Genomic Instability in Ageing and Cancer

衰老和癌症中的基因组不稳定性

• 批准号: 8391577
• 负责人: David J. Araten
• 金额: --
• 依托单位: VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391577
• 关键词: Address; Age; Aging-Related Process; Antioxidants; Biological; Blood specimen; Caring; Cell Line; Cell division; Cells; Chemoprevention; Clinical Chemoprevention; DNA biosynthesis; Data; Dehydroascorbic Acid; Demographic Aging; Drug Targeting; Elderly; Erythrocytes; Farnesyl Transferase Inhibitor; Flow Cytometry; Frequencies; Genes; Genomic Instability; Human; Incidence; Individual; Intervention; Investigation; Lamin Type A; Life; Link; Lymphoid Cell; Malignant Neoplasms; Measures; Membrane Proteins; Methods; Mutation; Neonatal; Normal Cell; Patients; Pharmaceutical Preparations; Phenotype; Population; Predisposition; Premature aging syndrome; Probability; Process; Progeria; Reactive Oxygen Species; Recruitment Activity; Sentinel; Somatic Mutation; Syndrome; T-Lymphocyte; Umbilical Cord Blood; United States; Veterans; X Chromosome; age group; age related; base; cancer cell; cancer risk; cohort; granulocyte; high risk; lymphoblastoid cell line; mutant; older patient; prevent; screening; trend; volunteer

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.

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，而且历史上在技术上一直很困难。测量 突变率（¿）——代表每个细胞的基因中发生新突变的概率 分裂——实际上是不可能的。尽管如此，f 和 ¿ 可能是老化的关键参数。为了 例如，假设： (i) ¿ 随着年龄的增长而增加； (ii) ¿ 在某些早产儿中升高 衰老综合症； (iii) 或许可以通过降低 ¿ 来减少与衰老相关的癌症。 我们现在已经开发出一种使用 PIG-A 基因测量这些参数的稳健方法，该方法 我们希望能够极大地促进这些假设的调查。 PIG-A位于X染色体上- 因此，单个失活突变可以产生突变表型。 PIG-A突变体缺乏所有 GPI 连接膜蛋白，有助于筛选具有 GPI 无效表型的稀有细胞 流式细胞术。通过这种方法，我们能够测量粒细胞中的 f，并且测量粒细胞中的 ¿ 和 f B 类淋巴母细胞系和来自血液样本的离体 T 淋巴细胞的扩增培养物。 最近，我们已经证明可以在额外的哨兵中测量人类红细胞中的 f 基因XK，具有与PIG-A类似的优点。具体目标 (a)：确定 ¿ 是否增加 人类随着年龄的增长； (b)：确定预防与衰老相关的措施是否可行 使用可以减少癌症的药物。对于目标（a），不同领域的志愿者受试者 将招募年龄组，并使用 PIG-A 作为 B 中的前哨基因直接测量 ¿ 类淋巴母细胞系和 T 淋巴细胞培养物与新生儿脐带血的比较 样品。 ¿ 也将根据对粒细胞频率的分析进行间接评估 PIG-A 突变和带有 XK 突变的红细胞与年龄的关系。对于目标 (b)，使用来自 正常老年人和早衰患者，对突变率的影响将是 确定了三种药理学方法：猝灭活性氧、调节 核纤层蛋白 A 使用法尼基转移酶抑制剂，并激活 SIRT1。初步数据： ¿ 在文化中 正常个体的淋巴细胞每次细胞分裂有 3 至 53 x 10-7 突变，并且 与年龄呈正相关。患有癌症倾向的患者的细胞中 ¿ 增加 综合征、早衰综合征和恶性细胞系。 减少 现在已经使用脱氢抗坏血酸证明了人类细胞的自发突变率 减少细胞内活性氧。 启示：除了解决非常重要的问题 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 对于规划临床化学预防研究很重要。如果 ¿ 在一生中保持不变，那么任何 减少突变的策略可能需要尽早开始。然而，如果正如所怀疑的那样，¿ 开始增加 随着年龄的增长，一旦癌症开始增加，就可以通过减少癌症来预防癌症。 目标 (b) 下的研究对于预测最佳药物靶点和药理学至关重要 临床上减少的策略。