Measurement & modulation of the mutation rate in humans

测量

• 批准号: 7258345
• 负责人: David J. Araten
• 金额: $ 26.28万
• 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/7258345
• 关键词: Antioxidants; Ascorbic Acid; Bacteria; Biological Assay; Blood specimen; Cell division; Cells; Chemoprevention; Data; Dehydroascorbic Acid; Exposure to; Family suidae; Flow Cytometry; Frequencies; Genes; Glycosylphosphatidylinositols; Human; In Vitro; Incubated; Individual; Interleukin-2; Lectin; Malignant Neoplasms; Measurement; Measures; Monoclonal Antibody HuM291; Morphologic artifacts; Muromonab-CD3; Mutation; Mutation Spectra; Phenotype; Population; Predisposition; Proteins; Radiation; Range; Rate; Reproducibility; Risk; Sentinel; Sorting - Cell Movement; Surface; Sus scrofa; Syndrome; T-Lymphocyte; Testing; Xp22.1; cancer genetics; cancer risk; link protein; lymphoblastoid cell line; mutant

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）：本项目旨在(1)确定人类血液样本细胞的突变率(f)；(2)判断抗氧化剂是否能还原f。F在患癌症的风险中是至关重要的，但在F首次在细菌中被测量60年后，仍然没有在人类细胞中测量这一参数的测试。在初步数据中，使用猪- a基因（Xp22.1）作为前哨，在人b淋巴母细胞样细胞系（BLCLs）中测量了f。广泛的突变使猪-A失活，并且具有突变表型的罕见细胞-缺乏糖基磷脂酰肌醇（GPI）连接蛋白（例如CD48， CD55和CD59）的表面表达-很容易通过流式细胞术识别。预先存在的猪- a突变体首先通过分选从群体中消除，然后进行体外扩增，然后确定缺乏gpl连接蛋白的细胞频率(f)， f计算为f = f/d，其中d =体外细胞分裂的#。在正常的blcl中，其范围为2.5至29.6 x 10-7突变/细胞分裂，并且在遗传癌症易感综合征和暴露于辐射时增加。虽然blcl可以从任何个体产生，但在原代细胞中测量f是至关重要的。这里假设：(a) f可以在T淋巴细胞中重复测量；(b) f可以接受药物调节。在优化实验的可重复性之后，f将在来自正常人血液样本的T细胞中进行测量，并通过凝集素，白细胞介素-2和抗cd3 /抗cd28刺激。抗氧化剂的作用将通过用脱氢抗坏血酸培养细胞来确定，脱氢抗坏血酸可以大大增加细胞内抗坏血酸（AA），并避免不需要的体外人工产物。初步数据表明，该试验将与blcl一样可靠，AA将降低f。这些研究将开始阐明f与癌症风险之间的关系，并为评估化学预防提供高度相关的体外参数。