The major emphasis of this project is to identify and investigate the mechanisms which regulate the expression of the mdr genes. We have established an inter-strain variability in xenobiotic induction of these genes in Fischer, Wistar and Sprague-Dawley rats and isolated hepatocytes. Although the parent compound 2-acetylaminofluorene (AAF) caused a muted response in Sprague-Dawley livers and hepatocytes, the electrophilic metabolites N-OH-AAF and AAAF caused a dose- dependent response in both Fischer and Sprague-Dawley livers and hepatocytes. This indicated a requirement for metabolic activation of this compound to mediate the induction of mdr expression. The 5' flanking region of the mdr2 gene has been cloned and a 1700 bp region directly adjacent to the mdr2 coding region has been sequenced. Primer extension experiments have indicated that this gene may utilize multiple transcription start sites, none of which correlate well with putative TATA elements. This region has been subcloned into a reporter gene vector to investigate promoter activity. An interesting point mutant was isolated which caused threefold lower promoter activity compared to the wild-type promoter. The promoter of the mdr1b gene has also been further characterized for cis acting elements critical to basal and xenobiotic inducible activity. Deletion constructs, gel mobility shift assays and DNaseI footprinting assays indicate that the region between -180 to -200 may be critical to basal promoter activity. The cDNAs for each the mdr1b and mdr2 have been subcloned into mammalian and bacculovirus expression vectors. Expression of the mdr1b cDNA in NIH3T3 cells has resulted in a multidrug resistant phenotype. These cells are currently being tested for a role of mdr expression in other cellular pathways.
