Murine Transgenic Cells Lacking DNA Topoisomerase IIβ Are Resistant to Acridines and Mitoxantrone: Analysis of Cytotoxicity and Cleavable Complex Formation
- 1School of Biochemistry and Genetics (F.E., E.W., C.A.A.) and 2Cancer Research Unit and Department of Child Health (M.J.T.), The Medical School, The Cookson Building, University of Newcastle upon Tyne, United Kingdom; 3Department of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York (L.L., G.L.);4Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts (W.L.); and 5Auckland Cancer Society Research Centre, University of Auckland School of Medicine, Auckland, New Zealand (B.C.B.)
Abstract
Murine transgenic cell lines lacking DNA topoisomerase II (topo II)β have been used to assess the importance of topo IIβ as a drug target. Western blot analysis confirmed that the topo IIβ −/− cell lines did not contain topo IIβ protein. In addition, both the topo IIβ +/+ and topo IIβ −/− cell lines contained similar levels of topo IIα protein. The trapped in agarose DNA immunostaining assay (TARDIS) was used to detect topo IIα and β cleavable complexes in topo IIβ −/− and topo IIβ +/+ cells. These results show that both topo IIα and β are in vivo targets for etoposide, mitoxantrone, and amsacrine (mAMSA) in topo IIβ +/+ cells. As expected, only the α-isoform was targeted in topo IIβ −/− cells. Clonogenic assays comparing the survival of topo IIβ −/− and topo IIβ +/+ cells were carried out to establish whether the absence of topo IIβ caused drug resistance. Increased survival of topo IIβ −/− cells compared with topo IIβ +/+ cells was observed after treatment with amsacrine (mAMSA), methylN-(4′-[9-acridinylamino]-2-methoxyphenyl) carbamate hydrochloride (AMCA), methylN-(4′-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride (mAMCA), mitoxantrone, and etoposide. These studies showed that topo IIβ −/− cells were significantly more resistant to mAMSA, AMCA, mAMCA, and mitoxantrone, than topo IIβ +/+ cells, indicating that topo IIβ is an important target for the cytotoxic effects of these compounds.
Footnotes
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Send reprint requests to: Dr. C. A. Austin, School of Biochemistry and Genetics, The Medical School, The Cookson Building, Newcastle upon Tyne, NE2 4HH, UK. E-mail: caroline.austin{at}ncl.ac.uk
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F. Errington is supported by a Gordon Piller Studentship from the Leukemia Research Fund (Grant 9683); Dr. E. Willmore is supported by the Leukemia Research Fund (Grant 9743); and Dr. M. J. Tilby is supported by the North of England Children's Cancer Research Fund.
- Abbreviations:
- topo
- topoisomerase
- mAMSA
- amsacrine
- AMCA
- methyl N-(4′-[9-acridinylamino]-phenyl)carbamate hydrochloride
- mAMCA
- methylN-(4′-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride
- FITC
- fluorescein isothiocyanate
- TARDIS
- trapped in agarose DNA immunostaining
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- Received June 7, 1999.
- Accepted September 15, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
