DNA STRAND BREAKS AND THE GENETIC BASIS OF LYMPHOMAS

哺乳动物细胞对DNA双链断裂的反应是通过激活 细胞周期停滞和DNA修复的途径。用于小区的信号 周期停滞需要ATM和P53基因，这两个基因在AT中突变 共济失调、毛细血管扩张和Li-Fraumeni综合征。DSB修复需要 四个基因：XRCC4、XRCC5(Ku86)、XRCC6(Ku70)和XRCC7(DNA-PKcs， DNA依赖蛋白激酶的催化亚单位)，后者 在SCID小鼠中发生突变。共济失调毛细血管扩张症与Li-Fraumeni 患者和SCID小鼠对淋巴瘤高度易感。因此， 一小部分淋巴瘤必须是由生殖系突变引起的 DSB反应基因。这项提议将检验这样一个假设： 很大一部分来自这些基因的体细胞突变。 具体目标是：1.1.检测淋巴瘤肿瘤的生化指标 对DSB反应的通路异常。淋巴瘤将是 筛查已知DSB反应基因中的生化异常。 这些检测快速且对这些基因和其他基因的突变敏感。 在DSB反应途径中。化验将测试dna末端结合。 DNA-PK在DNA末端的组装及其酶促Ku的活性 活动。免疫印迹将评估Ku、DNA-PKcs、P53和ATM 蛋白质，经常因突变而改变其大小的稳定性。 1.2.检测淋巴瘤肿瘤的通路中的遗传异常 响应DSB。淋巴瘤肿瘤将接受基因突变检测 DSB反应基因。寻找突变将由新的 技术，包括变性高性能液体 并且能够检测大于 98%的灵敏度比传统方法快得多。 1.3.DSB反应通路中的异常与 临床和其他实验室发现。令人惊讶的是，大多数弥漫性淋巴瘤 利用VH4.21免疫球蛋白基因。由于自动柜员机突变会导致 异常的V(D)J重组，这一提议将测试他们是否也导致了 对VH4.21的使用存在偏见。由于许多淋巴瘤没有p53突变， 这项提议将测试剩余的淋巴瘤是否有突变 在ATM中，ATM在相同的信号通路中起作用。自DSB响应以来 基因赋予关键抗癌药物抗药性，这项提议将 测试这些基因的突变是否会影响临床结果。 长期目标是定义与基因变化有关的 淋巴瘤的恶性进展。希望通过分子分析， 个别淋巴瘤总有一天会治愈更多的患者。

Mammalian cells respond to DNA double-strand breaks (DSBs) by activating pathways for cell cycle arrest and DNA repair. The signal for cell cycle arrest requires the ATM and p53 genes, which are mutated in at ataxia telangiectasia and Li-Fraumeni syndrome. DSB repair requires four genes: XRCC4, XRCC5 (Ku86), XRCC6 (Ku70), and XRCC7 (DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase), the latter of which is mutated in the scid mouse. Ataxia telangiectasia and Li-Fraumeni patients and the scid mouse are highly susceptible to lymphoma. Thus, a small fraction of lymphomas must arise from germ line mutations in one of the DSB response genes. This proposal will test the hypothesis that a significant fraction arises from somatic mutations in these genes. The specific aims are to: 1.1. Test lymphoma tumors for biochemical abnormalities in pathways responding to DSBs. Lymphomas will be screened for biochemical abnormalities in the known DSB response genes. The assays are rapid and sensitive to mutations in these and other genes in the DSB response pathway. The assays will test DNA end-binding activity for Ku, assembly of DNA-PK on DNA ends and its enzymatic activity. Immunoblots will evaluate the Ku, DNA-PKcs, p53, and ATM proteins, which are often altered in stability of size by mutations. 1.2. Test lymphoma tumors for genetic abnormalities in pathways responding to DSBs. Lymphoma tumors will be tested for mutations in the DSB response genes. The hunt for mutations will be facilitated by new technology, which consists of denaturing high performance liquid chromatography and is capable of detecting mutations with greater than 98 percent sensitivity much more rapidly than conventional methods. 1.3. Correlate abnormalities in pathways responding to DSBs with clinical and other lab findings. Surprisingly, most diffuse lymphomas utilize the VH4.21 immunoglobulin gene. Since ATM mutations lead to aberrant V(D)J recombination, this proposal will test if they also lead to biased VH4.21 usage. Since many lymphomas do not have p53 mutations, this proposal will test whether the remaining lymphomas have mutations in ATM, which acts in the same signaling pathway. Since DSB response genes confer resistance to key anticancer agents, this proposal will test whether mutations in these genes affect clinical outcome. The long term goal is to define the genetic changes that mediate malignant progression of lymphomas. Hopefully, molecular analysis of individual lymphomas will some day lead to the cure of more patients.