Scholl F G, Gamallo C, Quintanilla M
Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, Spain.
Lab Invest. 2000 Nov;80(11):1749-59. doi: 10.1038/labinvest.3780185.
PA2.26 antigen is a small mucin-type transmembrane glycoprotein induced in mouse epidermal keratinocytes during carcinogenesis. It is located at plasma membrane projections, such as microvilli and ruffles, where it interacts with the actin cytoskeleton. Previous studies revealed that ectopic expression of PA2.26 in epidermal MCA3D keratinocytes induces cell surface extensions and increased motility. Here, we show that PA2.26-expressing MCA3D (3D2.26) cell transfectants undergo a phenotypic conversion linked to the acquisition of malignant characteristics. The 3D2.26 cells down-regulate basal keratin K14 and up-regulate vimentin and keratin K8 expression. Immunofluorescence analysis in 3D2.26 cell cultures showed loss of cortical actin filaments and destabilization of adherens junctions mediated by E- and P-cadherin, although both cadherin mRNAs were expressed in the transfectants. When the cadherin protein levels were analyzed in Western blots, no P-cadherin protein or smaller polypeptide E-cadherin forms were detected, suggesting that E- and P-cadherin synthesized in 3D2.26 cells was unstable and proteolytically degraded. Transplantation of 3D2.26 cells into athymic nude mice induced tumors, whereas MCA3D cells and control (3DN) transfectants were not tumorigenic after 72 days postinjection. The phenotype of the tumors was undifferentiated, with mixed regions exhibiting a glandular differentiation pattern in which the presence of numerous surface microvilli was observed at the ultrastructural level. Interestingly, PA2.26 antigen was highly expressed in these microvillous cell surfaces. Tumor cells were vimentin- and K8-positive and showed an aberrant pattern of E-cadherin protein expression in which large cytoplasmic aggregates were found close to the nucleus. Infiltration of tumor cells into lymphatic vessels and the presence of frequent regional lymph node metastases were also observed in the tumors. These results indicate that expression of PA2.26 antigen in premalignant keratinocytes induces a fully transformed and metastatic phenotype, and they suggest an involvement of PA2.26 in malignant progression.
PA2.26抗原是一种在致癌过程中由小鼠表皮角质形成细胞诱导产生的小粘蛋白型跨膜糖蛋白。它位于质膜突起处，如微绒毛和褶皱处，在那里它与肌动蛋白细胞骨架相互作用。先前的研究表明，PA2.26在表皮MCA3D角质形成细胞中的异位表达会诱导细胞表面延伸并增加细胞运动性。在此，我们表明表达PA2.26的MCA3D（3D2.26）细胞转染子经历了与获得恶性特征相关的表型转化。3D2.26细胞下调基底角蛋白K14并上调波形蛋白和角蛋白K8的表达。对3D2.26细胞培养物进行的免疫荧光分析显示，皮质肌动蛋白丝减少，由E-钙黏蛋白和P-钙黏蛋白介导的黏附连接不稳定，尽管两种钙黏蛋白mRNA在转染子中均有表达。当通过蛋白质印迹分析钙黏蛋白的蛋白质水平时，未检测到P-钙黏蛋白或较小的多肽E-钙黏蛋白形式，这表明在3D2.26细胞中合成的E-钙黏蛋白和P-钙黏蛋白不稳定且被蛋白水解降解。将3D2.26细胞移植到无胸腺裸鼠中可诱导肿瘤形成，而MCA3D细胞和对照（3DN）转染子在注射后72天没有致瘤性。肿瘤的表型未分化，混合区域呈现腺管分化模式，在超微结构水平观察到大量表面微绒毛。有趣的是，PA2.26抗原在这些微绒毛细胞表面高度表达。肿瘤细胞波形蛋白和K8呈阳性，并显示出异常的E-钙黏蛋白蛋白表达模式，在细胞核附近发现大量细胞质聚集物。在肿瘤中还观察到肿瘤细胞浸润到淋巴管中以及频繁出现区域淋巴结转移。这些结果表明，PA2.26抗原在癌前角质形成细胞中的表达诱导了完全转化和转移的表型，并且提示PA2.26参与了恶性进展。
