Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene
Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene
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DOI：
10.1074/jbc.273.11.5997
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发表时间：
1998-03-13
影响因子：
4.8
通讯作者：
Kohno, K
中科院分区：
文献类型：
--
作者：
Ohga, T;Uchiumi, T;Kohno, K
编码P-糖蛋白的人类多药耐药1（MDR1）基因通常在化疗后的各种人类肿瘤中过度表达。在使用各种化疗药物治疗期间，MDR1 基因在转录水平被激活和/或扩增，导致过度表达。我们之前的研究表明，倒置CCAAT盒（Y盒）可能是调节UV或药物诱导的MDR1基因表达的关键顺式调控元件。我们现在已经从人头颈癌 KB 细胞中建立了各种细胞系，这些细胞系稳定转染了由各种 MDR1 启动子缺失构建体驱动的氯霉素乙酰转移酶 (CAT) 报告基因。通过蛋白质印迹和凝胶迁移率变动测定确定，反义 YB-1 表达构建体的瞬时转染导致 YB-1 蛋白水平和反向 CCAAT 盒的 DNA 结合活性降低。当用 UV 处理细胞时，还观察到由于反义 YB-1 构建体的表达而导致的有限表达和结合活性。经过紫外线照射以及顺铂和依托泊苷等基因毒性剂处理后，含有 Y-box 的构建体的 CAT 活性得到增强。此外，通过转染反义YB-1表达构建体，这种激活减少了50-80%。相反，反义YB-1表达构建体的转染对不包含Y盒的MDR1启动子构建体驱动的CAT活性没有影响。这些数据表明，YB-1 直接参与响应基因毒性应激的 MDR1 基因激活。
The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.