Oxidative DNA damage-induced tumorigenesis and its avoidance mechanism

DNA氧化损伤诱导肿瘤发生及其避免机制

基本信息

批准号：
12213098
负责人：
TSUZUKI Teruhisa
金额：
$ 49.66万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

项目摘要

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is the most abundant, and appears to play important roles in mutagenesis and carcinogenesis. Studies with Escherichia coli mutator mutants revealed that organisms possess elaborate mechanisms that prevent mutations caused by oxidation of the guanine base, in both DNA and free nucleotide forms. Enzymatic activities which may be responsible for preventing 8-oxoG-evoked mutations were identified in mammalian cells. We have focused on following the two enzymes. MTH1 (Mthl) protein is the mammalian counterpart of E. coli MutT protein, which hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. On the other hand, MUTYH (Mutyh) protein, a counterpart of E. coli MutY protein, having adenine/2-hydroxyadeni … More ne DNA glycosylase activity, is expected to prevent G : C to T : A transversions, by excising adenine from G : A mismatches induced by 8-oxoG and 2-OH-A. To analyze the function of the mammalian Mthl and Mutyh proteins in vivo, we established gene-knockout mice for these two enzymes by gene targeting, and investigated spontaneous tumorigenesis as well as mutagenesis.When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers of Mthl-deficient mice, as compared with wild-type mice (Tsuzuki, T. et al., 2001). Mutation frequencies on the rpsL transgene in spleen samples recovered at the age of 4 and 24 weeks, were determined. The spontaneous mutation frequency observed in spleen samples from Mthl-deficient mice showed no dramatic increase compared to the value of the one in wild-type mice. The site distribution of the mutations occurred on rpsL gene was slightly different between these to Mthl genotypes in spleen samples. In Mthl-deficient mice, there are 1-basepair frameshift mutations at the mononucleotide repeats those were not found in wild-type mice (Egashira, A. et al., 2002). When examined 18 months after birth, a greater number of tumors had formed in various tissues of Mutyh-deficient mice, as compared with wild-type mice. Especially, more small intestinal tumors were formed in Mutyh-deficient mice than in wild-type mice (in preparation). Mutation frequency observed in spleen samples from the Mutyh-deficient mice, at the age of 24 weeks, showed no apparent increase compared to the value of samples from wild-type mice. However, the site distribution of the mutations that occurred in the rpsL gene was significantly different between these two Mutyh genotypes ; an increase in frequency of G : C to T : A transversions was evident in Mutyh nullizygous mice (in preparation).Thus, both the Mthl-and Mutyh-deficient mice will provide useful models for investigating the roles of oxidative stress in human health. Less

氧自由基可通过正常细胞代谢产生，被认为在各种类型的氧化性 DNA 损伤中发挥重要作用，其中 8-oxo-7,8-二氢鸟嘌呤 (8-oxoG) 最为丰富。，并且似乎在诱变和致癌作用中发挥重要作用。对大肠杆菌突变突变体的研究表明，生物体具有复杂的机制，可以防止由氧化引起的突变。我们在哺乳动物细胞中鉴定了可能负责防止 8-oxoG 诱发突变的鸟嘌呤碱基，这两种酶是哺乳动物的对应物。大肠杆菌 MutT 蛋白，可将核苷酸库中的 8-oxo-dGTP 水解为单磷酸盐，从而防止颠换突变的发生。 (Mutyh) 蛋白是大肠杆菌 MutY 蛋白的对应物，具有腺嘌呤/2-羟基腺嘌呤 DNA 糖基化酶活性，预计可通过从 G : A 引起的错配中切除腺嘌呤来防止 G : C 到 T : A 颠换。 8-oxoG 和 2-OH-A 为了分析哺乳动物 Mthl 和 Mutyh 蛋白的体内功能，我们通过以下方法建立了这两种酶的基因敲除小鼠。基因靶向，并研究自发肿瘤发生和突变。在出生后 18 个月进行检查时，与野生型小鼠相比，Mthl 缺陷小鼠的肺、肝脏中形成了更多的肿瘤（Tsuzuki，T. 等） al., 2001) 测定了 4 周和 24 周时回收的脾脏样本中 rpsL 转基因的突变频率。与野生型小鼠相比，Mthl 缺陷型小鼠的 rpsL 基因突变位点分布与 Mthl 缺陷型小鼠的脾脏样本中的 Mthl 基因型略有不同。在单核苷酸重复中存在 1 个碱基对移码突变，这些突变在野生型小鼠中未发现（Egashira，A. 等人，2002 年在 18 个月后进行检查）。出生后，与野生型小鼠相比，Mutyh 缺陷型小鼠的各个组织中形成了更多的肿瘤，特别是，Mutyh 缺陷型小鼠比野生型小鼠（准备中）形成了更多的小肠肿瘤。在 24 周龄时，Mutyh 缺陷小鼠的脾脏样本中观察到的突变频率与野生型小鼠样本的值相比没有明显增加，但 rpsL 基因中发生的突变的位点分布却有所不同。这两种 Mutyh 基因型之间存在显着差异；Mutyh 无效小鼠（准备中）中 G : C 到 T : A 颠换的频率明显增加。因此，Mthl 和 Mutyh 缺陷小鼠都将为研究提供有用的模型氧化应激在人类健康中的作用。