Exposure Specific Mutation In Critical Target Genes

关键靶基因的暴露特异性突变

• 批准号: 6838351
• 负责人: JACK A TAYLOR
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6838351
• 关键词: biopsy; bladder neoplasm; bronchoscopy; cancer risk; chemical carcinogen; chemical carcinogenesis; clinical research; early diagnosis; endoscopy; environment related neoplasm /cancer; environmental exposure; fluoroscopy; gene environment interaction; gene mutation; human subject; lung neoplasms; molecular cloning; molecular oncology; neoplasm /cancer diagnosis; neoplasm /cancer genetics; neoplastic process; polymerase chain reaction; preneoplastic state; prognosis; single strand conformation polymorphism; tumor suppressor genes

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).

概括： 我的这一研究领域检验了这样的假设：环境暴露会在人类肿瘤中产生特定的基因突变模式。这种模式既可用于识别关键靶基因，又可用于提示环境因素导致癌症的突变机制。如果特定的致癌物在肿瘤中产生特征性的基因突变模式，那么对这些模式的检测将成为环境风险研究以及预防和早期诊断的有力工具。近年来，我们开始在正在进行的分子流行病学和临床研究中扩展这一概念，这些研究旨在研究种系突变，并使用我们开发的特殊技术来研究肿瘤前和正常组织的极小样本中的 DNA 损伤。随着实验室彗星测定的建立，这项技术使我们能够测量单个活细胞中的一般 DNA 损伤，我们现在正在开发一项新的临床实验研究，计划测量结肠连续活检中的 DNA 损伤水平当我们让人们接受不同的饮食习惯时，他们就会产生上皮细胞。长期目标是开发一种定量测量正常组织中 DNA 突变水平或“体细胞突变负荷”的方法。这样的指标可以提供一生中环境暴露的组织特异性测量，整合饮食、遗传易感性和修复，并且可以更精确地估计癌症、神经系统疾病、生殖疾病和其他 DNA 损伤发挥作用的疾病的风险。 荧光支气管镜检查和异常支气管病变的分子特征（LIFE 研究）： 我们目前正在进行的临床主要研究旨在检验以下假设：暴露与癌前和正常肺组织的突变模式相关，并且此类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，从因吸烟、职业暴露或家族史而患肺癌高风险的患者中收集正常、癌前和肿瘤组织样本。这些人经过两年的重复支气管镜检查和活检进行随访，使我们能够跟踪各个病变随时间的分子变化。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，旨在从因肺癌开胸手术的患者身上获取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 名患者并对其进行了支气管镜检查，使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。我们使用冷冻活检的激光捕获显微切割 (LCM) 来收集特定细胞，并开​​发了特殊的 PCR 方法，使我们能够仅使用 50 个细胞来检查一组选定基因座的杂合性丢失 (LOH)。此外，正在检查这些患者培养物中生长的细胞的细胞遗传学异常、端粒酶活性，并将进行 p53 和 p21 的免疫组织化学检查。这项工作在今年夏天举行的第十届世界肺癌大会上的三个独立演讲中提供了有关 LOH、细胞遗传学异常和端粒酶活性的初步结果。 膀胱癌中的环境暴露和 p53 突变模式：最近，我们的膀胱癌病因和机制的分子流行病学研究手稿被《癌症研究》接受发表。我们利用之前生成的致癌物代谢基因多态性数据对 143 个石蜡包埋肿瘤进行了新的 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，并从突变子集与代谢基因型的关联中寻找机制线索。主要观察结果是 GC>AT 转变 与未接触过环境的人相比，CpG 二核苷酸在有环境暴露的人（尤其是吸烟者）的肿瘤中出现的频率明显更高。加上编码链偏差的证据，以及可能与 NAT2 慢速相关的证据 乙酰化基因型，这可能是膀胱癌中烟草特征突变的第一个可信证据。 切尔诺贝利清理工人的种系突变：在最近完成的分子流行病学研究中，我们测试了这样的假设：切尔诺贝利事故清理工人在接触后比接触前具有更高的种系突变率。我们比较了接触前受孕的儿童和接触后受孕的儿童的 DNA 微卫星和小卫星突变率。我们证明了小卫星和微卫星位点的种系突变率相对较高，但在父亲暴露之前受孕的孩子与暴露后受孕的孩子之间的突变率没有显着差异。该手稿目前正在修改，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 Kunkel 合作研究镉对错配修复的影响。我们将少量 DNA 扩增技术扩展到单分子水平，以评估在环境相关镉浓度的培养物中生长的人类细胞是否会增加微卫星序列的突变率。这项工作的初步结果包含在《自然遗传学》论文中。我们希望在聘用一名新的博士后研究员来取代 Slebos 博士（目前在范德比尔特大学）时扩展这项工作。