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HUMAN MISMATCH REPAIR IN CHEMICAL CARCINOGENESIS

化学致癌过程中的人体错配修复

基本信息

批准号：
6376340
负责人：
Guo-Min Li
金额：
$ 12.89万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2003-07-31
项目状态：
已结题

项目摘要

DESCRIPTION: Dr. Li's long term goal is to understand the molecular mechanism of mismatch repair (MMR) and its role in cancer avoidance. MMR is a mutation avoidance system and plays an important role in maintaining genetic stability. It has been shown that defects in the human system confer a strong cancer predisposition including hereditary polyposis colorectal cancer. Environmental carcinogens covalently modify DNA to form carcinogen-DNA adducts, which induce mutations that initiate carcinogenesis. Recently, MMR components have been shown to recognize certain forms of carcinogen-DNA adducts that are previously thought to be only processed by nucleotide excision repair (NER). It is also documented that MMR-proficient cells are much more sensitive to the cytotoxic effects of N-methyl-N'-nitro-nitrosoguanidine (MNNG) compared to MMR-deficient cells. Given these findings, we hypothesize that MMR components either directly participate in repair of carcinogen-DNA adducts or function as a sensor to activate programmed cell death. To test this hypothesis, four lines of work are proposed in this application. First, using an electrophoretic mobility shift assay, purified hMutSa, a human mismatch recognition protein, will be tested for its ability to bind oligonucleotide duplexes containing site-specific adducts of acetylaminofluorene, benzo[a]pyrene diol epoxide, and MNNG, the three most critical chemical carcinogens. Second, cell extracts derived from tumor cells with different MMR backgrounds will be examined to process circular plasmid DNA containing site-specific adducts of the carcinogens listed above. Third, to investigate the physiological importance of the recognition and processing of carcinogen-DNA adducts by MMR, MMR-normal and mutant cells will be treated with carcinogens listed above or transfected with carcinogen-modified pZ189 plasmid, an SV40-based shuttle vector, and analyzed for apoptosis. Since MNNG-induced cell death of MMR-proficient cells has been attributed to their futile attempts to remove MMNG adducts in the template DNA strand, it is anticipated that replication of carcinogen-modified genomic or plasmid DNA will induce apoptosis in MMR-proficient cells. Finally, to determine if carcinogen adducts can be remove in vivo by MMRcriti, genomic DNA from MMR-proficient and MMR-deficient cells that are treated with carcinogens will be digested into mononucleosides, and followed by adduct detection and quantitation.

描述：李博士的长期目标是了解分子错配修复（MMR）机制及其在避免癌症中的作用。 MMR 是突变避免系统，在维持突变方面发挥着重要作用遗传稳定性。研究表明，人体系统存在缺陷具有强烈的癌症倾向，包括遗传性息肉病结直肠癌。环境致癌物共价修饰DNA形成致癌物-DNA 加合物，可诱导引发致癌作用的突变。最近，MMR 成分已被证明可以识别某些形式的以前认为致癌物-DNA 加合物只能由核苷酸切除修复（NER）。另据记载，精通 MMR 细胞对细胞毒性作用更加敏感 N-甲基-N'-硝基亚硝基胍 (MNNG) 与 MMR 缺陷细胞相比。鉴于这些发现，我们假设 MMR 成分要么直接参与致癌物-DNA 加合物的修复或作为传感器发挥作用激活程序性细胞死亡。为了检验这个假设，需要进行四项工作在本申请中提出。首先，使用电泳迁移率移位分析，纯化的 hMutSa，一种人类错配识别蛋白，将被测试了其结合寡核苷酸双链体的能力，其中包含乙酰氨基芴、苯并[a]芘二醇环氧化物的位点特异性加合物，和MNNG，三种最关键的化学致癌物。二、细胞来自具有不同 MMR 背景的肿瘤细胞的提取物将检查处理含有位点特异性加合物的环状质粒DNA 上面列出的致癌物质。三、考察生理情况致癌物-DNA 加合物识别和加工的重要性 MMR、MMR-正常和突变细胞将用列出的致癌物质处理上述或用致癌物修饰的 pZ189 质粒转染，这是一种基于 SV40 的质粒穿梭载体，并分析细胞凋亡。由于 MNNG 诱导的细胞死亡 MMR 熟练的细胞被归因于它们徒劳的尝试去除模板 DNA 链中的 MMNG 加合物，预计致癌物修饰的基因组或质粒 DNA 的复制会诱导 MMR 熟练细胞的凋亡。最后确定是否致癌加合物可以通过 MMRcriti 在体内去除，来自 MMR 熟练的基因组 DNA 用致癌物处理的 MMR 缺陷细胞将被消化转化为单核苷，然后进行加合物检测和定量。