Identification & genetic analysis of the human arsenite efflux pump

鉴别

• 批准号: 6442972
• 负责人: Toby G. Rossman
• 金额: $ 16.41万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6442972
• 关键词: arsenic; cell line; environmental toxicology; gene expression; genetic mapping; genetic susceptibility; hazardous substances; human tissue; membrane permeability; polymerase chain reaction; tissue /cell culture

Arsenic is of concern because it is a Superfund toxicant and a human carcinogen. It is one of the few human carcinogens which is not tumorigenic in rodents. Human cells are much more sensitive to the cytotoxic effects of arsenite (the likely carcinogenic form) compared to rodent cells, and there is evidence for heterogeneity in the response of humans to arsenic compounds. The main hypothesis to be tested is that cellular efflux of arsenic ("arsenic pump") is a primary mechanism of arsenic resistance, and heterogeneity in cellular efflux genes may underlie differences in susceptibility to arsenic compounds. Thus, the goals of this proposal are a molecular description of the pathway responsible for arsenite efflux in human cells and a determination of genetic heterogeneity in the genes encoding the proteins making up the pathways. We have previously shown that mammalian cells do have at least one arsenite efflux system. The product of a yeast (S. cerevisiae) gene, ACR3, is most likely an arsenite efflux pump with a function similar to the bacterial ArsB proteins. We have also demonstrated that the S. cerevisiae ACR2 gene is required for arsenate resistance and encodes an arsenate reductase which reduces arsenate to arsenite (substrate of the pump). No human homologues in human cells, we will use alternative methods which we have used to clone other resistance to arsenite (substrate of the pump). No human homologues to these sequences have been identified to date. A human liver cDNA library will be used to PCR amplify sequences with degenerate ACR3 primers. In the event that there are no ACR3 homologues in human cells, we will use alternative methods which we have to clone other resistance genes in this laboratory: expression cloning and/or PCR-select differential screening. We have isolated arsenite- resistance Chinese hamster and human osteosarcoma cells for use in these alternative strategies. We will also perform expression cloning by transfecting human cDNAs into yeast strains which have disrupted ACR2 and ACR3 genes. Any cDNAs isolated by any strategy will be transfected into wild type human cells in both sense and anti-sense orientation, and the resulting phenotypes will be characterized. Finally, we will compare arsenite efflux rates and genotypes in human cells from different donors which have different susceptibilities to arsenic compounds.

砷之所以关注，是因为它是一种超级基金有毒物质和人类致癌物。它是为数不多的人类致癌物之一，它在啮齿动物中不是肿瘤性。与啮齿动物细胞相比，人类细胞对砷（可能的致癌形式）的细胞毒性作用更为敏感，并且有证据表明人类对砷化合物的反应异质性。要测试的主要假设是，砷的细胞外排（“砷泵”）是砷耐药性的主要机制，细胞外排基因的异质性可能是对砷化合物易感性的差异。因此，该提案的目标是对负责人类细胞中砷外排的途径的分子描述，并确定编码构成途径的蛋白质的基因中的遗传异质性。我们以前已经表明，哺乳动物细胞确实至少具有一个砷排外系统。酵母（酿酒酵母）基因ACR3的产物很可能是一种具有类似于细菌ARSB蛋白的功能的砷外排泵。我们还证明，酿酒酵母ACR2基因是砷耐药性所必需的，并编码一种将砷酸盐还原为砷酸盐（泵的底物）。在人类细胞中没有人类同源物，我们将使用替代方法，我们用来克隆其他抗砷（泵的底物）的耐药性。迄今为止，尚未确定对这些序列的人类同源。人肝cDNA文库将用于使用退化的ACR3引物放大序列。如果人类细胞中没有ACR3同源物，我们将使用替代方法来克隆该实验室中的其他抗性基因：表达克隆和/或PCR选择差分筛选。我们已经孤立的砷耐性中国仓鼠和人骨肉瘤细胞用于这些替代策略。我们还将通过将人cDNA转染到破坏ACR2和ACR3基因的酵母菌菌株中来执行表达克隆。任何通过任何策略分离的cDNA都将在意义上和反义方向上转染到野生型人类细胞中，并且将表征所得的表型。最后，我们将比较来自不同供体的人类细胞中的砷外排率和基因型，这些供体对砷化合物具有不同的敏感性。