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Anomalous features of EMT during keratinocyte transformation.

Geiger T, Sabanay H, Kravchenko-Balasha N, Geiger B, Levitzki A - PLoS ONE (2008)

Bottom Line: Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration.We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa.Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Institute of Life Science, The Hebrew University, Jerusalem, Israel.

ABSTRACT
During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

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Related in: MedlinePlus

Representative immuno-fluorescence images of primary keratinotyes, early and late HF1 cells.A. Staining with anti-beta-Catenin antibody. B. Staining with anti-Paxillin antibody. Bar = 15 µm. Enlargements of focal adhesions are shown below.
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pone-0001574-g002: Representative immuno-fluorescence images of primary keratinotyes, early and late HF1 cells.A. Staining with anti-beta-Catenin antibody. B. Staining with anti-Paxillin antibody. Bar = 15 µm. Enlargements of focal adhesions are shown below.

Mentions: Down-regulation of desmosomes during transformation is at least partially attributable to reduced mRNA expression of desmosomal genes. Affymetrix cDNA results revealed a ∼10-fold reduction in the expression of the desmosomal cadherins: desmocollins and desmogleins, and in periplakin in late HF1 cells as compared to the primary keratinocytes (Table 2). Significant, changes were also observed in other desmosomal components, including plakoglobin, plakophillin and envoplakin. These changes were already observed in early HF1 cells, and correlated well with the apparent changes in the formation of desmosomes and cytokeratin filaments. The changes observed in adherens junctions were more modest. Our microarray data showed a 2-fold decrease in E-cadherin expression in late HF1 cells, compared to the primary keratinocytes (Table 2), and no change in the levels of other adherens junction components, including alpha and beta-catenin and P-cadherin. Immuno-staining of cells with anti-beta-catenin antibody showed intense staining of the primary keratinocytes, over large overlapping areas at the cell periphery, similar to plakoglobin staining (Figure 2A). This apparent overlap was reduced in early HF1 cells, and completely abolished in late HF1 cells. Nevertheless, most of the beta-catenin was still concentrated in cell-cell junctions, and no significant translocation of beta-catenin to the nucleus could be detected. Reduced cell-cell adhesion coincided with the partial dissociation of cells from the ECM. Upon monitoring of focal adhesions/complexes by paxillin staining, we noted a small decrease in focal adhesion number in early HF1 cells, compared to the primary keratinocytes, and a more dramatic decrease in the progression to late HF1 cells (Figure 2B).


Anomalous features of EMT during keratinocyte transformation.

Geiger T, Sabanay H, Kravchenko-Balasha N, Geiger B, Levitzki A - PLoS ONE (2008)

Representative immuno-fluorescence images of primary keratinotyes, early and late HF1 cells.A. Staining with anti-beta-Catenin antibody. B. Staining with anti-Paxillin antibody. Bar = 15 µm. Enlargements of focal adhesions are shown below.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2215777&req=5

pone-0001574-g002: Representative immuno-fluorescence images of primary keratinotyes, early and late HF1 cells.A. Staining with anti-beta-Catenin antibody. B. Staining with anti-Paxillin antibody. Bar = 15 µm. Enlargements of focal adhesions are shown below.
Mentions: Down-regulation of desmosomes during transformation is at least partially attributable to reduced mRNA expression of desmosomal genes. Affymetrix cDNA results revealed a ∼10-fold reduction in the expression of the desmosomal cadherins: desmocollins and desmogleins, and in periplakin in late HF1 cells as compared to the primary keratinocytes (Table 2). Significant, changes were also observed in other desmosomal components, including plakoglobin, plakophillin and envoplakin. These changes were already observed in early HF1 cells, and correlated well with the apparent changes in the formation of desmosomes and cytokeratin filaments. The changes observed in adherens junctions were more modest. Our microarray data showed a 2-fold decrease in E-cadherin expression in late HF1 cells, compared to the primary keratinocytes (Table 2), and no change in the levels of other adherens junction components, including alpha and beta-catenin and P-cadherin. Immuno-staining of cells with anti-beta-catenin antibody showed intense staining of the primary keratinocytes, over large overlapping areas at the cell periphery, similar to plakoglobin staining (Figure 2A). This apparent overlap was reduced in early HF1 cells, and completely abolished in late HF1 cells. Nevertheless, most of the beta-catenin was still concentrated in cell-cell junctions, and no significant translocation of beta-catenin to the nucleus could be detected. Reduced cell-cell adhesion coincided with the partial dissociation of cells from the ECM. Upon monitoring of focal adhesions/complexes by paxillin staining, we noted a small decrease in focal adhesion number in early HF1 cells, compared to the primary keratinocytes, and a more dramatic decrease in the progression to late HF1 cells (Figure 2B).

Bottom Line: Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration.We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa.Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Institute of Life Science, The Hebrew University, Jerusalem, Israel.

ABSTRACT
During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

Show MeSH
Related in: MedlinePlus