Molecular Pharmacology Unit, Dept. Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
Biochem Pharmacol. 2012 Jan 1;83(1):27-36. doi: 10.1016/j.bcp.2011.09.021. Epub 2011 Sep 29.
Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a unique function as it catalyzes the repair of topoisomerase I-mediated DNA damage. Thus, ovarian carcinoma cell lines exhibiting increased TDP1 levels and resistance to the topoisomerase I poisons campthotecins were used to clarify the role of this enzyme. The camptothecin gimatecan was employed as a tool to inhibit topoisomerase I because it produces a persistent damage. The resistant sublines displayed an increased capability to repair drug-induced single-strand breaks and a reduced amount of drug-induced double-strand breaks, which was enhanced following TDP1 silencing. In loss of function studies using U2-OS cells, we found that TDP1 knockdown did not produce a change in sensitivity to camptothecin, whereas co-silencing of other pathways cooperating with TDP1 in cell response to topoisomerase I poisons indicated that XRCC1 and BRCA1 were major regulators of sensitivity. No change in cellular sensitivity was observed when TDP1 was silenced concomitantly to RAD17, which participates in the stabilization of collapsed replication forks. The expression of dominant-negative PARP1 in cells with reduced expression of TDP1 due to a constitutively expressed TDP1 targeting microRNA did not modulate cell sensitivity to camptothecin. Mild resistance to gimatecan was observed in cells over-expressing TDP1, a feature associated with decreased levels of drug-induced single-strand breaks. In conclusion, since TDP1 alone can account for mild levels of camptothecin resistance, repair of topoisomerase I-mediated DNA damage likely occurs through redundant pathways mainly implicating BRCA1 and XRCC1, but not RAD17 and PARP1. These findings may be relevant to define novel therapeutic strategies.
酪氨酰 DNA 磷酸二酯酶 1（TDP1）具有独特的功能，因为它催化拓扑异构酶 I 介导的 DNA 损伤的修复。因此，使用卵巢癌细胞系来阐明这种酶的作用，这些细胞系表现出增加的 TDP1 水平和对拓扑异构酶 I 毒物喜树碱的抗性。喜树碱衍生物 gimatecan 被用作抑制拓扑异构酶 I 的工具，因为它会产生持续的损伤。耐药亚系显示出增加修复药物诱导的单链断裂的能力，并且药物诱导的双链断裂减少，这在 TDP1 沉默后得到增强。在使用 U2-OS 细胞进行的功能丧失研究中，我们发现 TDP1 敲低不会导致对喜树碱的敏感性改变，而 TDP1 在细胞对拓扑异构酶 I 毒物的反应中与其他途径的共沉默表明 XRCC1 和 BRCA1 是敏感性的主要调节剂。当 TDP1 与参与复制叉崩溃稳定的 RAD17 同时沉默时，细胞敏感性没有变化。由于持续表达的 TDP1 靶向 microRNA 导致 TDP1 表达减少的细胞中表达显性负 PARP1 不会调节细胞对喜树碱的敏感性。在 TDP1 过表达的细胞中观察到对 gimatecan 的轻度抗性，这与药物诱导的单链断裂水平降低有关。总之，由于 TDP1 本身可以解释轻度喜树碱抗性，因此拓扑异构酶 I 介导的 DNA 损伤的修复可能通过主要涉及 BRCA1 和 XRCC1 的冗余途径发生，而不是 RAD17 和 PARP1。这些发现可能与定义新的治疗策略有关。
