Protein-Associated DNA Breaks as Indicator of Topoisomerase Inhibition

DNA拓扑异构酶I和II （top1和top2）是癌症化疗的主要靶点，可以通过生理性和致癌性DNA修饰来抑制。拓扑异构酶毒素通过稳定酶连接的DNA断裂起作用，这种断裂可以在药物处理的细胞中检测到蛋白质相关的DNA断裂（切割复合物）。我们之前已经报道了各种DNA修饰，包括错配、碱性位点、氧化碱基损伤（8-氧鸟嘌呤）和致癌加合物（乙烯腺嘌呤和苯并[a]芘鸟嘌呤加合物），并首次证明了在暴露于苯并[a]芘二醇环氧化合物的致癌异构体的细胞中诱导top1-DNA加合物。这一观察结果表明，top1可能参与致癌暴露后观察到的突变。它还证明了小槽烷基化如何捕获top1，这与药物开发相关，因为我们发现小槽鸟嘌呤N2烷基化物ecteinascidin 743是top1毒药。我们还发现，在核苷类似物（阿糖胞嘧啶、吉西他滨）处理后，可以观察到top1切割复合物，这表明top1介导的DNA损伤有助于阿糖胞嘧啶和吉西他滨的抗增殖活性。我们建立了一种连接介导的PCR （LM-PCR）方法，在核苷酸水平上分析人结肠癌HT29细胞中由top1切割复合物诱导的复制介导的DNA双链断裂。我们发现，top1切割复合体转化为复制介导的DNA双链断裂仅在DNA合成的前导链上可检测到，这表明top1切割复合体在复制叉的两条臂上代谢的方式不对称。连接到LM-PCR引物不需要Taq DNA聚合酶的延伸，这表明DNA聚合酶在体内将3‘ DNA末端延伸到拓扑异构酶I切割复合物的5’末端。这些发现表明，复制介导的DNA双链断裂是由复制径流产生的。我们还发现，这些DNA双链断裂的5‘末端在体内被磷酸化，这表明DNA 5’激酶活性作用于复制径流产生的双链末端。在top1切割复合体停止后，复制介导的DNA双链断裂是快速可逆的，这表明存在有效的修复途径来去除核糖体DNA中top1-DNA共价加合物。我们对ecteinascidin 743进行了研究，目前处于II期临床试验，对肉瘤有反应。Ecteinascidin 743 （Et743, NSC 648766）不同于临床使用的其他抗癌药物，因为它在DNA小凹槽的特定鸟嘌呤上形成共价加合物。为了进一步阐明Et743的作用机制，我们培育了Et743耐药细胞株（HCT116/ER5）。HCT116/ER5细胞表现出增强的微卫星不稳定性和非整倍性。在HCT116/ER5细胞中发现，由于t（13; 14）易位不平衡导致染色体13q DNA拷贝数丢失，以及由于XPG在密码子240位发生移码突变导致停止密码子位于243位，导致13q33.3位点杂合性丢失，导致DNA修复基因XPG完全缺乏表达。转染XP-G cDNA恢复了HCT116/ER5细胞对Et743的敏感性，这表明参与核苷酸切除修复途径的蛋白质，如内切酶XPG，对Et743的抗增殖活性至关重要。Et743定义了一类新的抗癌药物，其中增强的抗增殖活性与增强的细胞dna修复能力并行。

DNA topoisomerase I and II (top1 and top2) are primary targets for cancer chemotherapy and can be inhibited by physiological and carcinogenic DNA modifications. Topoisomerase poisons act by stabilizing enzyme-linked DNA breaks which can be detected as protein-associated DNA breaks in drug-treated cells (cleavage complexes).We had previously reported that various DNA modifications including mismatches, abasic sites, oxidative base damage (8-oxoguanine) and carcinogenic adducts (ethenoadenine and benzo[a]pyrene guanine adducts), and demonstrated for the first time the induction of top1-DNA adducts in cells exposed to the carcinogenic isomer of benzo[a]pyrene diol epoxide. This observation suggests the possible involvement of top1 in the mutations observed after carcinogenic exposure. It also demonstrates how minor groove alkylation can trap top1, which is relevant for drug development as we found that the minor groove guanine N2 alkylator ecteinascidin 743 is a top1 poison.We have also found that top1 cleavage complexes can be observed after treatment with nucleoside analogs (cytosine arabinoside, gemcitabine), which suggests that top1-mediated DNA damage contributes to the antiproliferative activity of cytosine arabinoside and gemcitabine.We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by top1 cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of top1 cleavage complexes into replication- mediated DNA double-strand breaks is only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that top1 cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at top1 cleavage sites are produced by replication run-off. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication run-off. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the top1 cleavage complexes, suggesting the existence of efficient repair pathways for removal of top1-DNA covalent adducts in ribosomal DNA.We have pursued our studies with ecteinascidin 743, which is, at the present time in Phase II clinical trials with responses in sarcomas. Ecteinascidin 743 (Et743, NSC 648766) differs from other anticancer drugs in clinical use because it forms covalent adducts at specific guanines in the minor groove of DNA. To further elucidate the mechanism of action of Et743, we have generated an Et743-resistant cell line (HCT116/ER5). The HCT116/ER5 cells exhibit enhanced microsatellite instability and aneuploidy. Complete lack of expression of the DNA repair gene XPG was found in HCT116/ER5 cells as a result of chromosome 13q DNA copy-number loss, due to an unbalanced t(13; 14) translocation, and loss of heterozygosity at 13q33.3, due to a frameshift mutation of XPG at codon 240 resulting in a stop codon at position 243. Transfection of XP-G cDNA restored the sensitivity of the HCT116/ER5 cells to Et743 demonstrating that proteins involved in the nucleotide excision repair pathway such as the endonuclease XPG are essential for the antiproliferative activity Et743. Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability.