EL INTERES SUPERIOR DEL MENOR.

Transformation of breast epithelial cells with Ras results indirectly in increased tryosine phosphorylation o f p-catenin and p i 20^^^. These cells exhibit decreased cell-cell interactions and altered adherens junctions with less organized E-cadherin localization. In addition, tyrosine phosphorylated p-catenin does not interact with E-cadherin and shows increased detergent-solubility suggesting a decreased association w ith the actin cytoskeleton (Kinch et al., 1995).

It has been shown that the Ras target AF-6 can interact with ZO-1 in epithelial cells and has a distribution similar to ZO-1. AF-6 co-localizes with ZO-1 at the apical site sites of the cell-cell contact in MDCK cells. In cells lacking tight junctions such as non-epithelial cells, ZO-1 co-localizes with cadherin (Howarth et al., 1992, Itoh et al., 1993) and also with AF-6. The overexpression of Ras in R ati fibroblasts or PC12 cells, results in the

perturbation of intercellular contacts and decreased accumulation of AF-6 and ZO-1 at the cell surface. It is possible therefore that AF-6 participates in the regulation of intercellular contacts, including tight junctions, via direct interaction with ZO-1 downstream o f Ras (Yamamoto et al., 1997). Adhesion of epithelial cells to ECM has been previously shown to lead to protection from apoptosis via the activation of PI3K and protein kinase B (PKB) also named Akt. PKB acts downstream from PI3K in mediating cell survival in response to several stresses or growth factor deprivation (Alessi and Cohen, 1998). In addition, E-cadherin has been previously shown to mediate aggregation-dependent cell survival in a variety of experimental settings (Kantak and Kramer, 1998, Day et al., 1999).

Recently, it has been shown that E-cadherin-mediated cell adhesion promotes a PI3K- dependent increase in the activity of PKB and rapid translocation of PKB into the nucleus (Pece et al., 1999). PI3K mediates the translocation o f PKB into the nucleus, where it parlicipitates in the regulation of gene expression (Alessi and Cohen, 1998). In fact, E- cadherin physically associates with p85a in immunoprécipitation experiments and can stimulate the activation o f the PI3K/PKB cascade (Pece et al., 1999). Further evidence for a role o f E-cadherin in the regulation o f PKB activity was found in M DCK cells where PKB localizes not only to cell-matrix but also to cell-cell contacts where it co- localizes with p-catenin. This localization is disrupted when PI3K inhibitors are present. Consequently, it was proposed that PI3K mediated cell survival is not only dependent on cell-matrix but also on cell-cell interactions (Watton and Downward, 1999).

These data indicate that E-cadherin might have a dual role in regulating the relationship between intra- and extracellular environm ent as it controls the cell adhesion state simultaneously with the cell fate, cell death by apoptosis or cell survival. In addition, the observation that cadherin-mediated adhesion might transduce signals to the nucleus raises the intriguing possibility that changes in cell adhesion modulate gene expression and thus cell fate. Given the findings that gain of E-cadherin function blocks the transition from adenoma to carcinoma and that loss of cadherin-mediated cell adhesion induces invasion o f carcinoma cells (Perl et al., 1998), it could be speculated that these cell-adhesion- mediated signals govern complete genetic programmes, such as mesenchymal-epithelial conversion during embryonic development or progression from adenoma to carcinoma.

1.3.4.1 Regulation of adherens junctions by Rho GTPases

In epithelial cells, the effects of Rho GTPases seem contradictory, since they have been shown to be required for cell migration as well as for cell-cell adhesion. In kératinocytes, the activity of both Rho and Rac proteins is required for the assembly o f adherens

junctions during Ca^^-switch experiments (Braga et al., 1997, Takaishi et al., 1997). In polarized M DCK monolayers, R acl and RhoA regulate adherens junctions integrity and cell polarity (Jou and Nelson, 1998). In addition, Tiam-1 and V12Rac inhibit HGF/SF- induced scattering of MDCK cells by increasing E-cadherin-mediated cell-cell adhesion (Hordijk et al., 1997). In addition, Tiam-1-Rac signalling inhibits the motility and invasion of Ras-transformed M DCK cells due to restoration of E-cadherin-mediated adhesions. These data suggest that the activity of Rho-like GTPases is required to maintain cell-cell adhesion of a polarized epithelium. In contrast to these results are the findings that N17Rac inhibits the HGF/SF-induced membrane ruffling and lamellipodium formation in MDCK cells, suggesting a role for Rac in cell motility (Ridley et al., 1995). V 12Rac and V 12Cdc42 also stimulate the motihty of T47D mammary carcinoma cells in a manner which requires PI3K activity (Keely et al., 1997). Thus, in addition to a role for Rac in the establishment and maintenance o f E-cadherin mediated adhesions, Rac also plays an essential role in migratory responses of epithelial cells.

An explanation for these dual roles of Rac has been given by (Sander et al., 1998). They find that different Rac-mediated cellular responses are dependent on the cell substrate. They further suggest that Rac activation inhibits migration o f Ras-transformed MDCK cells by establishing E-cadherin-mediated cell-cell adhesion on fibronection and laminin- 1, but promotes motility on collagen substrates when E-cadherin mediated adhesions are weaker. Different intracellular localization of Rac could determine whether Rac stimulates E-cadherin mediated cell-cell adhesion or lamellipodial extension.

Finally, IQ G A Pl regulates cell-cell adhesion through Rac and Cdc42. Overexpresson of IQGAPl appears to induce the dissocation of a-catenin from the E-cadherin cell adhesion complex by competing with a-catenin's binding to p-catenin. This results in the loss of E- cadherin-mediated cell adhesion (Kuroda et al., 1998). However, it is possible that IQ G A Pl exerts its effects on cell-cell adhesion in this study primarily by titrating Cdc42 and Rac o f the system.

1.3.4.2 Regulation of tight junction integrity by Rho GTPases

Several studies have implicated Rho GTPases in the organization of tight junctions. For example, the organization of the perijunctional actin cytoskeleton and Z O -1 is disrupted after exposing T84 intestinal epithelial cells to C3 transferase (Nusrat et al., 1995). Concomitantly, the tight junction gate function is disturbed. Another study shows that constitutively expressed mutant RhoA genes do not change tight junction morphology in M DCK cells but the formation o f tight junctions is inhibited by the injection o f C3

transferase in Ca^+-switch experiments. However, ultrastructural or functional analyses o f the tight junctions are not included in this study (Takaishi et al., 1997).

In contrast, inducible expression of mutant Rho GTPases in MDCK cells shows that the tight junction gate function is disrupted upon expression of RhoA and R acl mutants in a dose-dependent and reversible m anner when monitored by TER m easurem ents. Constitutively active RhoA and R acl mutants induce disorganization of tight junction strands and protein (occludin, ZO-1, actin) distribution, while dominant negative RhoA and R acl mutants affect neither tight junction strand organization as revealed by freeze- fracture EM or protein distribution. However, dominant negative mutants were expressed to a lower level than activated mutants in these experiments (Jou et al., 1998).