We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.

Considerable evidence has accumulated indicating that overexpression of P-glycoproteins encoded by the multidrug-resistance (mdr) genes is responsible for the development of collateral resistance to a number of structurally and functionally dissimilar cytotoxic compounds in animal cells. There are three mdr genes (mdr1, mdr2, and mdr3) in the mouse genome and two (MDR1 and MDR2) in the human genome; however, only two mouse genes (mdr1 and mdr3) and one human gene (MDR1) can confer multidrug resistance upon transfection into otherwise drug-sensitive cells. Using RNase protection assay we report here that the steady-state levels of mdr1 and mdr3 messenger RNA were elevated in mouse hepatoma cells treated with dexamethasone (Dex); whereas no induction of mdr2 gene was found. Western blot analyses using anti-mdr1 and anti-mdr3 antibodies revealed that the encoded proteins appeared to be increased, but at much reduced levels. The induction was time and Dex concentration dependent. Nuclear run-on experiments demonstrated that the induction was at least in part by transcriptional control. The induction apparently required new protein synthesis since no increases in mdr1 and mdr3 transcripts was found when cultured cells were simultaneously treated with Dex and cycloheximide. Neither mdr1 nor mdr3 gene was induced in the Dex-treated nonhepatoma cell lines, LMtk- and NIH3T3. Similarly, MDR1 messenger RNA levels were elevated in the Dex-treated human hepatoma line, HepG2, but not in the nonhepatoma, HeLa. This study demonstrated that the hormonal regulation of mdr gene expression is gene and cell type specific.

The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during hepatocarcinogenesis. To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays of reporter constructs containing various 5-mdr1b flanking sequences in hepatoma and non-hepatoma cells. We found that nucleotides ؊233 to ؊116 preferentially enhanced the expression of reporter gene in mouse hepatoma cell lines in an orientation-and promoter context-independent manner. DNase I footprinting using nuclear extracts prepared from hepatoma and non-hepatoma cells identified four protein binding sites at nucleotides ؊205 to ؊186 (site A), ؊181 to ؊164 (site B), ؊153 to ؊135 (site C), and ؊128 to ؊120 (site D). Further analyses revealed that, while site B alone played a major part for the enhancer function, sites A and B combined conferred full enhancer activity. Site-directed mutagenesis results also supported these results. Gel retardation experiments using oligonucleotide competitors revealed that the site B contains a dominant binding protein. This is the first report demonstrating a cell type-specific enhancer in the mdr locus. The role of this enhancer in the activation of mdr1b gene during hepatocarcinogenesis is discussed.