GLUCOCORTICOID REGULATION OF GENE EXPRESSION IN M1.19 RAT HEPATOMA CELLS: ISOLATION OF CLONED DNA SEQUENCES COMPLEMENTARY TO SPECIFIC GLUCOCORTICOID INDUCED GENES

The structural and functional relationship of glucocorticoid steroid hormones to the genes that they regulate is a fundamental problem in cell biology. In target cells, steroid-protein receptor complexes bind in nuclei to a limited number of sites on chromatin or DNA, the consequence of which is an alteration in the transcriptional rates of specific messenger RNA\u27s. In HTC rat hepatoma cells, the expression of a small number of glucocorticoid responsive gene products, such as tyrosine aminotransferase, have been studied in detail. A clonal variant of HTC cells, the M1.19 cell line, contains integrated mouse mammary tumor proviral DNA sequences. The transcriptional rate of viral RNA is dramatically stimulated by glucocorticoids in these cells. M1.19 cells provide a unique model cell system for examining and comparing the glucocorticoid regulation of viral and HTC cellular genes. Recently, advances in recombinant DNA technology have made it feasible to clone glucocorticoid regulated genes and to study the hormone regulated expression of these genes. In the course of this project it was proven that: (1) a comparative colony hybridization analysis of a M1.19 cDNA library could be used to identify and isolate cDNA clones of hormonally induced mRNA\u27s; (2) the regulation of mRNA sequences represents specific cellular responses to hormone administration; and (3) isolated cDNA clones are derived from mRNA\u27s coding for previously identified rat hepatoma and liver cell proteins. Two M1.19 cDNA clones, p1394 and p255, were found to hybridize to a highly abundant 900 nucleotide mRNA in induced M1.19 and rat liver cells. This mRNA species codes for the acute phase reactant protein (alpha)(,1)-acid glycoprotein ((alpha)(,1)-AGP). Cycloheximide significantly reduces the accumulation of (alpha)(,1)-AGP mRNA but does not completely abolish the induction response. Southern blot analysis indicates that p1394 and p255 are complementary to different regions of the (alpha)(,1)-AGP gene. Two other cDNA clones, p655 and p333, hybridize to small, induced polyadenylated RNA sequences that contain highly repetitive genomic sequences. These cloned cDNA\u27s are being used to identify critical regulatory sites for glucocorticoid action within the coding or flanking regions of their complementary HTC cellular genes