| 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.
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