Murine Transgenic Cells Lacking DNA Topoisomerase IIβ Are Resistant to Acridines and Mitoxantrone: Analysis of Cytotoxicity and Cleavable Complex Formation

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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.

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