The regulation of glutathione synthesis by oxidative stresses

Research Project

Project/Area Number	09670158
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Pathological medical chemistry
Research Institution	Nagasaki University
Principal Investigator	URATA Yoshishige Nagasaki University School of Medicine, Asistant, 医学部, 助手 (30185087)
Project Period (FY)	1997 – 1998
Project Status	Completed (Fiscal Year 1998)
Budget Amount *help	¥700,000 (Direct Cost: ¥700,000) Fiscal Year 1998: ¥700,000 (Direct Cost: ¥700,000)
Keywords	Glutathione / NF-kB / gamma-Glutamylcysteine synthetase / γ-グルタミルシステインシンセターゼ / ルシフェラーゼ / トランスフェクション
Research 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-_kB). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by Nfi-_kB (Urata et al. J.Bio. Cheim., 1996). Here, we present direct evidence that NF-_kB 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 NP-_kB on an electrophoretic mobility shift assay and the induction of gamma-GCS mRNA on Northern blots and a nuclear run-on assay. DNA-binding activity of NF-_kB 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-_kB binding site, whereas not in that containing its mutated site. These results suggest that GSH synthesis is downregulation of the NF-KB-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.