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ABSTRACT: The mdr2 gene is highly expressed in liver and is involved in the translocation of phospholipid. To study the regulation of mdr2 expression, the promoter of the mdr2 gene has been isolated from a murine vinblastine-resistant cell line, J7.V2-1, and characterized. The 5' flanking region of this gene is GC-rich, has multiple transcription initiation sites as mapped by primer extension, and does not contain either TATA or CCAAT boxes. To test promoter activity, a 1.9-kb (-1867 to +37) DNA fragment was cloned in front of the luciferase reporter gene and transient transfection assays were done in a variety of cell lines. The promoter-luciferase construct displayed a 20- to 120-fold increase in activity compared to the promoterless vector. 5' and 3' deletion analysis using transient transfections revealed two major regulatory regions in the promoter, one located upstream and one situated downstream of the transcription start sites. The upstream region may be involved in basal expression and the downstream sequence may be involved in cell type-specific expression of the mdr2 gene. Gel mobility shift and DNA footprinting assays have identified a 29-bp sequence (-78 to -50) to which nuclear protein binds. Methylation interference analysis using this fragment has further determined that CTGGCAGCTCGCCC, within the 29-mer, contains the core sequence with which nuclear protein directly interacts. Mutation of the core sequence reduced basal promoter activity, indicating that it is involved in the basal expression of the mdr2 gene. Mutagenesis studies also suggested that the upstream and downstream sequences act independently in regulation of cell type-specific mdr2 expression.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 10/1996; 7(9):1227-37.
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ABSTRACT: The expression of mdr genes that encode P-glycoprotein, an integral membrane drug transporter, has been associated with the emergence of the multidrug resistance phenotype during treatment with cancer chemotherapeutic drugs. To understand the regulation of the mdr genes, the murine mdr1b promoter has been isolated and characterized in our laboratory. Three nuclear protein binding sites that interact with nuclear proteins present in both drug-sensitive and -resistant murine macrophage-like 1774.2 cells have been localized to the promoter. In this report, transcription factor NF-Y has been identified as binding to the Y-box sequence in site 1 and as a major factor in the regulation of the murine mdr1b promoter in the mouse adrenal cell line, Y-1, that endogenously expresses the mdr1b gene. The expression of CCAAT/enhancer binding protein beta (C/EBP beta) in Y-1 cells augmented mdr1b promoter activity and resulted in an increased level of mdr1b mRNA. The effect of C/EBP beta expression on mdr1b promoter activity was sensitive to mutations in the Y-box, suggesting that coordination of NF-Y with C/EBP beta is required for further activation of the mdr1b promoter. Our studies have indicated that NF-Y is a critical factor for the mdr1b promoter, and its coordination with other factors, such as C/EBP beta, could be an important mechanism involved in mdr1b gene expression.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 01/1996; 6(12):1505-12.
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ABSTRACT: Multidrug resistance genes (mdr) that encode P-glycoproteins (P-gp) are transcriptionally regulated in normal tissues and in some multidrug-resistant (MDR) cells. Several lines of evidence suggest that regulation of P-gp overexpression at the transcriptional level is also important in human tumors. In murine MDR cells, mdr1a and/or mdr1b genes are overexpressed and P-gp isoforms are overproduced. To identify the mdr1a promoter regions that are required for transcription, the promoter has been linked to the chloramphenicol acetyltransferase (CAT) gene in transient expression vectors. 5'-Deletions of the promoter sequences have demonstrated that the region between -155 to +89 bp is crucial for basal activity of the mdr1a gene. DNase I footprinting, methylation interference, and gel retardation assays identified two nuclear protein binding sites within these sequences. One of the nuclear protein binding sites contains an 11-bp DNA sequence that interacts with nuclear protein(s) and is conserved in the promoters of the murine mdr1a and mdr1b, hamster pgp1, and human MDR1 genes. The conserved SP1 site (5'-GGGCGGG-3') that is present further downstream was shown to interact with its nuclear factor. These observations suggest that at least part of mdr gene transcriptional regulation is mediated by conserved mdr cis-regulatory elements and common nuclear factors.
DNA and Cell Biology 07/1994; 13(6):641-9. · 2.07 Impact Factor
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ABSTRACT: Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.
Journal of Biological Chemistry 05/1993; 268(10):7520-6. · 4.77 Impact Factor
