• Molecules and Cells
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• v.21 no.3
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• pp.395-400
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• 2006

Glutathione S-transferase P1 (GSTP1) plays an important role in detoxification and the metabolism of xenobiotics. Here we show that GSTP1 also regulates the MEKK1-MKK7 signaling pathway. Over-expression of GSTP1 in HEK293 cells inhibited both ${\Delta}MEKK1$- and etoposide-induced apoptosis, and inhibited procaspase-3 activation and PARP cleavage. MEKK1- induced apoptosis requires both its kinase activity and proteolytic cleavage. ${\Delta}MEKK1$ activity was inhibited by over-expression of GSTP1 in vivo and MEKK1 kinase activity was also inhibited by GSTP1 in vitro when assayed with bacterially-expressed MKK7(KM) protein as substrate. GSTP1 inhibition of etoposide-induced cell apoptosis was mainly due to its ability to suppress MEKK1 kinase activity. The glutathione-conjugating activity of GSTP1 was essential for the above effects. These findings provide insight into the mechanism by which GSTP1 protects cells from genotoxin-induced apoptosis.

• Animal cells and systems
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• v.4 no.4
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• pp.389-394
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• 2000

The Tcf-1 (1-cell factor-1) protein binds to the T-cell specific enhancer sequences and plays an architectural role in the assembly of transcriptional machinery. One of the Tcf family proteins, Tcf-4, was found to be an important regulator for colon cancer development where it activates specific genes upon binding to $\beta$-catenin following Wnt signaling. We were interested in the transcriptional regulatory activities of Tcf-1 and Tcf-4 proteins in T-cells and colon cancer cells. Transactivation assay was developed using a reporter plasmid containing luciferase gene under the control of Tcf responsive elements. Luciferase activity was determined following co-transfection of the reporter along with Tcf-1 and/or $\beta$-catenin expressing plasmids. Transcription was significantly induced by $\beta$-catenin expression in all cells. Tcf-1 by itself did not induce transcription in the mature T-cell lines, but overexpressed Tcf-1 greatly activated transcription in the immature T-cell line. In addition, transfected $\beta$-catenin induced apoptosis, but co-transfected Tcf-1 suppressed apoptosis in HEK293 cells. These results suggest that Tcf-1 and $\beta$-catenin differently regulate transcription and apoptosis.

• BMB Reports
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• v.56 no.3
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• pp.172-177
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• 2023

BEST family is a class of Ca²⁺-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca²⁺-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca²⁺-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins.

• Journal of the Korean Wood Science and Technology
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• v.42 no.1
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• pp.68-77
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• 2014

A compound has been isolated from the methanol extract of Styrax japonica bark using conventional chromatographic methods including silica gel chromatography, TLC and HPLC. The molecular formula of Styraxlignolide F analyzed by spectrometric analyses using FAB-MS, NMR was found to be $C_{27}H_{34}O_{11}Na$. The cytotoxicity of the styralignolide F was showed 15.2% in $1.0mg/m{\ell}$ on human kidney cell (HEK 293). As anticancer activity of $CH_2Cl_2$ fraction, over 60% of AGS and MCF-7 cells were inhibited in concentration of $1.0mg/m{\ell}$. In the results of anticancer test using quantification of Bcl-2, $CH_2Cl_2$ fraction showed lower Bcl-2 and p53 expression than those of styraxlignolide F and other fractions. In apoptosis of human lung carcunoma cancer cell (A549), $CH_2Cl_2$ fraction showed the highest inhibition rate (46.9%) and styralignolide F was the next (43.5%). The $CH_2Cl_2$ fraction showed higher anti-cancer activities than isolated substance (styraxlignolide F), probably due to the crude extract showing synergic effects by other components.

• Bulletin of the Korean Chemical Society
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• v.32 no.6
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• pp.2051-2057
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• 2011

Several studies have demonstrated that nanoparticles (NPs) have toxic effects on cultured cell lines, yet there are no clear data describing the overall molecular changes induced by NPs currently in use for human applications. In this study, the in vitro cytotoxicity of three oxide NPs of around 100 nm size, namely, mesoporous silica (MCM-41), iron oxide ($Fe_2O_3$-NPs), and zinc oxide (ZnO-NPs), was evaluated in the human embryonic kidney cell line HEK293. Cell viability assays demonstrated that 100 ${\mu}g/mL$ MCM-41, 100 ${\mu}g/mL$ $Fe_2O_3$, and 12.5 ${\mu}g/mL$ ZnO exhibited 20% reductions in HEK293 cell viability in 24 hrs. DNA microarray analysis was performed on cells treated with these oxide NPs and further validated by real time PCR to understand cytotoxic changes occurring at the molecular level. Microarray analysis of NP-treated cells identified a number of up- and down-regulated genes that were found to be associated with inflammation, stress, and the cell death and defense response. At both the cellular and molecular levels, the toxicity was observed in the following order: ZnO-NPs > $Fe_2O_3$-NPs > MCM-41. In conclusion, our study provides important information regarding the toxicity of these three commonly used oxide NPs, which should be useful in future biomedical applications of these nanoparticles.

• Analytical Science and Technology
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• v.26 no.1
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• pp.106-112
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• 2013

PDE plays an important role in cAMP-mediated cellular signaling within the cells. The proper targeting of each PDE is mediated by unique N-terminal of each PDE isoform. It has been recently reported that supershort-, short- and long-forms of PDE4 in Aplysia were cloned in Aplysia. Long-form of ApPDE4 was localized at plasma membrane and presynaptic terminal in Aplysia sensory neurons. However, it remains elusive which part of ApPDE4 is minimal region for the proper targeting and what are the effects on the cell functions. Here, we identified that N-terminal 13 amino acids of ApPDE4 long-form is minimal regions for the plasma membrane targeting. In addition, overexpression of ApPDE4(N20)-mRFP could induce morphological changes in HEK293T cells. Interestingly, mRFP-$PLC{\delta}1$(PH), which selectively binds to PI4,$5P_2$, could induce morphological changes in similar with that by ApPDE4(N20)-mRFP. These results suggested that binding of ApPDE4(N20) to lipids including PI4,$5P_2$ might be responsible for targeting of ApPDE4 to plasma membrane and morphological changes in HEK293T cells.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.266.2-266.2
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• 2013

According to advanced nanotechnology, the nanostructured materials with various kinds and shape are synthesized easily or produced by process. Recently, researches about interaction between the nanostructured materials and biological system have been progressed actively. The surface topography may influence cellular responses, for example cell adhesion, cell morphology. In this work, we synthesized vertically aligned silicon nanowires (SiNWs) on the Au-covered Si(111) wafer by chemical vapor deposition (CVD) method. We accomplished to control of the SiNWs diameter by regulating thickness of Au film such as 1 nm and 10 nm. These substrates did not isolate cells and just provided surface topography for cell culture. Human Embryonic Kidney 293T cells (HEK 293T cells) were cultured on these substrates for 2 days. We studied the nanotopographical effects on cell morphology, adhesion, and growth which are evaluated on each SiNWs substrate comparing bare glass as control.

• Molecules and Cells
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• v.26 no.2
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• pp.193-199
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• 2008

The pro-apoptotic Bcl-2 family member Bim acts as a sensor for apoptotic stimuli and initiates apoptosis through the mitochondrial pathway. To identify novel regulators of Bim, we employed the yeast two-hybrid system and isolated the human gene encoding macrophage migration inhibitory factor (MIF), a ubiquitously expressed proinflammatory mediator that has also been implicated in cell proliferation, the cell cycle and carcinogenesis. The interaction between MIF and Bim was confirmed by both in vitro and in vivo protein interaction assays. Intriguingly, protein complexes between MIF and the three major Bim isoforms (BimEL/BimL/BimS) could be detected in HEK293 and K562 cells, especially in cells undergoing apoptosis. Moreover, exogenous expression of MIF partially inhibited Bim-induced apoptosis in HEK293 cells. SiRNA-mediated knockdown of MIF increased apoptosis in K562 cells exposed to the chemical oxidant diamide. Endogenous MIF may regulate the pro-apoptotic activity of Bim and inhibit the release of cytochrome c from mitochondria.

• Molecules and Cells
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• v.27 no.5
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• pp.577-582
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• 2009

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon ${\gamma}$, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

• Molecules and Cells
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• v.39 no.11
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• pp.821-826
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• 2016

The ${\beta}$-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, ${\beta}$-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of ${\beta}$-catenin. The level of ${\beta}$-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to ${\beta}$-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and ${\beta}$-catenin in HEK293T cells. To our knowledge, this is the first study to report that ${\beta}$-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated ${\beta}$-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active ${\beta}$-catenin via degradation, which stabilized SIAH-1 and increased its interaction with ${\beta}$-catenin. These results suggest that activated STAT3 regulates active ${\beta}$-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of ${\beta}$-catenin in HEK293T cells.