MECHANISMS OF ARSENITE RESISTANCE AND TOLERANCE

亚砷酸盐的抵抗和耐受机制

• 批准号: 2098090
• 负责人: Toby G. Rossman
• 金额: $ 15.5万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2098090
• 关键词: antimony; arsenic; autosomal dominant trait; chemical carcinogen; complementary DNA; drug resistance; drug tolerance; enzyme activity; enzyme induction /repression; enzyme inhibitors; gene complementation; genetic library; metal metabolism; metal poisoning; molecular cloning; nucleic acid sequence; phenotype; tissue /cell culture

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.

砷是一种人类致癌，其作用机理尚不清楚。 以前，我们证明了砷是DNA修复的抑制剂， 特别是结扎步骤。最近，阿森特的一个独特方面 我们发现砷治疗引起的发现显而易见 内源性DHFR基因的扩增，但不是SV4O序列的扩增。这 发现，以及砷的已知能力激活许多 规范（例如HSP，MDR）导致我们目前对砷诱导的兴趣 赋予砷耐受性的基因。许多稳定的砷 - 已经分离出V79细胞的抗性和敏感的子链。两个都 野生型和变体线可以通过砷诱导到瞬态 砷耐受状态，一个需要从头mRNA和蛋白质的过程 合成。初步数据表明，砷耐药线AS/R28A具有 独特的表型。它是抗杀伤力的抗性，但对其他 金属，并包含正常水平的GSH。 使用细胞融合技术， 砷耐药性比灵敏度的优势（使用敏感性 确定行为/s14a）。砷诱导的AS/R28A细胞将被使用 在创建cDNA减法库（来自砷诱导的AS/R28A的cDNA 从未诱导的AS/S14A）减去cDNA。向量PMSG，其中包含 可选的GPT标记，其中插入的序列正在控制 将使用糖皮质激素启动子。重组PMSG将是 转染为/si4a。转送者将与Hat +一起选择 地塞米松 +砷。 cDNA插入物将从 通过PCR扩增并测序的转染剂。其他表型 将抗砷矿和敏感的子序与 AS/R28A和AS/S14A。分析负责的基因产品 砷的抵抗和宽容将增加我们对 砷的毒性，可能对砷引起的重要见解 癌变也是如此。