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 radia tion activates transcriptional factors, such as nuclear factor kappa-B (NF-kappa B). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-kappa B (Urata et al. J. Biol. Ch em., 1996). Here, we present diner evidence that NF-kappa B activated by ionizing radiation induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme of GSH synthesis, us ing T98G human glioblastoma cells. T98G cells have approximately 14-ti mes the level of intracellular GSH of NB9 cells, radiation-sensitive n euroblastoma cells. In T98G cells, 30-Gy of ionizing radiation was req uired for the activation of NF-kappa B On an electrophoretic mobility shift assay and the induction of gamma-GCS mRNA on Northern blots and a nuclear run-on assay. However, when T98G cells were created with but hionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-bin ding activity of NF-kappa B and the expression of gamma-GCS. We constr ucted chimeric genes containing various regions of gamma-GCS promoter gene and the coding region for Luciferase. T98G cells transiently tran sfected with a plasmid containing the gamma-GCS promoter-luciferase co nstruct showed increased luciferase activity when treated with ionizin g radiation. The luciferase activity stimulated by ionizing radiation was found in the gamma-GCS promoter containing the NF-kappa B binding site, whereas not in that containing its mutated site. These results s uggest that GSH synthesis is upregulated by ionizing radiation mediate d by NF-kappa B and a high concentration of GSH in T98G cells causes d ownregulation of the NF-kappa B-DNA binding activity in response to io nizing radiation. The irresponsiveness of the intracellular signal tra nsduction cascade to irradiation may be a factor in the resistance of T98G cells to radiation therapy. (C) 1998 Elsevier Science Inc.