Fukudome Y, Yanagihara K, Takeichi M, Ito F, Shibamoto S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, Japan.
Int J Cancer. 2000 Nov 15;88(4):579-83. doi: 10.1002/1097-0215(20001115)88:4<579::aid-ijc10>3.0.co;2-u.
The cell-cell adhesion molecule E-cadherin plays an essential role in the maintenance and function of epithelial tissues. Altered expression of E-cadherin has been implicated in tumor invasion. Analysis of mutations of the human E-cadherin gene in gastric carcinoma of the diffuse type has revealed that deletion of exon 8 or 9 in its cDNA appears to be predominant. In this study, we carried out structural and functional analyses of a mutant form of E-cadherin in a cell line, HSC45-M2, established from a human signet ring-cell carcinoma. Although immunohistochemical analysis showed that the mutant cadherin was localized at cell-cell contact sites as usually seen with the wild type, these cells did not form compact colonies. HSC45-M2 cells expressed aberrant E-cadherin with an m.w. larger than that of the wild type. In these cells, we found deletion of the exon 9-intron 9 boundary including the splicing donor site in E-cadherin genomic DNA. RT-PCR indicated 2 transcripts, which appeared to be caused by the splicing defect. Northern blotting, however, showed that the transcript lacking exon 9 was predominantly detected in these cells. The electrophoretic mobilities on SDS-PAGE of the mutant E-cadherin protein in HSC45-M2 cells and the protein expressed from cDNA lacking exon 9 appeared identical. Analysis of the amino-terminal region of the mutant E-cadherin protein revealed that the cadherin was capable of becoming a mature form by removal of its amino-terminal peptide. However, the mutant E-cadherin was susceptible to trypsinization in the presence of Ca(2+), which is not the case for wild-type E-cadherin, suggesting that the mutant E-cadherin frequently found in diffuse-type gastric carcinoma may have lost its Ca(2+)-binding ability, leading to disruption of the tight cell-cell association.
细胞间黏附分子E-钙黏蛋白在上皮组织的维持和功能中起着至关重要的作用。E-钙黏蛋白表达的改变与肿瘤侵袭有关。对弥漫型胃癌中人E-钙黏蛋白基因的突变分析表明，其cDNA中外显子8或9的缺失似乎占主导地位。在本研究中，我们对从人印戒细胞癌建立的细胞系HSC45-M2中的一种E-钙黏蛋白突变体进行了结构和功能分析。尽管免疫组织化学分析表明，突变型钙黏蛋白定位于细胞间接触部位，这与野生型通常所见的情况相同，但这些细胞并未形成紧密集落。HSC45-M2细胞表达的异常E-钙黏蛋白分子量比野生型大。在这些细胞中，我们发现E-钙黏蛋白基因组DNA中包括剪接供体位点的外显子9-内含子9边界缺失。RT-PCR显示有2种转录本，这似乎是由剪接缺陷引起的。然而，Northern印迹分析表明，在这些细胞中主要检测到缺乏外显子9的转录本。HSC45-M2细胞中突变型E-钙黏蛋白的SDS-PAGE电泳迁移率与从缺乏外显子9的cDNA表达的蛋白相同。对突变型E-钙黏蛋白蛋白的氨基末端区域分析表明，该钙黏蛋白能够通过去除其氨基末端肽而成为成熟形式。然而，突变型E-钙黏蛋白在Ca(2+)存在下易被胰蛋白酶消化，而野生型E-钙黏蛋白则不然，这表明弥漫型胃癌中常见的突变型E-钙黏蛋白可能失去了其Ca(2+)结合能力，导致紧密的细胞间联系被破坏。
