Mucin synthesis in immortalized canine tracheal epithelial cells

To study the regulation of mucin synthesis in canine tracheal epithelial cells, it is desirable to establish a cell line which synthesizes mucin continuously. We adopted the approach of immortalizing canine tracheal epithelial cells using a vector encoding the human papillomavirus (type 18) E6 and E7 genes. The E6 and E7 genes are essential and sufficient for the immortalization of human genital keratinocytes, as well as human tracheal epithelium. Primary epithelial cells from dog trachea were transfected with a vector containing HPV18 genes E6 and E7. The resultant cells (CT1) were cloned and maintained in selective medium supplemented with growth factors and hormones. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the expression of the canine tracheal mucin (CTM) mRNA in these cells. The half-life of the CTM mRNA was found to be 45-60 min. Incorporation of labelled precursor (glucosamine) indicated that high-molecular-weight mucin glycoprotein was synthesized by these immortalized cells, which reacted with the antiserum to the native CTM. Equilibrium gradient centrifugation analysis showed that the buoyant density of the mucin synthesized in CT1 cells (1.486 g/ml) was similar to the reported value for native CTM (1.5 g/ml). Mucin which was isolated from immortalized cells was not a proteoglycan as chondroitinase treatment had no effect. These results suggest that CT1 cells synthesize a mucin glycoprotein which exhibits properties similar to native CTM. When characterized by immunostaining with a pool of monoclonal antibodies, these cells showed common epithelial antigens related to keratin expression. The CT1 cell line represents a unique resource for studying mucin biosynthesis and regulation.