Identification and characterization of three DNA-binding proteins on the promoter of the human MDR1 gene in drug-sensitive and -resistant cells.

In multidrug-resistant cell lines (K562/ADM), MDR1 was amplified and transcriptionally activated. But the mechanism of MDR1 expression is unknown in K562/ADM cells. A 131 bp DNA fragment upstream from the major cap site of MDR1 contained CAAT-like and GC-box-like motifs and showed promoter activity in a CAT expression assay, resulting in a 3.5-fold enhancement of MDR1 promoter activity in K562/ADM as compared with that in K562 cells. In the CAT assay using deletion mutants we found that sequences containing the CAAT-like motif and the GC-box-like motif were required for MDR1 proximal promoter activity. To understand the details of MDR1 transcription in K562/ADM cells we analyzed the interaction between the proximal promoter and DNA-binding protein(s). We have identified three DNA-binding proteins on the MDR1 promoter from K562/ADM and parental K562 cells. The first binding protein to the MDR1 promoter was NF-R1, which recognized the sequences containing the CAAT-like motif and the GC-box-like motif as revealed by using a gel mobility shift assay. The second protein was NF-R2, which bound to the sequence containing the CAAT-like motif. The third protein, which bound to the sequence containing the GC-box-like motif was designated NF-R3. NF-R2 and NF-R3 in the resistant cells formed different bands in the gel mobility shift assays as compared with those in the sensitive cells, respectively. The observed difference might be related to the transcriptional activation of MDR1 in K562/ADM cells.

chloramphenicol acetyl transferase

adriamycin

polymerase chain reac‐tion

phenylmethylsulfonyl fluoride

dithio‐threitol

sodium dodecyl sulfate

polyacrylamide gel electrophoresis

kilobase

kilodalton