Lung and other cancer etiological model of BER gene polymorphisms

描述(由申请人提供)：在散发性癌症中的一个关键观察是，不是所有的人在给定的环境致癌物暴露下都同样有可能患上癌症。癌症发病时间的差异最有可能归因于遗传变异，如人类基因组的多态。分子流行病学研究采用病例对照基因分型的统计分析以及生化和分子生物学功能分析，现在已经为这种联系提供了令人信服的证据，而在其他情况下，结果和结论是相互矛盾的。我们假设，碱基切除修复途径(BER)中涉及的基因以多态形式发生的各种功能变化，会导致人类群体对环境应激的遗传易感性的差异，这将导致疾病发生和进展的个体差异。检验这一假说的一个有效方法是将人类普遍存在的多态整合到小鼠基因组中，并观察模式生物是否会因为基因改变和衍生的分子事件而患上癌症。BER途径负责修复DNA碱基损伤。这一途径的缺陷可能对基因组产生两种不同的影响：1)突变基本位点的积累，这可能导致高突变频率；2)DNA损伤的积累，如由于中间体处理不当而造成的单链DNA缺口或缺口和双链DNA断裂。DNA损伤可能会导致受损细胞产生信号，阻止细胞周期的进展。众所周知，长时间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。在这一探索性拨款申请中，我们在APE1和POL2上选择了两个不同的多态性-两者都是两个关键的误码率成分。我们的初步数据表明，APE1 P311S缺乏内切酶活性，导致无法进一步处理基本位点，从而在下一轮DNA复制中诱导突变。DNA损伤诱导的突变表型会导致癌症。另一方面，我们发现Pol 2 R137Q显著降低了误码率能力，表现为聚合酶活性和增殖细胞核抗原相互作用能力的降低。我们预测，在这种情况下，尽管拆除受损基地的最初步骤不受影响，但由此产生的缺口将无法填补。这将导致基因组中未连接的缺口和随后的DNA断裂，进而扰乱细胞分裂，导致四倍体和非整倍体。在目前的应用中，我们将使用具有代表性的APE1和POL2突变小鼠模型来1)确定APE1 P311S或POL2 R137Q多态是否会导致敲入小鼠模型中癌症的高发；2)阐明癌症发生和发展的分子机制，包括通过生化分析评估总体的误码率能力、基因组稳定性状况、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、化学和辐射致癌物的敏感性。 公共卫生相关性：目前的应用旨在以人类BER基因多态为例，采用分子生物学和小鼠遗传分析相结合的方法，建立遗传改变、分子和细胞水平的功能缺陷以及癌症病理后果之间的全面关系。这些研究的成功完成将揭示绝大多数个体基因多态性在正常细胞向癌细胞转化过程中的作用机制，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.