MOLECULAR STUDIES OF CANCER SPECIFIC FRAGILE SITES

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的三种类型的脆性位点：FRA 3B，阿非迪霉素诱导的脆性位点，FRA 11B，叶酸敏感位点，和FRA 16 B，偏端霉素A诱导的位点，参与癌症的形成。 在FRA 11 B位点沿着与其他四个叶酸敏感位点一起发现的CCG重复序列已显示出抵抗组装成核小体，并且CpG甲基化的程度进一步按比例排除核小体组装。这些结果为脆性位点的性质提供了一个有趣的模型，其中在这些位点的核小体排斥将产生不寻常的染色质结构，以允许染色体重排和病毒整合，导致脆性位点特异性肿瘤发生。本论文的主要目标是：（1）在体外研究脆性位点DNA序列的染色质结构。 我们发现FRA 11B位点的CCG重复DNA不包括核小体。 我们将对另外两组不同的脆性位点FRA 3B和FRA 16 B采用相同的技术。此外，在三个脆性位点的大DNA上形成的核小体阵列将通过核酸酶探测和电子显微镜来表征，以确定在这些位点上形成的核小体的稳定性和物理结构。(2)将检查这三个脆性位点的体内染色质结构。 核小体位置将在来自表达脆性位点的个体或EBV游离型系统的细胞系中的脆性位点DNA上作图。 还将检查脆性位点诱导化学物质对核小体模式的影响。(3)脆性位点在高阶染色质结构域的位置将进行探讨。 将使用各种细胞系绘制每个脆性位点及其相邻区域相对于MAR/SAR的位置。 这种关联将进一步研究其细胞周期的依赖性，相互作用的性质，以及参与拓扑异构酶II裂解位点。这些实验将有助于了解这些脆弱位点的性质及其在癌症形成中的作用，并解决一些基本的生物学问题，如染色质结构的决定因素。

Instability of chromosomal fragile sites is related directly to many cancers. Three types of fragile sites generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA 16B, a distamycin-A-inducible site, are involved in the formation of cancers. The CCG repeating sequence found in the FRA11B site along with four other folate-sensitive sites, has been shown to resist assembly into nucleosomes and the degree of CpG methylation further excludes nucleosome assembly proportionally. These results provide an intriguing model for the nature of fragile sites in which nucleosome exclusion at these sites would create unusual chromatin structure to allow chromosomal rearrangement and viral integration, resulting in fragile site- specific tumorigenesis. Three goals are proposed : (1) The chromatin structure of DNA sequences derived from fragile sites will be investigated in vitro. We showed that the CCG repeating DNA of the FRA11B site excludes nucleosome. We will employ the same techniques for two other different groups of fragile sites, the FRA3B and FRA16B. Further, nucleosome arrays formed over large DNAs of three fragile sites will be characterized by nuclease probing and electron microscopy to determine the stability and physical structure of nucleosomes formed over these sites. (2) The in vivo chromatin structure of these three fragile sites will be examined. Nucleosome position will be mapped over fragile site DNAs in cell lines from fragile site-expressing individuals or an EBV episomal system. The effect of the fragile site-inducing chemicals on the nucleosome pattern will also be examined. (3) The location of fragile sites in higher-order chromatin domains will be explored. Various cell lines will be used to map the location of each fragile site and its adjacent region relative to MARs/SARs. This association will be further investigated for its cell-cycle dependency, the nature of the interaction, and the involvement of topoisomerase II cleavage sites. These experiments will contribute information about the nature of these fragile sites and their role in the formation of cancer, and also address some fundamental biological questions, such as determinants of chromatin structure.