Genotoxicity & Emergence of Genome Instability in Humans

遗传毒性

• 批准号: 6612527
• 负责人: BARRY Alan FINETTE
• 金额: $ 33.02万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612527
• 关键词: DNA damage; DNA repair; Hodgkin's disease; T cell receptor; T lymphocyte; acute lymphocytic leukemia; biomarker; cell proliferation; clinical research; flow cytometry; gel electrophoresis; gene environment interaction; gene expression; genome; human subject; hypoxanthine phosphoribosyltransferase; messenger RNA; polymerase chain reaction; tissue /cell culture

DESCRIPTION (provided by applicant): The objective of this research is to investigate the association between genotoxic exposure and the emergence of mutagenic mechanisms involved with the development of primary and secondary malignancies in humans. Our hypotheses are: 1) Genotoxic exposure leading to clonal proliferative bursts will result in the in vivo evolution of genomic instability in nonmalignant T cell clones from subjects with cancer or predisposed to cancer; and 2) T cell clones with genomic instability captured from these subjects are the result of altered gene function/expression in DNA repair pathways. Our specific aims are: 1) to isolate and characterize proliferating T cell clones with genomic instability from subjects with acute lymphocytic leukemia, Hodgkin's disease and chronic ulcerative colitis following therapeutic genotoxic exposure; and 2) to perform four functional assays for DNA repair pathways on T cell mutants with genomic instability to determine DNA repair pathway integrity. T cell clones with genomic instability will be isolated and characterized using the HPRT T cell biomarker system. T cell clonality will be determined by T cell receptor beta gene rearrangement mRNA analysis. Mechanistic studies of clones with genomic instability will initially include functional screening assays for DNA repair pathway defects. These assays include: a) microsatellite instability to test for mismatch repair defects; b) single cell gel electrophoresis and c) a plasmid based luciferase DNA repair assay to test for double strand break repair, nucleotide excision repair, and base excision repair capacity following repair specific genotoxic exposures; and d) chromosome instability by measuring frequency of chromosomal aberrations. Subsequent studies will include microarray gene expression and genotypic analysis of DNA repair defects in clones in which a functional DNA repair defect has been established. This research will provide insight into gene-environment interactions, genomic instability and malignant transformation in humans, In addition, these studies could eventually be translated to the clinical setting by leading to: 1) the direct screening of patients for inherited and acquired genetic defects associated with the initiation and progression of malignant transformation; 2) new therapeutic protocols and drug development that decreases the genotoxicity and the development of proliferative clones with genomic instability as a consequence of antineoplastic therapy; and 3) patient monitoring/screening for genetic mutations that are associated with the development of second malignant neoplasms.

描述（由申请人提供）：本研究的目的是调查基因毒性暴露与人类原发性和继发性恶性肿瘤发展所涉及的诱变机制的出现之间的关联。我们的假设是： 1) 导致克隆增殖爆发的基因毒性暴露将导致来自癌症或易患癌症受试者的非恶性 T 细胞克隆体内基因组不稳定性的进化； 2) 从这些受试者中捕获的基因组不稳定的 T 细胞克隆是 DNA 修复途径中基因功能/表达改变的结果。我们的具体目标是：1）从患有急性淋巴细胞白血病、霍奇金病和慢性溃疡性结肠炎的受试者中分离和表征具有基因组不稳定的增殖性T细胞克隆，这些受试者在治疗性基因毒性暴露后； 2) 对基因组不稳定的 T 细胞突变体进行 DNA 修复途径的四项功能测定，以确定 DNA 修复途径的完整性。将使用 HPRT T 细胞生物标志物系统分离和表征具有基因组不稳定的 T 细胞克隆。 T 细胞克隆性将通过 T 细胞受体 β 基因重排 mRNA 分析来确定。对基因组不稳定性克隆的机制研究最初将包括 DNA 修复途径缺陷的功能筛选分析。这些测定包括：a) 微卫星不稳定性，以测试错配修复缺陷； b) 单细胞凝胶电泳和 c) 基于质粒的荧光素酶 DNA 修复测定，用于测试修复特异性基因毒性暴露后的双链断裂修复、核苷酸切除修复和碱基切除修复能力； d) 通过测量染色体畸变频率来确定染色体不稳定性。后续研究将包括微阵列基因表达和克隆中 DNA 修复缺陷的基因型分析，其中已建立功能性 DNA 修复缺陷。这项研究将深入了解人类基因与环境的相互作用、基因组不稳定性和恶性转化。此外，这些研究最终可以转化为临床环境，从而：1）直接筛查患者的遗传性和获得性遗传缺陷相关随着恶性转化的开始和进展； 2) 新的治疗方案和药物开发，减少抗肿瘤治疗导致的基因毒性和基因组不稳定的增殖性克隆的发展； 3) 患者监测/筛查与第二种恶性肿瘤的发展相关的基因突变。