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1.
Biochem Biophys Rep ; 15: 13-18, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29998192

RESUMO

Transcription factor lymphoid-enhancer-binding factor 1 (LEF-1) is a key molecule in the Wnt/ß-catenin signaling pathway. Slug is one of the Wnt/ß-catenin target genes and can induce epithelial-mesenchymal transition (EMT). Previously, we have shown that not only wild-type LEF-1 but also LEF-1 lacking the amino-terminal ß-catenin-binding region can induce EMT, suggesting that LEF-1 acts independently of ß-catenin. Because it has been reported that LEF-1 interacts with ß-catenin outside the amino-terminal domain, namely, in the middle part of the molecule, the possible participation of ß-catenin has not been formally ruled out. To determine the involvement of ß-catenin in the LEF-1-induced EMT, we produced MDCK cells with a deletion of the ß-catenin gene and then expressed LEF-1 in the cells. We found that LEF-1 induced EMT in those cells. In the absence of ß-catenin, γ-catenin has been shown to take over the role of ß-catenin. To examine this possibility, we first established MDCK cells with a double knockout of ß-catenin and γ-catenin genes and then expressed LEF-1 in these cells. We found that LEF-1 can induce EMT in these cells; therefore, we conclude that neither ß-catenin nor γ-catenin expression is necessary for the LEF-1-mediated induction of EMT.

2.
Int J Mol Med ; 37(6): 1521-7, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27121032

RESUMO

The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10-15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells.


Assuntos
Linhagem da Célula/genética , Células Epiteliais/metabolismo , Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Adenoviridae/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Transformada , Desmogleína 2/genética , Desmogleína 2/metabolismo , Células Epiteliais/citologia , Células Epiteliais/virologia , Células HEK293 , Humanos , Queratina-5/genética , Queratina-5/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Rim , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/virologia , Ocludina/genética , Ocludina/metabolismo , Suínos , Vimentina/genética , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
3.
Biochem Biophys Rep ; 8: 120-126, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28955947

RESUMO

The epithelial-mesenchymal transition (EMT) is a fundamental characteristic of carcinoma cells. EMT is generally associated with a change in cellular morphology from cobblestone to spindle shape, reduced expression of epithelial markers such as E-cadherin, and enhanced expression of mesenchymal markers such as N-cadherin. This EMT-associated reciprocal expression of E-cadherin and N-cadherin has been called the "cadherin switch". Downregulation of E-cadherin enables cells to dissociate from colonies while upregulation of N-cadherin is associated with increased invasiveness. The transcription factor Snail1 induces these changes in various epithelial cell lines, including canine MDCK cells and human A431 cells. In the present study, we introduced a Snail1 expression vector into human DLD-1 cells and isolated stable transfectants. These cells showed changes in morphology, reduced expression of epithelial marker E-cadherin and occludin, and elevated invasion and migration. However, neither expression of N-cadherin protein nor its corresponding mRNA was detected. Therefore, elevated N-cadherin expression is not required for invasiveness of the cells.

4.
Int J Mol Med ; 36(1): 166-72, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25998899

RESUMO

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail­induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the downregulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.


Assuntos
Caderinas/biossíntese , Agregação Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Homeodomínio/biossíntese , Fatores de Transcrição/biossíntese , Animais , Caderinas/genética , Bovinos , Adesão Celular/fisiologia , Linhagem Celular , Metilação de DNA , Desmogleínas/biossíntese , Cães , Regulação Neoplásica da Expressão Gênica , Células Madin Darby de Rim Canino , Regiões Promotoras Genéticas/genética , Fatores de Transcrição da Família Snail , Dedos de Zinco/fisiologia
5.
Biochem Biophys Res Commun ; 458(3): 608-613, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25681770

RESUMO

The epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. Snail is an EMT-inducer whose expression in several different epithelial cells, e.g., Madin-Darby canine kidney (MDCK), leads to EMT. To further understand EMT induced by Snail expression, the Cre-loxP site-specific recombination system was used to investigate its reversibility. Transfection of MDCK cells with loxP-flanked Snail (Snail-loxP) resulted in EMT induction, which included the acquisition of a spindle-shaped fibroblastic morphology, the downregulation of epithelial markers, and the upregulation of mesenchymal markers. DNA methylation of the E-cadherin promoter, which often occurs during E-cadherin downregulation, was not observed in Snail+ cells. After Cre-mediated excision of Snail-loxP, the cells reacquired an epithelial morphology, upregulated epithelial markers, and downregulated mesenchymal markers. Thus, EMT induced by Snail expression was reversible.


Assuntos
Bacteriófago P1/enzimologia , Transição Epitelial-Mesenquimal , Integrases/genética , Fatores de Transcrição/genética , Animais , Caderinas/genética , Linhagem Celular , Metilação de DNA , Cães , Expressão Gênica , Vetores Genéticos/genética , Humanos , Células Madin Darby de Rim Canino , Regiões Promotoras Genéticas , Fatores de Transcrição da Família Snail , Transgenes
6.
FEBS J ; 281(24): 5479-97, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25283713

RESUMO

Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction.


Assuntos
Motivos de Aminoácidos , Sequência Conservada , Farnesil-Difosfato Farnesiltransferase/metabolismo , Methylococcus capsulatus/enzimologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Farnesil-Difosfato Farnesiltransferase/química , Cinética , Mutagênese Sítio-Dirigida
7.
PLoS One ; 9(8): e105313, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25121615

RESUMO

The downregulation of E-cadherin function has fundamental consequences with respect to cancer progression, and occurs as part of the epithelial-mesenchymal transition (EMT). In this study, we show that the expression of the Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domain in cells inhibited the cell surface localization of endogenous E-cadherin, leading to morphological changes, the inhibition of junctional assembly and cell dissociation. These changes were associated with increased cell migration, but were not accompanied by the down-regulation of epithelial markers and up-regulation of mesenchymal markers. Thus, these changes cannot be classified as EMT. The cadherin cytoplasmic domain interacted with ß-catenin or plakoglobin, reducing the levels of ß-catenin or plakoglobin associated with E-cadherin, and raising the possibility that ß-catenin and plakoglobin sequestration by these constructs induced E-cadherin intracellular localization. Accordingly, a cytoplasmic domain construct bearing mutations that weakened the interactions with ß-catenin or plakoglobin did not impair junction formation and adhesion, indicating that the interaction with ß-catenin or plakoglobin was essential to the potential of the constructs. E-cadherin-α-catenin chimeras that did not require ß-catenin or plakoglobin for their cell surface transport restored cell-cell adhesion and junction formation.


Assuntos
Caderinas/metabolismo , Desmossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Junções Íntimas/metabolismo , Animais , Caderinas/química , Caderinas/genética , Adesão Celular , Comunicação Celular/genética , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Transição Epitelial-Mesenquimal/genética , Expressão Gênica , Genes Reporter , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico , beta Catenina/metabolismo , gama Catenina/metabolismo
8.
Biochem Biophys Res Commun ; 442(1-2): 133-8, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24269234

RESUMO

The epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. LEF-1 is a member of the lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) family of DNA-binding transcription factors, which interact with nuclear ß-catenin and act as central transcriptional mediators of Wnt signaling. To investigate the role of LEF-1 in EMT, we generated stable LEF-1 transfectants using MDCK cells. The transfectants had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. These EMT changes were accompanied by the downregulation of an epithelial marker protein, E-cadherin, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin. Consistent with these observations, the mRNA levels of Slug, ZEB1, and ZEB2-EMT-related transcription factors-increased significantly. Although the N-terminally deleted mutant LEF-1 cannot interact with ß-catenin, it retained the ability to induce EMT. Consistent with these observations, neither the expression of a dominant negative ß-catenin/engrailed chimera, nor the expression of a cytoplasmic domain of E-cadherin that sequesters ß-catenin from binding to LEF/TCF, reversed LEF-1-induced EMT. Together, these data indicated that the nuclear function of ß-catenin was not necessary for the induction of Slug, ZEB1, and ZEB2 expression leading to EMT.


Assuntos
Transição Epitelial-Mesenquimal/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , beta Catenina/metabolismo , Animais , Cães , Células HEK293 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Células Madin Darby de Rim Canino , Camundongos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Dedos de Zinco , beta Catenina/antagonistas & inibidores , beta Catenina/genética
9.
Biochem Biophys Res Commun ; 430(3): 1078-82, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23261444

RESUMO

Cutaneous spindle cell squamous cell carcinoma (SCC) is a rare, but highly malignant variant of SCC. The presence of spindle-shaped cells with a sarcomatous appearance, which are derived from squamous cells, suggests that these cells are produced as a result of epithelial-mesenchymal transition (EMT). EMT is a complex process in which epithelial cells lose their polarity and cell-cell contacts, while also acquiring increased motility and invasiveness. Snail regulates EMT by binding to proximal E-boxes in the promoter region of E-cadherin and repressing its transcription. When examining the expression of EMT markers and Snail in spindle cell SCCs, we found that cyclooxygenase-2 (COX-2) expression was down-regulated. Since it has been shown that COX-2 is constitutively overexpressed in a variety of malignancies, including colon, gastric, and lung carcinomas, the down-regulation of COX-2 expression was unexpected. The presence of E-box-like sequences in the promoter region of COX-2 prompted us to perform a more detailed analysis. We introduced a Snail expression vector into keratinocyte-derived cell lines (HaKaT, HSC5, and A431 cells), and isolated stable transfectants. We determined that COX-2 expression was down-regulated in cells expressing Snail. Consistent with these observations, reporter assays revealed that COX-2 promoter activity was repressed upon Snail overexpression. Thus Snail down-regulates COX-2 in these cells.


Assuntos
Carcinoma de Células Escamosas/patologia , Ciclo-Oxigenase 2/genética , Transição Epitelial-Mesenquimal/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Dedos de Zinco , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Regiões Promotoras Genéticas , Neoplasias Cutâneas/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
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