Exposure Specific Mutation In Critical Target Genes

摘要： 我的这一研究领域测试了这样一个假设，即环境暴露会在人类肿瘤中产生特定的基因突变模式。这种模式既可以用来识别关键的靶基因，也可以用来建议环境因素导致癌症的突变机制。如果特定致癌物在肿瘤中产生特有的基因突变模式，那么对这些模式的检测将是研究环境风险以及用于预防和早期诊断的有力工具。近年来，我们已经开始在我们正在进行的分子流行病学和临床研究中扩展这一概念，这些研究旨在研究生殖系突变，并使用我们开发的特殊技术来观察癌前病变和正常组织中非常小的样本中的DNA损伤。随着彗星试验在实验室的建立，这是一种允许我们测量单个活细胞中一般DNA损伤的技术，我们现在正在开发一项新的临床实验研究，我们计划测量人们在不同饮食方案下进行的结肠上皮连续活检中的DNA损伤水平。一个长期目标是开发一种定量测量正常组织中DNA突变水平的方法，即“体细胞突变负荷”。这样的衡量标准可以提供终身环境暴露的组织特异性衡量标准，整合了饮食、遗传易感性和修复，并可能提供对癌症、神经、生殖和其他DNA损伤起作用的疾病风险的更准确估计。 荧光支气管镜检查和异常支气管病变的分子特征(LIFE研究)： 我们目前正在临床上进行的主要研究旨在测试以下假设：暴露与癌前和正常肺组织中的突变模式相关，并且这些突变可能对肺癌的发展具有预后意义。我们正在使用肺成像荧光内窥镜(LIFE)，这是一种新开发的支气管镜检查技术，可以从吸烟、职业暴露或有家族史的肺癌高危患者身上收集正常、癌前和肿瘤组织样本。对这些患者进行为期两年的重复支气管镜检查和活检，使我们能够跟踪单个病变随着时间的推移的分子变化。此外，我们还有一个与北卡罗来纳大学联合资助的小型试点项目，目的是从接受开胸手术的肺癌患者身上获得最佳收集的肿瘤和正常组织。 去年的进展： LIFE肺癌研究：去年我们又招募了12名患者接受支气管镜检查，使我们目前的患者总数达到40名，其中许多人接受了第二次支气管镜检查。我们使用激光捕获显微切割(LCM)冰冻切片来收集特定的细胞，并开发了特殊的聚合酶链式反应方法，允许我们只处理50个细胞，以便检查一组选定基因座的杂合性丢失(LOH)。此外，在这些患者的培养中生长的细胞正在接受细胞遗传学异常和端粒酶活性的检查，并将接受免疫组织化学检查p53和p21。关于杂合性缺失、细胞遗传学异常和端粒酶活性的初步结果由这项工作在今年夏天举行的第10届世界肺癌会议上的三个不同的报告中提供。 膀胱癌的环境暴露和P53突变模式：我们最近接受了我们关于膀胱癌病因和机制的分子流行病学研究的手稿，发表在癌症研究杂志上。我们利用了我们之前产生的关于致癌物代谢基因多态的数据，以及对143例石蜡包埋肿瘤的新的p53突变分析。使用病例对照设计，我们在突变谱中寻找环境、职业和烟草诱变剂的证据，并从突变亚群与代谢基因型的关联中寻找机制线索。主要的观察结果是GC&>AT转变 在CpG，有环境暴露的人，特别是吸烟者，比那些没有暴露的人的肿瘤中出现二核苷酸的频率要高得多。再加上编码链偏向的证据，以及可能与Nat2 Slow有关的证据 乙酰化基因，这可能是膀胱癌中烟草标志性突变的第一个可信的证据。 切尔诺贝利事故清理工人的生殖系突变：在最近完成的这项分子流行病学研究中，我们检验了这样一个假设，即切尔诺贝利事故清理工人在接触切尔诺贝利事故后，生殖系突变率高于暴露前。我们比较了暴露前受孕的儿童和暴露后受孕的儿童的DNA微卫星和小卫星突变率。我们证明了微小和微卫星基因座的胚系突变率相对较高，但在父亲暴露之前怀孕的孩子和暴露后怀孕的孩子之间没有显著差异。这份手稿目前正在修改中，以便重新提交给突变研究。 镉错配修复抑制和微卫星突变：我们与Mike Resnick和Tom Kukel合作，研究镉对错配修复的影响。我们扩展了扩增少量DNA的技术，向下延伸到单分子水平，以评估在环境相关浓度的镉培养中生长的人类细胞是否增加了微卫星序列的突变率。这项工作的初步结果包括在由此产生的《自然遗传学》论文中。我们希望在聘请一名新的博士后研究员来接替斯莱博斯博士(现供职于范德比尔特大学)时，延长这项工作。

Summary: This area of my research tests the hypothesis that environmental exposures produce specific patterns of gene mutation in human tumors. Such patterns can be used both to identify critical target genes and to suggest mutational mechanisms by which an environmental agent causes cancer. If specific carcinogens produce characteristic patterns of gene mutation in tumors, the detection of those patterns would be a powerful tool in studies of environmental risk and for use in prevention and early diagnosis. In recent years we have begun to extend this concept in our ongoing molecular epidemiologic and clinical studies designed to look at germline mutation and, using special techniques that we have developed, to look at DNA damage in very small samples of preneoplastic and normal tissue. With the establishment of the Comet assay in the lab, a technique that allows us to measure general DNA damage in individual living cells, we are now developing a new clinical-experimental study where we plan to measure levels of DNA damage in sequential biopsies of colon epithelium from people as we subject them to different dietary regimes. A long term goal is to develop a quantitative measure of the level of DNA mutation in normal tissue or "somatic mutational load". Such a metric could provide a tissue specific measure of lifetime environmental exposure, integrated across diet, genetic susceptibility, and repair, and might offer a more precise estimate of risk for cancer, neurologic, reproductive, and other diseases where DNA damage plays a role. Fluorescence Bronchoscopy and Molecular Characterization of Abnormal Bronchial Lesions (LIFE Study): Our major study that is currently in the clinic is designed to test the hypothesis that exposure correlates with the pattern of mutation in premalignant and normal lung tissues and that such mutations may have prognostic significance for lung cancer development. We are using the Lung Imaging Fluorescent Endoscope (LIFE), a newly developed bronchoscopy technique to collect normal, premalignant, and neoplastic tissue samples from patients at high risk of lung cancer from smoking, occupational exposures, or because of family history. These people are followed over a 2 year period with repeat bronchoscopies and biopsy allowing us to follow the molecular changes in individual lesions over time. In addition we have a small pilot project, jointly funded with UNC, to obtain optimally-collected tumor and normal tissue from patients undergoing thoracotomy for lung cancer. Last Year's Progress: LIFE lung cancer study: We have enrolled and bronchoscoped an additional 12 patients in the last year bringing our current total to 40 patients, many of whom have undergone second bronchoscopies. We use laser capture microdissection (LCM) of frozen biopsies to collect specific cells, and have developed special PCR methods that allow us to work with only 50 cells in order to examine loss of heterozygosity (LOH) at a panel of selected loci. In addition, cells grown in culture from these patients are being examined for cytogenetic abnormalities, telomerase activity, and will be examined immunohistochemically for p53 and p21. Preliminary results on LOH, cytogenetic abnormalities, and telomerase activity are being provided from this work in three separate presentations at the 10th World Conference on Lung Cancer this summer. Environmental exposure and p53 mutation patterns in bladder cancer: We have recently had our manuscript of our molecular epidemiology study of the causes and mechanisms of bladder cancer accepted for publication in Cancer Research.. We utilized our previously-generated data on carcinogen metabolism gene polymorphisms along with new p53 mutation analyses of 143 paraffin-embedded tumors. Using a case-control design we sought evidence of environmental, occupational, and tobacco mutagens in the mutation spectrum and mechanistic clues from associations of mutation subsets with metabolic genotypes. The principal observation is that GC>AT transitions at CpG dinucleotides occur significantly more often in tumors from people with environmental exposure, particularly smokers, than those without exposure. Coupled with evidence of a coding strand bias, and a possible association with NAT2 slow acetylator genotype, this is perhaps the first plausible evidence of a tobacco signature mutation in bladder cancer. Germline mutations in Chernobyl cleanup workers: In this recently completed molecular-epidemiology study we tested the hypothesis that Chernobyl accident cleanup workers had higher rates of germline mutations after their exposure than before their exposure. We compared rates of DNA microsatellite and minisatellite mutation in children conceived before their exposure to children conceived after their exposure. We demonstrate a relatively high rate of germline mutation in mini and microsatellite loci, but show no significant difference in rates between children conceived prior to their father's exposure vs those conceived post exposure. This manuscript is currently being revised for resubmission to Mutation Research. Cadmium mismatch repair inhibition, and microsatellite mutation: We entered into a collaboration with Mike Resnick and Tom Kunkel to investigate the effect of cadmium on mismatch repair. We extended out technique for amplifying small quantities of DNA down to the single molecule level in order to evaluate whether human cells grown in culture with environmentally-relevant concentrations of cadmium had increase mutation rates in microsatellite sequences. Preliminary results of this work were included in the resulting Nature Genetics paper. We are hoping to extend this work when a new postdoctoral fellow is hired to replace Dr. Slebos (now at Vanderbilt U).