This proposal describes plans to characterize a well differentiated variant (RSP-) of the rat hepatoma cell line Fao that we have recently isolated. RSP-, however, fails to secrete a small set, at least four, of serum proteins that are secreted by the parental cell line. These proteins include albumin, Gc globulin and two other, as yet, unidentified proteins. Southern and slot blot hybridization analyses of the DNA and RNA isolated from these cells show that they contain an intact albumin gene and that this gene is not expressed. Other experiments measuring the relative efficiencies with which the rat serum albumin promoter drives the expression of the luciferase gene in Fao and RSP- indicate that a trans acting factor that mediates expression of the albumin gene is differentially expressed in these cell lines. The known trans acting factors produced in liver cells that appear to be required to maintain the differentiated state seem to affect expression of most if not all liver specific genes. On the other hand, trans acting factors produced in non- liver cells have been described which repress expression of defined sets of liver specific genes. Thus it seems possible that the RSP- phenotype may result from activation of a normally silent gene that encodes a protein involved in repressing expression of a set of genes specifying the four proteins that fail to be secreted or that a mutation of a gene specifying a trans acting factor that activates a small set of liver specific genes has occurred. To establish which, if either, of these explanations is correct I will: 1) utilize DNase I footprint analyses to identify sequences in the promoter regions of the albumin and Gc globulin genes that are differentially protected by nuclear proteins extracted from Fao and RSP- cells and 2) establish whether somatic cell hybrids formed by fusion of RSP- and Hep G2 (a human hepatoma line) retain the RSP- phenotype. The results of these experiments will allow evaluation of the possibility that a trans acting factor is differentially expressed in Fao and RSP- cells and, if so, demonstrate whether or not this factor acts as a positive or a negative regulator.
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