We propose to investigate the molecular aspects of amylase regulation in a well-defined cultured cell line. The expression of the Amy-1 and Amy-2 genes in vivo is limited to specific tissues: Amy-1, or salivary amylase, is a major product in the parotid gland and a minor product in the liver, and Amy-2, pancreatic amylase, is a major product in the pancreas. We have identified a hepatoma cell line in which both genes can simultaneously be expressed. In these cells, the production and secretion of salivary amylases is induced more than 30-fold by dexamethasone. The expression of Amy-2 appears to be independently controlled by other components of the hormonally supplemented serum-free culture medium devised for our studies. These cells provide an excellent system for analysis of the molecular mechanisms involved in hormonal regulation of the amylase genes. The effects of dexamethasone on transcription and stability of the Amy-1 and Amy-2 mRNAs will be determined. We propose to identify the promoters which are used for expression of Amy-1 and Amy-2 in hepatoma cells, and to compare them with the well-characterized promoters used for in vivo expression in parotid, liver or pancreas. Transfection of cloned genes and their modified derivatives will be employed to identify the sequences required for induction by dexamethasone. We will analyze short term cultures of primary hepatocytes to determine whether both sets of amylase genes are expressed in normal liver, a question which has been difficult to resolve in vivo. We will further examine the expression of Amy-1 and Amy-2 in fetal mouse liver and pancreas during gestation to determine the developmental sequence of their expression. Additional hepatoma lines will be tested to determine the generality of amylase expression in hepatomas. These studies will contribute to our understanding of the molecular basis for tissue specific expression and hormonal regulation of mammalian genes.
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