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.
Tsukatani
Y
Y
Department of Biochemistry, Kanagawa Cancer Center Research Institute, Yokohama, Japan.
Suzuki
K
K
Takahashi
K
K
D000185:Actinin / Q000378:metabolism; D000911:Antibodies, Monoclonal; D001940:Breast; D015820:Cadherins / Q000276:immunology / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015534:Trans-Activators; D014638:Vanadates / Q000494:pharmacology; D051178:alpha Catenin; D051176:beta Catenin
D000911:Antibodies, Monoclonal; C495273:CTNNA1 protein, human; C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D051178:alpha Catenin; D051176:beta Catenin; D000185:Actinin; D014638:Vanadates; D011505:Protein-Tyrosine Kinases