Katsuharu Mori

Nuclear Factor Kappa B Dependent Induction of Gamma Glutamylcysteine Synthetase by Ionizing Radiation in T98G Human Glioblastoma Cells

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.

Regulation of γ-glutamycysteine synthetase expression in response to oxidative stress

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gammaglutamylcysteine synthetase (γ-GCS) is the rate limiting enzyme for the GSH synthesis. γ-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of γ-GCS gene, in the present study, we show evidences that γ-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-α mediated by nuclear factor-κB (NF-κB), and impairment of the expression of γ-GCS by TNF-α in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-κB subunits.

Co-expression of gamma-glutamylcysteine synthetase sub-units in response to cisplatin and doxorubicin in human cancer cells

Glutathione (γ‐glutamylcysteinyl glycine, GSH) is believed to be important in the acquisition of resistance to anti‐cancer drugs such as cisplatin (CDDP) and doxorubicin (DOX). γ‐Glutamylcysteine synthetase (γ‐GCS) is a key enzyme for maintaining intracellular GSH levels; it is composed of a catalytic heavy (γ‐GCSh) and a regulatory light (γ‐GCSl) sub‐unit. In the present study, the expression of γ‐GCS sub‐units was examined in human cancer cell lines. The levels of GSH, the expression of γ‐GCSh and γ‐GCSl mRNAs and proteins in human cancer cells were higher in CDDP‐resistant cells and DOX‐resistant cells than in the drug‐sensitive cells. Treatment of CDDP/DOX‐resistant human colonic‐cancer cells (HCT8DDP) with CDDP and DOX caused simultaneous induction of the expression of γ‐GCSh and γ‐GCSl mRNAs. The transcriptional regulation of γ‐GCS was studied and it was observed that the DNA‐binding activity of activator protein 1 (AP‐1) is induced by CDDP and DOX using an electrophoretic mobility shift assay. We constructed chimeric genes containing various regions of the γ‐GCSh‐promoter gene and the coding region for luciferase. The HCT8DDP cells transiently transfected with a plasmid containing an AP‐1 site of the γ‐GCSh‐promoter‐luciferase construct showed increased luciferase activity when treated with CDDP and DOX. These results suggest that stimulation of the expression of γ‐GCSh by CDDP and DOX is mediated by AP‐1, which relates to the resistance of cancer cells to the drug. Int. J. Cancer 82:405–411, 1999.

Dose optimization and indication of linac radiosurgery for brain metastases

PURPOSE The authors have examined treatment effects of linear accelerator based radiosurgery for brain metastases. Optimal doses and indications were determined in an attempt to improve the quality of life for terminal cancer patients. METHODS AND MATERIALS Ninety-two patients with 162 lesions were treated with Linac radiosurgery for brain metastases between April 1993 and September 1998. To determine prognostic factors, risk factors for recurrence, and appearance of new lesions, univariate and multivariate analyses were performed. To compare the local control between the high-dose (minimum dose > or =25 Gy: prescribed to the 50-80% isodose line) and low-dose (minimum dose <25 Gy) irradiated groups, matched-pairs analysis was performed. RESULTS Median survival time was 11 months. In univariate analysis, extracranial tumor activity (p<0.001) and Karnofsky Performance Status (KPS) (p = 0.036) were two significant predictors of survival. In multivariate analysis, the status of an extracranial tumor was the single significant predictor of survival (p = 0.005). Minimum dose was the single most significant predictor of local recurrence in univariate, multivariate, and matched-pairs analyses (p<0.05). As to the appearance of new lesions, activity of extracranial tumors was a significant predictor (p<0.05). Side effects due to radiosurgery were experienced in 4 of 92 cases (4.3%). CONCLUSIONS We concluded that brain metastases patients should be irradiated with > or =25 Gy, when extracranial lesions are stable and longer survival is expected. Combined surgery and conventional radiation may have little advantage over radiosurgery alone when metastatic brain tumors are small and extracranial tumors are well controlled. When extracranial tumors are progressive, the rate of appearance of new lesions in other nonirradiated locations becomes higher. In such cases, careful follow-up is required and a combination with whole brain irradiation should be considered.

Mitogen-activated protein kinase, ERK1/2, is essential for the induction of vascular endothelial growth factor by ionizing radiation mediated by activator protein-1 in human glioblastoma cells

Vascular Endothelial Growth Factor (VEGF)/Vascular Permeability Factor plays an important role in angiogenesis and cell proliferation of cancer cells. Glioblastoma cells are most malignant and show resistance to radiation therapy inducing VEGF to cause angiogenesis and brain edema. In the present study, the regulatory mechanism of the expression of VEGF by ionizing radiation was studied in three human glioblastoma cells. Induction of VEGF mRNA by ionizing radiation was dependent on dose and incubation time. Activator protein-1 (AP-1) was activated by 10 Gy of ionizing radiation in 1 h in T98G glioblastoma cells on an electrophoretic mobility shift assay. We constructed chimeric genes containing various regions of the VEGF promoter gene and the coding region for chloramphenicol acetyltransferase (CAT) and transiently transfected them to T98G cells. CAT assay with the VEGF promoter gene containing an AP-1 site demonstrated that the promoter activity of the VEGF gene was enhanced by ionizing radiation. Immunological analysis of the activity of mitogen-activated protein kinase, ERK1/2, showed that this activity is up-regulated by ionizing radiation. These results suggest that ERK1/2 pathway is involved in the up-regulation of VEGF expression ionizing radiation mediated by AP-1, which may lead to further neovascularization and proliferation of glioblastoma cells resistant to radiation therapy.

Striatal dopamine in acute cerebral ischemia of stroke-resistant rats.

We devised the present experiments to assess the effects of ischemia on the production of dopamine in the caudate nucleus of spontaneously hypertensive stroke-resistant rats. Ringers solution was continuously perfused at a rate of 10 microliters/min through 0.2-mm-diameter dialysis tubing implanted in the rats caudate nucleus. After bilateral occlusion of the common carotid artery, perfusate was collected at 20-minute intervals for 120 minutes and was analyzed for monoamines and their metabolites using high-performance liquid chromatography and an electrochemical detection system. The extracellular concentration of dopamine increased abruptly approximately 3 minutes after the ischemic insult, reached a maximum at between 20 and 40 minutes after the insult, and subsequently decreased. During the 120 minutes, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole-3-acetic acid concentrations decreased significantly, whereas 5-hydroxytryptamine was not detected. Our results indicate that during cerebral ischemia a large increase in extracellular dopamine concentration in the caudate nucleus occurs, probably as a result of energy failure of the cell membranes. This leakage of dopamine may be a causal factor in the neuronal damage associated with cerebral ischemia.

Ruptured aneurysm at the choroidal branch of the posterior inferior cerebellar artery: a case report and review of the literature

BACKGROUND The authors present a rare case of a ruptured aneurysm at the choroidal branch of the posterior inferior cerebellar artery (PICA). CASE DESCRIPTION A 77-year-old female was admitted to our institute because of sudden onset of severe headache and vomiting. Radiologic examination revealed intraventricular hemorrhage caused by rupture of the aneurysm at the choroidal branch of the PICA. The fusiform aneurysm was resected after ligation via a midline suboccipital approach. CONCLUSIONS The conclusions drawn from this experience and a review of the literature include the following: (1) the aneurysm at the branch of the PICA is frequently associated with anomalies of the vascular structure, particularly in hypoplasty of the contralateral PICA; (2) hemodynamic stress is speculated to be a causative factor of these lesions; (3) cases with hypoplasty of the contralateral PICA have the possibility of developing nonmycotic peripheral aneurysms at the branch of the PICA; (4) these aneurysms should be managed immediately because of the high risk of rebleeding.

Hammerhead ribozyme against γ-glutamylcysteine synthetase attenuates resistance to ionizing radiation and cisplatin in human T98G glioblastoma cells

Glioblastoma cells are highly malignant and show resistance to ionizing radiation, as well as anti‐cancer drugs. This resistance to cancer therapy is often associated with a high concentration of glutathione (GSH). In this study, the effect of continuous down‐regulation of γ‐glutamylcysteine synthetase (γ‐GCS) expression, a rate‐limiting enzyme for GSH synthesis, on resistance to ionizing radiation and cisplatin (CDDP) was studied in T98G human glioblastoma cells. We constructed a hammerhead ribozyme against a γ‐GCS heavy subunit (γ‐GCSh) mRNA and transfected it into T98G cells. (1) The transfection of the ribozyme decreased the concentration of GSH and resulted in G1 cell cycle arrest of T98G cells. (2) The transfection of the ribozyme increased the cytotoxicity of ionizing radiation and CDDP in T98G cells. Thus, hammerhead ribozyme against γ‐GCS is suggested to have potential as a cancer gene therapy to reduce the resistance of malignant cells to ionizing radiation and anti‐cancer drugs.