Characterization of an altered DNA topoisomerase ii-alpha from a mitoxantrone resistant Mammalian-cell line hypersensitive to DNA cross-linking agents.

To further define the molecular basis for drug resistance to mitoxantrone, a Chinese hamster ovary cell line (MXN(4)) was selected in the presence of 25 nM mitoxantrone and fully characterized. This cell line is 20-fold resistant to mitoxantrone, cross-resistant to several other topoisomerase II poisons, and 2- to 3-fold collaterally sensitive to cisplatin, carboplatin and BCNU. Neither an alteration in cellular uptake of topoisomerase II inhibitor nor overexpression of P-glycoprotein contribute to the drug resistance of MXN(4) cells. Immunoblotting demonstrates equivalent amounts of topoisomerase II alpha and beta in the wild-type and drug resistant cell lines, suggesting that a quantitative alteration in topoisomerase II is not the mechanism of resistance of MXN(4) cells. Mitoxantrone-induced DNA double strand breaks measured in situ were attenuated 28-fold in the drug resistant cell line. Nuclear extracts of MXN(4) cells, as well as topoisomerase II alpha purified to homogeneity from these cells, were found to be markedly resistant to drug-induced covalent DNA: topoisomerase II complex formation. The catalytic activity of purified MXN(4) topoisomerase II was the same as wild-type activity. Thus, the resistance of MXN(4) cells to mitoxantrone involves the expression of a topoisomerase II alpha with altered DNA cleavage activity. The hypersensitivity of this cell line to platinum analogs is due to an apparent increased uptake of these drugs which results in augmented DNA interstrand crosslinking.