about 47 kDa. Overexpression of the mouse Nck transformed a mouse fibroblast cell line, NIH3T3. The results clearly indicate that normal nck gene has transforming ability and provide an argument against a suggested possibility that the transforming ability of the human nck gene is due to a mutation(s) in the gene.

• 생명과학회지
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• 제13권5호
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• pp.551-558
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• 2003

C형 간염바이러스는 간암을 야기하는 심각한 바이러스이다. C형 간염바이러스의 core 단백질의 과발현은 섬유아세포를 암화시키는 것으로 알려져 있다. Phospholipase D (PLD)의 효소활성이 세포증식 신호전달에 의해 활성화되어 있으며, 사람의 암조직에서 과발현 및 활성이 증가되어 있는 것으로 알려져 있다. 본 연구의 목적은, core 단백질에 의해 암화된 세포에서 PLD가 어떻게 조절되는지를 이해하고자 하는 것이다. 자극이 없는 상태에서뿐만 아니라 PMA에 의해 유도되는 PLD효소활성은, 암화된 세포에서 더 증가하였으며, control 세포와 core 단백질에 의해 암화된 세포에서 PLD와 PKC 단백질의 발현은 서로 유사하였다. PKC 특이적인 억제제와 PKC의 세포막으로의 이동에 관한 실험을 통해서, PKC-d가 암화된 세포에서 PMA에 의해 유도되는 PLD활성의 증가에 중요하게 관여하고 있음을 밝혔다. 이러한 결과는, PLD가 core 단백질에 의해 유도되는 세포의 암화과정에 관여하고 있을 것으로 추정된다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권5호
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• pp.1833-1837
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• 2015

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6-tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine $IC_{10}$, $IC_{20}$ and $IC_{50}$ levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the $IC_{50}$ levels of $26.0{\pm}4.45$ and $51.3{\pm}2.05{\mu}M$, respectively. For murine normal fibroblast NIH3T3 cells, the $IC_{50}$ concentration was $81.3{\pm}10.1{\mu}M$. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

• BMB Reports
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• 제29권3호
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• pp.205-209
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• 1996

To investigate the role of heparin in keratinocyte growth factor (KGF) and acidic fibroblast growth factor (aFGF) high affinity binding to the KGF receptor (KGFR), a cell free system was established which utilized a secreted chimeric molecule between the KGFR extracellular domain and the immunoglobulin heavy chain Fc domain (KGFR-HFc). KGFR-HFc was purified from NIH 3T3 cells and demonstrated the binding of $[^3H]-heparin$ as well as heparin Sepharose. Scatchard analysis showed that the dissociation constant for heparin binding to KGFR-HFc was 140 nM. High affinity KGF and aFGF binding to KGFR-HFc remained unchanged after treatment with 0.6 M NaCl, which is the concentration sufficient to release any bound heparin to the KGFR-HFc. These results strongly suggest that although the KGFR interacts with heparin, the presence of heparin is not absolutely required for high affinity binding of either KGF or aFGF to the KGFR.

• 대한임상검사과학회지
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• 제42권3호
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• pp.116-124
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• 2010

This study was aimed to clerify the cytotoxicity of some plant extracts such as Hosta longissima HONDA (HL), Hemerocallis fulva var. Kwanso REGL (HFVK), Hemerocallis fulva L (HF), Macrocapium officinale NAKAI (MO) and Mentha canadensis var. piperascens HARA (MCVP), the cultured NIH3T3 fibroblasts were treated with 25, 50, 100, 150 and $200{\mu}g/mL$ of five kinds of plant extracts for 48 hours, respectively. The cytotoxicity of plant extracts was measured by MTT and NR assays for the cell viability, and XTT assay for the cell adhesion activity. In this study, HL, MO and FHVK extracts showed the range of midtoxic-non toxic by the criteria of chemical cytotoxicity. While, the HF and MCVP extracts showed midtoxic. In the extract cytotoxicity, HL, MO and FHVK extracts showed non-toxic by the criteria of extract cytotoxicity. While, HF extract was determined as lower-toxic. In the responsive sensitivity of each plant extract on colorimetric assays, HF extract was sensitive to mitochondrial enzyme by MTT assay, lysosomal enzyme by NR assay and mitochondrial nucleus by XTT assay. While, MCVP extract was sensitive to mitochondrial enzyme by MTT assay and lysosomal enzyme by NR assay than other assays. While, HL, HFVK and MO extracts were most sensitive to NR assay. Cell culture is one of useful materials in the screening of cytotoxic and recovary effect on the putative chemical agents or plant extract. And also, colorimetric assay is regarded as very useful tools for quantitative measurement of cytotoxic effect on plant extracts in vitro.

• BMB Reports
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• 제52권8호
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• pp.490-495
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• 2019

Using tunneling nanotubes (TNTs), various pathological molecules and viruses disseminate to adjacent cells intercellularly. Here, we show that the intracellular invasion of Mycoplasma hyorhinis induces the formation of actin- and tubulin-based TNTs in various mammalian cell lines. M. hyorhinis was found in TNTs generated by M. hyorhinis infection in NIH3T3 cells. Because mycoplasma-free recipient cells received mycoplasmas from M. hyorhinis-infected donor cells in a mixed co-culture system and not a spatially separated co-culture system, direct cell-to-cell contact via TNTs was necessary for the intracellular dissemination of M. hyorhinis. The activity of Rac1, which is a small GTP binding protein, was increased by the intracellular invasion of M. hyorhinis, and its pharmacological and genetic inhibition prevented M. hyorhinis infection-induced TNT generation in NIH3T3 cells. The pharmacological and genetic inhibition of Rac1 also reduced the cell-to-cell dissemination of M. hyorhinis. Based on these data, we conclude that intracellular invasion of M. hyorhinis induces the formation of TNTs, which are used for the cell-to-cell dissemination of M. hyorhinis.

• Journal of Ginseng Research
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• 제41권3호
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• pp.411-418
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• 2017

Background: Recently, protein from ginseng was studied and used for the treatment of several kinds of diseases. However, the effect of ginseng total protein (GTP) on proliferation and wound healing in fibroblast cells remains unclear. Methods: In this study, cell viability was analyzed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cell cycle distribution was analyzed by flow cytometer. The levels of transforming growth factor ${\beta}1$, vascular endothelial growth factor, and collagens were analyzed by enzyme-linked immunosorbent assay and immunofluorescence staining. The expressions of cyclin A, phosphorylation of extracellular signal-related kinase (p-ERK1/2), and ERK1/2 were analyzed by Western blotting. Results: Our results showed that GTP promoted cell proliferation and increased the percentage of cells in S phase through the upregulation of cyclin A in NIH/3T3 cells. We also found that GTP induced the secretion of type I collagen, and promoted the expression of other factors that regulate the synthesis of collagen such as transforming growth factor ${\beta}1$ and vascular endothelial growth factor. In addition, the phosphorylation of ERK1/2 at Thr202/Tyr204 was also increased by GTP. Conclusion: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

• 동의생리병리학회지
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• 제23권6호
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• pp.1314-1319
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• 2009

In previous study, we have shown that continentalic acid (CA) isolated from Aralia continentalis induced the growth inhibition and apoptosis in HepG2 cells. In this study, we examine the effects of CA from A. continentalis on growth inhibition and apoptosis induction in human leukemia HL-60 and mouse fibroblast NIH 3T3 cell lines. The results demonstrated that CA decreased cell growth of leukemia HL-60 cells but not human HaCaT keratinocytes, assessed with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and LDH (lactate dehydrogenase) assay. Flow cytometric analysis of mouse fibroblast cell lines exposed to CA showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with CA decreased the number of normal cells and increased the number of early apoptotic and late apoptotic cells in a dose-dependent manner. The induction of apoptosis in mouse cell lines by CA was mediated through the activation of caspase-3, Bak, and Bax and the down-regulation of Bcl-2. Our results suggest that CA efficiently induces apoptosis in human leukemia cells.

• Macromolecular Research
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• 제10권3호
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• pp.150-157
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• 2002

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a microbial storage polymer with biodegradable properties. In order to improve the cell compatibility of PHBV surfaces, the physicochemical treatments have been demonstrated. In this study, physical method was corona discharge treatment and chemical method was chloric acid mixture solution treatment. The physicochemically treated PHBV film surfaces were characterized by the measurement of water contact angle, electron spectroscopy for chemical analysis, and scanning electron microscopy (SEM). The water contact angle of the physicochemically treated PHBV surfaces decreased from 75 to 30～40 degree, increased hydrophilicity. due to the introduction of oxygen-based functional group onto the PHBV backbone chain. The mouse NIH/3T3 fibroblasts cultured onto the physicochemically treated PHBV film surfaces with different wettability. The effect of the PHBV surface with different wettability was determined by SEM as counts of cell number and [$^3$H]thymidine incorporation as measures of cell proliferation. As the surface wettability increased, the number of the cell adhered and proliferated on the surface was increased. The result seems closely related with the serum protein adsorption on the physicochemically treated PHBV surface. In conclusion, this study demonstrated that the surface wettabilily of biodegradable polymer as the PHBV plays an important role for cell adhesion and proliferation behavior for biomedical application.

• Restorative Dentistry and Endodontics
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• 제29권4호
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• pp.370-377
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• 2004

The purpose of this study was to regenerate human dental pulp tissues similar to native pulp tissues. Using the mixture of type I collagen solution, primary cells collected from the different tissues (pulp, gingiva, and skin) and NIH 3T3 ($1{\;}{\times}{\;}10^5{\;}cells/ml/well$) were cultured at 12-well plate at $37^{\circ}C$ for 14 days. Standardized photographs were taken with digital camera during 14 days and the diameter of the contracted collagen gel matrix was measured and statistically analyzed with student t-test. As one of the pulp tissue engineering, normal human dental pulp tissue and collagen gel matrix cultured with dental pulp cells for 14 days were fixed and stained with Hematoxyline & Eosin. According to this study, the results were as follows: 1. The contraction of collagen gel matrix cultured with pulp cells for 14 days was significantly higher than other fibroblasts (gingiva, skin) (p < 0.05), 2. The diameter of collagen gel matrix cultured with pulp cells was reduced to 70.4% after 7 days, and 57.1% after 14 days. 3. The collagen gel without any cells did not contract, whereas the collagen gel cultured with gingiva and skin showed mild contraction after 14 days (88.1% and 87.6% respectively). 4. The contraction of the collagen gel cultured with NIH 3T3 cells after 14 days was higher than those cultured with gingival and skin fibroblasts, but it was not statistically significant (72.1%, p > 0.05). 5. The collagen gel matrix cultured with pulp cells for 14 days showed similar shape with native pulp tissue without blood vessels. This approach may provide a means of engineering a variety of other oral tissue as well and these cell behaviors may provide information needed to establish pulp tissue engineering protocols.