Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

Abstract

To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation-and epithelialspecific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 ,LM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for >250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression. (6,7).In contrast to the relatively large number of studies describing cells from other organs (recently reviewed in refs. 1 and 2), only three reports indicate transformation of respiratory tract cells (8-10). Bronchial epithelial cells immortalized with v-Ha-ras display anchorage-independent growth and form tumors in nude mice. However, these cells were poorly differentiated and did not contain tight junctions or keratins characteristic of differentiated epithelial cells. The epithelial origin of the cells was verified by the presence of keratin. A somewhat greater level of differentiation was retained by diethylnitrosamine-transformed fetal tracheal cells, which contained mucin. These cells also expressed carcinoembryonic antigen and wool merokeratin and had enhanced, although limited, growth capacity (9). A third study primarily described methods for transfection and showed expression of the SV40 large tumor antigen (T antigen) three to four subcultures posttransfection (10).A goal of the present study was to develop a transformed human tracheal epithelial (HTE) cell line for detailed studies of the mechanisms regulating Cl ion transport. This aspect of epithelial function is essential for rehydration of airway mucus. Abnormalities in the Cl conductance pathway also appear critical to disease processes such as those occurring in cystic fibrosis (11,12).Phenotypic characteristics have been well maintained in SV40-transformed fibroblasts (13-15). In SV40-transformed epithelial cells (16)(17)(18)(19), certain aspects of phenotype are retained better than others (17,18,20,21). A plasmid containing a SV40 genome defective in the origin of replication (pSVori-) has been shown to enhance the transformation efficiency of human fibroblasts (22). The defective origin of replication precludes background ...

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