E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation-sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism oftranscriptional inactivation of some genes. We found that E-cadherin expression-negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E-cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property.

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Reduced expression of E-cadherin has been regarded as one of the main molecular events involved in dysfunction of the cell-cell adhesion system, triggering cancer invasion and metastasis. However, even with a sufficient amount of E-cadherin, cell-cell adhesion is sometimes lost in "diffusely invasive" human carcinomas. Ten human cancer cell lines, showing growth characterized morphologically by loose cell-cell adhesion, were analyzed for possible structural abnormalities of their expressed E-cadherin. Four of the cell lines showed strong mRNA and protein expression with no nudeotide sequence abnormalities, and mRNA was absent in four other cell lines. mRNA sequence was abnormal in the remaining two gastric carcinoma cell lines. In MKN45 (poorly differentiated adenocarcinoma), this involved a 12-bp in-frame deletion with strong expression of mRNA and protein. In KATO-HI (signet ring cell carcinoma), there were four mRNA species with insertions of different sizes, among which the major transcripts (with a 7-bp insertion) caused a frameshift, and expression of both mRNA and protein was markediy reduced. In these two cell lines, DNA mutations were detected around exon-intron junctions, revealing that aberrant RNA splicing was the cause ofthe mRNA abnormalities. In addition, the wild-type allele of the E-cadherin locus was lost, suesting that the E-cadherin gene had been inactivated by two hits (mutation and allele loss), similar to the mechanism for inactivation of tumor suppressor genes.Elucidation of the molecular events involved in cancer invasion and metastasis, which determine the clinical prognosis of cancer patients, is of the utmost importance in cancer research. These phenomena should be triggered by the dissociation of cancer cells from the primary tumor through breakdown of the cell-cell adhesion system, of which one of the main component molecules is E-cadherin (for review, see ref.

We identified three novel human type-II classic cadherins, cadherin-7, -9 and -10, by cDNA cloning and sequencing, and confirmed that they interact with catenins and function in cell-cell adhesion as do other classic cadherins. Cell-cell binding activities of the eight human type-II classic cadherins, including the three new molecules, were evaluated by long-term cell-aggregation experiments using mouse L fibroblast clones transfected with the individual cadherins. The experiments indicated that all the type-II cadherins appeared to possess similar binding strength, which was virtually equivalent to that of E-cadherin. We next examined the binding specificities of the type-II cadherins using the mixed cell-aggregation assay. Although all of the type-II cadherins exhibited binding specificities distinct from that of E-cadherin, heterophilic interactions ranging from incomplete to complete were frequently observed among them. The combinations of cadherin-6 and -9, cadherin-7 and -14, cadherin-8 and -11, and cadherin-9 and -10 interacted in a complete manner, and in particular cadherin-7 and -14, and cadherin-8 and -11 showed an indistinguishable binding specificity against other cadherin subclasses, at least in this assay system. Although these data were obtained from an in vitro study, they should be useful for understanding cadherin-mediated mechanisms of development, morphogenesis and cell-cell interactions in vivo.

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