Loss of density-dependent growth inhibition and dissociation of alpha-catenin from E-cadherin.

Normal human breast epithelial (HBE) cells at early (9th) passage ceased growth and formed a monolayer when they reached confluence. Immunostaining and Western blotting revealed that alpha- and beta-catenins colocalized and coprecipitated with E-cadherin, suggesting a complex formation of E-cadherin with alpha- and beta-catenins in early passage cells. In contrast, HBE cells at late (12-13th) passage did not cease growth after confluence but stratified. The late passage cells exhibited enhanced colony forming ability in soft agar compared with early passage cells, however, they had a definite proliferating lifespan and were primarily diploid. In late passage cells grown as multilayers, alpha-catenin was expressed but did not colocalize or coprecipitate with E-cadherin, suggesting its dissociation from E-cadherin. Coimmunoprecipitation of alpha-actinin with alpha-catenin suggested an indirect link between the E-cadherin-beta-catenin complex and alpha-actinin via alpha-catenin in early, but not in late passage cells. Beta-Catenin in late passage cells was tyrosine phosphorylated and was not dephosphorylated following the addition of inhibitors of tyrosine kinases. Inhibition of dephosphorylation of beta-catenin in early passage cells by vanadate, an inhibitor of protein tyrosine phosphatases, caused overgrowth of cells beyond the saturation density and loss of alpha-catenin from the E-cadherin-beta-catenin complex. The results suggest that E-cadherin requires its association with alpha-actinin-associated alpha-catenin to maintain epithelial monolayers and accomplish the density-dependent inhibition of growth. In addition, association between E-cadherin and alpha-catenin is suggested to be prevented by the presence of tyrosine phosphorylated beta-catenin which associates with E-cadherin.