Activation of the nuclear transcription factor kappaB (NFkappaB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine.
OBJECTIVE
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
The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 microM 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 microM WR-1065 resulted in a marked activation of NFkappaB between 30 min and 1 h after treatment. Expression of MnSOD, 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.
Kataoka
Yasushi
Y
Department of Radiation and Cellular Oncology, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.
Murley
Jeffrey S
JS
Khodarev
Nikolai N
NN
Weichselbaum
Ralph R
RR
Grdina
David J
DJ
D004999:Amifostine / Q000494:pharmacology*; D018946:Chemokine CCL5 / Q000378:metabolism; D005786:Gene Expression Regulation; D020570:Genes, myb / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D005910:Glioma / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000187:drug effects / Q000235:genetics; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit; D008624:Mercaptoethylamines / Q000494:pharmacology; D016328:NF-kappa B / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D011853:Radiobiology; D018123:Receptors, Interleukin / Q000187:drug effects / Q000235:genetics; D013482:Superoxide Dismutase / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018946:Chemokine CCL5; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D008624:Mercaptoethylamines; D016328:NF-kappa B; D011837:Radiation-Protective Agents; D018123:Receptors, Interleukin; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane; D013482:Superoxide Dismutase; D005982:Glutathione Transferase; D004999:Amifostine