DNA REPAIR AND NITROSAMINE-INDUCED CARCINOGENESIS

DNA 修复和亚硝胺诱发的致癌作用

• 批准号: 3169811
• 负责人: Sankar Mitra
• 金额: $ 16.74万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-15 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3169811
• 关键词: DNA repair; Escherichia coli; HeLa cells; X ray crystallography; adduct; alkylating agents; animal tissue; antineoplastics; chemical carcinogen; chemical carcinogenesis; cytotoxicity; enzyme mechanism; gene expression; genetic library; genetic manipulation; genetic mapping; genetically modified animals; human genetic material tag; human tissue; laboratory mouse; methyltransferase; molecular cloning; monoclonal antibody; mutagen testing; mutagens; natural gene amplification; nitrosamines; nucleic acid hybridization; nucleic acid probes; nucleic acid repetitive sequence; nucleic acid sequence; regulatory gene; transcription factor; transfection

O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous protein in bacteria and higher organisms, is responsible for repair of the carcinogenic and mutagenic adduct O6-alkylguanine induced in DNA by various simple alkylating carcinogens (e.g., N-alkylnitrosamines) and antitumor drugs (Procarbazine and haloethyl-N-nitrosoureas). MGMT, a stoichiometric protein without turnover, is strictly regulated in both bacteria and mammals. The level of mammalian MGMT activity is tissue- and cell type-specific and is modulated during the cell cycle. Some cultured cell lines called Mex-/Mer-, and often derived from tumors, have no detectable MGMT activity even though they appear to have the MGMT gene. In contrast, some drug-resistant tumor xenografts and lines have higher levels of MGMT than their progenitor cells. With the recent availability of cDNAs of human and mouse MGMT genes and of antibodies against the human protein, the broad objective of the project is a comprehensive understanding of the regulation of MGMT gene in mammals and the impact of this regulation on mutagenesis and carcinogenesis induced by alkylating agents and on antitumor activity of alkylating drugs. specifically, the following topics are to be addressed in both individual and collaborative efforts. (1) The organization of human and mouse MGMT genes. (2) Identification of regulatory elements of these genes. (3) Molecular basis of extinction of MGMT in Mex- cells. (4) Induction of MGMT gene and its amplification, particularly in drug-resistant human tumors. (5) Involvement of trans-acting factors in MGMT regulation and their characterization. (6) Construction of expression vectors for large-scale production of human and mouse MGMT for subsequent biochemical studies and X ray crystallographic analysis of the 3-D structure of the MGMT polypeptides. (7) Construction of mammalian expression vectors to be used in the creation of transgenic cells and mice with modulated expression of human MGMT in order to investigate the role of O6-alkylguanine in mutagenesis, carcinogenesis, toxicity, and other biological endpoints. (8) Construction of gene-targeting vectors containing mouse MGMT gene sequences for use in targeted mutagenesis of embryonic stem (ES) cells. (9) Insertion of the ES cells into blastocysts for subsequent production of heterozygous and homozygous mice with null mutation in the MGMT gene in order to study the essentiality of MGMT in development and in the prevention of germ-line mutation and tumorigenesis.

O6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT)，一种普遍存在的蛋白质 细菌和高等生物，负责修复 DNA 中诱导的致癌和致突变加合物 O6-烷基鸟嘌呤 各种简单的烷基化致癌物（例如，N-烷基亚硝胺）和 抗肿瘤药物（丙卡巴肼和卤乙基-N-亚硝基脲）。 MGMT，一个 没有周转的化学计量蛋白质，在两者中均受到严格监管 细菌和哺乳动物。 哺乳动物 MGMT 活性水平是组织和 细胞类型特异性并在细胞周期中受到调节。 有的有文化 称为 Mex-/Mer- 的细胞系，通常源自肿瘤，没有 即使它们似乎具有 MGMT 基因，也可检测到 MGMT 活性。 相反，一些耐药肿瘤异种移植物和细胞系具有更高的耐药性。 MGMT 水平高于其祖细胞。 随着最近的可用性 人类和小鼠 MGMT 基因的 cDNA 以及针对人类的抗体 蛋白质，该项目的总体目标是综合 了解哺乳动物中 MGMT 基因的调控及其影响 这种对烷基化诱导的突变和致癌的调节 剂和烷化药物的抗肿瘤活性。具体来说， 以下主题将以个人和协作方式解决 努力。 (1)人和小鼠MGMT基因的组织。 (2) 鉴定这些基因的调控元件。 (3)分子基础 Mex 细胞中 MGMT 的消退。 (4) MGMT基因及其诱导 扩增，特别是在耐药人类肿瘤中。 (5) 参与 MGMT 调节中的反式作用因子及其特征。 (6) 构建用于大规模生产人类和 小鼠 MGMT 用于后续生化研究和 X 射线晶体学 MGMT 多肽的 3-D 结构分析。 (七) 施工 用于创建转基因的哺乳动物表达载体 调节人类 MGMT 表达的细胞和小鼠，以便 研究 O6-烷基鸟嘌呤在突变、致癌、 毒性和其他生物学终点。 (8) 建设 含有小鼠 MGMT 基因序列的基因靶向载体，用于 胚胎干（ES）细胞的定向诱变。 (9) ES的插入 细胞进入囊胚，随后产生杂合子和 MGMT 基因无效突变的纯合小鼠，以研究 MGMT 在发育和预防生殖细胞系中的重要性 突变和肿瘤发生。