Lung and other cancer etiological model of BER gene polymorphisms

描述（由申请人提供）：散发性癌症中的一个关键观察结果是，并非所有个体在暴露于特定环境致癌物的情况下都同样可能患上癌症。癌症发病时间的差异最有可能归因于遗传变异，例如人类基因组中的多态性。分子流行病学研究采用病例对照基因分型的统计分析和生物化学和分子生物学功能分析，现在提供了令人信服的证据，这种关联，而在其他情况下，结果和结论是矛盾的。我们假设，在碱基切除修复途径（BER）中涉及的基因的各种功能改变，在多态性的形式，导致在人群中的遗传易感性的差异，环境压力，这将随后导致疾病的开始和进展的时间的个体差异。一个强有力的方法来测试这一假设是将人类普遍的多态性纳入小鼠基因组，并观察模型生物是否会发展癌症由于遗传改变和衍生的分子事件。BER途径负责修复DNA碱基损伤。该途径中的缺陷可能对基因组具有两种不同的影响：1）诱变性脱碱基位点的积累，这可能导致高突变频率，以及2）DNA损伤的积累，如ssDNA切口或缺口以及由于中间体加工不当而导致的dsDNA断裂。DNA损伤可能导致受损细胞产生信号以阻止细胞周期进程。已知延长的细胞周期停滞会导致四倍体和非整倍体，这是癌症的标志。在这个探索性的赠款申请，我们选择两个不同的多态性APE 1和Pol 2？两者都是两个关键的BER分量。我们的初步数据表明，APE 1 P311 S是缺乏核酸内切酶活性，导致无法进一步处理脱碱基位点，并随后诱导突变的下一轮的DNA复制。DNA损伤诱导的突变子表型导致癌症。另一方面，我们发现Pol 2 R137Q在降低聚合酶活性以及PCNA相互作用能力方面显著降低BER能力。我们预测，在这种情况下，虽然拆除受损基座的最初步骤不受影响，但由此产生的缺口将无法填补。这将导致基因组中的未连接的间隙和随后的DNA断裂，这反过来将干扰细胞分裂，诱导四倍体和非整倍体。在本申请中，我们将使用代表性的APE 1和Pol 2突变小鼠模型来1）确定APE 1 P311S或Pol 2 R137Q多态性是否将使敲入小鼠模型中的癌症的高发病率易感，以及2）阐明癌症起始和发展的分子机制，包括通过生物化学测定评估的总体BER能力，基因组稳定性的状态，突变频率、四倍体和非整倍体的形成、突变细胞的细胞转化以及小鼠变异细胞对DNA损伤剂和化学及辐射致癌物的易感性。 公共卫生相关性：本申请旨在以人BER基因多态性为例，采用分子生物学和小鼠遗传分析的组合方法，建立遗传改变、分子和细胞水平的功能缺陷和癌症病理后果之间的综合关系。该研究的成功完成将揭示个体多态性在正常细胞向癌细胞转化过程中的功能机制的绝大部分信息，并为开发新的治疗方案和个性化药物提供参考。

DESCRIPTION (provided by applicant): A critical observation in sporadic cancers is that not all individuals are equally likely to develop cancers with a given exposure to an environmental carcinogen. The difference in the timing of cancer disease onset is most likely attributable to genetic variations, such as polymorphisms in the human genome. Molecular epidemiological studies employing statistical analysis of case-control genotyping and biochemical and molecular biological function analyses have now provided convincing evidence for such an association, while in other cases, the results and conclusions are contradictive. We hypothesize that various functional alterations in genes involved in the base excision repair pathway (BER), in the form of polymorphisms, result in differences in genetic susceptibilities to environmental stresses in the human population, which will subsequently leading to individual differences in the timing of disease initiation and progression. A powerful approach to test this hypothesis is to incorporate the human prevalent polymorphisms into the mouse genome and observe if the model organisms will develop cancer due to the genetic alteration and derived molecular events. The BER pathway is responsible for repair DNA base damage. Defects in this pathway could have two distinct impacts on the genome: 1) accumulation of mutagenic abasic sites, which can result in high mutation frequency, and 2) accumulation of DNA damage such as ssDNA nicks or gaps and dsDNA breaks due to improper processing of the intermediates. DNA damage may cause damaged cells to generate signals to arrest the cell cycle progression. Prolonged cell cycle arrest is known to cause tetraploidy and aneuploidy, a hallmark of cancer. In this exploratory grant application, we choose two distinct polymorphisms on APE1 and Pol 2 ?both are two key BER components. Our preliminary data indicate that APE1 P311S is deficient in endonuclease activity, leading to failure to further process abasic sites, and subsequently inducing mutations in the next round of DNA replication. The DNA damage-induced mutator phenotype causes cancer. On the other hand, we found that Pol 2 R137Q significantly reduced BER capacity in terms of decreased polymerase activity as well as PCNA interaction capacity. We predict that, in this case, although the initial steps for the removal of the damaged base is not affected, the resulting gap will not be able to be filled. This will lead to un-ligated gaps with subsequent DNA breaks in the genome that, in turn, will disturb cell division, inducing tetraploidy and aneuploidy. In the current application, we will use the representative APE1 and Pol 2 mutant mouse models to 1) determine if the APE1 P311S or Pol 2 R137Q polymorphism will predispose a high incidence of cancer in knock-in mouse models and 2) elucidate the molecular mechanisms underlying cancer initiation and development, including overall BER capacity, as evaluated by biochemical assays, the status of genome stability, mutation frequency, formation of tetraploidy and aneuploidy, cellular transformation of mutant cells, and the susceptibility of mouse variant cells to DNA damage agents and chemical and radiation carcinogens. PUBLIC HEALTH RELEVANCE: The current application aims to establish a comprehensive relationship among genetic alterations, functional deficiency at molecular and cellular levels, and cancer pathological consequences, using human BER gene polymorphisms as an example and employing a combined approach of molecular biological and mouse genetic analyses. Successful completion of the proposed studies will reveal a vast majority of information of functional mechanisms of individual polymorphisms during transformation from a normal cell to a cancer cell and provide a reference in developing new therapeutic regimens and personalized medicine.