Measurement & modulation of the mutation rate in humans

描述(申请人提供)：该项目旨在(1)测定人体血液样本中细胞的突变率(F)；以及(2)确定抗氧化剂是否能降低f。F对罹患癌症的风险至关重要，但在f首次在细菌中测量60年后，仍然没有测试来测量人类细胞中的这一参数。在初步数据中，使用PIG-A基因(Xp22.1)作为前哨，在人B淋巴母细胞系(BLCL)中测量了f。广泛的突变使PIG-A失活，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仪鉴定。预先存在的PIG-A突变体首先通过分选从种群中消除，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的计算公式为f=f/d，其中d=细胞在体外分裂的次数。在正常BLCL中，f在2.5-29.6×10-7突变/细胞分裂之间，并且在遗传性癌症易感综合征和辐射暴露中增加。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；以及(B)f是受药物调节的。在优化了检测的重复性后，将在凝集素、白介素2和抗CD3/抗CD28的刺激下，从正常人的血液样本中检测T细胞中的f。抗氧化剂的效果将通过与脱氢抗坏血酸孵育细胞来确定，脱氢抗坏血酸可极大地增加细胞内抗坏血酸(AA)，并避免体外不受欢迎的人工制品。初步数据表明，这项检测将与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，并提供一个高度相关的体外参数来评估化学预防。

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.