METABOLIC DETERMINANTS OF SPONTANEOUS MUTAGENESIS

描述：本提案的目标是评估 抗氧化剂维生素C（抗坏血酸盐）、维生素E（α-生育酚）和 金属硫蛋白（MT），以减少自发的频率和频谱 HPRT基因座的突变。 抗坏血酸有望减少DNA损伤 由含氮碱、α-生育酚通过 通过脂质过氧化产物阻断DNA加合物的形成， 金属硫蛋白通过清除细胞自由基。 的光谱 自发突变和DNA损伤模式将在三个 人细胞系：HCT 116，一种错配修复缺陷型结肠癌细胞 细胞系; HCT 116/ch 3，和HCT 116衍生物，其中完整的人 3号染色体已被引入，而SW 480，一种擅长错配修复的 结肠癌细胞系 细胞将与两种中的一种一起孵育， 抗氧化剂或用金属硫蛋白表达载体转染， 用锌孵育，收集HPRT突变体，并观察突变谱和DNA 已确定损坏情况。 此外，错配修复缺陷细胞 将评价一个品系的一组未描述的微卫星标记 用于确定氧化损伤是否是 微卫星突变

DESCRIPTION: The goal of this proposal is to evaluate the ability of the antioxidants Vitamin C (ascorbate), Vitamin E (a-tocopherol) and metallothionein (MT) to reduce the frequency and spectrum of spontaneous mutations at the HPRT locus. Ascorbate is anticipated to reduce DNA damage resulting from direct oxidation of the nitrogenous bases, a-tocopherol by blocking DNA adduct formation by lipid peroxidation products and metallothionein by scavenging cellular free radicals. The spectra of spontaneous mutations and pattern of DNA damage will be evaluated in three human cell lines: HCT116, a mismatch repair deficient colon carcinoma cell line; HCT116/ch3, and HCT116 derivative into which an intact human chromosome 3 has been introduced, and SW480, a mismatch repair proficient colon carcinoma cell line. Cells will be incubated with one of the two antioxidants or transfected with a metallothionein expression vector and incubated with Zn, HPRT mutants collected and the mutational spectra and DNA damage profile determined. In addition, the mismatch repair deficient cell line will be evaluated for an undescribed panel of microsatellite markers for determination of whether oxidative damage is a principal cause of microsatellite mutations.