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Mechanistic study of fragile site instability by investigating RET/PTC rearrangements, a common cause of papillary thyroid carcinoma.

机译：通过研究RET / PTC重排，即甲状腺乳头状癌的常见原因，对脆弱的部位不稳定进行机理研究。

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摘要

Chromosomal fragile sites are non-random regions of the genome with a predisposition to the formation of DNA breaks. The goal of this work is to investigate the mechanism of common fragile site breakage, the role it plays in the formation of cancer-causing chromosomal translocations, and how this knowledge can be utilized to tailor the treatment of patients.;To provide direct evidence for the role of fragile sites in the formation of oncogenic chromosomal translocations, RET/PTC rearrangements, a common cause of papillary thyroid carcinoma (PTC), were examined since the genes involved in the two most common subtypes, RET/PTC1 and RET/PTC3, are all located within common fragile sites. Furthermore, thyroid cancer rates are rapidly increasing, especially in women where it is the fastest growing cancer, and increased PTC incidences are almost entirely responsible for this upsurge. A large number of PTC tumors containing RET/PTC rearrangements are sporadic in nature, and may be the result of fragile site instability. The location of RET, CCDC6, and NCOA4 genes participating in RET/PTC1 and RET/PTC3 rearrangements, in fragile sites and their instability when exposed to fragile site-inducing chemicals was confirmed by Fluorescence In Situ Hybridization (FISH) and ligation mediation-PCR (LM-PCR). More importantly, treatment of thyroid cells with these chemicals resulted in the formation of RET/PTC1 rearrangements, providing direct evidence for fragile site involvement in the formation of a cancer-causing chromosomal translocation.;While a role for fragile sites in cancer development is strongly supported, the initial events leading to breakage at these sites are not well understood. Although no consensus sequence has been identified for common fragile sites, several characteristics are shared among many fragile sites studied to date, including the formation of stable DNA secondary structures, which are believed to impede replication fork progression and result in genomic instability. DNA topoisomerases I and II maintain chromosome structural integrity during replication and transcription by transiently inducing DNA breaks, and these enzymes have been shown to recognize and preferentially cleave at DNA secondary structures. Using the RET oncogene, initial events of fragile site breakage following treatment with the fragile site-inducing chemical aphidicolin (APH), were investigated. The location of DNA breaks within intron 11 of RET, the major breakpoint cluster region observed in PTC patients with RET/PTC rearrangements, was determined following treatment of thyroid cells with APH using LM-PCR. These breakpoints were located at or near predicted DNA topoisomerase I and/or II cleavage sites. Furthermore, these breakpoints were predicted to coincide with DNA structural features recognized by topoisomerases I and II. Using topoisomerase catalytic inhibitors in combination with APH treatment, the rate of APH-induced DNA breakage at RET and FHIT, also located at an APH-induced common fragile site, was significantly decreased, confirming the involvement of DNA topoisomerases I and II in initiating DNA breakage at these common fragile sites.;Overall, these results strongly support the role of fragile sites in the formation of cancer-causing chromosomal translocations, specifically the RET/PTC1 rearrangement in PTC. The location of fragile site breakage within the RET oncogene and the DNA secondary structure prediction analysis of chromosome 10, provide valuable insight into the initial events of common fragile site breakage, supporting a model of fragile site instability whereby the formation of stable DNA secondary structures impede replication fork progression and DNA topoisomerases I and II initiate DNA breakage at these sites through recognition of secondary structures. Furthermore, the ability of benzene and DEN to induce fragile site-specific breakage at the RET oncogene supports fragile site involvement in sporadic PTC tumors. Significant levels of double-strand DNA breakage at RET in normal thyroid tissue from RET/PTC-positive patients further supports this finding, suggesting this property could be used as a potential diagnostic assay to tailor the treatment of cancer patients with chemotherapeutic drugs or monitor cancer susceptibility in individuals exposure to high levels of external fragile site-inducing agents. (Abstract shortened by UMI.).
机译：染色体易碎位点是基因组的非随机区域，容易形成DNA断裂。这项工作的目的是研究常见的易碎部位断裂的机制，其在致癌染色体易位形成中的作用，以及如何利用这些知识来定制患者的治疗方法。研究了脆性位点在致癌染色体易位形成，RET / PTC重排（乳头状甲状腺癌（PTC）的常见原因）中的作用，因为涉及两种最常见亚型的基因RET / PTC1和RET / PTC3都位于常见的易碎地点内。此外，甲状腺癌的发病率正在迅速增加，尤其是在癌症增长最快的女性中，PTC发病率的上升几乎完全是造成这种疾病的原因。包含RET / PTC重排的大量PTC肿瘤本质上是零星的，可能是脆弱的部位不稳定的结果。通过荧光原位杂交（FISH）和连接介导PCR证实了参与RET / PTC1和RET / PTC3重排的RET，CCDC6和NCOA4基因在易碎位点中的位置以及当暴露于易碎位点诱导化学物质时它们的不稳定性（LM-PCR）。更重要的是，用这些化学药品处理甲状腺细胞会导致RET / PTC1重排的形成，为脆弱位点参与形成致癌染色体易位的形成提供了直接证据。虽然脆弱位点在癌症发展中的作用很强受支持的情况下，导致这些站点破裂的初始事件尚不十分清楚。尽管尚未确定常见脆弱位点的共有序列，但迄今研究的许多脆弱位点共有一些特征，包括形成稳定的DNA二级结构，这被认为会阻碍复制叉的发展并导致基因组不稳定。 DNA拓扑异构酶I和II通过瞬时诱导DNA断裂来维持复制和转录过程中的染色体结构完整性，并且这些酶已显示出识别并优先切割DNA二级结构的能力。使用RET致癌基因，研究了易碎位诱导化学蚜虫（APH）处理后易碎位断裂的初始事件。在RET / PTC重排的PTC患者中观察到的主要断裂点簇区域是RET内含子11内的DNA断裂位置，是在使用LM-PCR对APH处理甲状腺细胞后确定的。这些断点位于预测的DNA拓扑异构酶I和/或II切割位点处或附近。此外，这些断点预计与拓扑异构酶I和II识别的DNA结构特征相符。将拓扑异构酶催化抑制剂与APH处理结合使用时，同样位于APH诱导的常见脆弱位点的APH诱导的RET和FHIT DNA断裂速率显着降低，这证实了DNA拓扑异构酶I和II参与了起始DNA总的来说，这些结果强烈支持了脆弱位点在致癌染色体易位形成中的作用，特别是PTC中的RET / PTC1重排。 RET致癌基因内易碎位点断裂的位置以及10号染色体的DNA二级结构预测分析，为常见的易碎位点断裂的初始事件提供了有价值的见解，支持了易碎位点不稳定的模型，从而阻碍了稳定DNA二级结构的形成复制叉的进行以及DNA拓扑异构酶I和II通过识别二级结构在这些位点引发DNA断裂。此外，苯和DEN在RET致癌基因上诱导脆弱的位点特异性断裂的能力支持了散发PTC肿瘤中的脆弱位点参与。来自RET / PTC阳性患者的正常甲状腺组织中RET处双链DNA断裂的显着水平进一步支持了这一发现，表明该特性可以用作潜在的诊断测定方法，以量身定制化疗药物或监测癌症患者的癌症暴露于高水平的外部易碎部位诱导剂的个体的易感性。 （摘要由UMI缩短。）。

著录项

  • 作者

    Dillon, Laura Williams.;

  • 作者单位

    Wake Forest University.;

  • 授予单位 Wake Forest University.;
  • 学科 Biology Molecular.;Biology Genetics.;Chemistry Biochemistry.;Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 211 p.
  • 总页数 211
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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