title: Nuclear factor kappa B dependent induction of gamma glutamylcysteine synthetase by ionizing radiation in T98G human glioblastoma cells.
abstract: 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-kappa B). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-kappa B (Urata et al. J. Biol. Chem., 1996). Here, we present direct 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, 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-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 treated with buthionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-binding activity of NF-kappa B and the expression of gamma-GCS. We constructed chimeric genes containing various regions of gamma-GCS promoter gene and the coding region for Luciferase. T98G cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with ionizing 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 suggest that GSH synthesis is upregulated by ionizing radiation mediated by NF-kappa B and a high concentration of GSH in T98G cells causes downregulation of the NF-kappa B-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.
authors.affiliation: Department of Neurosurgery, Nagasaki University School of Medicine, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Iwanaga
authors.forename: K
authors.initials: K
authors.lastname: Mori
authors.forename: T
authors.initials: T
authors.lastname: Iida
authors.forename: Y
authors.initials: Y
authors.lastname: Urata
authors.forename: T
authors.initials: T
authors.lastname: Matsuo
authors.forename: A
authors.initials: A
authors.lastname: Yasunaga
authors.forename: S
authors.initials: S
authors.lastname: Shibata
authors.forename: T
authors.initials: T
authors.lastname: Kondo
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004790:Enzyme Induction / Q000528:radiation effects; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000532:radiotherapy*; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000975:Antioxidants; D004273:DNA, Neoplasm; D016328:NF-kappa B; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D019328:Buthionine Sulfoximine; D008156:Luciferases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D000111:Acetylcysteine