RESPONSE TO DNA DAMAGE--COLON VS SMALL INTESTINE

对 DNA 损伤的反应——结肠与小肠

• 批准号: 6512984
• 负责人: JOANNE R LUPTON
• 金额: $ 22.68万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6512984
• 关键词: DNA damage; DNA repair; adduct; age difference; alkylating agents; alkylation; apoptosis; colon neoplasms; electron transport; free radical oxygen; guanine analog; immunocytochemistry; intestine neoplasm; laboratory rat; mitochondrial membrane; oxidation; oxidizing agents; small intestines

Colon cancer is the second leading cause of death from cancer in the United States today whereas cancer of the small intestine is a relatively rare event. Intestinal tumors develop from a series of somatic mutations subsequent to an initiating event such as DNA-alkylation or oxidation. Our primary hypothesis is that the small intestine (SI) and large intestine (LI) respond differently to these two important forms of DNA damage (alkylation and oxidation), which is a key reason for the difference in tumor incidence at these two sites. Our secondary hypothesis is that SI cells are protected from tumor induction because they produce less reactive oxygen species (ROS) and less oxidative damage to DNA and LI cells. In specific aim # 1 we will inject rats with the DNA alkylating agent azoxymethane and measure in vivo DNA damage, repair and apoptosis in SI and LI over the 48 h period post injection. DNA damage and repair are measured by quantitative immunohistochemistry of O6-methylguanine adducts and its repair enzyme; and apoptosis is detected by the TUNEL assay. In specific aim #2 we will determine in vivo response to the DNA oxidizing agent dextran sodium sulfate in rat SI and LI within the first 48 h after removal of the oxidizing agent. DNA damage is measured by quantitative immunohistochemistry of 8oxodG adducts; activity of the repair enzyme specific for 8oxodG by an endonuclease assay; and apoptosis by the TUNEL assay. In specific aim #3 we will determine steady state levels of oxidative DNA damage and ROS generation in SI and LI in young and old rats and whether or not ROS generation in SI and LI is due to differences in mitochondrial electron transport. DNA damage will be assessed by the FLARE assay and ROS production by oxidation of the vital dye 2',7'-dichlorofluorescein in cells incubated with and without electron transport inhibitors.

结肠癌是癌症死亡的第二大原因 当今美国，而小肠癌是相对的 罕见事件。肠道肿瘤是由一系列体细胞突变发展而来的 在启动事件之后，例如DNA-烷基化或氧化。我们的 主要假设是小肠（SI）和大肠（LI） 对这两种重要形式的DNA损伤（烷基化和 氧化），这是肿瘤发生率差异的关键原因 这两个站点。我们的次要假设是SI细胞受到保护 肿瘤诱导是因为它们产生较少的活性氧（ROS）和 对DNA和LI细胞的氧化损伤较少。在特定的目标＃1中，我们将注入 带有DNA烷基化剂甲氧基烷的大鼠，并测量体内DNA损伤， 在注射后48小时内，SI的修复和凋亡。脱氧核糖核酸 损伤和维修是通过定量免疫组织化学来衡量的 O6-甲基鸟氨酸加合物及其修复酶；并通过 TUNEL分析。在特定的目标＃2中，我们将确定体内对 DNA氧化剂葡萄剂硫酸钠在大鼠Si中和LI中的第一个48 去除氧化剂后H。通过定量测量DNA损伤 8氧加合物的免疫组织化学；修复酶特异性的活性 对于核酸内切酶测定法进行8氧；和TUNEL分析的凋亡。在 特定目的＃3我们将确定氧化DNA损伤的稳态水平 在SI和Li的Ros Generation和年轻的老鼠中以及ROS是否是ROS Si和Li的产生是由于线粒体电子的差异 运输。 DNA损伤将通过耀斑测定和ROS产生评估 与细胞中生命染料2'，7'-二氯荧光的氧化 并且没有电子传输抑制剂。