CF TS TRANSFORMANTS--BIOCHEMICAL AND GENETIC ANALYSIS

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The goal of this project is establishment and characterization of permanent cultures of cystic fibrosis (CF) airway epithelial cells. Primary cultures of proliferating CF epithelial cells will be transfected with the ptsA209ori- plasmid. The plasmid contains an SV40 genome defective in the origin of replication and has a temperature-sensitive mutation in the large T-antigen. The plasmid will be introduced by calcium phosphate precipitation. Clones of transformed CF epithelial cells will be derived from trachea, bronchi, submucosa, and nasal polyps. Transformants will initially be assayed for their epithelial origin, transformation- specific antigens, and defective Cl ion transport. Individual colonies will be analyzed at the permissive (33 degrees C) and nonpermissive (40 degrees C) temperatures and compared to normal epithelial cells. Cells will be monitored and comprehensively analyzed for ion transport, secretion, and oncogene expression. SV40 T-antigen expression, keratin production, and secretory antigens expressed in serous, mucous, or goblet cells will be determined by immunocytochemical staining. Electron microscopy will be used to characterize the morphological features of the cells, e.g., junction formation, microvilli, and secretory granules. Chloride ion transport will be measured by patch clamp, by the uptake of 36Cl, with Ussing chambers, and fluorescent Cl- sensitive quinolinium dyes. The cells will also be analyzed for beta-adrenergic stimulation of cAMP and the role oncogene products play in this pathway as well as in ion transport. Changes in oncogene expression after treatment with phorbol esters, retinoic acid, and dimethyl sulfoxide will be compared to changes in ion transport and secretion. Immortal CF cells will be used to construct cDNA libraries which will be screened with pulsed- field gel fragments. Candidate clones isolated by screening and pulsed-field gel fragments will be used in complementation studies.