The purpose of this project is to establish and characterize permanent clonal populations of airway epithelial cells from healthy individuals and individuals with cystic fibrosis (CF). Cultures of replicating primary epithelial cells and diploid fibroblasts will be exposed to oncogenes through gene transfer. Oncogene transformation will be induced by transfection with the pSVori- plasmid into nontransformed cells. Specific emphasis will be given to calcium phosphate and microinjection techniques. Cells will then be grown in culture and monitored for transformation, i.e. loss of contract inhibition and anchorage independent growth. Permanent cell lines will be characterized for C1 ion transport, DNA replication, and DNA repair C1 ion transport will be measured with Using chambers, by the uptake of 36C1, and by measuring the quenching of fluorescent C1-sensitive quinolinium dye. The rate of DNA synthesis will be determined from the incorporation of 3H- thymidine. Replicon initiation and replication fork displacement will be analyzed by velocity gradient sedimentation. Cytotoxicity and DNA repair will be assessed following exposure to DNA damaging agents and subsequent incubation in radioactive DNA precursors. DNA repair will be measured autoradiographically or by isopyknic gradient analysis. Modulation of exocrine gene expression will be monitored by RNA blot hybridization or in situ hybridization. Oncogene expression will be determined after exposure to agents that effect cellular metabolism such as phorbol esters and retinoid. Complementation studies with immortal CF cells will be undertaken to further characterize the CF gene. Transformed CF cells will be transfected with human genomic cosmid library from a normal individual. In addition, a plasmid containing and inducible G-protein gene will be used to assess G-protein involvement in C1 ion transport in CF cells.
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