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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK039619-01
Application #
3239443
Study Section
Diabetes and Digestive and Kidney Diseases Special Grants Review Committee (DDK)
Project Start
1988-03-01
Project End
1991-02-28
Budget Start
1988-03-01
Budget End
1989-02-28
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Gruenert, D C; Finkbeiner, W E; Widdicombe, J H (1995) Culture and transformation of human airway epithelial cells. Am J Physiol 268:L347-60
Cozens, A L; Yezzi, M J; Kunzelmann, K et al. (1994) CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10:38-47
Kunzelmann, K; Schwiebert, E M; Zeitlin, P L et al. (1993) An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the delta F508 CFTR mutation. Am J Respir Cell Mol Biol 8:522-9
Cozens, A L; Yezzi, M J; Chin, L et al. (1992) Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells. Proc Natl Acad Sci U S A 89:5171-5
Schwiebert, E M; Kizer, N; Gruenert, D C et al. (1992) GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells. Proc Natl Acad Sci U S A 89:10623-7
Cozens, A L; Yezzi, M J; Yamaya, M et al. (1992) A transformed human epithelial cell line that retains tight junctions post crisis. In Vitro Cell Dev Biol 28A:735-44
Rasola, A; Galietta, L J; Gruenert, D C et al. (1992) Ionic selectivity of volume-sensitive currents in human epithelial cells. Biochim Biophys Acta 1139:319-23
Borson, D B; Gruenert, D C (1991) Glucocorticoids induce neutral endopeptidase in transformed human tracheal epithelial cells. Am J Physiol 260:L83-9
Gruenert, D C; Cozens, A L (1991) Inheritance of phenotype in mammalian cells: genetic vs. epigenetic mechanisms. Am J Physiol 260:L386-94
Galietta, L J; Rasola, A; Barone, V et al. (1991) A forskolin and verapamil sensitive K+ current in human tracheal cells. Biochem Biophys Res Commun 179:1155-60

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