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The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Izawa G, Kobayashi W, Haraguchi M, Sudo A, Ozawa M - Int. J. Mol. Med. (2015)

Bottom Line: 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.Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT.However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
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 Snail‑induced 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.

No MeSH data available.


Related in: MedlinePlus

Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.
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f3-ijmm-36-01-0166: Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.

Mentions: Previous analysis of the E-cadherin gene revealed that its proximal promoter contains CpG islands, which are targets for methylation during TGF-β-induced EMT (26,27). Therefore, in this study, we examined the methylation status of the E-cadherin promoter. No significant de novo DNA methylation was detected at the E-cadherin promoter in the Snail cells as compared to the control neo cells, as measured by bisulfite sequencing (Fig. 3). These results were consistent with the observation that no significant downregulation of E-cadherin expression was detected in the Snail cells.


The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Izawa G, Kobayashi W, Haraguchi M, Sudo A, Ozawa M - Int. J. Mol. Med. (2015)

Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494602&req=5

f3-ijmm-36-01-0166: Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.
Mentions: Previous analysis of the E-cadherin gene revealed that its proximal promoter contains CpG islands, which are targets for methylation during TGF-β-induced EMT (26,27). Therefore, in this study, we examined the methylation status of the E-cadherin promoter. No significant de novo DNA methylation was detected at the E-cadherin promoter in the Snail cells as compared to the control neo cells, as measured by bisulfite sequencing (Fig. 3). These results were consistent with the observation that no significant downregulation of E-cadherin expression was detected in the Snail cells.

Bottom Line: 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.Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT.However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
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 Snail‑induced 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.

No MeSH data available.


Related in: MedlinePlus