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， 非常少量的哨兵基因，并且在历史上一直是技术上困难的。测量 突变率（<$）--表示每个细胞中基因发生新突变的概率 分裂几乎是不可能的然而，f和<$可能是老化的关键参数。为 例如，假设：（i）随着年龄的增长，（ii）在某些早产儿中， 衰老综合征;以及（iii）通过减少衰老相关的癌症可能会减少。 我们现在已经开发出一种强大的方法来测量这些参数使用PIG-A基因， 我们希望这将极大地促进对这些假说的研究。PIG-A在X染色体上 因此，单个失活突变可以产生突变表型。PIG-A突变体缺乏所有 GPI连接的膜蛋白，有助于通过以下方法筛选具有GPI无效表型的罕见细胞： 流式细胞仪通过这种方法，我们已经能够测量粒细胞中的f，以及粒细胞中的f和f。 B类淋巴母细胞系和来自血液样品的离体T淋巴细胞的扩增培养物。 最近，我们已经表明，它是可能的测量f在人类红细胞在一个额外的哨兵 XK基因具有与PIG-A相似的优势。具体目标（a）：确定是否增加 （B）：确定是否可以预防与衰老相关的 用药物治疗癌症可以减少。就目标（a）而言， 将招募年龄组，并使用PIG-A作为B中的哨兵基因直接测量 淋巴母细胞系和T淋巴细胞培养，与新生儿脐带血比较 样品也将根据对粒细胞频率的分析间接评估， PIG-A突变和带有XK突变的红细胞作为年龄的函数。对于目标（B），使用来自 正常老年人和早衰症患者，对突变率的影响将是 确定了三种药理学方法：淬灭活性氧，调节 核纤层蛋白A使用法尼基转移酶抑制剂，并激活SIRT 1。初步数据： 来自正常个体的淋巴样细胞的范围为每次细胞分裂3至53 × 10-7个突变， 与年龄正相关。在癌症易感性患者的细胞中， 综合征、早衰综合征和恶性细胞系。 的减少 现在已经使用脱氢抗坏血酸证明了人类细胞中的自发突变率， 减少细胞内活性氧。 影响：除了解决非常重要的 关于衰老和癌症风险的基本生物学问题，目标（a）下的研究包括： 这对临床化学预防研究的规划很重要。如果它在整个生命中是恒定的，那么任何 减少突变的战略可能需要尽早开始。然而，如果像怀疑的那样， 随着年龄的增长，一旦癌症开始增加，就有可能通过在以后的年龄减少来预防癌症。 目标（B）下的研究对于预测最佳药物靶点和药理学作用至关重要。 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.