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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

Disruption of Actin organization in late HF1 cells is associated with reduced Rac1 activity.A. Representative images of phalloidin staining for F-Actin in the primary keratinocytes, early and late HF1 cells. Bar = 15 µm. B. Western blot analysis of Rac1. Upper panel shows activated, GTP-loaded Rac1 pulled-down with GST-PBD. Lower panel shows total Rac1 levels in whole cell lysates before pull down. Graph shows average active, GTP-loaded Rac1. Error bars represent standard errors of three independent experiments.
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pone-0001574-g005: Disruption of Actin organization in late HF1 cells is associated with reduced Rac1 activity.A. Representative images of phalloidin staining for F-Actin in the primary keratinocytes, early and late HF1 cells. Bar = 15 µm. B. Western blot analysis of Rac1. Upper panel shows activated, GTP-loaded Rac1 pulled-down with GST-PBD. Lower panel shows total Rac1 levels in whole cell lysates before pull down. Graph shows average active, GTP-loaded Rac1. Error bars represent standard errors of three independent experiments.

Mentions: Both cell spreading and migration are highly dependent on the dynamics of the actin cytoskeleton. Staining of the primary keratinocytes for F-actin, using phalloidin, showed ordered circumferential actin fibers forming lamellipodial protrusions from the cell surface (Figure 5A). Progression to early HF1 cells induced some disorder in actin organization, yet it did not affect the enrichment of actin along the cell periphery. A dramatic change occurred upon progression to late HF1 cells, which was characterized by poor actin organization, a marked absence of lamellipodial protrusions, and appearance of tangled microvilli (Figure 5A). Lamellipodia formation is known to be mediated primarily by the activity of the Rho-family GTPase Rac1. We therefore measured Rac1 activity by a pull-down assay, using PAK1 binding domain (PBD) fused to GST. We detected a 2-fold drop in Rac1 activity in late HF1 cells, compared to either the primary keratinocytes or early HF1 cells (Figure 5B).


Anomalous features of EMT during keratinocyte transformation.

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

Disruption of Actin organization in late HF1 cells is associated with reduced Rac1 activity.A. Representative images of phalloidin staining for F-Actin in the primary keratinocytes, early and late HF1 cells. Bar = 15 µm. B. Western blot analysis of Rac1. Upper panel shows activated, GTP-loaded Rac1 pulled-down with GST-PBD. Lower panel shows total Rac1 levels in whole cell lysates before pull down. Graph shows average active, GTP-loaded Rac1. Error bars represent standard errors of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001574-g005: Disruption of Actin organization in late HF1 cells is associated with reduced Rac1 activity.A. Representative images of phalloidin staining for F-Actin in the primary keratinocytes, early and late HF1 cells. Bar = 15 µm. B. Western blot analysis of Rac1. Upper panel shows activated, GTP-loaded Rac1 pulled-down with GST-PBD. Lower panel shows total Rac1 levels in whole cell lysates before pull down. Graph shows average active, GTP-loaded Rac1. Error bars represent standard errors of three independent experiments.
Mentions: Both cell spreading and migration are highly dependent on the dynamics of the actin cytoskeleton. Staining of the primary keratinocytes for F-actin, using phalloidin, showed ordered circumferential actin fibers forming lamellipodial protrusions from the cell surface (Figure 5A). Progression to early HF1 cells induced some disorder in actin organization, yet it did not affect the enrichment of actin along the cell periphery. A dramatic change occurred upon progression to late HF1 cells, which was characterized by poor actin organization, a marked absence of lamellipodial protrusions, and appearance of tangled microvilli (Figure 5A). Lamellipodia formation is known to be mediated primarily by the activity of the Rho-family GTPase Rac1. We therefore measured Rac1 activity by a pull-down assay, using PAK1 binding domain (PBD) fused to GST. We detected a 2-fold drop in Rac1 activity in late HF1 cells, compared to either the primary keratinocytes or early HF1 cells (Figure 5B).

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