ANTINEOPLASTIC v LEUKEMOGENIC EPIPODOPHYLLOTOXIN EFFECTS

描述(由申请人提供)：这项工作的目的是了解在用DNA拓扑异构酶II抑制剂进行抗癌治疗后，导致白血病MLL易位的DNA损伤的性质。CYP3A4基因启动子是多态的，且CYP3A4基因是易感基因。CYP3A4将依托泊苷转化为依托泊苷邻苯二酚；邻苯二酚很容易被氧化成苯二酚。这些代谢物是遗传毒素。MLL与许多伴侣基因中的一个结合形成易位。基因组断点连接序列包含DNA损伤和修复的证据。一些基因组断点连接序列显示了精确或接近精确的染色体间DNA重组，但额外断点的克隆对于识别损伤谱是必不可少的。依托泊苷及其代谢产物在体外诱导MLL及其配对基因的DNA拓扑异构酶II在易位断裂点发生裂解。我们认为依托泊苷及其代谢产物可以刺激一系列不同的DNA损伤，这些损伤被修复形成断点连接，基因组断点连接序列的异质性反映了损伤及其分辨率的异质性。待检测的DNA损伤包括依托泊苷母药直接诱导DNA拓扑异构酶II的裂解，依托泊苷醌或活性氧物种诱导DNA加合物形成DNA拓扑异构酶II的裂解，复制叉与DNA拓扑异构酶II共价复合体的碰撞，以及DNA拓扑异构酶II非依赖性损伤。目的1将使用质谱学方法检测MLL BCR DNA底物中依托泊苷代谢产物的DNA加合物的光谱并量化其相对重要性。目的2利用DNA阵列技术研究依托泊苷和依托泊苷代谢产物在人CD34+造血祖细胞中诱导MLL和基因组中功能性DNA拓扑异构酶II共价复合体的作用。为了回答在断点连接的产生过程中是否发生了精确的重组、核外溶解、大的缺失、插入、倒置、重复和非同源末端连接，以及发生的频率和程度，Aim 3将描述患者白血病中两条衍生染色体的基因组序列。解决DNA拓扑异构酶II抑制剂的白血病发生机制与有针对性地预防这种通常是致命的抗癌治疗并发症高度相关。

DESCRIPTION (provided by applicant): The objective of this work is to understand the nature of the DNA damage leading to MLL translocations in leukemias following anticancer treatment with DNA topoisomerase II inhibitors. The CYP3A4 promoter is polymorphic and CYP3A4 genotype confers susceptibility. CYP3A4 converts etoposide to etoposide catechol; the catechol is readily oxidized to a quinone. These metabolites are genotoxins. MLL joins with one of many partner genes to form the translocations. The genomic breakpoint junction sequences contain evidence of DNA damage and repair. Several genomic breakpoint junction sequences indicate precise or near-precise interchromosomal DNA recombinations, but the cloning of additional breakpoints is essential to discern the damage spectrum. Etoposide and its metabolites induce DNA topoisomerase II cleavage at the translocation breakpoints in MLL and in its partner genes in vitro. We propose that etoposide and its metabolites can stimulate a series of different DNA lesions, which are repaired to form the breakpoint junctions, and that the heterogeneity in genomic breakpoint junction sequences reflects heterogeneity in the damage and its resolution. The DNA lesions to be tested include the direct induction of DNA topoisomerase II cleavage by etoposide parent drug, induction of DNA topoisomerase II cleavage from DNA adduct formation by etoposide quinone or reactive oxygen species, replication fork collisions with DNA topoisomerase II covalent complexes and DNA topoisomerase II-independent damage. Aim 1 will examine the spectrum and quantify the relative importance of DNA adducts from etoposide metabolites in an MLL bcr DNA substrate using mass spectrometry. Aim 2 will investigate the induction of functional DNA topoisomerase II covalent complexes in MLL and in the genome by etoposide and etoposide metabolites in human CD34+ hematopoietic progenitor cells using DNA arrays. To answer whether, how often and to what degree precise recombinations, exonucleolytic nibbling, large deletions, insertions, inversions, duplications and nonhomologous end-joining have occurred in creation of the breakpoint junctions, Aim 3 will characterize the genomic sequences of both derivative chromosomes in the leukemias in patients. Solving the mechanism of leukemogenesis of the DNA topoisomerase II inhibitors is highly relevant to the targeted prevention of this usually fatal complication of anticancer treatment.