Molecular Studies of Cancer Specific Fragile Sites

描述(申请人提供)：染色体脆性部位的不稳定性与许多癌症直接相关。在三种不同的培养条件下产生了三种类型的脆性位点：FRA3B，一个由Aphidiclin诱导的脆性位点，FRA11B，一个叶酸敏感的位点，以及FRA16B，一个地塞霉素-A诱导的位点，参与了癌症的形成。在脆性部位发现了紧密的染色质结构和不寻常的DNA序列。此外，脆弱站点会显示复制延迟，并可以逃脱依赖ATR的复制检查点。这些观察为脆弱部位的性质提供了一个有趣的模型，在这些部位的染色质和DNA结构会暂停复制分叉的进程，并扰乱细胞周期检查点途径，从而允许染色体重排和病毒整合，导致脆弱部位特异性肿瘤发生。提出了三个目标：(1)确定脆性部位特定染色质的独特决定因素。染色质免疫沉淀(CHIP)分析将被用来识别涉及这些位置的表观遗传标记。此外，通过在所有三个脆弱DNA上重组染色质，将分析脆弱部位特定的染色质结构，并将识别参与形成脆弱染色质的必要成分。(2)建立参与脆性部位表达的细胞周期检查点通路。利用芯片实验和RNA干扰产生的突变细胞，将检测几种细胞周期检查点蛋白参与脆性位点表达的情况，以确定它们与脆性DNA的关联。(3)利用SV40复制模型系统确定影响脆性位点不稳定性的顺式和反式因素。通过操纵脆性DNA(长度、序列、复制方向和相对于复制起点的位置)和细胞周期检查点组件，将评估脆性位点的不稳定性(断裂位点的生成和重复长度的变化)。含有脆性DNA的复制中间体也将被描述为提供有关脆性位点复制延迟的直接信息。这项建议阐述了脆弱部位如何在三个连续步骤中导致致癌损害。因此，这些实验将进一步促进对这些脆弱部位的性质及其在癌症形成中的作用的了解，并解决基本的生物学问题，如染色质结构的决定因素。

DESCRIPTION (provided by applicant): Instability of chromosomal fragile sites is directly related to many cancers. Three types of fragile sites are generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA16B, a distamycin-A-inducible site, are involved in the formation of cancers. Compact chromatin structures and unusual DNA sequences have been found in fragile sites. Also, fragile sites display replication delay and can escape the ATR-dependent replication checkpoint. These observations provide an intriguing model for the nature of fragile sites, in which chromatin and DNA structures at these sites would pause the progress of the replication fork, and disrupt cell cycle checkpoint pathways to allow chromosomal rearrangement and viral integration, resulting in fragile site-specific tumorigenesis. Three goals are proposed: (1) Identify unique determinants for the fragile site-specific chromatin. Chromatin immunoprecipitation (CHIP) assays will be employed to identify epigenetic marks involved in these sites. Further, by reconstituting chromatin over all three fragile DNAs, fragile site-specific chromatin structure will be analyzed, and the essential components involved in the formation of fragile chromatin will be identified. (2) Establish cell cycle checkpoint pathways involved in the expression of fragile sites. Using CHIP assay and mutant cells created by RNA interference, the involvement of several cell cycle checkpoint proteins in the expression of fragile sites will be examined for their association with fragile DNA. (3) Determine cis- and trans-factors affecting fragile site instability by using an SV40 replication model system. By manipulating fragile DNA (length, sequences, replication direction, and location relative to replication origin) and cell cycle checkpoint components, fragile site instability (generation of break sites and changes in repeat length) will be evaluated. Replication intermediates containing fragile DNAs will also be characterized to provide direct information about replication delay of fragile sites. This proposal addresses how fragile sites lead to oncogenic lesions at three sequential steps. Therefore, these experiments will further advance knowledge about the nature of these fragile sites and their role in the formation of cancer, and also address fundamental biological questions, such as determinants of chromatin structure.