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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

Individual cell motility in primary keratinocytes, early and late HF1 cells.Cell motility movies were created by acquiring live-cell images every 15 min throughout 14 h of the experiment. Cell tracks, in yellow, were determined by marking cell nuclei in every frame of the movie. The figure shows the same cells at the beginning of the experiment (0 h), after 7 h, and at the end of the movie (14 h). Cell velocity was calculated by marking the cell nucleus in every frame, and following cell movement. Average velocity was calculated by an application within the UCSF PRIISM environment ((http://msg.ucsf.edu/ive). Errors represent standard error of >100 cells examined in 3 independent experiments.
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pone-0001574-g003: Individual cell motility in primary keratinocytes, early and late HF1 cells.Cell motility movies were created by acquiring live-cell images every 15 min throughout 14 h of the experiment. Cell tracks, in yellow, were determined by marking cell nuclei in every frame of the movie. The figure shows the same cells at the beginning of the experiment (0 h), after 7 h, and at the end of the movie (14 h). Cell velocity was calculated by marking the cell nucleus in every frame, and following cell movement. Average velocity was calculated by an application within the UCSF PRIISM environment ((http://msg.ucsf.edu/ive). Errors represent standard error of >100 cells examined in 3 independent experiments.

Mentions: One of the hallmarks of EMT is increased cell migration. We, on the other hand, noticed a marked reduction in the motility of cells during the progression from the primary keratinocytes and early HF1 to late HF1 cells. We examined individual cell motility by live-cell imaging, in which we followed live cells for 14 hours, acquiring phase contrast images every 15 min (Figure 3 and Movies S1, S2, S3). Tracking the migratory pathways, we calculated migration velocity for each cell type, based on randomly chosen cells (>100 cells). While the primary keratinocytes and early HF1 cells were highly motile (typical mean migration velocity: 0.35 µm/min), late HF1 cells barely moved throughout the experiment. Cell spreading, another feature that usually increases following EMT was also inhibited during the progression from the normal keratinocytes to late HF1 cells. We monitored cell spreading by acquiring live cell images every minute over two hours from cell plating until cells became fully spread on the plates. Figure 4A (and Movies S4, S5, S6) shows a representative cell, from each cell type at four time points starting before the beginning of attachment, to full spreading after 2 hours. Quantification of cell-spreading of ∼30 cells, showed that the highest spreading values were noted in the primary keratinocytes, and the lowest values were measured for late HF1 cells (Figure 4B). Early HF1 cells showed higher initial cell area, and the area of fully spread cells was similar to or higher than that of the primary keratinocytes (not shown), but the fold change is similar to that of late HF1 cells.


Anomalous features of EMT during keratinocyte transformation.

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

Individual cell motility in primary keratinocytes, early and late HF1 cells.Cell motility movies were created by acquiring live-cell images every 15 min throughout 14 h of the experiment. Cell tracks, in yellow, were determined by marking cell nuclei in every frame of the movie. The figure shows the same cells at the beginning of the experiment (0 h), after 7 h, and at the end of the movie (14 h). Cell velocity was calculated by marking the cell nucleus in every frame, and following cell movement. Average velocity was calculated by an application within the UCSF PRIISM environment ((http://msg.ucsf.edu/ive). Errors represent standard error of >100 cells examined in 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215777&req=5

pone-0001574-g003: Individual cell motility in primary keratinocytes, early and late HF1 cells.Cell motility movies were created by acquiring live-cell images every 15 min throughout 14 h of the experiment. Cell tracks, in yellow, were determined by marking cell nuclei in every frame of the movie. The figure shows the same cells at the beginning of the experiment (0 h), after 7 h, and at the end of the movie (14 h). Cell velocity was calculated by marking the cell nucleus in every frame, and following cell movement. Average velocity was calculated by an application within the UCSF PRIISM environment ((http://msg.ucsf.edu/ive). Errors represent standard error of >100 cells examined in 3 independent experiments.
Mentions: One of the hallmarks of EMT is increased cell migration. We, on the other hand, noticed a marked reduction in the motility of cells during the progression from the primary keratinocytes and early HF1 to late HF1 cells. We examined individual cell motility by live-cell imaging, in which we followed live cells for 14 hours, acquiring phase contrast images every 15 min (Figure 3 and Movies S1, S2, S3). Tracking the migratory pathways, we calculated migration velocity for each cell type, based on randomly chosen cells (>100 cells). While the primary keratinocytes and early HF1 cells were highly motile (typical mean migration velocity: 0.35 µm/min), late HF1 cells barely moved throughout the experiment. Cell spreading, another feature that usually increases following EMT was also inhibited during the progression from the normal keratinocytes to late HF1 cells. We monitored cell spreading by acquiring live cell images every minute over two hours from cell plating until cells became fully spread on the plates. Figure 4A (and Movies S4, S5, S6) shows a representative cell, from each cell type at four time points starting before the beginning of attachment, to full spreading after 2 hours. Quantification of cell-spreading of ∼30 cells, showed that the highest spreading values were noted in the primary keratinocytes, and the lowest values were measured for late HF1 cells (Figure 4B). Early HF1 cells showed higher initial cell area, and the area of fully spread cells was similar to or higher than that of the primary keratinocytes (not shown), but the fold change is similar to that of late HF1 cells.

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