Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 6988951
• 负责人: Kyungjae Myung
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6988951
• 关键词: DNA binding protein; DNA damage; DNA repair; Saccharomyces cerevisiae; cancer risk; cell component structure /function; cell cycle; cell growth regulation; chromosome aberrations; fungal genetics; fungal proteins; gene expression; gene mutation; gene rearrangement; human tissue; intermolecular interaction; molecular oncology; molecular pathology; neoplasm /cancer genetics; neoplastic transformation; protein structure function; telomere

Genome instability is a characteristic of cancer cells. Different types of genome instability such as the accumulation of mutations, genome rearrangements and aneuploidy have been observed in different genetic disorders including cacners. There is growing evidence supporting the idea that acquisition of a mutator phenotype is required to account for the high rate of accumulating genetic changes in cancer cells. Some of the many examples are cancer susceptibility genes such as ATM, NBS, BLM, BRCA1 and BRCA2 whose protein products have been linked to defects in DNA damage responses and/or DNA repair. In some cases, tumors and even normal blood cells from mutation-bearing individuals show an abnormally high frequency of chromosomal aberrations. While there are many observations of genetic cancer syndromes associated with genome instability, there is little work linking these gene defects to the molecular events that cause genome instability. To understand the mechanisms of genome instability, we have studied 1) The role of Ku protein in suppression of GCR through telomere maintenance, 2) The role of mitotic checkpoint to increase the GCRs and 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. 1) The role of Ku protein in suppression of GCR through telomere maintenance. Telomeres are the terminal structures of linear chromosomes. Telomeres appear to perform at least two functions; a) they allow for the replication of the ends of chromosomes and b) they stabilize chromosomes by keeping them from recombining with one another. Ku86 plays a key role in nonhomologous end joining (NHEJ) in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We found that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs that additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length. Furthermore, we investigated the mechanism how Ku proteins suppress genome instability by using yeast GCR assay system. We found that the overexpression of yeast Ku70-Ku86 heterodimer suppressed the GCR formation either spontaneously generated or induced by treatments with different DNA damaging agents, which are sometimes used for radio- and chemotherapies for cancer. The suppression of GCR formation by the yKu70-yKu86 overexpression was disappeared only when the DNA damage checkpoint is despaired suggesting that the GCR suppression by Ku protein is its interaction through the DNA damage checkpoint not through its role in NHEJ. The Ku overexpression caused cell growth delay, which is dependent on intact Okazaki fragment maturation proteins. Furthermore, the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression arrested cell cycle at S phase in DNA damage checkpoint dependent fashion. 2) The role of mitotic checkpoint to increase the GCRs. Checkpoints are surveillance mechanisms designed to ensure correct transmission of genetic information during cell division. There are a number of checkpoints that respond to DNA damage and as well as aberrant DNA structures that occur when DNA replication is blocked. The DNA damage checkpoint arrests the cell cycle eithher in G1 or G2 in response to DNA damage and also results in the slowing of DNA replication when DNA damage occurs during S phase; this latter checkpoint response is sometimes called the intra-S checkpoint. The DNA replication checkpoint arrests cell cycle progression and suppresses the firing of late replication origins in response to blocked DNA replication. The mitotic checkpoints respond to the failure of spindle assembly and arrests the cell cycle at M phase. Genetic defects in various DNA damage and S-phase checkpoints have been demonstrated to result in differing degrees of increased spontaneous GCR rates, increased chromosome loss and increased recombination. However, compared to the significant increases in GCR rates caused by defects in S phase checkpoints including the replication checkpoint and intra-S checkpoints, defects in the mitotic checkpoint did not appear to increase GCR rates. The mitotic checkpoint, also known as the spindle checkpoint, ensures proper chromosome segregation by arresting the cell cycle at mitosis by responding to improper or incomplete spindle assembly. In S. cerevisiae the genes that function in the mitotic checkpoint include MAD1, 2, 3, BUB1, 3 and MPS1 and these in part function through the anaphase-promoting complex (APC). Bub2 functions in the MEN by inhibiting the degradation of mitotic cyclins and other regulators of the exit from mitosis. Mitotic exit is achieved by inactivation of Tem1 by conversion of bound GTP to GDP by the Bub2 and Bfa1 GTPase activating proteins. Mutations in genes encoding mitotic checkpoint and MEN proteins lead to increased missegregation of chromosomes even in the absence of spindle damages and failure of mitotic cell cycle arrest in response to spindle depolymerizing drugs such as nocodazole or benomyl. We found that defects in the mitotic checkpoint and the mitotic exit network (MEN) often result in the suppression of GCRs in strains containing defects that increase the GCR rate. These data strongly suggest that functional mitotic checkpoints can play an important role in the formation of genome rearrangements. 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. Mutation in any of genes in the MRX complex (MRE11, RAD50 or XRS2) causes sensitivity to alkylating agents and ionizing radiation, defects in mitotic and meiotic recombination and NHEJ and also results in a decrease in telomere length. The fact that the mammalian MRX complex equivalent forms foci at the site of DNA damage suggests that the MRX complex functions in cell cycle checkpoints and DNA repair in mammalian cells. Furthermore, mutations of genes encoding these subunits have been identified in many cancer prone syndromes including Ataxia Telangiectasia Like Disorder (ATLD) and Nijmegen Breakage Syndrome (NBS). Null mutations in any of the MRX genes increased the GCR rate up to 1000 fold. However, because of the multiple functions and biochemical activities of the MRX complex, it is unclear which functions and what biochemical activities of the MRX complex are important for suppression of GCRs. To understand which function of MRX complex is the main function of GCR suppression, we generated different types of point mutations that specifically inactivate different MRX functions. We found that at least three different activities of the MRX complex are important for suppression of GCRs. The nuclease activity of Mre11, an activity related to MRX complex formation and the telomere maintenance function of the MRX complex are important for the suppression of GCRs. An activity related to MRX complex formation is especially important for the suppression of translocation type of GCRs.

基因组不稳定性是癌细胞的特征。在包括CACNER在内的不同遗传疾病中，已经观察到了不同类型的基因组不稳定性，例如突变，基因组重排和非整倍性的积累。越来越多的证据支持这样一种观念，即需要对突变器表型的获取来说明癌细胞积累遗传变化的高率。众多例子中有一些是癌症易感性基因，例如ATM，NBS，BLM，BRCA1和BRCA2，其蛋白质产物与DNA损伤反应和/或DNA修复的缺陷有关。在某些情况下，来自突变个体的肿瘤甚至正常血细胞表现出异常高的染色体畸变频率。尽管有许多与基因组不稳定性相关的遗传癌综合征的观察结果，但几乎没有将这些基因缺陷与引起基因组不稳定性的分子事件联系起来的工作。为了了解基因组不稳定性的机制，我们研究了1）KU蛋白通过端粒维持抑制GCR的作用，2）有丝分裂检查点增加GCR的作用和3）MRX（MRE11-RAD50-XRS2）的表征（MRE11-RAD50-XRS2）在GCR抑制GCR的作用方面的表征。 1）KU蛋白通过端粒维持抑制GCR的作用。 端粒是线性染色体的末端结构。端粒似乎至少执行两个功能。 a）它们允许复制染色体的末端，b）通过防止它们相互重组来稳定染色体。 KU86在非同源末端连接（NHEJ）中起着关键作用，如细菌和人类的进化上不同。在真核细胞中，KU86也与端粒长度的调节有关，尽管Ku86突变的作用在物种之间差异很大。实际上，端粒要么显着缩短，要么稍微缩短，保持不变，要么分别在发芽的酵母，裂变酵母，鸡肉细胞或植物中显着延长，而KU86表达的无效。因此，尚不清楚哪种模型系统与人类最相关。我们发现，即使是人类体细胞中Ku86单个等位基因的功能失活也导致端粒丧失，这伴随着染色体融合，易位和基因组不稳定性的增加。总之，这些实验表明，KU86与其在非同源末端连接中的作用分开，通过调节端粒长度来抑制基因组不稳定性，在人类体细胞中执行了其他功能。此外，我们研究了KU蛋白如何使用酵母GCR测定系统抑制基因组不稳定性的机制。我们发现，酵母KU70-KU86异二聚体的过表达抑制了用不同DNA损伤剂的处理自发产生或诱导的GCR形成，有时用于癌症的放射性和化学疗法。仅当DNA损伤检查点绝望地表明KU蛋白抑制GCR是通过DNA损伤检查点而不是通过其在NHEJ中的作用，才表明其抑制GCR的抑制作用表明，YKU70-YKU86过表达抑制GCR形成。 KU过表达导致细胞生长延迟，这取决于完整的Okazaki碎片成熟蛋白。此外，端粒酶抑制剂的灭活，PIF1与KU过表达一起在DNA损伤检查点依赖性的情况下以S相阻止了细胞周期。 2）有丝分裂检查点增加GCR的作用。 检查点是旨在确保细胞分裂期间遗传信息正确传输的监视机制。有许多检查点对DNA损伤以及DNA复制被阻断时发生的异常DNA结构响应。 DNA损伤检查点会响应DNA损伤，使G1或G2中的细胞周期在S期发生DNA损伤时会减慢DNA复制。后一种检查点响应有时称为Intra-S检查点。 DNA复制检查点会阻止细胞周期的进展，并抑制后期复制起源的发射，以响应阻塞的DNA复制。有丝分裂检查点响应纺锤体组件的故障，并在M期停止细胞周期。已经证明，各种DNA损伤和S期检查点中的遗传缺陷导致自发GCR率增加，染色体损失增加和重组增加。但是，与S相检查点缺陷引起的GCR率的显着增加相比，包括复制检查点和S Intra-S检查点，有丝分裂检查点中的缺陷似乎并没有增加GCR率。 有丝分裂检查点，也称为主轴检查点，通过响应不当或不完整的纺锤体组件来阻止有丝分裂时的细胞周期来确保适当的染色体分离。在酿酒酵母中，有丝分裂检查点功能的基因包括MAD1、2、3，BUB1、3和MPS1，以及这些基因通过后期促进复合物（APC）的部分功能。 BUB2通过抑制有丝分裂出口的有丝分裂细胞周期蛋白和其他调节剂的降解来发挥作用。通过BUB2和BFA1 GTPase激活蛋白将结合的GTP转化为GDP，通过将TEM1灭活来实现有丝分裂出口。编码有丝分裂检查点和男性蛋白质的基因突变，即使在没有纺锤体损伤的情况下，染色体的错误隔离也会增加，而有丝分裂细胞周期的失败因响应纺锤体去聚合药物（例如诺科唑唑或苯丙核基）而导致丝分裂细胞周期停滞。我们发现，有丝分裂检查点和有丝分裂出口网络（MEN）中的缺陷通常会导致抑制GCR的菌株，这些缺陷含有增加GCR率的缺陷。这些数据强烈表明功能性有丝分裂检查点可以在基因组重排的形成中起重要作用。 3）MRX（MRE11-RAD50-XRS2）复合物的表征是其抑制GCR的作用。 MRX复合物中任何基因的突变（MRE11，RAD50或XRS2）都会引起对烷基化剂的敏感性以及电离辐射，有丝分裂和减数分裂重组和NHEJ的缺陷，也导致端粒长度的降低。哺乳动物MRX复合物在DNA损伤部位处的哺乳动物MRX复合物等效灶表明，MRX复合物在细胞周期检查点和哺乳动物细胞中的DNA修复中起作用。此外，在许多易癌症综合征中已经鉴定出编码这些亚基的基因的突变，包括毛道症状（ATLD）和Nijmegen Breakage综合征（NBS）。任何MRX基因中的无效突变将GCR速率提高到1000倍。但是，由于MRX复合物的多种功能和生化活性，尚不清楚MRX复合物的哪些功能和哪些生化活性对于抑制GCR很重要。为了了解MRX复合物的哪个功能是GCR抑制的主要功能，我们产生了不同类型的点突变，这些突变特别使不同的MRX函数失活。我们发现，MRX复合物的至少三种不同的活动对于抑制GCR很重要。 MRE11的核酸酶活性，与MRX复合物形成和MRX复合物的端粒维持功能有关的活性对于抑制GCR很重要。与MRX复合物形成有关的活性对于抑制GCR的易位类型尤为重要。