Genetic Determinants of Epithelial DNA Damage in Smokers

虽然大多数肺癌患者是吸烟者，但只有少数吸烟者真正患上肺癌。大量证据表明，遗传因素在决定哪些吸烟者患肺癌方面起着重要作用。该项目的总体目标是确定吸烟者对上皮细胞DNA损伤的个体易感性的遗传决定因素，这是肺癌发生的基础。 在对肺癌患者的初步研究中，我们从几个通常与吸烟相关的癌症有关的部位采集了上皮细胞样本。 这些研究已经证明了杂合性缺失（洛）频率的广泛的受试者间差异，杂合性缺失是烟草烟雾诱导的DNA损伤的指标，并且来自气道、口腔和膀胱样品的上皮细胞中相同等位基因的频繁缺失。 我们将在更大的患者群体中证实这些结果，方法是在两个吸烟人群中检测洛缺失，这两个人群在遗传上具有更高的肺癌发病风险;慢性阻塞性肺疾病受试者及其吸烟兄弟姐妹，以及年龄小于60岁的肺癌患者及其吸烟兄弟姐妹。 我们将关联洛缺失的频率、洛缺失的染色体位点以及从先证者及其吸烟同胞中获得的颊和膀胱上皮DNA中丢失的等位基因。 由于基因组DNA是从口腔刮片和尿液样本中获得的，它使我们能够以非侵入性的方式对大量吸烟者进行采样。此外，我们已经验证了在我们的高通量方法中测量洛缺失的灵敏度，该方法使用荧光标记的标记物和连续稀释的DNA和放射性标记物。 我们将患者组织样本中的观察结果与替代模型系统中的诱导效应相关联。 为了确定潜在的分子机制负责频率和位点的洛，我们将测量洛淋巴细胞传代几代后，从相同的主题暴露于烟草致癌物在体外。 我们将确定洛缺失的数量是否与DNA修复能力相关，特定等位基因的缺失是否代表遗传的易感DNA序列，以及诱导或印记的DNA甲基化模式在确定参与洛缺失的等位基因中的作用。 我们将用来自临床样本的上皮细胞证实体外数据。 我们假设吸烟的兄弟姐妹在数量、位点和等位基因丢失方面表现出高度一致性，这可能与本研究中探索的遗传因素有关。 我们相信，这些研究的结果将使基因组上的遗传改变的热点的本地化，从而加快烟草烟雾衍生的致癌物诱导的肺癌过程中的靶向基因的失活的鉴定。 从长远来看，这些靶基因可以作为疾病的治疗干预、诊断、预后和管理的节点。

Although most patients with lung cancer are smokers, only a minority of smokers actually develop lung cancer. Considerable evidence suggests that genetic factors play an important role in determining which smokers develop lung cancer. The overall goal of this project is to define the genetic determinants for individual susceptibility to epithelial cell DNA damage in smokers, which underlie the genesis of lung cancer. In preliminary studies of patients with lung cancer, we have sampled epithelial cells from several sites commonly involved in smoking related cancer. These studies have demonstrated wide inter- subject variations in the frequency of loss of heterozygozity (LOH), an indicator of tobacco-smoke induced DNA damage, and frequent loss of the same allele in epithelial cells from the airways, buccal and bladder samples. We will confirm these results in a larger group of patients by examining LOH in two populations of smokers who are genetically at increased risk for developing lung cancer; subjects with chronic obstructive lung disease and their smoking siblings, and lung cancer patients less than 60 years of age and their smoking siblings. We will correlate the frequency of LOH, chromosomal sites of LOH, and alleles lost in buccal and bladder epithelial DNA obtained from probands and their smoking siblings. Since the genomic DNA is obtained from buccal scrapings and urine samples, it allows us to sample a large number of smokers in a non-invasive fashion. Furthermore, we have already validated the sensitivity of measuring LOH in our high throughput method using fluorescent labeled markers with serial dilution of the DNA and radiolabeled markers. We would correlate observations made in patient tissue samples with inducible effects in a surrogate model system. To define potential molecular mechanisms responsible for frequency and sites of LOH, we will measure LOH in lymphocytes passaged for several generations from the same subjects after exposure to tobacco carcinogens in vitro. We will determine whether the amounts of LOH correlate with DNA repair proficiency and specific alleles lost represent susceptible DNA sequences that are inherited as well as the role of DNA methylation patterns induced or imprinted in determining the allele(s) involved in LOH. We will substantiate the in vitro data with epithelial cells from clinical samples. We postulate that smoking siblings will display a high degree of concordance for amounts, sites and alleles lost and that may relate to the heritable factors being explored in this proposal. We believe that the findings from these studies will enable the localization of hot spots for genetic alterations on the genome and hence accelerate the identification of genes targeted for inactivation during tobacco smoke derived carcinogen induced lung cancer. In the long-term, these target genes may serve as nodal points for therapeutic intervention, diagnosis, prognosis and management of the disease.