The Role of Fragile Sites in RET/PTC Rearrangement

描述(申请人提供)：染色体重排在许多类型的人类癌症中很常见，在癌症的发生中起着重要的作用。然而，癌细胞中的重排机制仍然知之甚少，甲状腺癌是研究这些机制的一个很好的模型，因为涉及RET基因的染色体重排是一种常见的称为RETIPTC的现象，并且与电离辐射引起的DNA损伤有良好的联系。然而，辐射暴露只占所有甲状腺肿瘤的一小部分，这为研究其他因素在重排产生中的作用提供了机会。这项拟议的研究旨在直接测试DNA的脆弱性是否参与了人类细胞中染色体重排的产生。我们的初步结果表明，脆性位点诱导化学物质可以诱导RET基因的断裂，并且在脆性位点诱导条件下可以产生重排。为了进一步研究这一点，我们将首先检查甲状腺细胞中涉及RETIPTC的一个或多个染色体区域是否为脆性部位的一部分。具体地说，我们将直接确定脆性位点与RET、H4和ELE1基因之间的物理关系。我们还将发现在用脆弱的位点诱导化学物质处理后，这些区域的DMA断裂的频率。然后，我们将测试脆性位点的表达是否可以直接导致人类甲状腺细胞中RETIPTC重排的产生。我们将直接测试在化学诱导脆弱部位以及ATR或BRCA1失活后，RET/PTC1和RET/PTC3重排在人HTori-3甲状腺细胞中的诱导情况，这已被证明显著增加了脆弱部位的表达。最后，我们将确定在RETIPTC重排涉及的区域中是否存在脆性位点的两个特征，即晚期DNA复制和二级结构形成，从而导致这些区域的DNA断裂。这些研究将确定脆性部位是否参与了甲状腺细胞中RETIPTC重排的产生，并允许探索这些区域的脆性机制。这将扩大我们对癌细胞中染色体重排的分子机制的理解，并最终可能导致通过染色体重排的机制开发新的治疗或预防恶性肿瘤的措施。

DESCRIPTION (provided by applicant): Chromosomal rearrangements are .common in many types of human cancer and play an important role in carcinogenesis. However, the mechanisms of rearrangement in cancer cells remain poorly understood, thyroid cancer represents an excellent model to study these mechanisms because of a common occurrence of chromosomal rearrangements involving the RET gene, called RETIPTC, and well-established association with DMA damage induced by ionizing radiation. However, radiation exposure accounts only for a small portion of all thyroid tumors, offering a chance to study the role of other factors in the generation of rearrangements. The proposed study aims to test directly if DMA fragility participates in the generation of chromosomal rearrangements in human cells. Our preliminary results showed that fragile site-inducing chemicals can induce breaks in the RET gene, and the rearrangement can be generated under fragile-site inducing conditions. To further investigate this, .we will first examine whether one or more chromosomal regions involved in RETIPTC in thyroid cells are part of fragile sites. Specifically, we will determine directly the physical relationship between the fragile sites and the RET, H4, and ELE1 genes. We will also find the frequency of DMA breaks in these regions after treatment with fragile site-inducing chemicals. Then, we will test whether fragile site expression can lead directly to the generation of RETIPTC rearrangements in human thyroid cells. We will test directly the induction of RET/PTC1 and RET/PTC3 rearrangements in human HTori-3 thyroid cells after chemical induction of fragile sites, and after inactivation of ATR or BRCA1, which has been shown to increase fragile site expression dramatically. Finally, we will determine whether two characteristic features of fragile sites, late DNA replication and secondary structure formation, exist in the regions involved in RETIPTC rearrangements, contributing to the DNA breakage in these areas. These studies will determine whether fragile sites participate in the generation of RETIPTC rearrangement in thyroid cells and allow to explore the mechanisms of fragility in these regions. This will extend our understanding of the molecular mechanisms of chromosomal rearrangements in cancer cells, and may eventually lead to the development of novel therapeutic or preventive measures for malignant tumors developing thro.ugh the mechanism of chromosomal rearrangements.