Loss of E-Cadherin-Dependent Cell-Cell Adhesion due to Mutation of the b-Catenin Gene in a Human

Detachment of cell-cell adhesion is indispensable for the first step of invasion and metastasis of cancer. This mechanism is frequently associated with the impairment of either E-cadherin expression or function. However, mechanisms of such abnormalities have not been fully elucidated. In this study, we demonstrated that the function of E-cadherin was completely abolished in the human gastric cancer cell line HSC-39, despite the high expression of E-cadherin, because of mutations in one of the E-cadherin-associated cytoplasmic proteins, b-catenin. Although immunofluorescence staining of HSC-39 cells by using an anti-E-cadherin antibody (HECD-1) revealed the strong and uniform expression of E-cadherin on the cell surface, cell compaction and cell aggregation were not observed in this cell. Western blotting (immunoblotting) using HECD-1 exhibited a 120-kDa band which is equivalent to normal E-cadherin. Northern (RNA) blotting demonstrated a 4.7-kb band, the same as mature E-cadherin mRNA. Immunoprecipitation of metabolically labeled proteins with HECD-1 revealed three bands corresponding to E-cadherin, a-catenin, and g-catenin and a 79-kDa band which was apparently smaller than that of normal b-catenin, indicating truncated b-catenin. The 79-kDa band was immunologically identified as b-catenin by using immunoblotting with anti-b-catenin antibodies. Examination of b-catenin mRNA by the reverse transcriptase-PCRmethod revealed a transcript which was shorter than that of normal b-catenin. The sequencing of PCR product for b-catenin confirmed deletion in 321 bases fro