Loss of E-Cadherin-Dependent Cell-Cell Adhesion due to Mutation of the β-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, ␤-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, ␣-catenin, and ␥-catenin and a 79-kDa band which was apparently smaller than that of normal ␤-catenin, indicating truncated ␤-catenin. The 79-kDa band was immunologically identified as ␤-catenin by using immunoblotting with anti-␤-catenin antibodies. Examination of ␤-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal ␤-catenin. The sequencing of PCR product for ␤-catenin confirmed deletion in 321 bases from nucleotides ؉82 to ؉402. Southern blotting of ␤-catenin DNA disclosed mutation at the genomic level. Expression vectors of ␤-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 immunofluorescence staining. From these results, it was concluded that in HSC-39 cells, impaired cell-cell adhesion is due to mutations in ␤-catenin which results in the dysfunction of E-cadherin.

Mutations of the β - and γ -catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas

“…However, our results also indicate that the β-catenin deletion is sufficiently large to eliminate detectable message. Deletion of the NH 2 -terminal part of β-catenin was described for 2 cell lines originating from signet ring cell carcinomas of the stomach (Oyama et al, 1994;Kawanishi et al, 1995), however, neither deletions nor mutations of exon 3 of the β-catenin gene were detected in the other 133 cell lines examined. β-catenin mutations within armadillo repeats 2 and 3 and outside the NH 2 -terminal regulatory domain were also described in two colorectal carcinoma cell lines with mutant APC (Ilyas et al, 1997).…”

Tyrosine phosphorylation of 100-130 kDa proteins in lung cancer correlates with poor prognosis

“…f-catenin is one of the components of the cadherin-catenin cell adhesion system and appears to play a crucial role in cadherin-dependent cell adhesion. In the huiman gastric cancer cell line, HSC-39, mutation in the f-catenin gene that resulted in complete abolishment of E-cadherin-dependent cell-cell adhesion was observed, and transfection of the f-ctenin gene in the cells fully recovered the cell-cell adhesion (Kawanishi et al, 1995). We have previously reported (Matsuyoshi et al, 1992;Hamaguchi et al, 1993a) that cadherin dependent cell-cell adhesion was strongly erturbed upon tyrosine phosphorylation of f-catenin in RSV-transformed cells where cadherins and catenins were expressed and formed complexes as normal cells.…”

IQGAP1, a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A

“…Accordingly, overexpression of IQGAP1 may be an another mechanism for the dissociation of α-catenin from the E-cadherin/ -catenin complex, resulting in disruption of the E-cadherin-mediated cell adhesion system. Interestingly, in HSC39 cells -one of the cell lines that overexpressed IQGAP1 -impaired cell-cell adhesion and the complete abolition of E-cadherin function have been reported (Oyama et al 1994;Kawanishi et al 1995;Jawhari et al 1999). One of the mechanisms of this disruption of Ecadherin function is due to -catenin mutation, resulting in protein truncation with loss of the α-catenin-binding site (Kawanishi et al 1995).…”