Identification of DNA-protein interactions in the 5 ' flanking and 5 ' untranslated regions of the human multidrug resistance protein (MRP1) gene: Evaluation of a putative antioxidant response element/AP-1 binding site

Overexpression of the multidrug resistance protein, MRP1, confers resistanc e to multiple natural product-type chemotherapeutics. MRP1 amplification is observed in some multidrug-resistant cell lines, while in others, increase d transcription occurs in the absence of gene amplification. To investigate mechanisms influencing MRP1 transcription, three small cell lung cancer ce ll lines were examined: drug sensitive H69 cells with two apparently normal MRP1 alleles, highly resistant H69AR cells in which MRP1 is amplified and low level resistant H69PR cells that contain only one MRP1 allele. Deoxyrib onuclease 1 footprinting and gel mobility shift assays were undertaken usin g nuclear extracts from the three cell lines and a 1 kb region encompassing the 5 ' flanking region of MRP1. Thirteen protein binding sites were ident ified of which six were sequence specific. Differences in levels of protein binding occurred with a putative antioxidant response element (ARE)/AP-1 b inding site at -511 to -477. Levels of protein binding to this site were 2. 5- to 3.0-fold higher in H69AR nuclear extracts versus extracts from H69 or H69PR cells. The AP-1 sequence is required for binding and c-Jun and JunD were identified as components of the protein complex. The ARE/AP-1 element functioned as a transcriptional enhancer but did not mediate induction of a luciferase reporter gene upon beta -naphthoflavone treatment. (C) 2001 Aca demic Press.