MECHANISMS OF ARSENITE RESISTANCE AND TOLERANCE

亚砷酸盐是一种人类致癌物，其作用机制尚不清楚。 此前，我们证明了亚砷酸盐是一种DNA修复的抑制物， 尤其是结扎这一步。最近，亚砷酸盐的一个独特方面 在我们发现亚砷酸盐治疗诱导 扩增内源性DHFR基因，但不扩增SV4O序列。这 随着亚砷酸盐已知的能力的发现，激活了一些 调节子(如HSP、MDR)导致了我们目前对亚砷酸盐诱导的兴趣 赋予亚砷酸盐耐受性的基因。一些稳定的亚砷酸盐- 已分离出耐药和敏感的V79细胞亚系。两者都有 亚砷酸盐可诱导野生型和变异型品系发生瞬变 亚砷酸盐耐受状态，一个需要从头开始的mRNA和蛋白质的过程 综合。初步数据表明，抗亚砷品系AS/R28A 一种独特的表型。它对锑有交叉抵抗力，但对其他 金属，并含有正常水平的谷胱甘肽。利用细胞融合技术， 亚硝酸盐抗性对敏感性的优势(使用敏感性 品系AS/S14A)。将使用亚砷酸盐诱导的AS/R28A细胞 在创建cDNA消减文库时(从亚砷酸盐诱导的AS/R28A中获得 从未诱导的AS/S14A中减去cDNA)。矢量pmsg，它包含 可选择的gpt标记，其中插入的序列受 将使用糖皮质激素促进剂。重组pMSG将被 转化AS/SI4A细胞。转基因人员将通过HAT+进行选择 地塞米松+亚砷酸盐。该cDNA插入物将从 通过聚合酶链式反应扩增并测序。其他品种的表型 耐亚砷酸盐和敏感的亚硝酸盐亚系将与 AS/R28A和AS/S14A。对致病基因产物的分析 亚砷酸盐的抵抗力和耐受性将增加我们对 亚砷酸盐毒性，并可能对砷诱导的研究提供重要的见解 致癌也是如此。

Arsenite is a human carcinogen whose mechanism of action is unknown. Previously, we demonstrated that arsenite is an inhibitor of DNA repair, especially of the ligation step. Recently, a unique aspect of arsenite's activity became apparent in our finding that arsenite treatment induces amplification of the endogenous dhfr gene, but not of SV4O sequences. This finding, along with the known ability of arsenite to activate a number of regulons (e.g. hsp, MDR) led to our current interest in arsenite-inducible genes which confer arsenite tolerance. A number of stable arsenite- resistant and sensitive sublines of V79 cells have been isolated. Both wild-type and variant lines can be induced by arsenite to a transient arsenite-tolerant state, a process requiring de novo mRNA and protein synthesis. Preliminary data shows that arsenite resistant line As/R28A has a unique phenotype. It is cross-resistant to antimonite but not to other metals, and contains normal levels of GSH. Using cell fusion techniques, the dominance of arsenite resistance over sensitivity (using sensitive line As/S14A) was ascertained. Arsenite-induced As/R28A cells will be used in creating a cDNA subtractive library (cDNA from arsenite-induced As/R28A minus cDNA from uninduced As/S14A). Vector pMSG, which contains the selectable gpt marker and in which inserted sequences are under control of the glucocorticoid promoter, will be used. Recombinant pMSG will be transfected into As/SI4A. Transfectants will be selected with HAT + dexamethasone + arsenite. The cDNA insert will be rescued from the transfectants by PCR amplification and sequenced. The phenotypes of other arsenite-resistant and sensitive sublines will be compared to those of As/R28A and As/S14A. Analysis of the gene products responsible for arsenite resistance and tolerance will increase our understanding of arsenite toxicity and may give important insights into arsenic-induced carcinogenesis as well.