Measurement & modulation of the mutation rate in humans

测量

• 批准号: 7121132
• 负责人: David J. Araten
• 金额: $ 27.06万
• 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/7121132
• 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（由申请人提供）：该项目的目的是（1）确定人类血液样本细胞中的突变率（F）； （2）确定是否可以通过抗氧化剂降低F。 F对于患癌症的风险至关重要，但是在F中首次测量F之后60年，仍未进行测量人类细胞中该参数的测试。在初步数据中，使用PIG-A基因（XP22.1）作为前哨，在人的B膜细胞细胞系（BLCL）中测量F。广泛的突变型PIG-A和具有突变表型的稀有细胞 - 糖基磷脂酰肌醇（GPI）二联蛋白（例如CD48，CD55和CD59）的表面表达 - 易于鉴定。首先通过排序，然后在体外扩张，然后测定缺乏GPL连接蛋白的细胞的频率（F），F = f = f/d，其中d =＃d =＃细胞分裂。在正常的BLCLS中，F范围为2.5至29.6 x 10-7突变/细胞分裂，遗传癌症易感性综合征和暴露于辐射中。虽然可以从任何个体产生BLCL，但在原代细胞中测量F至关重要。在这里假设：（a）可以在T淋巴细胞中重复测量F； （b）F适合药理调节。在优化测定的可重复性后，F将在来自正常个体的血液样本中得出的T细胞中测量，并被凝集素，白介素-2和抗CD3/抗CD28刺激。抗氧化剂的作用将通过将细胞与脱氢抗坏血酸孵育，从而大大增加细胞内抗坏血酸（AA）并避免不希望的体外人工伪像。初步数据表明，该测定法与BLCL一样可靠，并且AA将减少f。这些研究将开始阐明F与癌症风险之间的关系，并提供高度相关的体外参数，以评估化学预防。