M. Iwanaga et al., NUCLEAR FACTOR KAPPA-B DEPENDENT INDUCTION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE BY IONIZING-RADIATION IN T98G HUMAN GLIOBLASTOMA CELLS, Free radical biology & medicine, 24(7-8), 1998, pp. 1256-1268
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.