The Rho/Rho-kinase and the phosphatidylinositol 3-kinase pathways are essential for spontaneous locomotion of Walker 256 carcinosarcoma cells

Signal transduction pathways controlling spontaneous locomotion of Walker c arcinosarcoma cells are not well understood. We have therefore investigated the role of signalling proteins in development of polarity and locomotion of these cells, Treatment of the cells with 100 ng/ml pertussis toxin had n o significant effect on the percentage of polarized cells, In contrast, 2 d ifferent phosphatidylinositol 3-kinase inhibitors (wortmannin and LY-294002 ) markedly reduced the proportion of polarized cells. Spontaneous locomotio n of the cells was also significantly inhibited by these two inhibitors. in agreement with these data, we observed localization of the p85 alpha subun it of phosphatidylinositol 3-kinase predominantly in the membrane fraction of Walker carcinosarcoma cells, indicating constitutive activation of this enzyme, We also investigated a role of Rho family proteins. Spontaneous dev elopment of polarity was almost completely suppressed by electroporation of the cells in the presence of 4 mug/ml C-3 exoenzyme, which specifically AD P-ribosylates and inactivates Rho, Two downstream targets of Rho, the Rho-a ctivated kinases I and II, were also detected predominantly in the particul ate membrane fraction, suggesting constitutive activation. A specific Rho-k inase inhibitor (Y-27632) blocked spontaneous polarization and migration in a concentration-dependent manner. Our results indicate that constitutive a ctivation of the Rho/Rho kinase pathway and of phosphatidylinositol 3-kinas e plays an essential part in spontaneous development of polarity and cell l ocomotion of these cells. (C) 2001 Wiley-Liss. Inc.