Genomic Instability in Ageing and Cancer

描述(由申请人提供)： 背景：在人类中，测量携带突变的细胞的频率(F)对于非常少量的前哨基因是可能的，并且在历史上一直是技术上的困难。测量突变率(代表每个细胞分裂的基因发生新突变的可能性)几乎是不可能的。然而，f和lt；可能是衰老的关键参数。例如，它假设：(I)随着年龄的增长，&lt；会增加；(Ii)在某些早衰综合征中，&lt；会升高；以及(Iii)可能会通过减少&lt；来减少与衰老有关的癌症。我们现在已经开发出一种健壮的方法来使用PIG-A基因来测量这些参数，我们希望这将极大地促进这些假说的研究。PIG-A位于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突变体缺乏所有GPI连接的膜蛋白，便于通过流式细胞术筛选具有GPI零表型的罕见细胞。通过这种方法，我们已经能够测量粒细胞中的f，以及B淋巴母细胞系和体外培养的血液样本中T淋巴细胞的f。最近，我们已经证明了在人类红细胞中通过另一个前哨基因XK来测量f是可能的，XK具有与PIG-A相似的优点。具体目标(A)：确定人类是否随着年龄的增长而增加；(B)：确定是否可以用可以降低&lt；的药物来预防与衰老相关的癌症。对于目标(A)，将招募不同年龄段的志愿者，并将直接使用PIG-A作为哨点基因在B淋巴母细胞系和T淋巴细胞培养中进行测量，并与新生儿脐带血样本进行比较。还将根据对携带PIG-A突变的粒细胞和带有XK突变的红细胞随年龄变化的频率的分析，间接对其进行评估。对于目标(B)，利用来自正常老年人和早衰症患者的细胞，将确定三种药物方法对突变率的影响：猝灭活性氧物种，使用法尼基转移酶抑制剂调节层蛋白A，以及激活SIRT1。初步数据：在正常人的淋巴细胞培养中，每个细胞分裂的突变范围从3到53×10-7，并且与年龄呈正相关。在癌症易感综合征、早衰综合征和恶性细胞系患者的细胞中，&lt；增加。现在已经证明，使用脱氢抗坏血酸来减少细胞内的活性氧物种，可以降低人类细胞的自发突变率。影响：除了解决与衰老和癌症风险有关的非常重要的基本生物学问题外，AIM(A)项下的研究对于规划临床化学预防研究也很重要。如果&lt；在一生中是恒定的，那么任何减少突变的策略都可能需要及早开始。然而，如果正如人们所怀疑的那样，随着年龄的增长而开始增加，那么一旦癌症开始增加，就有可能通过在较晚的年龄减少&lt；来预防癌症。根据AIM(B)进行的研究将对预测临床上最佳的药物靶点和减少药物的药理策略至关重要。 公共卫生相关性： 叙述：退伍军人事务部照顾着一大批老年患者，他们因为年迈而容易患上癌症。更好地了解衰老和突变率之间的关系是非常必要的，以计划在这一人群中预防癌症的研究。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.