Genotoxicity & Emergence of Genome Instability in Humans

遗传毒性

• 批准号: 6743250
• 负责人: BARRY Alan FINETTE
• 金额: $ 33.71万
• 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/6743250
• 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(申请人提供)：这项研究的目的是调查遗传毒性暴露和突变机制的出现之间的联系，这些机制涉及人类原发和继发性恶性肿瘤的发展。我们的假设是：1)基因毒性暴露导致克隆性增殖爆发将导致来自癌症患者或癌症易感人群的非恶性T细胞克隆的基因组不稳定性在体内进化；2)从这些对象获得的具有基因组不稳定性的T细胞克隆是DNA修复途径中基因功能/表达改变的结果。我们的具体目标是：1)从急性淋巴细胞白血病、霍奇金病和慢性溃疡性结肠炎患者中分离和鉴定基因组不稳定的增殖性T细胞克隆；以及2)对基因组不稳定的T细胞突变进行四种DNA修复途径的功能分析，以确定DNA修复途径的完整性。具有基因组不稳定性的T细胞克隆将被分离，并使用HPRT T细胞生物标记系统进行鉴定。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。具有基因组不稳定性的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。这些检测方法包括：a)微卫星不稳定性以检测错配修复缺陷；b)单细胞凝胶电泳法和c)基于质粒的荧光素酶DNA修复实验以检测双链断裂修复、核苷酸切除修复和修复后的碱基切除修复能力；以及d)通过测量染色体异常频率来检测染色体不稳定性。后续的研究将包括微阵列基因表达和DNA修复缺陷的基因分析，在克隆中已经确定了功能性DNA修复缺陷。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洞察，此外，这些研究最终可能转化为临床环境，从而导致：1)直接筛查患者与恶性转化的启动和进展相关的遗传和获得性遗传缺陷；2)新的治疗方案和药物开发，降低遗传毒性和由于抗肿瘤治疗而导致的具有基因组不稳定性的增殖性克隆的发展；以及3)患者监测/筛查与第二种恶性肿瘤的发展有关的基因突变。