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。 以前，我们证明了亚砷酸盐是DNA修复的抑制剂， 尤其是连接步骤。最近，亚砷酸盐的一个独特方面 在我们的发现中，亚砷酸盐处理诱导 内源性dhfr基因扩增，但SV 4 O序列未扩增。这 发现，沿着已知的能力，亚砷酸盐激活一些 调节子（如热休克蛋白，MDR）导致我们目前的兴趣亚砷酸盐诱导 赋予砷耐受性的基因。一些稳定的亚砷酸盐- 已经分离出V79细胞的抗性和敏感亚系。两 野生型和变异株系可被亚砷酸盐诱导为瞬时突变， 砷耐受状态，这一过程需要从头mRNA和蛋白质 合成.初步数据表明，抗砷系As/R28 A具有 一种独特的表型对辉锑矿有交叉抗性，对其它矿物无交叉抗性 金属，并含有正常水平的GSH。 利用细胞融合技术， 抗砷性优于敏感性（使用敏感性 As/S14 A线）。将使用亚砷酸盐诱导的As/R28 A细胞 在构建cDNA消减文库（来自砷诱导的As/R28 A的cDNA 减去来自未诱导的As/S14 A的cDNA）。载体pMSG，其含有 可选择的GPT标记，并且其中插入的序列在 将使用糖皮质激素启动子。重组pMSG将 转染到AS/SI 4A中。将使用HAT +选择转染子 地塞米松+亚砷酸盐。cDNA插入片段将从 通过PCR扩增转染子并测序。其他的表型 将亚砷酸盐抗性和敏感亚系与 As/R28A和As/S14A。基因产物的分析 亚砷酸盐抗性和耐受性将增加我们对 砷的毒性，并可能提供重要的见解，砷引起的 致癌作用也是如此。

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.