ABSTRACT
Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines. Of the 11 CF clones isolated, 6 developed a polarized phenotype with vectorial ion transport and membrane-specific expression of histamine and purinergic receptors. The ion transport properties of these lines differed from the normal lines and approximated those of primary CF airway epithelial cell cultures more closely than do those of cell lines transformed with the simian virus 40 large T gene. When transplanted into denuded tracheal grafts, these cells can differentiate into ciliated and secretory phenotypes. We conclude that HPV-18 E6 and E7 genes are sufficient to transform human airway epithelial cells and that the resultant cell lines express differentiated phenotypic properties that approximate those of the native epithelium.
Subject(s)
Cystic Fibrosis/physiopathology , DNA-Binding Proteins , Papillomaviridae/genetics , Trachea/physiopathology , Transfection , Adult , Animals , Bradykinin/pharmacology , Calcium/metabolism , Carbachol/pharmacology , Cell Differentiation , Cell Division , Cell Line, Transformed , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Chloride Channels , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator , Epithelium/physiology , Humans , Male , Membrane Potentials/drug effects , Membrane Proteins/biosynthesis , Membrane Proteins/isolation & purification , Membrane Proteins/physiology , Oncogene Proteins, Viral/analysis , Phenotype , Polymerase Chain Reaction , Rats , Rats, Inbred F344 , Trachea/pathology , Trachea/transplantationABSTRACT
Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.
