title: Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39.
abstract: 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, beta-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, alpha-catenin, and gamma-catenin and a 79-kDa band which was apparently smaller than that of normal beta-catenin, indicating truncated beta-catenin. The 79-kDa band was immunologically identified as beta-catenin by using immunoblotting with anti-beta-catenin antibodies. Examination of beta-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal beta-catenin. The sequencing of PCR product for beta-catenin confirmed deletion in 321 bases from nucleotides +82 to +402. Southern blotting of beta-catenin DNA disclosed mutation at the genomic level. Expression vectors of Beta-catenin were introduced into HSC-39 cells by transfection. In the obtained transfectants, E-cadherin-dependent cell-cell adhesiveness was recovered, as revealed by cell compaction, cell aggregation, and immunoflourescence staining. From these results, it was concluded that in HSC-39 cells, impaired cell-cell adhesion is due to mutations in beta-catenin which results in the dysfunction of E-cadherin.
authors.affiliation: Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Kawanishi
authors.forename: J
authors.initials: J
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Sasaki
authors.forename: S
authors.initials: S
authors.lastname: Fujii
authors.forename: N
authors.initials: N
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Niitsu
mesh_terms: D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis / Q000096:biosynthesis / Q000502:physiology*; D002448:Cell Adhesion; D002449:Cell Aggregation; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013274:Stomach Neoplasms; D015534:Trans-Activators; D014162:Transfection; D014407:Tumor Cells, Cultured; D051176:beta Catenin
chemical_list: D000911:Antibodies, Monoclonal; C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015534:Trans-Activators; D051176:beta Catenin