Human 5T4 oncofoetal antigen defined by the murine 5T4 monoclonal antibody is a highly glycosylated protein expressed by trophoblast and a few specialized adult epithelia. Up-regulation of 5T4 expression in some cancers is associated with poor clinical outcome; overexpression of human 5T4 cDNA in epithelial cells can alter their morphology and motility, supporting a role for such functions in cancer and development. A murine model to study 5T4 biology and tumour immunology would be useful. The production of m5T4-specific antibodies, their use in establishing transfected cells and documenting their biological properties in vitro are described. A rat monoclonal antibody specific for mouse 5T4 molecules by ELISA, flow cytometry, immunohistochemistry and immunoprecipitation was isolated and epitope mapped. Similar to its human counterpart, murine 5T4 antigen is a 72kDa glycoprotein (immunoprecipitation and Western blot analysis) and exhibits punctate cell surface expression, dependent upon the integrity of the actin cytoskeleton. Likewise, overexpression of autologous murine 5T4 by B16 F10 melanoma cells and A9 L fibroblasts accentuates the 5T4 phenotype, which is characterized by a spindle-like morphology, increased motility, and reduced adhesion and proliferation rate. Immunohistochemical analysis of adult mouse tissues shows a restricted pattern of expression similar to that of human 5T4 antigen. The murine 5T4 antigen-expressing cell lines and antibody reagents are now being used to explore novel immunotherapies in pre-clinical models and the biology of 5T4 in development.
- cell biology
- leucine-rich repeat
Abbreviations used: CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; hm, human—mouse; HRP, horseradish peroxidase; h5T4, human 5T4; LRR, leucine-rich repeat; MAb5T4, monoclonal antibody 5T4; α-MEM, α-minimal essential medium; mh, mouse—human; m5T4, mouse 5T4; m5T4ex, extracellular domain of m5T4; m5T4vac WR, recombinant m5T4 expressing Western Reserve vaccinia virus; Rabαm5T4, rabbit anti-m5T4-Fc polyclonal antiserum; 5T4-Fc, extracellular domain of 5T4 fused to the Fc domain of human IgG1.
- The Biochemical Society, London ©2002