Nuclear Factor Kappa B Dependent Induction of Gamma Glutamylcysteine Synthetase by Ionizing Radiation in T98G Human Glioblastoma Cells

Glioblastoma is one of the most malignant of all neoplasms, and often shows resistance to chemotherapy and radiation therapy. Ionizing radiation activates transcriptional factors, such as nuclear factor kappa-B (NF-κB). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-κB (Urata et al. J. Biol. Chem., 1996). Here, we present direct evidence that NF-κB activated by ionizing radiation induces the expression of γ-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme of GSH synthesis, using T98G human glioblastoma cells. T98G cells have approximately 14-times the level of intracellular GSH of NB9 cells, radiation-sensitive neuroblastoma cells. In T98G cells, 30-Gy of ionizing radiation was required for the activation of NF-κB on an electrophoretic mobility shift assay and the induction of γ-GCS mRNA on Northern blots and a nuclear run-on assay. However, when T98G cells were treated with buthionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-binding activity of NF-κB and the expression of γ-GCS. We constructed chimeric genes containing various regions of γ-GCS promoter gene and the coding region for Luciferase. T98G cells transiently transfected with a plasmid containing the γ-GCS promoter-luciferase construct showed increased luciferase activity when treated with ionizing radiation. The luciferase activity stimulated by ionizing radiation was found in the γ-GCS promoter containing the NF-κB binding site, whereas not in that containing its mutated site. These results suggest that GSH synthesis is upregulated by ionizing radiation mediated by NF-κB and a high concentration of GSH in T98G cells causes downregulation of the NF-κB-DNA binding activity in response to ionizing radiation. The irresponsiveness of the intracellular signal transduction cascade to irradiation may be a factor in the resistance of T98G cells to radiation therapy.