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有毒物质，也是一种人类致癌物。它是为数不多的对啮齿动物没有致瘤性的人类致癌物之一。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）的细胞毒性作用要敏感得多，而且有证据表明，人类对砷化合物的反应存在异质性。待验证的主要假设是，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易感性差异的基础。因此，本提案的目标是对人体细胞中负责亚砷酸盐外排的途径进行分子描述，并确定编码构成该途径的蛋白质的基因的遗传异质性。我们之前已经证明哺乳动物细胞至少有一个亚砷酸盐排出系统。酵母（酿酒酵母）基因ACR3的产物很可能是亚砷酸盐外排泵，其功能类似于细菌的ArsB蛋白。我们还证明酿酒葡萄球菌ACR2基因是砷酸盐抗性所必需的，并编码一种砷酸盐还原酶，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，我们将使用替代方法克隆其他对亚砷酸盐（泵的底物）的抗性。到目前为止，还没有发现与这些序列相似的人类同源物。人类肝脏cDNA文库将用于用退化的ACR3引物进行PCR扩增。如果在人类细胞中没有ACR3同源物，我们将使用我们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。我们还将通过将人类cdna转染到破坏ACR2和ACR3基因的酵母菌株中来进行表达克隆。通过任何策略分离的任何cdna都将以正义取向和反义取向转染到野生型人类细胞中，并对所产生的表型进行表征。最后，我们将比较不同供体对砷化合物具有不同敏感性的人体细胞的亚砷酸盐外排率和基因型。

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.