HUMAN MISMATCH REPAIR IN CHEMICAL CARCINOGENESIS

化学致癌过程中的人体错配修复

• 批准号: 6172855
• 负责人: Guo-Min Li
• 金额: $ 12.42万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6172855
• 关键词: DNA binding protein; DNA damage; DNA repair; N methyl N' nitro N nitrosoguanidine; acetylaminofluorene; adduct; apoptosis; autoradiography; benzopyrenediol epoxide; binding proteins; chemical carcinogen; chemical carcinogenesis; environmental toxicology; gel mobility shift assay; gene mutation; high performance liquid chromatography; human tissue; molecular oncology; molecular shape; nuclear magnetic resonance spectroscopy; site directed mutagenesis; thin layer chromatography

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）机制及其在癌症避免中的作用。 寄存器是 突变避免系统，并在维持 遗传稳定性 研究表明，人类系统的缺陷 赋予强烈的癌症倾向，包括遗传性息肉病 结肠直肠癌 环境致癌物共价修饰DNA形成 致癌物-DNA加合物，其诱导启动致癌作用的突变。 最近，MMR组件已被证明可以识别某些形式的 致癌物-DNA加合物，以前认为仅由 核苷酸切除修复（NER）。 还记录了MMR熟练 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缺陷型细胞相比。 鉴于这些发现，我们假设MMR成分直接 参与修复致癌物-DNA加合物或作为传感器发挥作用， 激活程序性细胞死亡 为了验证这一假设，四条工作线 在本申请中提出。 首先，使用电泳迁移率 转移测定，纯化的hMutSa，一种人错配识别蛋白，将被用于 测试其结合寡核苷酸双链体的能力，所述寡核苷酸双链体含有 乙酰氨基芴、苯并[a]芘二醇环氧化物 和MNNG，三种最关键的化学致癌物。 二、细胞 来自不同MMR背景的肿瘤细胞的提取物将 检查以处理含有以下的位点特异性加合物的环状质粒DNA 上面列出的致癌物质。 第三，调查生理 识别和处理致癌物-DNA加合物的重要性 MMR、MMR正常细胞和突变细胞将用列出的致癌物处理 以上或用致癌物修饰的pZ 189质粒转染， 穿梭载体，并分析细胞凋亡。 由于MNNG诱导的细胞死亡 的MMR-熟练细胞已被归因于他们徒劳的尝试， 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的复制将诱导 MMR-熟练细胞中的凋亡。 最后，为了确定致癌物质 加合物可以通过MMRcriti在体内去除，来自MMR-proficient的基因组DNA 用致癌物处理的MMR缺陷细胞将被消化 转化为单核苷酸，然后进行加合物检测和定量。