EPITHELIAL CELL LINES FROM CF AND NORMAL TRACHEAS

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Cystic fibrosis (CF) is the most common lethal genetic disease among the Caucasian population. Its major clinical symptoms are all related to epithelial of exocrine glands including the submucosal glands and mucosal secretory cells of the major airways. Recently, major advances have occurred in defining the genetics of CF and characterizing the defects in various epithelia with regards to electrolyte and fluid transport and mucus secretion. Despite these advances, CF research is now reaching an impasse. Major impediments includes the scarcity of tissues and cells to work with and the 'finite' life span of human epithelial cells propagated in vitro. The development of immortalized cell lines capable of maintaining the differentiated CF phenotypes would greatly facilitate biochemical and physiological studies with these cells. The major objectives of this proposal are the following: propagate in serum-free medium, low passage epithelial cells derived from submucosal glands and surface mucosa of trachea obtained from CF patients; characterize the cells as epithelial on the basis of histochemical, ultrastructural, immunocytochemical, and biochemical properties; Immortalize the CF and normal epithelial cells (already established) using oncogenic viruses, transfection with viral DNAs, and by modifying culture conditions. Specifically, we plan to use procedures involving transfecting with subgenomic segments containing the intact transforming early regions of Ad12-SV40, and plasmid vectors containing oncogenes such as the v-Ha-ras. Integration of viral DNA sequences into host cell chromosomes and the expression of viral genes will be examined by Southern blot and immunocytochemical analyses, respectively. If these procedures are not successful, we will test procedures involving infection of the cell for the activation of chloride channels. For this, single channel patch clamp techniques will be used. In normal cells, the C1 channels should be activated by 8-bromo-cAMP in the cell- attached mode of recording, but channels should not be activated in CF cells. After detaching the membrane patch, inside-out mode Cl- channels should be activated in normal and CF cells subsequently by using 180 nM Ca++ in the bath; determine the growth kinetics (doubling times, cell cycle parameters) of low passage and immortalized cells derived from normal and CF tissues; compare the nature of mucoproteins and cytokeratins in normal and CF cells at a low passage and in immortalized cells; establish procedures for freezing the cells and distributing them to investigators interested in CF research. The availability of normal and CF tracheal epithelial cells to the research community would facilitate studies on different aspects of CF research and may be instrumental in discovering new therapies for the lethal disease.