P-glycoprotein (pgp) is a plasma membrane protein located prominently in liver which participates in the removal of a variety of organic substances from the cell. However, despite knowledge that pgp participates in cellular detoxification a systematic study of pgp mRNA regulation by environmental agents has not been undertaken. The overall objective of this proposal is to elucidate the molecular biological mechanisms responsible for xenobiotic and hormonal regulation of the pgp gene in normal rat liver. The studies outlined in this proposal will use to advantage significant, recent technological breakthroughs in rat hepatocyte cell culture and molecular biology. Toward this end I have isolated and sequenced the rat liver pgp cDNA and isolated its genomic clone. I will use this cDNA to systematically examine hormonal and polycyclic aromatic hydrocarbon (PAH) regulation of pgp gene expression at the posttranscriptional and transcriptional level. The pgp gene will permit me to identify the cis-acting sequences which control basal pgp gene expression and those sequences involved in xenobiotic activation and hormonal suppression of pgp mRNA expression. This will be accomplished by transfecting rat hepatocytes in culture with recombinant 5' pgp gene deletion constructs . The pgp gene deletions will be linked to the CAT reporter gene. Thereafter. DNA protein interactions will be assessed using gel retardation assays, DNAse sensitivity and southwestern blots. Specifically. the role that trans-acting factors play in activation of the pgp gene will be assessed by performing DNA-protein binding studies using nuclear extracts from hepatocytes. These studies will determine whether common DNA regulatory elements exist between the pgp gene family and genes from Phase and Phase II detoxification systems. These studies will be the first studies in normal cultured hepatocytes to examine the mechanism of xenobiotic and hormonal regulation of the pgp gene which has an important role in cellular detoxification.
