Differences in the longevity of topo IIα and topo IIβ drug-stabilized cleavable complexes and the relationship to drug sensitivity
Differences in the longevity of topo IIα and topo IIβ drug-stabilized cleavable complexes and the relationship to drug sensitivity
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DOI：
10.1007/s00280-003-0701-1
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发表时间：
2004-02-01
影响因子：
3
通讯作者：
Austin, CA
中科院分区：
文献类型：
--
作者：
Errington, F;Willmore, E;Austin, CA
目的. DNA拓扑异构酶II（topo II）是化疗药物的重要细胞靶点。人类细胞具有两种拓扑异构体II（α和β），并且两者都被化疗剂依托泊苷、安吖啶（mAMSA）和米托蒽醌抑制。已知topo II alpha或topo II beta药物诱导的复合物的细胞毒性重要性取决于存在哪种药物。本研究旨在（a）评估拓扑异构酶II α和β可裂解复合物的异构体特异性形成和逆转，以及（B）确定任一异构体的细胞毒性重要性是否与复合物寿命的差异相关。方法.使用小鼠胚胎成纤维细胞（MEFs）来研究对topo II毒物依托泊苷、米托蒽醌和mAMSA的细胞应答。使用TARDIS测定确定拓扑异构酶II α和β复合物的寿命。这种免疫荧光检测可以区分拓扑异构体II，从而使我们能够调查的持久性和重要性的拓扑异构体II α和β复合物的第一次。结果在用足叶乙甙处理的MEFs中，50%的拓扑II α复合物在40分钟内解离，而拓扑II β复合物的解离仅需20分钟。复合物的消失是一个较慢的过程米托蒽醌处理的细胞。将拓扑异构酶II α和拓扑异构酶II β可裂解复合物减少50%所需的时间分别为10和6小时。相比之下，mAMSA稳定的topo II alpha和topo II beta可切割复合物同样稳定（两种亚型在15分钟内解离）。使用体外试验证实了这些稳定性数据。结论.我们以前证明，拓扑II α是依托泊苷和米托蒽醌的主要目标，但拓扑II α和拓扑II β是重要的mAMSA的细胞毒性。因此，此处显示的拓扑异构酶II α和β可裂解复合物的寿命是确定任一亚型对这些药物的细胞毒性敏感性的重要因素。
Purpose. DNA topoisomerase II (topo II) is an important cellular target for chemotherapeutic agents. Human cells have two isoforms of topo II (alpha and beta), and both are inhibited by the chemotherapeutic agents etoposide, amsacrine (mAMSA) and mitoxantrone. It is known that the cytotoxic importance of topo IIalpha or topo IIbeta drug-induced complexes differs depending on which drug is present. This study was designed to (a) assess isoform-specific formation and reversal of topo IIalpha and beta cleavable complexes, and (b) determine whether the cytotoxic importance of either isoform was related to differences in the longevity of the complexes. Methods. Mouse embryonic fibroblasts (MEFs) were used to study the cellular response to the topo II poisons etoposide, mitoxantrone and mAMSA. The longevity of topo IIalpha and beta complexes was determined using the TARDIS assay. This immunofluorescence assay can differentiate between the topo II isoforms and thus allowed us to investigate the persistence and importance of topo IIalpha and beta complexes for the first time. Results. In MEFs treated with etoposide, 50% of topo IIalpha complexes dissociated within 40 min whereas dissociation of topo IIbeta complexes took only 20 min. Disappearance of complexes was a slower process for mitoxantrone-treated cells. The time taken to reduce topo IIalpha and topo IIbeta cleavable complexes by 50% was 10 and 6 h, respectively. In contrast, mAMSA-stabilized topo IIalpha and topo IIbeta cleavable complexes were equally stable (dissociation within 15 min for both isoforms). These stability data were confirmed using an in vitro assay. Conclusions. We previously demonstrated that topo IIalpha is the major target for etoposide and mitoxantrone but that both topo IIalpha and topo IIbeta are important for mAMSA cytotoxicity. The longevity of the topo IIalpha and beta cleavable complexes shown here is therefore an important factor in determining the cytotoxic sensitivity of either isoform to these drugs.