Identification & genetic analysis of the human arsenite efflux pump

砷是令人关注的，因为它是一种超级基金有毒物质和人类致癌物质。它是少数几种在啮齿动物中不致癌的人类致癌物之一。与啮齿类动物细胞相比，人类细胞对亚砷酸盐（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具有异质性。要测试的主要假设是，砷的细胞外排（“砷泵”）是砷抗性的主要机制，细胞外排基因的异质性可能是对砷化合物敏感性差异的基础。因此，该建议的目标是负责在人类细胞中的亚砷酸盐流出的途径的分子描述和确定的基因编码的蛋白质组成的途径的遗传异质性。我们以前已经表明，哺乳动物细胞至少有一个亚砷酸盐外排系统。酵母的产物（S.酿酒酵母）基因，ACR 3，最有可能是具有与细菌ArsB蛋白类似的功能的亚砷酸盐外排泵。我们还证明了S。酿酒酵母ACR 2基因是砷酸盐抗性所必需的，并且编码砷酸盐还原酶，其将砷酸盐还原为亚砷酸盐（泵的底物）。在人类细胞中没有人类同源物，我们将使用替代的方法，我们已经用来克隆其他抗亚砷酸盐（底物的泵）。迄今尚未发现这些序列的人类同源物。人肝脏cDNA文库将用于使用简并ACR 3引物PCR扩增序列。如果人类细胞中没有ACR 3同源物，我们将使用我们必须在本实验室克隆其他抗性基因的替代方法：表达克隆和/或PCR选择差异筛选。我们已经分离出抗砷的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转染到已破坏ACR 2和ACR 3基因的酵母菌株中来进行表达克隆。将通过任何策略分离的任何cDNA以正义和反义方向转染到野生型人细胞中，并表征所得表型。最后，我们将比较不同供体的人类细胞中亚砷酸盐的外排率和基因型，这些供体对砷化合物具有不同的敏感性。

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.