Differences in the longevity of topo IIα and topo IIβ drug-stabilized cleavable complexes and the relationship to drug sensitivity
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DNA topoisomerase II (topo II) is an important cellular target for chemotherapeutic agents. Human cells have two isoforms of topo II (α and β), and both are inhibited by the chemotherapeutic agents etoposide, amsacrine (mAMSA) and mitoxantrone. It is known that the cytotoxic importance of topo IIα or topo IIβ drug-induced complexes differs depending on which drug is present. This study was designed to (a) assess isoform-specific formation and reversal of topo IIα and β cleavable complexes, and (b) determine whether the cytotoxic importance of either isoform was related to differences in the longevity of the complexes.
Mouse embryonic fibroblasts (MEFs) were used to study the cellular response to the topo II poisons etoposide, mitoxantrone and mAMSA. The longevity of topo IIα and β 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 IIα and β complexes for the first time.
In MEFs treated with etoposide, 50% of topo IIα complexes dissociated within 40 min whereas dissociation of topo IIβ complexes took only 20 min. Disappearance of complexes was a slower process for mitoxantrone-treated cells. The time taken to reduce topo IIα and topo IIβ cleavable complexes by 50% was 10 and 6 h, respectively. In contrast, mAMSA-stabilized topo IIα and topo IIβ cleavable complexes were equally stable (dissociation within 15 min for both isoforms). These stability data were confirmed using an in vitro assay.
We previously demonstrated that topo IIα is the major target for etoposide and mitoxantrone but that both topo IIα and topo IIβ are important for mAMSA cytotoxicity. The longevity of the topo IIα and β cleavable complexes shown here is therefore an important factor in determining the cytotoxic sensitivity of either isoform to these drugs.
KeywordsDNA Topoisomerase II Etoposide mAMSA Mitoxantrone Cleavable complex
Trapped in agarose DNA immunostaining
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