The goal of this project is to produce airway epithelial cell lines with well characterized phenotypes and the Cystic Fibrosis (CF) genotype. An immortalization protocol designed to introduce minimal oncogenic transformation using defective retroviral vectors will be employed. Immortalizing human cells with little alteration of differentiated function is possible because 1) mouse and human cell lines with a reduced transformation phenotype have been made this way, 2) several immortalizing genes have been cloned into expression vectors, 3) transformation is quantitatively related to phenotype, 4) these genes and vectors can be genetically manipulated, and 5) phenotypic analysis can be quantified. Significant progress in CF research has been made at the cellular and molecular level by identification of abnormal ion transport regulation in epithelial cells of the airways, and the finding that these properties are retained when airway cells are cultured in the laboratory. Complete understanding of the CF defect at the molecular and cellular level progresses slowly due to scarcity of tissue and the short life time of cultured cells. Well characterized cell lines will relieve this bottle-neck. Advances in organic virology and tumor biology have identified several viral and cellular genes that control cell growth and differentiation that, when abnormally expressed, will transform normal cells in a step-wise manner to a tumorigenic state. One independent step in this process is cell immortalization defined as the ability to escape normal senescence. Several studies have indicated that introduction and expression of a single gene (from a list of several) will immortalize tissue specific cell types that retain most if not all of the differentiated phenotype expressed at the time of immortalization. It also seems clear, especially from analysis of leukemias and glial cell lines, that immortal cells are arrested at a differentiation stage along a pathway leading to a terminal phenotype. Therefore, cell lines can be made that express tissue-specific, differentiation-specific and transformation-specific phenotypes. The details of an individual cell line's composite phenotype, are likely governed by which immortalizing gene is used, its expression level, the differentiation state of the cell at the time of immortalization, and genotype. This proposal describes a program to 1) immortalize airway epithelial cells from non-CF and CF tissue using a defective retroviral vector system, 2) assess the level of CF, epithelial, and differentiated phenotype retained as well as the level of transformation phenotype induced, and 3) use phenotype information to genetically modify the immortalizing vectors to reduce the transformed phenotype to a minimum. Modifications of vectors include switching oncogenes, using mutant alleles, and/or modulation of oncogene expression by switching promoters or reinfecting with antisense oncogene RNA vectors.
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