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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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Src dependent Rac1 activity is essential for cell motility.A. Late HF1 cells were transfected with active Rac1 (Rac1L61) or with GFP as control. Cell motility movies were created by acquiring live-cell images every 15 min throughout 5 h of the experiment. 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). Graph shows fold change in velocity, normalized to the control cells. Errors represent standard error of >100 cells. B. Primary keratinocytes were treated with 5 µM PP1 or 5 µM LY294002 for 4 h before cell lysis and Rac1 activity assay. Graphs show quantification of Rac1-GTP. Error bars represent standard deviations of two experiments. C. Quantification of cell motility in the presence of PP1 or LY294002. Migration rate was examined as in A. Error bars represent standard error of >50 cells.
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pone-0001574-g006: Src dependent Rac1 activity is essential for cell motility.A. Late HF1 cells were transfected with active Rac1 (Rac1L61) or with GFP as control. Cell motility movies were created by acquiring live-cell images every 15 min throughout 5 h of the experiment. 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). Graph shows fold change in velocity, normalized to the control cells. Errors represent standard error of >100 cells. B. Primary keratinocytes were treated with 5 µM PP1 or 5 µM LY294002 for 4 h before cell lysis and Rac1 activity assay. Graphs show quantification of Rac1-GTP. Error bars represent standard deviations of two experiments. C. Quantification of cell motility in the presence of PP1 or LY294002. Migration rate was examined as in A. Error bars represent standard error of >50 cells.

Mentions: In order to examine whether low Rac1 activity in late HF1 cells is responsible for the reduced migration of these cells as compared to the primary keratinocytes, we transfected late HF1 cells with active-Rac (Rac1L61) and examined their migration velocity. Comparison of Rac1L61-transfected HF1 cells to GFP-transfected controls showed 1.4 fold increase in migration velocity (Figure 6A). When we consider that transfection efficiency was ∼30%, we can estimate a ∼2 fold increase in velocity as a result of active Rac1 expression. We further examined the signaling components, which may affect Rac1 activity. Rac1 is modulated by the activities of its exchange factors (GEFs) [22], which are regulated mainly by Src family kinases and by PI3K [22], [23]. In order to examine which of these kinases control Rac1 activity in our system, we examined the effect of the PI3K inhibitor LY294002 or of the Src inhibitor PP1 on Rac1 activity. Src inhibition with PP1 induced an over 2-fold decrease in Rac1 activity, whereas LY294002 had no apparent effect (Figure 6B). In agreement with these results, we found that PP1, but not LY294002, inhibited the migration of the primary keratinocytes (Figure 6C). Therefore Src activity in the primary keratinocytes is responsible for Rac1 activation and cell migration.


Anomalous features of EMT during keratinocyte transformation.

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

Src dependent Rac1 activity is essential for cell motility.A. Late HF1 cells were transfected with active Rac1 (Rac1L61) or with GFP as control. Cell motility movies were created by acquiring live-cell images every 15 min throughout 5 h of the experiment. 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). Graph shows fold change in velocity, normalized to the control cells. Errors represent standard error of >100 cells. B. Primary keratinocytes were treated with 5 µM PP1 or 5 µM LY294002 for 4 h before cell lysis and Rac1 activity assay. Graphs show quantification of Rac1-GTP. Error bars represent standard deviations of two experiments. C. Quantification of cell motility in the presence of PP1 or LY294002. Migration rate was examined as in A. Error bars represent standard error of >50 cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215777&req=5

pone-0001574-g006: Src dependent Rac1 activity is essential for cell motility.A. Late HF1 cells were transfected with active Rac1 (Rac1L61) or with GFP as control. Cell motility movies were created by acquiring live-cell images every 15 min throughout 5 h of the experiment. 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). Graph shows fold change in velocity, normalized to the control cells. Errors represent standard error of >100 cells. B. Primary keratinocytes were treated with 5 µM PP1 or 5 µM LY294002 for 4 h before cell lysis and Rac1 activity assay. Graphs show quantification of Rac1-GTP. Error bars represent standard deviations of two experiments. C. Quantification of cell motility in the presence of PP1 or LY294002. Migration rate was examined as in A. Error bars represent standard error of >50 cells.
Mentions: In order to examine whether low Rac1 activity in late HF1 cells is responsible for the reduced migration of these cells as compared to the primary keratinocytes, we transfected late HF1 cells with active-Rac (Rac1L61) and examined their migration velocity. Comparison of Rac1L61-transfected HF1 cells to GFP-transfected controls showed 1.4 fold increase in migration velocity (Figure 6A). When we consider that transfection efficiency was ∼30%, we can estimate a ∼2 fold increase in velocity as a result of active Rac1 expression. We further examined the signaling components, which may affect Rac1 activity. Rac1 is modulated by the activities of its exchange factors (GEFs) [22], which are regulated mainly by Src family kinases and by PI3K [22], [23]. In order to examine which of these kinases control Rac1 activity in our system, we examined the effect of the PI3K inhibitor LY294002 or of the Src inhibitor PP1 on Rac1 activity. Src inhibition with PP1 induced an over 2-fold decrease in Rac1 activity, whereas LY294002 had no apparent effect (Figure 6B). In agreement with these results, we found that PP1, but not LY294002, inhibited the migration of the primary keratinocytes (Figure 6C). Therefore Src activity in the primary keratinocytes is responsible for Rac1 activation and cell migration.

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