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，一个重要的观察结果是，并不是所有的个体在暴露于特定的环境致癌物中时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，例如人类基因组的多态性。采用病例对照基因分型统计分析以及生化和分子生物学功能分析的分子流行病学研究现已为这种关联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，参与碱基切除修复途径（BER）的基因以多态性的形式发生各种功能改变，导致人类对环境压力的遗传易感性存在差异，这将随后导致疾病发生和进展时间的个体差异。验证这一假设的有力方法是将人类普遍的多态性纳入小鼠基因组，并观察模式生物是否会因遗传改变和衍生的分子事件而发展为癌症。BER通路负责修复DNA碱基损伤。该通路的缺陷可能对基因组产生两种不同的影响：1)诱变基本位点的积累，这可能导致高突变频率；2)由于中间产物加工不当，导致DNA损伤的积累，如ssDNA的缺口或缺口和dsDNA断裂。DNA损伤可能导致受损细胞产生信号来阻止细胞周期进程。已知细胞周期阻滞延长会导致四倍体和非整倍体，这是癌症的标志。在这个探索性拨款申请中，我们选择了APE1和Pol 2 ？两者都是两个关键的误码率组件。我们的初步数据表明，APE1 P311S缺乏内切酶活性，导致无法进一步加工基本位点，并随后在下一轮DNA复制中诱导突变。DNA损伤引起的突变表型导致癌症。另一方面，我们发现Pol 2 R137Q在降低聚合酶活性和PCNA相互作用能力方面显著降低了BER容量。我们预测，在这种情况下，尽管移除受损底座的初始步骤不受影响，但产生的间隙将无法填补。这将导致基因组中随后的DNA断裂产生未连接的间隙，进而干扰细胞分裂，诱发四倍体和非整倍体。在目前的应用中，我们将使用具有代表性的APE1和Pol 2突变小鼠模型来1)确定APE1 P311S或Pol 2 R137Q多态性是否会在敲入小鼠模型中导致高癌症发病率；2)阐明癌症发生和发展的分子机制，包括总体BER能力，通过生化分析评估，基因组稳定性状态，突变频率，四倍体和非整倍体的形成。突变细胞的细胞转化，以及小鼠突变细胞对DNA损伤剂和化学和辐射致癌物的易感性。

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.