The long term objectives of the research outlined in this proposal are to understand function, structure, and mechanisms of tissue-specific gene regulation in salivary glands. The specific goals are 1) to develop immortalized rat salivary gland epithelial cell lines, 2) to characterize immortalized cell lines and their secretions with particular attention to salivary cell-specific properties, and 3) to determine the mechanism(s) responsible for the increased levels of proline-rich protein mRNAs in cycloheximide-treated rat cells. Rat parotid and submandibular gland cells will be fused to rat hepatoma (FTO-2B) cells using polyethylene glycol. The biochemical and morphological characteristics of the immortalized cells will be analyzed to determine the extent to which hybrid cells retain salivary gland-specific properties. Biochemical markers for parotid and submandibular gland functions will include proline-rich proteins, alpha- amylase and glutamine-rich proteins. Immunohistochemical techniques and electron microscopy will be used to characterize the morphology and structural differentiation of the cells. Cycloheximide treatment causes an increase in the levels of proline-rich protein mRNAs in primary cultures of hamster parotid cell's, parotid/FTO-2B fusion cells, and FTO-2B cells. The increase in proline-rich protein mRNAs in response to cycloheximide treatment may result from an increase in the stability of the RNA (decreased turnover), an increase in the rate of transcription, or both. These possibilities will be investigated using nuclear runoff transcription and RNA stability assays.
