A knowledge of the molecular mechanisms governing gene expression in the salivary glands (SGs) is fundamental to an understanding of the function of these glands in health and disease. Cell lines capable of expressing salivary protein genes are crucial to the study of gene regulation, since they can be grown in quantity free of other cell types, and they allow the analysis of the regulatory elements controlling genes and the purification and characterization of transcription factors. Many of the protective salivary proteins are synthesized and secreted by the acinar cells of the SG. Unfortunately, no cell lines exist that adequately express SG acinar cell-specific genes. A potentially powerful route to deriving cell lines that retain the ability to express markers of differentiation is to construct transgenic mice where the transgene comprises the regulatory sequences from a marker gene coupled to the SV40 large T antigen (Tag), thereby targeting expression of this potent oncogene to the cell type of interest. The murine SPT gene family encodes a class of abundant salivary protein, and members of this gene family are expressed at high levels in the acinar cells of the mouse submandibular glands. This proposal is based on the hypothesis that transgenic mice carrying SPT-Tag constructs will develop tumors derived from submandibular gland acinar cells, from which immortalized cell lines can be derived that retain the ability to express SPT, and perhaps other acinar cell-specific genes. To test this hypothesis, four Specific Aims are proposed: 1. Isolated a genomic clone carrying 5'and 3' flanking sequences of the SPT-1 gene, and prepare a 5'-SPT-1/Tag/3-SPT1 construct. 2. Generate transgenic mouse lines carrying the 5'-SPT-1/Tag/3'-SPT1 construct in the germline, and monitor for salivary gland tumors. 3. Derive cell lines from salivary gland tumors, and screen for expression of acinar cell specific genes. 4. Test the transfectability of the derived cell lines with a control reporter plasmid. The overall goal of this project is to develop cell lines that will provide a powerful tool with which to perform a detailed analysis of the mechanisms regulating gene expression in the acinar cells of salivary glands. Since the regulatory systems are often conserved across large evolutionary distances, it is likely that human genes will be functional in these cell lines. This project will also provided the P.I with exposure to transgenic mouse technology. Collectively, completion of this project will provide the foundation for future studies aimed at elucidating the molecular programs that govern development of functional human salivary glands. Further, transgenic mice susceptible to SG tumors could provide a valuable model system for studies of salivary gland cancer.
