Activation of the nuclear transcription factor κB (NFκB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine

<UNL TYPE="BAR" STYLE="S">Purpose</UNL>: 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 κB (NFκB) activation and subsequent gene expression in U87 glioma cells.<UNL TYPE="BAR" STYLE="S">Methods and Materials</UNL>: The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 μM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFκB 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.<UNL TYPE="BAR" STYLE="S">Results</UNL>: Exposure of U87 cells to 40 μM WR-1065 resulted in a marked activation of NFκB between 30 min and 1 h after treatment. Expression of MnSOD, an NFκB-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 NFκB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFκB 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 NFκB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065.<UNL TYPE="BAR" STYLE="S">Conclusions</UNL>: The redox-sensitive transcription factor NFκB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFκB 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 NFκB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFκB-responsive elements. [Copyright &y& Elsevier]