ANTINEOPLASTIC v lEUKEMOGENIC EPIPODOPHYLLOTOXIN EFFECTS

描述（由申请人提供）：这项工作的目的是了解DNA拓扑异构酶II抑制剂抗癌治疗后白血病中导致MLL易位的DNA损伤的性质。CYP3A4启动子是多态性的，CYP3A4基因型赋予易感性。CYP3A4将依托泊苷转化为依托泊苷儿茶酚；儿茶酚很容易被氧化成醌。这些代谢物是基因毒素。MLL与许多伙伴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表明精确或接近精确的染色体间DNA重组，但是克隆额外的断点对于辨别损伤谱是必不可少的。依托泊苷及其代谢物在体外诱导MLL及其伴侣基因易位断点处的DNA拓扑异构酶II裂解。我们提出依托泊苷及其代谢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解决的异质性。待检测的DNA损伤包括依托泊苷母体药物直接诱导DNA拓扑异构酶II裂解，依托泊苷醌或活性氧诱导DNA拓扑异构酶II裂解形成DNA加合物，复制叉与DNA拓扑异构酶II共价复合物碰撞以及DNA拓扑异构酶II非依赖性损伤。目的1将使用质谱法检查光谱并量化来自MLL bcr DNA底物中依托泊苷代谢物的DNA加合物的相对重要性。目的2将利用DNA阵列研究依托泊苷和依托泊苷代谢物在人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共价复合物的作用。为了回答在断点连接的形成过程中是否发生了精确的重组、外核溶解啃咬、大缺失、插入、倒位、重复和非同源末端连接，以及发生的频率和程度，Aim 3将描述白血病患者中两个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靶向预防这种通常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.