Genetic Determinants of Epithelial DNA Damage in Smokers

虽然大多数肺癌患者都是吸烟者，但实际上只有少数吸烟者会患上肺癌。大量证据表明，遗传因素在决定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。在肺癌患者的初步研究中，我们从吸烟相关癌症的几个常见部位取样上皮细胞。这些研究表明，杂合性缺失（LOH）的频率（烟草烟雾引起的DNA损伤的一个指标）和气道、口腔和膀胱样本上皮细胞中相同等位基因的频繁缺失在受试者之间存在广泛的差异。我们将在更大的患者群体中证实这些结果，通过检查两组肺癌遗传风险增加的吸烟者的LOH；慢性阻塞性肺病患者及其吸烟的兄弟姐妹，60岁以下的肺癌患者及其吸烟的兄弟姐妹。我们将从先证者及其吸烟兄弟姐妹中获得LOH的频率、LOH的染色体位点和口腔和膀胱上皮DNA中丢失的等位基因。由于基因组DNA是从口腔刮痕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，使用连续稀释DNA的荧光标记标记物和放射性标记物来测量LOH的灵敏度。我们将在患者组织样本中观察到的结果与替代模型系统中的诱导效应相关联。为了确定导致LOH频率和位点的潜在分子机制，我们将在体外暴露于烟草致癌物质的同一受试者的几代传代淋巴细胞中测量LOH。我们将确定LOH的数量是否与DNA修复能力相关，丢失的特定等位基因是否代表遗传的易感DNA序列，以及诱导或印迹的DNA甲基化模式在确定LOH相关等位基因中的作用。我们将用临床样本的上皮细胞来证实体外数据。我们假设兄弟姐妹中吸烟的人在数量、位置和等位基因丢失方面表现出高度的一致性，这可能与本研究中正在探索的遗传因素有关。我们相信，这些研究结果将有助于定位基因组遗传改变的热点，从而加速鉴定烟草烟雾源性致癌物诱导肺癌过程中失活的靶向基因。从长远来看，这些靶基因可以作为治疗干预、诊断、预后和疾病管理的节点。

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.