Abstract
NF‐R2 is a DNA‐binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF‐R2 binds within the promoter's — 126 and — 102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF‐R2 from the nuclear extract of K562/ADM cells, a multidrug‐resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S‐300, DEAE‐Sepharose, heparin‐Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF‐R2 runs as a single protein of 75 kDa on SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF‐R2 binding. These results suggest that NF‐R2 may be involved in the positive regulation of the MDR1 gene transcription.
Keywords: Multidrug resistance, MDR1 gene, Promoter, DNA‐binding protein
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