Miyazaki M, Kohno K, Uchiumi T, Tanimura H, Matsuo K, Nasu M, Kuwano M
Department of Biochemistry, Oita Medical University, Japan.
Biochem Biophys Res Commun. 1992 Sep 16;187(2):677-84. doi: 10.1016/0006-291x(92)91248-o.
The multidrug resistance (MDR1) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents. Various environmental stresses including heat shock can induce the expression of endogenous MDR1 genes. In order to study the regulatory mechanisms of MDR1 gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1 promoter. Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDR1 promoter of -2 kilobases. The MDR1 gene promoter contains a typical heat shock element (HSE) motif located -152 bp to -178 bp from the initiation site. Heat shock at 45 degrees C for 90 min significantly induced CAT activity in Kst-6 cells. Northern blot analysis showed a 4-5 fold increase in CAT mRNA levels in Kst-6 cells. Deletion analysis of the MDR1 promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDR1 promoter construct, pMDRCAT7. However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing -76 approximately +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between -136 and -76. Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at -152 bp to -178 bp in the MDR1 promoter. We also found that one distinct DNA-protein complex formed specifically within the MDR1 promoter region -99 to -66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells. In our present assay system, activation of the MDR1 promoter in response to heat shock appears to be mediated through both a new heat shock responsive element and MDR1 specific transcription factor.
多药耐药（MDR1）基因编码一种P-糖蛋白，它催化抗癌药物的能量依赖性外排。包括热休克在内的各种环境应激可诱导内源性MDR1基因的表达。为了研究MDR1基因表达的调控机制，我们建立了人癌细胞KB细胞系，该细胞系能稳定整合由不同长度MDR1启动子驱动的细菌氯霉素乙酰转移酶（CAT）基因。Kst-6整合了质粒pMDRCAT1，其含有-2千碱基的人MDR1启动子。MDR1基因启动子包含一个典型的热休克元件（HSE）基序，位于起始位点上游-152 bp至-178 bp处。45℃热休克90分钟显著诱导了Kst-6细胞中的CAT活性。Northern印迹分析显示Kst-6细胞中CAT mRNA水平增加了4至5倍。对MDR1启动子的缺失分析表明，在含有缺失HSE的MDR1启动子构建体pMDRCAT7的Kxh-14细胞中观察到了CAT活性的诱导。然而，进一步的缺失分析表明，热休克不能在含有启动子-76至+121碱基序列的Khp-1细胞中诱导CAT活性，这表明一个新的热休克反应元件位于-136至-76之间。凝胶迁移实验表明，热休克因子（HSF）能与MDR1启动子中位于-152 bp至-178 bp处的HSE基序结合。我们还发现，在热休克细胞的核提取物中，在温和变性条件下，MDR1启动子区域-99至-66内特异性形成的一种独特的DNA-蛋白质复合物没有显著增加，但相对更稳定。在我们目前的检测系统中，MDR1启动子对热休克的激活似乎是通过一个新的热休克反应元件和MDR1特异性转录因子介导的。
