Abstract

Glucose-6-phosphate dehydrogenase is the first key enzyme in the pentose phosphate pathway. Its main physiological function is to produce NADPH and ribose-5-phosphate which are essential for reductive biosynthesis and nucleic acid synthesis. The pentose phosphate pathway is especially important in red blood cells because it is the only way to produce NADPH. The major role of NADPH in erythrocytes has been reported to regenerate the reduced form of glutathione (GSH), which plays an important role in detoxification of hydrogen peroxide and organic peroxides. However, the physiological function of G6PD in non-erythroid cells is less well-characterized.

In order to study the function of G6PD in non-erythroid cells, we examined the sensitivity of NIH3T3 cells transfected with a plasmid containing human G6PD cDNA to tert-butyl hydroperoxide (TBH) and paraquat. Two transfected clones which had a sixteen fold (H7 clone) and six fold (H6 clone) increase in their intracellular G6PD activity, were compared with control cells transfected with a vector alone. Cells with high-level expression of human G6PD were 2.3 (H6) to 3.7 (H7) times more resistant to TBH than control cells. The antioxidant (anti-TBH) abilities in H6 and H7 cells were revealed by (1) a significant increase in the intracellular level of NADPH and glutathione, (2) a reduction of fluorescent intensity of the oxidant-sensitive dye, 2*,7*-dichlorofluorescin diacetate, and (3) a significant reduction in the production of oxidized adducts generated by lipid peroxidation. In contrast, cells overexpressing G6PD were very sensitive to paraquat, a superoxide-producing herbicide. The concentrations of paraquat required to produce a 50% decrease in cell viability of H7, H6 and control cells were 0.80 mM, 1.14 mM and 2.19 mM, respectively. The cytotoxicity of paraquat correlated with the expression level of NADPH in the cells. In this study, overexpression of human G6PD in NIH3T3 cells had different effects on the toxicity of TBH versus paraquat. Reduction of NADP+ to NADPH by G6PD protects cells from oxidative damage by TBH, but appears to enhance the toxicity of paraquat.

In addition to the role of G6PD in antioxidation systems, G6PD activity was reported to be positively correlated with active cell growth. In normal cells, G6PD expression is tightly controlled, however, regulation of its expression is altered, resulting in a significant increase of G6PD activity in many tumors. To investigate the potential role of G6PD in tumorigenesis, we study the growth properties and transforming ability of the two G6PD-overexpressing cell clones, H6 and H7. Both showed altered cell morphology and exhibited tumorigenic properties. In contrast to the control cells or cells transfected with mutated G6PD cDNA, cells with high level expression of G6PD were not contact inhibited and exhibited anchorage-independent growth. They divided more quickly and induced rapidly growing, large fibrosarcomas in nude mice. The formation of new blood vessels was observed in the G6PD-induced tumors. Moreover, the induced tumorigenic properties were positively correlated with the level of the intracellular G6PD activity. H7 cells had a much higher saturation density and formed foci and colonies more efficiently than H6. Moreover, H7-induced tumors had a larger tumor mass and exhibited a shorter latency period. This is the first report to describe that G6PD is a proto-oncogene. Our results provide a model to examine the causative role of G6PD misregulation in cellular proliferation and the interaction of G6PD with other gene products in the development of tumors.
封面
目錄
摘要
Abstract
第一章 緒論
Ⅰ、G6PD的介紹
(1)Glucose-6-phosphate dehydrogenase
(2)G6PD缺乏症
Ⅱ、研究目的
第二章 材料和方法
Ⅰ.細胞培養
Ⅱ.G6PD的基因轉染
Ⅲ.利用native gel elctrophoresis system分析人類表現
Ⅳ.Paraquat及TBH的細胞毒性測試
Ⅴ.細胞中酵素的活性
Ⅵ.測量lipid peroxidation的產物
Ⅶ.利用Flow cytometer測定細胞中的活性氧物質
Ⅷ.統計分析
Ⅸ.細胞的生長特質(growth properties)分析
Ⅹ.致癌性測試(tumorigenic assay)
第三章 G6PD在細胞抗氧化系統中所扮演的角色
Ⅰ、簡介
Ⅱ、結果
(1)人類G6PD在NTH 3T3細胞中的大量表現
(2)TBH和 paraquat 的細胞毒性
(3)細胞中抭氧化酵素的活性及NADPH和glutathione的濃度
(4)TBH處理後、細胞中NADPH和GSH濃度的變化
(5)TBH或paraquat處理後,細胞中氧化物的生成
(6)TBH所誘發的脂質過氧化
Ⅲ、討論
第四章 G6PD的高量表現造成NIH 3T3細胞的致癌化轉型
Ⅰ、簡介
Ⅱ、結果
(1)細胞的型態及生長曲線分析
(2)G6PD高量表現的細胞會形成transformed focus
(3)G6PD高量表現的細胞具有anchorage-independent growth的能力
(4)G6PD高量表現的細胞具有誘發癌症的能力
(5)G6PD在tumor中的持續高量表現
(6)大量表現突變過的G6PD無法造成細胞的轉型
(7)G6PD所造成的細胞致癌性轉型,可能是經由改變細胞中的氧化還原態
Ⅲ、討論
第五章、論文總結
Ⅰ、結論
Ⅱ、未來計畫
Tables & Figures
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