Murine transgenic cells lacking DNA topoisomerase IIβ are resistant to acridines and mitoxantrone:: Analysis of cytotoxicity and cleavable complex formation

Murine transgenic cell lines lacking DNA topoisomerase II (topo II)beta have been used to assess the importance of topo II beta as a drug target. Western blot analysis confirmed that the topo II beta -/- cell lines did not contain topo II beta protein. In addition, both the topo II beta +/+ and topo II beta -/- cell lines contained similar levels of topo II alpha protein. The trapped in agarose DNA immunostaining assay (TARDIS) was used to detect topo II alpha and beta cleavable complexes in topo II beta -/- and topo II beta +/+ cells. These results show that both topo II alpha and beta are in vivo targets for etoposide, mitoxantrone, and amsacrine (mAMSA) in topo II beta +/+ cells. As expected, only the alpha-isoform was targeted in topo II beta -/- cells. Clonogenic assays comparing the survival of topo II beta -/- and topo II beta +/+ cells were carried out to establish whether the absence of topo II beta caused drug resistance. Increased survival of topo II beta -/- cells compared with topo II beta +/+ cells was observed after treatment with amsacrine (mAMSA), methyl N-(4'-[9-acridinylamino]-2-methoxyphenyl) carbamate hydrochloride (AMCA), methyl N-(4'-[9-acridinylamino]- 2-methoxyphenyl) carbamate hydrochloride (mAMCA), mitoxantrone, and etoposide. These studies showed that topo II beta -/- cells were significantly more resistant to mAMSA, AMCA, mAMCA, and mitoxantrone, than topo II beta +/+ cells, indicating that topo II beta is an important target for the cytotoxic effects of these compounds.