Molecular Studies of Cancer Specific Fragile Sites

描述（由申请人提供）：染色体脆性位点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是阿菲霉素诱导的脆性位点，FRA11B是叶酸敏感位点，FRA16B是distamycin- a诱导的脆性位点，它们参与了癌症的形成。紧凑的染色质结构和不寻常的DNA序列已经发现在脆弱的位置。此外，脆弱站点显示复制延迟，并且可以逃避依赖atr的复制检查点。这些观察结果为脆弱位点的本质提供了一个有趣的模型，其中这些位点上的染色质和DNA结构会暂停复制叉的进程，并破坏细胞周期检查点途径，以允许染色体重排和病毒整合，导致脆弱位点特异性肿瘤发生。提出了三个目标：(1)确定脆弱位点特异性染色质的独特决定因素。染色质免疫沉淀（CHIP）测定将用于鉴定这些位点涉及的表观遗传标记。此外，通过在所有三种脆弱dna上重构染色质，将分析脆弱位点特异性染色质结构，并确定脆弱染色质形成的基本成分。(2)建立参与脆弱位点表达的细胞周期检查点通路。利用CHIP实验和RNA干扰产生的突变细胞，将检查几个细胞周期检查点蛋白在脆弱位点表达中的参与，以确定它们与脆弱DNA的关联。(3)利用SV40复制模型系统确定影响脆弱站点不稳定性的顺式和反式因素。通过操纵脆弱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，将评估脆弱位点的不稳定性（断裂位点的产生和重复长度的变化）。含有脆弱dna的复制中间体也将被表征，以提供有关脆弱位点复制延迟的直接信息。这一建议阐述了脆弱部位如何在三个连续的步骤中导致致癌病变。因此，这些实验将进一步推进对这些脆弱位点的性质及其在癌症形成中的作用的认识，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.