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）：这项研究的目的是研究遗传毒性暴露与与人类主要和继发性恶性肿瘤发展有关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遗传毒性暴露将导致来自癌症患者或患有癌症的受试者的非态度T细胞克隆的基因组不稳定性的体内进化； 2）从这些受试者捕获的基因组不稳定性的T细胞克隆是DNA修复途径中基因功能/表达改变的结果。我们的具体目的是：1）与患有急性淋巴细胞性白血病，霍奇金病和治疗性基因毒性暴露后的急性淋巴细胞性白血病，霍奇金病和慢性溃疡性结肠炎的受试者分离和表征具有基因组不稳定性的增殖T细胞克隆； 2）对具有基因组不稳定性的T细胞突变体的DNA修复途径进行四个功能测定，以确定DNA修复途径完整性。具有基因组不稳定性的T细胞克隆将使用HPRT T细胞生物标记系统进行分离和表征。 T细胞克隆性将由T细胞受体β基因重排mRNA分析确定。具有基因组不稳定性克隆的机械研究最初将包括针对DNA修复途径缺陷的功能性筛选测定法。这些测定法包括：a）测试错配维修缺陷的微卫星不稳定性； b）单细胞凝胶电泳和c）基于质粒的荧光素酶DNA修复测定法，以测试双链断裂修复，核苷酸切除修复和基本切除修复能力后修复特定的基因毒性暴露； D）通过测量染色体畸变的频率来染色体不稳定性。随后的研究将包括对已经建立功能性DNA修复缺陷的克隆中DNA修复缺陷的微阵列基因表达和基因型分析。这项研究将提供对人类基因环境相互作用，基因组不稳定性和恶性转化的见解，此外，这些研究最终可以通过以下方式转化为临床环境：1）1）患者直接筛查与恶性转化的遗传和获得的遗传缺陷有关的遗传和获得的遗传缺陷； 2）新的治疗方案和药物开发降低了由于抗肿瘤治疗而具有基因组不稳定性的遗传毒性和增殖克隆的发展； 3）患者监测/筛查与第二种恶性肿瘤发展有关的基因突变。