Identification & genetic analysis of the human arsenite efflux pump

砷之所以令人担忧，是因为它是一种超级基金毒物和人类致癌物。它是为数不多的不会在啮齿动物身上致癌的人类致癌物质之一。与啮齿动物细胞相比，人类细胞对亚砷酸盐(可能致癌的形式)的细胞毒性作用更敏感，有证据表明人类对砷化合物的反应具有异质性。需要检验的主要假设是，细胞外排砷(“砷泵”)是砷抵抗的主要机制，细胞外排基因的异质性可能是导致对砷化合物敏感性差异的原因。因此，这项建议的目标是对导致人体细胞亚砷酸盐外流的途径进行分子描述，并确定构成该途径的蛋白质编码基因的遗传异质性。我们之前已经证明，哺乳动物细胞确实至少有一个亚砷酸盐外排系统。酵母(酿酒链霉菌)基因ACR3的产物很可能是一种亚砷酸盐外排泵，其功能类似于细菌的ARSB蛋白。我们还证明了酿酒酵母ACR2基因对砷的抗性是必需的，它编码一种砷酸还原酶，将砷还原为亚砷酸盐(泵的底物)。在人类细胞中没有人类同源物的情况下，我们将使用我们已经使用的替代方法来克隆对亚砷酸盐(泵的底物)的其他抗性。到目前为止，还没有发现这些序列的人类同源物。用简并的ACR3引物对人肝cDNA文库进行PCR扩增。如果在人类细胞中没有ACR3同源基因，我们将使用在本实验室克隆其他抗性基因的替代方法：表达克隆和/或聚合酶链式反应-选择差异筛选。我们已经分离了抗亚砷酸盐的中国仓鼠和人骨肉瘤细胞，用于这些替代策略。我们还将通过将人的cDNA导入破坏ACR2和ACR3基因的酵母菌株来进行表达克隆。通过任何策略分离的任何cDNA都将以正义和反义方向导入野生型人类细胞，并对所产生的表型进行表征。最后，我们将比较不同捐赠者对砷化合物的易感性不同的人体细胞亚砷酸盐外排速度和基因类型。

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.