三氧化二砷激發氧化自由基之分子機轉

Abstract

在吞噬性白血球中,三氧化二砷可以刺激NADPH 氧化脢進而激發superoxide 之形成, 這是免疫反應中很重要的一環.但是三氧化二砷刺激NADPH 氧化脢的分子機轉卻不甚 明瞭.在此研究中,我們證實三氧化二砷在各種白血病細胞中均有刺激NADPH 氧化脢 並激發superoxide 之能力.而大量的superoxide 似乎有增加NADPH 氧化脢活化之現象. 分化的吞噬性白血球有很強的NADPH 氧化脢,但三氧化二砷並未促使細胞分化,因此, 細胞分化不是三氧化二砷刺激NADPH 氧化脢的原因.我們並製備了可以產生 superoxide 的XCGD 細胞(XCGD-gp91),在細胞分化時,此細胞可以產生大量之 superoxide,而XCGD 細胞仍無任何可偵測的superoxide.我們相信這是進一步研究氧化 自由基生物學的一個重要的工具.我們進一步運用luciferase assay 來嘗試探討三氧化二 砷刺激NADPH 氧化脢的原因.我們把p47 PHOX (此乃NADPH 氧化脢之一個成分)之 promoter 接到Renilla luciferase reporter 之上游,並在三氧化二砷給予前後來測試 luciferase 之活性.結果顯示雖然三氧化二砷可以增加p47 PHOX 之表達,但是並未明顯提 升luciferase 之活性.這個矛盾可以以下列三種可能性來解釋: (1)雖然三氧化二砷可以 增加p47 PHOX 之表達,但是p47 PHOX 之transcription 並未增加,而是p47 PHOX 之半衰期被 三氧化二砷所延長.我們將運用actinomycin D 來阻斷新的mRNA 之合成,並測量 p47 PHOX 之半衰期,來驗證此假說. (2)三氧化二砷增加p47 PHOX 之表達之幅度可能不夠 高,使得p47 PHOX promoter-luciferase 之增加(if any)不夠明顯.為了證實這假設,我們 將用藥物促使細胞分化,此時之NADPH 氧化脢(包括p47 PHOX )之表達已大幅提升,再 運用這個系統去測試p47 PHOX promoter-luciferase 是否有上升. (3) p47 PHOX promoter-luciferase 之reporter assay 太過於人工化,無法運用於解答我們的問題.我們 相信下一年度是關鍵的一年.在過去的一年中,很多技術上的問題已經克服.只要再給 予我們支持,我們相信很快會有令人振奮的結果.

Arsenic trioxide (ATO) can induce superoxide production by up-regulation of NADPH oxidase, an enzyme complex in phagocytes for bactericidal effects via “respiratory burst ”. However, the mechanisms of NADPH oxidase activation remain to be defined. In this report, we showed that arsenic trioxide can induce superoxide in various kinds of leukemia cells via activation of NADPH oxidase. Reciprocally, large amount of superoxide produced by cell differentiation can further augment the activation of NADPH oxidase. Thus, there seems to be a positive feedback loop of NADPH oxidase and superoxide production. Furthermore, we show that ATO did not induce leukemia cell differentiation, thus excluding the possibility of cellular differentiation as a mechanism of NADPH oxidase activation and superoxide formation. We also generate a cell line, XCGD-gp91, which replenishes NADPH oxidase function in XCGD cells. Upon differentiation, this cell line exhibits huge amount of superoxide production while the parental cells still do not have detectable superoxide. We believe this pair of cell lines can be an excellent tool for research of biological effects of superoxide. Finally, we encounter a problem in matching the induction of in vivo NADPH oxidase and in vitro luciferase reporter activity. Several possibilities exist, but we have solutions to solve this problem. We have solved the technical problems in the past year, and we are ready to collect the data by use of these techniques. We believe there will soon be exciting data coming up by your further support next year.