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).

总结： 我的这一研究领域验证了一个假设，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，也可以用来提示环境因子导致癌症的突变机制。如果特定的致癌物在肿瘤中产生基因突变的特征模式，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，这些研究旨在观察生殖系突变，并使用我们开发的特殊技术，观察肿瘤前和正常组织中非常小样本的DNA损伤。随着彗星试验在实验室中的建立，一种使我们能够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种新的临床实验研究，我们计划测量结肠上皮连续活检中的DNA损伤水平，因为我们使人们接受不同的饮食制度。长期目标是开发正常组织中DNA突变水平或“体细胞突变负荷”的定量测量。这种度量可以提供终生环境暴露的组织特异性测量，整合饮食，遗传易感性和修复，并可能提供癌症，神经系统，生殖和其他疾病的风险更精确的估计，其中DNA损伤起作用。 荧光支气管镜检查和异常支气管病变的分子表征（LIFE研究）： 我们目前在临床上的主要研究旨在检验暴露与癌前和正常肺组织中的突变模式相关的假设，以及这些突变可能对肺癌发展具有预后意义。我们正在使用肺成像荧光内窥镜（LIFE），一种新开发的支气管镜检查技术，从吸烟、职业暴露或家族史导致肺癌高风险的患者中收集正常、癌前病变和肿瘤组织样本。这些人随访2年，反复进行支气管镜检查和活检，使我们能够跟踪单个病变随时间的分子变化。此外，我们还有一个小型的试点项目，与联合国共同资助，以获得最佳收集的肿瘤和正常组织，从病人接受开胸肺癌。 去年的进展： LIFE肺癌研究：去年，我们又招募了12名患者并进行了支气管镜检查，使我们目前的患者总数达到40名，其中许多患者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）的冷冻活检收集特定的细胞，并已开发出特殊的PCR方法，使我们的工作只有50个细胞，以检查杂合性丢失（洛）在一组选定的基因座。此外，这些患者培养的细胞正在检查细胞遗传学异常、端粒酶活性，并将进行p53和p21的化学检测。在今年夏天的第10届世界肺癌会议上，这项工作的洛、细胞遗传学异常和端粒酶活性的初步结果将在三个独立的报告中提供。 膀胱癌中的环境暴露和p53突变模式：我们最近在《癌症研究》杂志上发表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。我们利用了我们先前产生的致癌物代谢基因多态性的数据沿着新的p53突变分析143例石蜡包埋肿瘤。使用病例对照设计，我们寻求证据的环境，职业和烟草诱变剂的突变谱和机制的线索，从关联的突变子集与代谢基因型。主要观察到GC>AT跃迁 CpG二核苷酸在有环境暴露的人，特别是吸烟者的肿瘤中比没有暴露的人更常见。再加上编码链偏好的证据，以及与NAT2慢 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生殖系突变：在这项最近完成的分子流行病学研究中，我们测试了一个假设，即切尔诺贝利事故清理工人在暴露后的生殖系突变率高于暴露前。我们比较了暴露前怀孕的儿童与暴露后怀孕的儿童的DNA微卫星和小卫星突变率。我们证明了一个相对较高的率在迷你和微卫星基因座的种系突变，但显示没有显着差异，在他们的父亲的曝光之前与那些怀孕后曝光的儿童之间的利率。目前正在修订这份手稿，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。我们将扩增少量DNA的技术扩展到单分子水平，以评估在环境相关浓度的镉培养中生长的人类细胞是否会增加微卫星序列的突变率。这项工作的初步结果被收录在《自然遗传学》的论文中。我们希望在聘请一位新的博士后研究员来取代Slebos博士（现在在范德比尔特大学）时扩展这项工作。