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Differences in the longevity of topo IIα and topo IIβ drug-stabilized cleavable complexes and the relationship to drug sensitivity

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Abstract

Purpose

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.

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 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.

Results

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.

Conclusions

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.

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Abbreviations

FITC:

Fluorescein isothiocyanate

mAMSA:

Amsacrine

PBS:

Phosphate-buffered saline

TARDIS:

Trapped in agarose DNA immunostaining

topo:

Topoisomerase

References

  1. Austin CA, Marsh KL (1998) Eukaryotic DNA topoisomerase IIβ. Bioessays 20:215

    CAS  PubMed  Google Scholar 

  2. Austin CA, Sng J, Patel S, Fisher LM (1993) Novel HeLa topoisomerase II is the IIβ isoform: complete coding sequence and homology with other type II topoisomerases. Biochim Biophys Acta 1172:283

    Article  CAS  PubMed  Google Scholar 

  3. Bailly JD, Skladanowski A, Bettaieb A, Mansat V, Larsen AK, Laurent G (1997) Natural resistance of acute myeloid leukemia cell lines to mitoxantrone is associated with lack of apoptosis. Leukemia 11:1523

    Article  CAS  PubMed  Google Scholar 

  4. Caldecott K, Banks G, Jeggo P (1990) DNA double-strand break repair pathways and cellular tolerance to inhibitors of topoisomerase II. Cancer Res 50:5778

    CAS  PubMed  Google Scholar 

  5. Capranico G, Binaschi M (1998) DNA sequence selectivity of topoisomerases and topoisomerase poisons. Biochim Biophys Acta 1400:185

    Article  CAS  PubMed  Google Scholar 

  6. Chen C, Fuscoe JC, Liu O, Relling MV (1996) Etoposide causes illegitimate V (D) J recombination in human lymphoid leukemic cells. Blood 86:2210

    Google Scholar 

  7. Covey JM, Kohn KW, Kerrigan D, Tilchen EJ, Pommier Y (1988) Topoisomerase II-mediated DNA damage produced by 4′-(9-acridinylamino)-methanesulfon-m-anisidide and related acridines in L1210 cells and isolated nuclei: relation to cytotoxicity. Cancer Res 48:860

    CAS  PubMed  Google Scholar 

  8. Cowell IG, Willmore E, Chalton D, Marsh KL, Jazrawi E, Fisher LM, Austin CA (1998) Nuclear distribution of human DNA topoisomerase IIβ: a nuclear targeting signal residues in the 116-residue C-terminal tail. Exp Cell Res 243:232

    Article  CAS  PubMed  Google Scholar 

  9. Errington F, Willmore E, Tilby MJ, Li L, Li G, Li W, Baguley BC, Austin CA (1999) Murine transgenic cells lacking DNA topoisomerase IIβ are resistant to acridine and mitoxantrone: analysis of cytotoxicity and cleavable complex formation. Mol Pharmacol 56:1309

    CAS  PubMed  Google Scholar 

  10. Fox ME, Smith PJ (1990) Long-term inhibition of DNA synthesis and the persistence of trapped topoisomerase II complexes in determining the toxicity of the antitumor DNA intercalators mAMSA and mitoxantrone. Cancer Res 50:5813

    CAS  PubMed  Google Scholar 

  11. Frank AJ, Procter SJ, Tilby MJ (1996) Detection and quantification of melphalan-DNA adducts at the single cell level in hematopoietic tumor cells. Blood 88:977

    CAS  PubMed  Google Scholar 

  12. Harker WG, Slade DL, Drake FH, Parr RL (1991) Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase IIβ isoform. Biochemistry 30:9953

    CAS  PubMed  Google Scholar 

  13. Hazelhurst LA, Foley NE, Gleason-Guzman MC, Hacker MP, Cress AE, Greenberger LW, de Jong MC, Dalton WS (1999) Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line. Cancer Res 59:1021

    CAS  PubMed  Google Scholar 

  14. Hsiang, Y, Liu LF (1989) Evidence for the reversibility of cellular DNA lesion induced by mammalian topoisomerase II poisons. J Biol Chem 264:9713

    CAS  PubMed  Google Scholar 

  15. Long BH, Musial ST, Brattain MG (1985) Single- and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide. Cancer Res 45:3106

    CAS  PubMed  Google Scholar 

  16. Osheroff N (1989) Effect of antineoplastic agents on the DNA cleavage/religation of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide. Biochemistry 28:6157

    CAS  PubMed  Google Scholar 

  17. Pommier Y, Zwelling LA, Kao-Shan C, Whang-Peng J, Bradley MO (1985) Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster cells. Cancer Res 45:3143

    CAS  PubMed  Google Scholar 

  18. Pommier Y, Leteurtre F, Fesen MR, Fujimori A, Bertrand R, Solary E, Kohlhagen G, Kohn KW (1994) Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors. Cancer Invest 12:530

    CAS  PubMed  Google Scholar 

  19. Strumberg D, Nitiss JL, Rose A, Nicklaus MC, Pommier Y (1999) Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions. J Biol Chem 274:7292–7301

    Article  CAS  PubMed  Google Scholar 

  20. Suzuki H, Tarumoto Y, Ohsawa M (1997) Topoisomerase II inhibitors fail to induce chromosome-type aberrations in etoposide-resistant cells: evidence for essential contribution of the cleavable complex formation to the induction of chromosome-type aberrations. Mutagenesis 12:29

    CAS  PubMed  Google Scholar 

  21. Tan KB, Dorman TE, Falls KM, Chung TDY, Mirabelli CK, Crooke ST, Mao J (1992) Topoisomerase IIα and topoisomerase IIβ genes: characterization and mapping to human chromosome 17 and 3, respectively. Cancer Res 52:231

    CAS  PubMed  Google Scholar 

  22. Wang JC (1996) DNA Topoisomerases. Annu Rev Biochem 65:635

    CAS  PubMed  Google Scholar 

  23. Willmore E, Frank AJ, Padget K, Tilby MJ, Austin CA (1998) Etoposide targets topoisomerase IIα and IIβ in leukemic cells: isoform-specific cleavable complexes visualised and quantified in situ by a novel immunofluorescence technique. Mol Pharmacol 53:78

    Google Scholar 

  24. Zwelling LA, Michaels S, Erickson LC, Ungerleider RS, Nichols M, Kohn KW (1981) Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4′-(9-acridinylamino)methanesulfon-m-anisidide and adriamycin. Biochemistry 20:6553

    Google Scholar 

  25. Zwelling LA, Estey E, Bakic M, Silberman L, Chan D (1987) Topoisomerase II as a target of antileukemic drugs. NCI Monographs 4:79

    PubMed  Google Scholar 

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Acknowledgements

This work was funded by the Leukemia Research Fund grants 9743 (E.W.) and 9683 (F.E.) and the Wellcome Trust (grant reference 056232). We would like to thank Professor J.C. Wang for the murine mTOP2β-4 topo IIβ+/+ and mtop2β-5 topo IIβ−/− cells.

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Authors and Affiliations

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

    Fiona Errington, Elaine Willmore, Chrysoula Leontiou & Caroline A. Austin

  2. Northern Institute for Cancer Research The Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE2 4HH, UK

    Michael J. Tilby

Authors
  1. Fiona Errington
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  2. Elaine Willmore
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  3. Chrysoula Leontiou
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  4. Michael J. Tilby
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  5. Caroline A. Austin
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Correspondence to Caroline A. Austin.

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Errington, F., Willmore, E., Leontiou, C. et al. Differences in the longevity of topo IIα and topo IIβ drug-stabilized cleavable complexes and the relationship to drug sensitivity. Cancer Chemother Pharmacol 53, 155–162 (2004). https://doi.org/10.1007/s00280-003-0701-1

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  • DOI: https://doi.org/10.1007/s00280-003-0701-1

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