The regulation of glutathione synthesis by oxidative stresses

胶质母细胞瘤是所有肿瘤中恶性程度最高的肿瘤之一，通常对化疗和放射治疗具有抵抗力。电离辐射可激活转录因子，如核因子kappaB(NFkB)。以前我们发现谷胱甘肽(GSH)的合成是由NFI-kB介导的细胞因子诱导的(Urata等人)。J.Bio。Cheim，1996)。在这里，我们提出了直接的证据，核因子-kB被电离辐射激活诱导GSH合成的限速酶-γ-谷氨酰半胱氨酸合成酶(γ-GCS)的表达，使用T98G人胶质母细胞瘤细胞。T98G细胞的细胞内GSH水平大约是NB9细胞的14倍，NB9细胞是对辐射敏感的神经母细胞瘤细胞。在T98G细胞中，需要30GY的电离辐射才能激活NP-kB，并在Northern杂交和核连续实验中诱导γ-GCS mRNA的表达。构建了含有不同区域的γ-GCS启动子基因和荧光素酶编码区的嵌合基因。瞬时转染含有GCS启动子-荧光素酶基因的T98G细胞，在电离辐射作用下，其荧光素酶活性增强。在含有核因子-kB结合位点的γ-GCS启动子中发现了电离辐射刺激的荧光素酶活性，而在含有突变位点的启动子中则没有。这些结果表明，GSH的合成是对电离辐射的反应，下调了核因子-KB-DNA的结合活性。细胞内信号转导通路对辐射的无反应性可能是T98G细胞耐受放射治疗的一个因素。

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.