• 제목/요약/키워드: HEK-293 cells

검색결과 244건 처리시간 0.029초

Macrophage Migration Inhibitory Factor (MIF) Interacts with Bim and Inhibits Bim-mediated Apoptosis

  • Liu, Lingfeng;Chen, Jinzhong;Ji, Chaoneng;Zhang, Jiayi;Sun, Junlei;Li, Yao;Xie, Yi;Gu, Shaohua;Mao, Yumin
    • Molecules and Cells
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    • 제26권2호
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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.

Knock-down of human MutY homolog (hMYH) decreases phosphorylation of checkpoint kinase 1 (Chk1) induced by hydroxyurea and UV treatment

  • Hahm, Soo-Hyun;Park, Jong-Hwa;Ko, Sung-Il;Lee, You-Ri;Chung, In-Sik;Chung, Ji-Hyung;Kang, Lin-Woo;Han, Ye-Sun
    • BMB Reports
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    • 제44권5호
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    • pp.352-357
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    • 2011
  • The effect of human MutY homolog (hMYH) on the activation of checkpoint proteins in response to hydroxyurea (HU) and ultraviolet (UV) treatment was investigated in hMYH-disrupted HEK293 cells. hMYH-disrupted cells decreased the phosphorylation of Chk1 upon HU or UV treatment and increased the phosphorylation of Cdk2 and the amount of Cdc25A, but not Cdc25C. In siMYH-transfected cells, the increased rate of phosphorylated Chk1 upon HU or UV treatment was lower than that in siGFP-transfected cells, meaning that hMYH was involved in the activation mechanism of Chk1 upon DNA damage. The phosphorylation of ataxia telangiectasia and Rad3-related protein (ATR) upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells. Co-immunoprecipitation experiments showed that hMYH was immunoprecipitated by anti-ATR. These results suggest that hMYH may interact with ATR and function as a mediator of Chk1 phosphorylation in response to DNA damage.

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy

  • Byoung Kwon Kang;Yong-Tae Jung
    • Journal of Microbiology and Biotechnology
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    • 제34권2호
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    • pp.280-288
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    • 2024
  • The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Conditional Replication of a Recombinant Adenovirus Studied Using Neomycin as a Selective Marker

  • Xue, Feng;Qi, Yi-Peng;Joshua, Mallam Nock;Lan, Ping;Dong, Chang-Yuan
    • BMB Reports
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    • 제36권3호
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    • pp.275-281
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    • 2003
  • An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.

Identification of HUGT1 as a Potential BiP Activator and a Cellular Target for Improvement of Recombinant Protein Production Using a cDNA Screening System

  • Ku, Sebastian Chih Yuan;Lwa, Teng Rhui;Giam, Maybelline;Yap, Miranda Gek Sim;Chao, Sheng-Hao
    • Molecules and Cells
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    • 제27권5호
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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.

STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells

  • Shin, Minkyung;Yi, Eun Hee;Kim, Byung-Hak;Shin, Jae-Cheon;Park, Jung Youl;Cho, Chung-Hyun;Park, Jong-Wan;Choi, Kang-Yell;Ye, Sang-Kyu
    • Molecules and Cells
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    • 제39권11호
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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.

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone displays suppression of inflammatory responses via inhibition of Src, Syk, and NF-κB

  • Kim, Yong;Jeong, Eun Jeong;Han Lee, In-Sook;Kim, Mi-Yeon;Cho, Jae Youl
    • The Korean Journal of Physiology and Pharmacology
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    • 제20권1호
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    • pp.91-99
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    • 2016
  • (E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-${\alpha}$. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-${\kappa}B$. Furthermore, the activation of a series of upstream signals for NF-${\kappa}B$ activation, composed of Src, Syk, Akt, and $I{\kappa}B{\alpha}$, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-${\kappa}B$ inhibitory properties via the suppression of Src and Syk.

Ginsenoside Rc from Panax ginseng exerts anti-inflammatory activity by targeting TANK-binding kinase 1/interferon regulatory factor-3 and p38/ATF-2

  • Yu, Tao;Yang, Yanyan;Kwak, Yi-Seong;Song, Gwan Gyu;Kim, Mi-Yeon;Rhee, Man Hee;Cho, Jae Youl
    • Journal of Ginseng Research
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    • 제41권2호
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    • pp.127-133
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    • 2017
  • Background: Ginsenoside Rc (G-Rc) is one of the major protopanaxadiol-type saponins isolated from Panax ginseng, a well-known medicinal herb with many beneficial properties including anticancer, anti-inflammatory, antiobesity, and antidiabetic effects. In this study, we investigated the effects of G-Rc on inflammatory responses in vitro and examined the mechanisms of these effects. Methods: The in vitro inflammation system used lipopolysaccharide-treated macrophages, tumor necrosis $factor-{\alpha}/interferon-{\gamma}-treated$ synovial cells, and HEK293 cells transfected with various inducers of inflammation. Results: G-Rc significantly inhibited the expression of macrophage-derived cytokines, such as tumor necrosis $factor-{\alpha}$ and $interleukin-1{\beta}$. G-Rc also markedly suppressed the activation of TANK-binding kinase $1/I{\kappa}B$ kinase ${\varepsilon}/interferon$ regulatory factor-3 and p38/ATF-2 signaling in activated RAW264.7 macrophages, human synovial cells, and HEK293 cells. Conclusion: G-Rc exerts its anti-inflammatory actions by suppressing TANK-binding kinase $1/I{\kappa}B$ kinase ${\varepsilon}/interferon$ regulatory factor-3 and p38/ATF-2 signaling.

Induction of Two Mammalian PER Proteins is Insufficient to Cause Phase Shifting of the Peripheral Circadian Clock

  • Lee, Joon-Woo;Cho, Sang-Gil;Cho, Jun-Hyung;Kim, Han-Gyu;Bae, Ki-Ho
    • Animal cells and systems
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    • 제9권3호
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    • pp.153-160
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    • 2005
  • Most living organisms exhibit the circadian rhythm in their physiology and behavior. Recent identification of several clock genes in mammals has led to the molecular understanding of how these components generate and maintain the circadian rhythm. Many reports have implicated the photic induction of either mPer1 or mPer2 in the hypothalamic region called the suprachiasmatic nucleus (SCN) to phase shift the brain clock. It is now established that peripheral tissues other than the brain also express these clock genes and that the clock machinery in these tissues work in a similar way to the SCN clock. To determine the role of the two canonical clock genes, mPer1 and mPer2, in the peripheral clock shift, stable HEK293EcR cell lines that can be induced and stably express these proteins were prepared. By regulating the expression of these proteins, it could be shown that induction of the clock genes, either mPer1 or mPer2 alone is not sufficient to cause clock phase shifting in these cells. Our real-time PCR analysis on these cells indicates that the induction of mPER proteins dampens the expression of the clock-specific transcription factor mBmal1. Altogether, our present data suggest that mPer1 and mPer2 may not function in clock shift or take part in differential roles on the peripheral circadian clock.

Synthesis, characterization, and toxicity of multi-walled carbon nanotubes functionalized with 4-hydroxyquinazoline

  • Tahermansouri, Hasan;Mirosanloo, Atieh;Keshel, Saeed Heidari;Gardaneh, Mossa
    • Carbon letters
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    • 제17권1호
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    • pp.45-52
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    • 2016
  • The attachment of 2-aminobenzamide to carboxylated multi-wall carbon nanotubes (MWCNTs)-COOH was achieved through the formation of amide bonds. Then, the functionalized MWCNTs, MWCNT-amide, were treated by phosphoryl chloride to produce MWCNT-quin. The products were characterized by Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, thermogravimetric analysis, derivative thermogravimetric, steady-state fluorescence spectroscopy, and solubility testing. MWCNT-quin showed photo-electronic properties, which is due to the attachment of the 4-hydroxyquinazoline groups to them as proved by steady-state fluorescence spectroscopy. This suggests intramolecular interactions between the tubes and the attached 4-hydroxyquinazoline. The toxicity of the samples was evaluated in human embryonic kidney HEK293 and human breast cancer SKBR3 cell lines, and the viable cell numbers were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) after the cells were cultured for 24 h. Cellular investigations showed that the modified MWCNTs, particularly MWCNT-quin, have considerably significant toxic impact on SKBR3 as compared to HEK293 at the concentration of 5 µg/mL.