Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio.
Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio
Mol Pharmacol. 2018 May;93(5):515-525. doi: 10.1124/mol.117.111567. Epub 2018 Mar 7.
DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2/90 expression decreases drug-induced TOP2-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2/170. Alternative processing of TOP2 pre-mRNA, and subsequent synthesis of TOP2/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2/170-DNA covalent complexes in response to TOP2-targeting agents.
DNA 拓扑异构酶 II（170 kDa，TOP2/170）通过在染色体浓缩、复制和分离过程中解决 DNA 拓扑缠结，对于增殖细胞是必不可少的。我们之前已经对一种 C 端截断的异构体（TOP2/90）进行了特征描述，该异构体可在人白血病 K562 细胞中检测到，但在具有获得性抗癌症药物依托泊苷耐药性的克隆亚系 K/VP.5 中表达更为丰富。TOP2/90（786 个氨基酸）是保留加工过的内含子 19 的 TOP2 mRNA 的翻译产物。TOP2/90 缺乏产生双链 DNA 断裂所需的活性位点酪氨酸-805，以及 TOP2/170 异构体中存在的核定位信号（1531 个氨基酸）。在这里，我们发现 TOP2/90 与 TOP2/170 一样，可在 K562 和 K/VP.5 细胞的核和细胞质中检测到。内源性 TOP2/90 和 TOP2/170 的共免疫沉淀表明这些异构体的异二聚化。在 K562 细胞中强制表达 TOP2/90 可抑制，而在 K/VP.5 细胞中 siRNA 介导的 TOP2/90 敲低可增强依托泊苷介导的 DNA 链断裂，与分别用空载体或对照 siRNA 转染的类似处理细胞相比。此外，在 K562 细胞中强制表达 TOP2/90 可通过集落形成测定抑制依托泊苷细胞毒性。qPCR 和免疫测定法证明了正常人类组织/细胞和患者白血病细胞中的 TOP2/90 mRNA 和蛋白表达。总之，结果强烈表明，TOP2/90 的表达降低了药物诱导的 TOP2-DNA 共价复合物的形成，并且通过与 TOP2/170 的异二聚化相关的显性负效应，成为化学抗性的决定因素。TOP2 前 mRNA 的替代加工，以及随后的 TOP2/90 合成，可能是一种重要的机制，可调节拓扑异构酶 2 靶向药物作用下细胞毒性 TOP2/170-DNA 共价复合物的形成和/或稳定性。
