Activation of the nuclear transcription factor κB (NFκB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine
Activation of the nuclear transcription factor κB (NFκB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine
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DOI：
10.1016/s0360-3016(01)02820-6
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发表时间：
2002-05-01
影响因子：
7
通讯作者：
Grdina, DJ
中科院分区：
文献类型：
--
作者：
Kataoka, Y;Murley, JS;Grdina, DJ
目的：氨磷汀已被批准作为一种治疗方法，用于降低接受头颈部癌症术后放射治疗的患者中重度口干症的发生率。氨磷汀作为一种能够参与细胞内还原/氧化过程的还原剂，可能影响氧化还原敏感型转录因子和基因表达，研究了氨磷汀的活性游离硫醇WR-1065对U87胶质瘤细胞核转录因子kappaB(NFkappaB)激活和随后基因表达的影响。方法与材料：将人脑胶质瘤细胞系U87培养到融合状态，然后用40微米的WR-1065作用30min~24 h，用流式细胞仪检测细胞周期的变化。凝胶移位实验检测WR-1065对NFkappaB活化的影响。用Northern印迹和Atlas Human cDNA表达阵列(Clontech，Palo Alto，CA)测定基因表达随暴露于WR-1065的时间的变化。结果：U87细胞暴露于40um WR-1065后，NFkappaB在处理后30min~1h内显著激活。NFkappaB应答基因WnSOD的表达在处理16h后增加2倍以上，并在处理后24h继续升高，在此期间没有观察到细胞周期分布的变化。为了评估作为WR-1065暴露的函数的NFkappaB反应基因的表达水平的变化，使用了包含49个被鉴定为具有NFkappaB的DNA结合基序的基因的cDNA阵列。只有5个基因在治疗1、4和/或16小时时受到显著影响。GST-3和c-myc分别被抑制2倍和4倍。相比之下，IL-2ra、RANTES和c-myb的表达水平分别增强了14倍、3倍和2倍。其余启动子区域含有NFkappaB反应元件的基因未表达(20个基因)或不受WR-1065影响(24个基因)。结论：细胞保护剂氨磷汀的活性硫醇形式可激活U87胶质瘤细胞中氧化还原敏感转录因子NFkappaB。抗氧化剂WR-1065激活NFkappaB的同时，癌基因c-myc的表达减少，抗氧化剂基因MnSOD的表达增强，该基因在肿瘤细胞中的表达相对较低，但当过度表达时，已与抑制恶性表型相关。然而，WR-1065激活NFkappaB会导致包含NFkappaB反应元件的基因表达发生选择性变化，而不是全局变化。(C)2002年爱思唯尔科学公司。
Purpose: Amifostine has been approved as a therapy to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation treatment for head-and-neck cancer. As a reducing agent capable of participating in intracellular reductive/oxidative processes, it has the potential to affect redox-sensitive transcription factors and gene expression, Amifostine's active free thiol WR-1065 was investigated to determine its effect on nuclear transcription factor kappaB (NFkappaB) activation and subsequent gene expression in U87 glioma cells.Methods and Materials: The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 muM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFkappaB activation was determined by a gel shift assay. Changes in gene expression as a function of time of exposure to WR-1065 were determined by Northern blot and the Atlas Human cDNA Expression Array (Clontech, Palo Alto, CA). Changes in gene expression using the Atlas Array were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers.Results: Exposure of U87 cells to 40 muM WR-1065 resulted in a marked activation of NFkappaB between 30 min and 1 h after treatment. Expression of WnSOD, an NFkappaB-responsive gene, was enhanced by over 2-fold after 16 h of treatment and remained elevated at 24 h. During this period of time, no changes in cell cycle distribution were observed. To assess changes in the expression levels of NFkappaB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFkappaB were used. Only five genes were found to be significantly affected at 1, 4, and/or 16 h of treatment. GST-3 and c-myc were repressed up to 2- and 4-fold, respectively. The expression levels of IL-2Ra, RANTES, and c-myb, in contrast, were enhanced up to 14-, 3-, and 2-fold, respectively. The remaining genes having NFkappaB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065.Conclusions: The redox-sensitive transcription factor NFkappaB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFkappaB by the antioxidant WR-1065 is accompanied by a reduced expression of the oncogene c-myc and an enhanced expression of the antioxidant gene MnSOD, a gene whose expression in tumor cells is relatively low, but when overexpressed has been correlated with a suppression of the malignant phenotype. Activation of NFkappaB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFkappaB-responsive elements. (C) 2002 Elsevier Science Inc.