Isolation and characterization of the murinemdr1b gene promoter

Abstract

Expression of mdr genes that encode P-glycoproteins, integral membrane drug transporters that are associated with multidrug resistance, are often seen in humans undergoing cancer chemotherapy. Murine multidrug resistant cell lines derived from a parental macrophage-like cell line, J774.2, overexpress mdr1a and/or mdr1b genes and have served as model systems to explore gene expression. The murine mdr1b promoter was isolated from a mouse liver genomic library and analyzed by gel mobility shift and DNase I footprinting assays. These analyses revealed three nuclear protein binding sites, from {dollar}-{dollar}82 to {dollar}-{dollar}59 (site 1), from {dollar}-{dollar}123 to {dollar}-{dollar}101 (site 2) and from {dollar}-{dollar}272 to {dollar}-{dollar}249 (site 3), in the mdr1b promoter. The nuclear proteins binding to these sites are present in both sensitive and drug-resistant J774.2 cells. Site 1 contains a partially conserved AP-2 site and a Y-box consensus sequence. A variety of studies including mutagenesis demonstrated that the Y-box sequence is sufficient for both nuclear protein binding and transactivation. However, results from gel mobility shift assays using competitors indicated that the nuclear protein binding to site 1 is not a known Y-box binding protein. The Y-box sequence in site 1 is conserved in the human mdr1 promoter. The nuclear protein binding to site 1 was also found in human Hela cells. Site 2 is conserved in the murine mdr1a, human mdr1 and hamster pgp1 and pgp2 promoters. The nuclear protein binding to site 2 was present in human Hela cells. Mutation studies done with site 2 identified an 8 bp sequence that is required for nuclear protein binding and enhancer activity. This core sequence has homology to the binding site for nuclear factor E2F. Further studies suggested that the nuclear protein biding to site 2 may be an E2F-like protein. Site 3 contains a conserved AP-1 site that is recognized by human AP-1 protein, indicating that the nuclear protein binding to site 3 is AP-1 or an AP-1-like protein. These results provide the evidence to suggest that murine mdr1b gene expression could be transcriptionally regulated by the interaction of the mdr1b promoter with the nuclear proteins identified, and also that a common mechanism may be involved in the expression of human mdr1 and murine mdr1b genes. The presence of nuclear proteins binding to sites 1, 2 and 3 in both sensitive and drug-resistant cells suggested that the activation of the nuclear proteins may be a post-translational event. The studies in this thesis provide insight into the possible mechanisms of murine mdr1b gene regulation.