Molecular Mechanisms in Neoplasia

描述（由申请人提供）：Myc是一种重要的致癌基因，在模型系统中被广泛研究，与许多人类癌症有关。我们已经发现了鸡法氏囊中强大的遗传不稳定表型的证据，包括DNA回文形成和基因拷贝数变化，与构成性Myc癌基因过表达和由此产生的淋巴瘤发生有关。我们建议更完整地描述这一现象。为了实现这一总体目标，我们开发了几种基因组分析工具，包括用于基于阵列的比较基因组杂交（Array CGH）的鸡cDNA微阵列，以及用于分析淋巴瘤衍生细胞系和体内肿瘤法氏囊发育早期和晚期组织的回文形成全基因组分析（GAPF）。特定的计划实验采用DT40，这是一种表达myc-over的法氏囊淋巴瘤衍生细胞系，在不同的克隆分离株中表现出广泛的DNA拷贝数变化和回文形成。我们将：(a)确定这些变化发生的时间尺度，(b)尝试测试低c- Myc表达水平对稳定性的影响，以及(c)测试删除几个DNA修复基因的影响，包括那些被证明对DT40重组很重要的基因：已知Myc在转录调控区和其他未知功能区域的许多位点上结合DNA。我们将绘制DT40基因组中Myc结合位点，并确定Myc结合位点与回文形成位点之间是否存在关系。我们观察到法氏囊淋巴瘤的许多回文形成位点和DT40都以RNA的形式表达。我们将采用基于阵列的技术来确定肿瘤发生过程中形成的DNA回文表达的RNA是否具有双链特征。这些dsRNAs是形成功能性小干扰RNA的潜在底物，在肿瘤发展过程中可能在基因沉默中发挥作用。最后，我们将确定myc诱导的不稳定性是否发生在哺乳动物系统中。将评估Emu-c-myc转基因小鼠发生淋巴瘤时的回文形成情况。在Myc可以通过实验调节的人P493-6 b细胞中，我们将确定Myc的诱导是否会导致Myc结合位点的回文形成。通过这种方式，我们希望确定myc诱导的基因组不稳定性是否值得在人类肿瘤中进行更广泛的探索。

DESCRIPTION (provided by applicant): Myc is an important oncogene widely studied in model systems and implicated in many human cancers. We have uncovered evidence for a robust genetic instability phenotype, involving DNA palindrome formation and gene copy number change, associated with constitutive Myc oncogene over expression, and resultant lymphomagenesis, in the chicken bursa of Fabricius. We propose to characterize this phenomenon more completely. To accomplish this general goal we have developed several tools for genomic analysis including a chicken cDNA microarray effective for array-based comparative genome hybridization (Array CGH) and genome-wide analysis of palindrome formation (GAPF) for analysis of lymphoma derived cell lines, and of tissues representing early and late stages of neoplastic bursal development in vivo. Specific planned experiments employ DT40, a myc-over expressing bursal lymphoma-derived cell line that demonstrates extensive DNA copy number change and palindrome formation that differs in different clonal isolates. We will:(a) determine the time scale over which these changes occur,(b) attempt to test effects of lower c-myc expression levels on stability, and (c) test the effects of deletion of several DNA repair genes including those demonstrated to be important for recombination in DT40: Myc is known to bind DNA at many loci, both in transcriptional regulatory regions and other regions of unknown function. We will map sites of Myc binding in the DT40 genome and determine if there is a relationship between Myc binding sites and sites of palindrome formation. We have observed that many sites of palindrome formation in bursal lymphomas and DT40 are expressed as RNA. We will adapt our array-based technology to determine whether RNA expressed from DNA palindromes formed during tumorigenesis has double-stranded character. Such dsRNAs are potential substrates for the formation of functional small interfering RNA which could play a role in gene silencing during tumor development. Finally, we will determine if Myc-ihduced instability occurs in mammalian systems. Palindrome formation will be assessed in Emu-c-myc transgenic mice which develop lympomas. In human P493-6 B-cells, in which Myc can be experimentally regulated, we will determine if induction of Myc results in palindrome formation at sites of Myc binding. In this fashion we hope to establish whether Myc-induced genomic instability merits wider exploration in human neoplasm.