METALLOTHIONEIN--EFFECTS ON MUTAGENESIS

本提案的主要目的是检验以下假设： 金属硫蛋白（MT）能够影响致突变事件的水平 由遗传毒性致癌物引起的。 有一些相互矛盾的数据 关于MT表达水平不同的细胞的敏感性， 对致癌物和化疗药物毒性作用的敏感性 剂. 然而，尚未对突变进行研究。 这些细胞中的频率。 我们开发了一系列的中国仓鼠 V79转基因细胞系含有单拷贝的E.大肠杆菌谷丙转氨酶 基因 其中之一，G12，已被证明具有靶基因（gpt）， 通常可被烷基化剂改变， 引起氧化损伤（例如X射线），其不产生许多基因 大多数系统中的突变。 像许多永久性细胞系一样，G12具有低的细胞毒性。 组成型和诱导型MT表达水平。 我们用G12细胞 通过将小鼠MT-I基因转染到 pBPV载体。 初步结果显示，具有 最高水平的表达具有升高的自发突变， 与其亲本G12相比， 然而，这些结果是 由于染色体外复制质粒的存在而混淆， 这可能会导致遗传不稳定。 我们建议建造 转基因株系，使用G12作为宿主，其含有稳定整合的 Mouser MT-I的副本。 此外，我们将创建一个MT反义 表达载体，以敲除宿主MT-I表达。 这些 线也将用于测量自发突变频率 作为用一些金属处理后的诱发突变， （结晶硫酸镍和铬酸钙）、物理（UV和x-射线） 射线）和有机（MNU和MNNG）致癌物和化学治疗剂 （顺铂、博来霉素、mAMSA）。 亚砷酸盐、Cd（II）和 还将评估Pb（II）作为共诱变剂的作用。 如果影响 发现诱变或共诱变随MT表达而变化， MT水平对DNA修复酶（需要锌）的影响将是 在透化细胞中研究。

The main goal of this proposal is to test the hypothesis that metallothionein (MT) is able to effect the level of the mutagenic events induced by genotoxic carcinogens. There is some contradictory data concerning the sensitivity of cells with varying levels of MT expression sensitivity to the toxic effects of carcinogens and chemotherapeutic agents. However, no studies have yet been carried out on mutation frequencies in such cells. We have developed a series of Chinese hamster V79-transgenic cell lines containing single copies of the E. coli gpt gene. One of these, G12, has been shown have a target gene (gpt) which is normally mutable by alkylating agents and highly mutable by agents causing oxidative damage (e.g. x-rays) which do not produce many gene mutations in most systems. Like many permanent cell lines, G12 has a low level of constitutive and induced MT expression. We have used G12 cells to create MT-overproducing cells by transfection of a mouse MT-I gene in a pBPV vector. Preliminary results show that the MT transfectant with the highest level of expression has an elevated spontaneous mutation frequency compared to its parent, G12. However, these results are confounded by the presence of extrachromosomally replicating plasmid, which might confer genetic instability. We propose to construct transgenic lines, using G12 as host, which contain stably integrated copies of mouser MT-I. In addition, we will create an MT anti-sense expression vector in order to knock out host MT-I expression. These lines will be used to measure the spontaneous mutation frequency as well as the induced mutagenesis after treatment with a number of metal (crystalline nickel sulfate and calcium chromate), physical (UV and x- rays) and organic (MNU and MNNG) carcinogens and chemotherapeutic agents (cisplatin, bleomycin, mAMSA). The abilities of arsenite, Cd(II), and Pb(II) to act as comutagens will also be evaluated. If effects on mutagenesis or comutagenesis are found to vary with MT expression, effects of MT levels on DNA repair enzymes (which require zinc) will be studied in permeabilized cells.