Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene

Lijia Yu, Dalia Cohen, Richard L. Piekarz, Susan Band Horwitz

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 over-produce 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.

Original languageEnglish (US)
Pages (from-to)7520-7526
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number10
StatePublished - Apr 5 1993

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MDR Genes
Nuclear Proteins
Protein Binding
Genes
Binding Sites
Gene expression
Deoxyribonuclease I
Cells
Transcription Factor AP-1
Multiple Drug Resistance
P-Glycoprotein
Gene Expression
Proteins
Cell Line
Chloramphenicol O-Acetyltransferase
Genomic Library
Thymidine Kinase
Vinblastine
Macrophages
5' Flanking Region

ASJC Scopus subject areas

  • Biochemistry

Cite this

Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene. / Yu, Lijia; Cohen, Dalia; Piekarz, Richard L.; Band Horwitz, Susan.

In: Journal of Biological Chemistry, Vol. 268, No. 10, 05.04.1993, p. 7520-7526.

Research output: Contribution to journalArticle

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