ABSTRACT
Two monoclonal antibodies (MAbs) were established in 20 clones of MAbs generated against cytokeratin fraction extracted from canine squamous cell carcinoma to investigate the expression of intermediate filament proteins during tumorigenesis. These MAbs were confirmed to react with purified cytokeratin by ELISA. One monoclonal antibody, MAb32 reacted all layers of epidermis except the cornified layer and mammary myoepithelial cells but not any epithelial cells. Another antibody named MAb24 exclusively reacted the basal monolayer of epidermis, the stratum germinativum. Any positive reactions with MAb24 were not detected in normal mammary gland. From the analysis by two-dimensional gel electrophoresis followed by immunoblotting, it was revealed that MAb24-recognizing cytokeratin subunit gave a molecular weight of 57 kilodalton and a isoelectric-pH value of pI5.1, indicating type I (acidic) cytokeratin. In intraductal papillomas developed in canine mammary glands, most tumor cells were positively stained with MAb32 in addition of myoepithelial cells while no positive reaction with MAb24 was seen. In ductal carcinomas, MAb24-positive cytokeratin was begun to express by tumor cells with positive reaction of MAb32 where these cells showed infiltrative growth into the stroma. We therefore proposed that 57 kilodalton-type I cytokeratin was a molecular marker for malignant transformation in canine mammary tumor and these antibodies could be useful tools to investigate the change of cytokeratin expression during tumorigenesis.