Measurement & modulation of the mutation rate in humans

测量

• 批准号: 6931691
• 负责人: David J. Araten
• 金额: $ 27.72万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-02 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931691
• 关键词: CD28 molecule; CD3 molecule; T lymphocyte; antioxidants; ascorbate; cancer risk; chemoprevention; clinical research; electrochemistry; flow cytometry; gene mutation; genetic regulation; human subject; interleukin 2; lectin; lymphocyte proliferation; membrane proteins; neoplasm /cancer genetics

DESCRIPTION (provided by applicant): This project aims to (1) determine the mutation rate (f) in cells from human blood samples; and (2) determine whether f can be reduced by anti-oxidants. f is critical in the risk of developing cancer, but 60 years after f was first measured in bacteria, there still has been no test to measure this parameter in human cells. In preliminary data f was measured in human B-lymphoblastoid cell lines (BLCLs), using the PIG-A gene (Xp22.1) as a sentinel. A broad spectrum of mutations inactivate PIG-A, and rare cells with the mutant phenotype--lack of surface expression of glycosylphosphatidylinositol (GPI)-Iinked proteins (e.g. CD48, CD55, and CD59)--are readily identified by flow cytometry. Pre-existing PIG-A mutants are first eliminated from the population by sorting, followed by in vitro expansion, and then determination of the frequency (f) of cells lacking GPl-linked proteins, f is calculated as f = f/d, where d = # of cell divisions in vitro. In normal BLCLs f ranged from 2.5 to 29.6 x 10-7 mutations/cell division, and was increased in genetic cancer predisposition syndromes and by exposure to radiation. While BLCLs can be produced from any individual, it is critical to measure f in primary cells. Here it is hypothesized that: (a) f can be reproducibly measured in T lymphocytes; and (b) that f is amenable to pharmacologic modulation. After optimization of the reproducibility of the assay, f will be measured in T cells derived from blood samples from normal individuals and stimulated by lectin, interleukin-2, and anti-CD3/anti-CD28. The effects of antioxidants will be determined by incubating cells with dehydroascorbic acid, which greatly increases intracellular ascorbic acid (AA) and avoids undesired in vitro artefacts. Preliminary data suggests that this assay will be as reliable as with BLCLs and that AA will reduce f. These studies will begin to elucidate the relationship between f and cancer risk and provide a highly relevant in vitro parameter with which to evaluate chemoprevention.

描述（申请人提供）：本项目旨在（1）确定人血液样本细胞中的突变率（f）;（2）确定抗氧化剂是否可以降低f。氟在患癌症的风险中至关重要，但在首次在细菌中测量氟60年后，仍然没有在人体细胞中测量这一参数的测试。在初步数据中，使用PIG-A基因（Xp22.1）作为哨兵，在人B淋巴母细胞样细胞系（BLCL）中测量了f。广泛的突变会使PIG-A失活，而具有突变表型的罕见细胞--缺乏糖基磷脂酰肌醇（GPI）相关蛋白质（例如CD 48、CD 55和CD 59）的表面表达--很容易通过流式细胞术进行鉴定。首先通过分选从群体中消除预先存在的PIG-A突变体，然后进行体外扩增，然后测定缺乏GPl连接蛋白的细胞的频率（f），f计算为f = f/d，其中d =体外细胞分裂的次数。在正常BLCL中，f范围为2.5至29.6 x 10-7突变/细胞分裂，并且在遗传性癌症易感综合征和暴露于辐射中增加。虽然BLCL可以从任何个体产生，但测量原代细胞中的f至关重要。这里假设：（a）f可以在T淋巴细胞中重复测量;（B）f可以进行药理学调节。在优化试验重现性后，将在正常个体血液样本来源的T细胞中测量f，并通过凝集素、白细胞介素-2和抗CD 3/抗CD 28刺激。抗氧化剂的作用将通过将细胞与脱氢抗坏血酸一起孵育来确定，这大大增加了细胞内抗坏血酸（AA）并避免了不希望的体外伪影。初步数据表明，该测定与BLCL一样可靠，AA将降低f。这些研究将开始阐明f和癌症风险之间的关系，并提供一个高度相关的体外参数，以评估化学预防。