IMMORTALIZATION OF HUMAN SALIVARY EPITHELIAL CELL LINESS

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Salivary glands consist of relatively heterogenous cell populations of serous, mucous, myoepithelial, and duct cells that exhibit specific markers of differentiation functions. Little however, is known about the biochemical and molecular mechanisms by which the production and secretion of the specialized molecules are coordinated and regulated. Such studies are hindered by a lack of availability of homogeneous cell populations that exhibit salivary specific differentiation. Thus the main objective of this project is to establish immortalized populations of serous, mucous, ductal and myoepithelial cells, from the human salivary glands,using oncogenic viruses, transfection with viral DNA, and by modifying culture conditions. Specifically, we plan to use procedures involving transfecting cells with subgenomic segments containing the intact transforming early regions of mutant of AD12-SV/40 hybrid virus, of wild-type SV40 virus, and of the tsA255 temperature sensitive mutant of 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 cultures with SV40- virus and Ad12-SV40 hybrid virus. The cells will be characterized by ultrastructural parameters; immunocytochemical localization of cell specific markers i.e. amylase, lactoferrin, secretory component, lysozyme, and mucin secretion; and by detecting cell specific mRNAs. Additionally cDNA libraries will be prepared and used in identifying genes expressed in specific salivary gland cells. The genes will be characterized by DNA sequencing and their expression in specific cells will be confirmed by PCR and Northern blot analyses. The availability of well characterized human salivary gland epithelial cell lines to the research community would facilitate studies on the molecular mechanisms that regulate secretions of the various cell types.