Genomic Instability in Ageing and Cancer

描述（由申请人提供）： 背景资料：在人类中，测量携带突变的细胞的频率（f）对于非常少量的哨兵基因是可能的，并且历史上在技术上是困难的。测量突变率（<）-代表每次细胞分裂中基因发生新突变的概率-几乎是不可能的。然而，f和<可能是老化的关键参数。例如，假设：（i）<随着年龄的增长而增加;（ii）<在某些过早衰老综合征中升高;以及（iii）通过降低<可能减少与衰老相关的癌症。 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，我们希望这将大大促进这些假设的调查。PIG-A位于X染色体上，因此单个失活突变可产生突变表型。PIG-A突变体缺乏所有GPI连接的膜蛋白，便于通过流式细胞术筛选具有GPI无效表型的罕见细胞。通过这种方法，我们已经能够测量粒细胞中的f，以及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中的δ和f。 最近，我们已经表明，它是可能的测量f在人类红细胞中的一个额外的哨兵基因，XK，它具有类似的优势PIG-A。具体目标（a）：以确定是否随着人类年龄的增长而增加;（B）：以确定是否可以用可以减少的药理学试剂来预防与衰老相关的癌症。对于目的（a），将招募不同年龄组的志愿受试者，并将使用PIG-A作为B淋巴母细胞系和T淋巴细胞培养物中的哨兵基因直接测量，与新生儿脐带血样本进行比较。还将基于具有PIG-A突变的粒细胞和具有XK突变的红细胞的频率作为年龄的函数的分析来间接评估。对于目的（B），使用来自正常老年个体和患有早衰症的患者的细胞，将确定三种药理学方法对突变率的影响：淬灭活性氧物质、使用法尼基转移酶抑制剂调节核纤层蛋白A和激活SIRT 1。初步数据：在正常个体的淋巴细胞培养物中，每个细胞分裂的突变数为3至53 × 10 - 7，并且与年龄呈正相关。在来自患有癌症易感综合征、早衰综合征的患者的细胞中以及在恶性细胞系中， 现在已经证明使用脱氢抗坏血酸减少细胞内活性氧物质可以降低人类细胞中的自发突变率。 含义：除了解决有关衰老和癌症风险的非常重要的基本生物学问题外，目标（a）下的研究对规划临床化学预防研究也很重要。如果<在整个生命过程中是恒定的，那么任何减少突变的策略都需要尽早开始。然而，如果像怀疑的那样，<随着年龄的增长而开始增加，那么一旦<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减少临床<的药理学策略至关重要。 公共卫生相关性： 叙述：退伍军人事务部关心一大批老年患者，他们因为年老而容易患癌症。更好地了解衰老和突变率之间的关系是非常必要的，以计划研究，以预防这一人群的癌症。这里提出的研究将有助于预测在什么年龄可能需要干预以减少突变。这些研究还可能确定可能预防突变和癌症的药物。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Narrative: The Department of Veterans' Affairs cares for a large cohort of elderly patients who are prone to cancer because they are elderly. A better understanding of the relationship between ageing and the mutation rate is very much needed to plan studies to prevent cancer in this population. The studies proposed here will help predict at what age intervention to decrease mutations might be required. These studies may also identify drugs that are likely to prevent mutations and cancer.