拓扑异构酶IIα和拓扑异构酶IIβ药物稳定的可裂解复合物的寿命差异及其与药物敏感性的关系。

Differences in the longevity of topo IIalpha and topo IIbeta drug-stabilized cleavable complexes and the relationship to drug sensitivity.

作者信息

Errington Fiona, Willmore Elaine, Leontiou Chrysoula, Tilby Michael J, Austin Caroline A

机构信息

School of Cell and Molecular BioSciences, The Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE2 4HH, UK.

出版信息

Cancer Chemother Pharmacol. 2004 Feb;53(2):155-62. doi: 10.1007/s00280-003-0701-1. Epub 2003 Sep 23.

DOI:10.1007/s00280-003-0701-1

PMID:14504921

Abstract

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.

摘要

目的

DNA拓扑异构酶II（拓扑异构酶II）是化疗药物的重要细胞靶点。人类细胞有两种拓扑异构酶II同工型（α和β），二者均受化疗药物依托泊苷、安吖啶（mAMSA）和米托蒽醌抑制。已知拓扑异构酶IIα或拓扑异构酶IIβ药物诱导复合物的细胞毒性重要性因存在的药物不同而有所差异。本研究旨在（a）评估拓扑异构酶IIα和β可裂解复合物的同工型特异性形成及逆转情况，以及（b）确定任一同工型的细胞毒性重要性是否与复合物寿命的差异有关。

方法

使用小鼠胚胎成纤维细胞（MEF）研究细胞对拓扑异构酶II毒药依托泊苷、米托蒽醌和mAMSA的反应。使用TARDIS检测法确定拓扑异构酶IIα和β复合物的寿命。这种免疫荧光检测法能够区分拓扑异构酶II同工型，从而使我们首次能够研究拓扑异构酶IIα和β复合物的持久性及重要性。

结果

在用依托泊苷处理的MEF中，50%的拓扑异构酶IIα复合物在40分钟内解离，而拓扑异构酶IIβ复合物的解离仅需20分钟。对于米托蒽醌处理的细胞，复合物的消失过程较为缓慢。将拓扑异构酶IIα和拓扑异构酶IIβ可裂解复合物减少50%所需的时间分别为10小时和6小时。相比之下，mAMSA稳定的拓扑异构酶IIα和拓扑异构酶IIβ可裂解复合物同样稳定（两种同工型均在15分钟内解离）。这些稳定性数据通过体外检测得到了证实。