Etiology of translocations in hematopoietic cells

描述(申请人提供)：这项研究的长期目标是了解特定于造血的发育计划对一种类型的DNA损伤修复的影响-双链断裂(DSB)-以及导致易位的初始分子事件，这是白血病、淋巴瘤和软组织肉瘤的标志。染色体双链断裂是在正常代谢过程中形成的，包括发育中的淋巴细胞的DNA复制和抗原受体重排，以及暴露于DNA损伤剂之后。某些化疗药物与继发性白血病之间存在关联，例如拓扑异构酶II抑制剂的治疗和涉及1lq23的重排，断裂点的序列分析表明DNA损伤参与了易位过程。然而，在最初的重排形成中，特定的易位发生在发育中的造血亚群中的机制仍然不清楚。这些问题可以通过在小鼠ES细胞中开发的基于稀有切割酵母内切酶I-SCE I的遗传系统来解决，以在定义的基因组位点引入DSB并在分子水平上分析重组修复事件。该系统还对可能导致的易位、重复和缺失进行评分。这项建议将使I-SCE I系统适用于检测DSB修复和重组在造血早期祖细胞和髓系细胞谱系中，以及这种类型的损伤促进非法重组的可能性。这些方法将：(1)确定双链断裂修复和染色体间重组的潜力，以促进特定谱系内的基因组重组；(2)确定MLL和AF-4基因断点簇区内双链断裂修复导致t(4；11)易位的潜力；以及(3)使用基因组学微阵列的方法，表征造血发育阶段或损伤剂对细胞DNA损伤反应的特异性的影响。这些研究将为DSB在造血细胞亚群中重新加入的生物学提供重要的见解，以及负责正常抑制基因组重排并最终导致肿瘤发生的因素。解开易位的病因和后果可能导致新的治疗和预防方法。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the influence of hematopoietic-specific developmental programs on the repair of one type of DNA damage--a double strand break (DSB)--and the initial molecular events that lead to translocations, which are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas. Chromosomal DSBs are formed during normal metabolic processes including DNA replication and antigen receptor rearrangement in developing lymphoid cells, as well as following exposure to DNA damaging agents. There is an association between certain chemotherapeutic agents and secondary leukemia, e.g. treatment with topoisomerase II inhibitors and rearrangements involving 1lq23, and sequence analysis of breakpoints suggests that DNA damage is involved in the translocation process. However, the mechanisms by which specific translocations occur within developing hematopoietic subpopulations in the initial formation of rearrangements remain unclear. These questions can be addressed by adaptation of a genetic system developed in murine ES cells based on the rare-cutting yeast endonuclease I-Sce I to introduce DSBs at defined genomic loci and analyze recombinant repair events at the molecular level. This system also scores for translocations, duplications, and deletions that may result. This proposal will adapt the I-Sce I system to examine DSB repair and recombination in hematopoietic early progenitor and myeloid cell lineages and the potential for this type of damage to promote illegitimate recombination. Approaches will: (1) determine the potential for repair of DSBs and interchromosomal recombination to promote genome rearrangements within specific lineages; (2) determine the potential for repair of DSBs within the breakpoint cluster regions of the MLL and AF-4 genes to result in t(4;11) translocations similar to those observed in the clinical setting; and (3) use a genomics microarray-based approach to characterize the influence of the stage of hematopoietic development or the damaging agent on the specificity of a cell's DNA damage response. These studies will provide important insight into the biology of DSB rejoining in hematopoietic cell subpopulations, and the factors responsible for the normal suppression of genome rearrangements and, ultimately, tumorigenesis. Unraveling the etiology and consequences of translocations may lead to new approaches to therapy and prevention.