Ikeguchi M, Teeter L D, Eckersberg T, Ganapathi R, Kuo M T
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
DNA Cell Biol. 1991 Nov;10(9):639-49. doi: 10.1089/dna.1991.10.639.
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.
我们之前已经表明，多药耐药/ P -糖蛋白基因mdr3/mdr1a在小鼠肝细胞癌（HCC）中被激活。在本研究中，我们发现，在一些源自HCC的细胞系（Hepa1c1c、Hepa1c1c - BprC1和Hepa1 - 6）中，mdr3高水平表达。为了研究这些细胞中mdr3的转录调控，我们分离出了一个包含小鼠mdr3基因5'部分的DNA片段，并对其启动子进行了功能分析。使用不同长度的启动子序列指导氯霉素乙酰转移酶（CAT）报告基因表达的瞬时转染试验表明，位于小鼠mdr3转录起始位点上游 - 94个核苷酸处的序列在小鼠肝癌细胞中起负调控元件的作用。从以下观察结果可知，位于 - 117处的典型AP - 1结合序列TGA - GTCA至少部分地导致了这种负效应：（i）通过定点诱变改变该AP - 1序列可增强CAT表达。（ii）当在共转染实验中使用含有AP - 1序列而非突变序列的双链DNA作为竞争剂时，CAT报告基因的表达升高。（iii）在使用表达与AP - 1序列相互作用的c - jun/c - fos蛋白的重组质粒DNA进行的共转染实验中，也观察到了CAT表达的增强。有趣的是，仓鼠pgp1启动子的近端区域与小鼠mdr3基因的近端区域具有显著的序列相似性，包括AP - 1位点，但仓鼠启动子中的AP - 1位点作为正调控因子。尽管先前的研究已经证明，正负转录因子可通过与c - jun/c - fos相互作用来调节基因表达，但这是第一项表明AP - 1位点在基因表达调控中作为负顺式元件发挥作用的研究。
