Genomic Instability in Ageing and Cancer

背景：在人类中，测量携带突变的细胞的频率(F)是可能的 前哨基因的数量非常少，从历史上看一直是技术上的困难。测量 突变率(？)--代表每个细胞中一个基因发生新突变的概率 分裂--几乎是不可能的。然而，f和？可能是衰老的关键参数。为 例如，它被假设：(I)随着年龄的增长而增加；(Ii)在某些早产儿中升高 老龄化综合症；以及(3)有可能通过减少与衰老有关的癌症。 我们现在已经开发了一种使用PIG-A基因测量这些参数的可靠方法，它 我们希望这将极大地促进对这些假说的研究。猪A在X染色体上-- 因此，一个单一的失活突变就能产生突变表型。PIG-A突变体缺乏一切 GPI连接的膜蛋白，便于通过以下方式筛选具有GPI零表型的罕见细胞 流式细胞术。通过这种方法，我们已经能够测量粒细胞中的f，以及 B淋巴母细胞系和外周血T淋巴细胞体外扩增培养。 最近，我们已经证明了用一个额外的哨点来测量人类红细胞中的f是可能的。 基因，XK，与PIG-A具有相似的优势。具体目标(A)：确定是否增加 (B)：确定预防与衰老有关的疾病是否可行 使用可以减少的药物的癌症。对于目标(A)，不同的志愿者受试者 将招募不同年龄段的人，并使用猪A作为B的哨兵基因直接进行测量 淋巴母细胞系和T淋巴细胞培养与新生儿脐带血的比较 样本。也将根据对粒细胞频率的分析进行间接评估 PIG-A突变和具有XK突变的红细胞是年龄的函数。对于目标(B)，使用来自 正常的老年人和早衰症患者，对突变率的影响会是 测定三种药理方法：猝灭活性氧自由基，调节 使用法尼基转移酶抑制剂，并激活SIRT1。初步数据：在文化中 正常人的淋巴样细胞每个分裂的突变范围从3到53×10-7，并且是 与年龄呈正相关。癌症易感性患者的细胞内IS增加 综合征、早衰综合征和恶性细胞系。减少了 人类细胞中的自发突变率现已被证明使用脱氢抗坏血酸 减少细胞内的活性氧。影响：除了解决非常重要的问题外 关于衰老和癌症风险的基本生物学问题，目标(A)下的研究是 对计划临床化学预防研究很重要。如果？是终生不变的，那么任何 减少突变的策略可能需要及早开始。然而，如果如怀疑的那样，？开始增加 随着年龄的增长，一旦癌症开始增加，就有可能通过晚年减少癌症的发病率来预防癌症。 目标(B)项下的研究对于预测最佳药物靶点和药理作用至关重要。 临床上减少的策略。

Background: In humans, measuring the frequency (f) of cells harboring a mutation is possible for a very small number of sentinel genes and historically has been technically difficult. Measuring the mutation rate (¿)--which represents the probability of new mutations occurring in a gene per cell division-- has been virtually impossible. Nevertheless, f and ¿ may be key parameters in ageing. For example, it is hypothesized: (i) that ¿ increases with age; (ii) that ¿ is elevated in certain premature ageing syndromes; and (iii) that it might be possible to reduce ageing related cancers by decreasing ¿. We have now developed a robust method to measure these parameters using the PIG-A gene, which we hope will greatly facilitate the investigation of these hypotheses. PIG-A is on the X-chromosome- therefore a single inactivating mutation can produce the mutant phenotype. PIG-A mutants lack all GPI-linked membrane proteins, facilitating screening for rare cells with the GPI-null phenotype by flow cytometry. By this approach, we have been able to measure f in granulocytes, and both ¿ and f in B lymphoblastoid cell lines and expanding cultures of ex vivo T lymphocytes from blood samples. Recently, we have shown that it is possible to measure f in human red cells in an additional sentinel gene, XK, which has similar advantages as PIG-A. Specific Aims (a): to determine whether ¿ increases in humans as they age; (b): to determine whether it would be feasible to prevent ageing related cancers with pharmacologic agents that could decrease ¿. For aim (a), volunteer subjects in different age groups will be recruited, and ¿ will be measured directly using PIG-A as a sentinel gene in B lymphoblastoid cell lines and T lymphocyte cultures, in comparison with neonatal cord blood samples. ¿ will also be assessed indirectly, based on analysis of the frequency of granulocytes with PIG-A mutations and red cells with XK mutations as a function of age. For aim (b), using cells from normal older individuals and patients with progeria, the effect on the mutation rate will be determined for three pharmacologic approaches: quenching reactive oxygen species, modulating lamin A using farnesyltransferase inhibitors, and activating SIRT1. Preliminary data: ¿ in cultures of lymphoid cells from normal individuals ranges from 3 to 53 x 10-7 mutations per cell division and is positively correlated with age. ¿ is increased in cells from patients with cancer predisposition syndromes, premature ageing syndromes, and in malignant cell lines. A reduction in the spontaneous mutation rate in human cells has now been demonstrated using dehydroascorbic acid to reduce intracellular reactive oxygen species. Implications: Apart from addressing very important fundamental biological questions regarding ageing and cancer risk, studies under aim (a) are important for planning clinical chemoprevention studies. If ¿ is constant throughout life, then any strategy to reduce mutations may need to start early. If however, as suspected, ¿ starts to increase with age, it may be possible to prevent cancer by reducing ¿ at a later age, once it starts to increase. Studies under aim (b) will be critical for predicting the optimal drug targets and pharmacologic strategy for reducing ¿ clinically.