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 mitoxantr one, a Chinese hamster ovary cell line (MXN(4)) was selected in the pr esence of 25 nM mitoxantrone and fully characterized. This cell line i s 20-fold resistant to mitoxantrone, cross-resistant to several other topoisomerase II poisons, and 2- to 3-fold collaterally sensitive to c isplatin, carboplatin and BCNU. Neither an alteration in cellular upta ke of topoisomerase II inhibitor nor overexpression of P-glycoprotein contribute to the drug resistance of MXN(4) cells. Immunoblotting demo nstrates equivalent amounts of topoisomerase II alpha and beta in the wild-type and drug resistant cell lines, suggesting that a quantitativ e 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. Nucle ar extracts of MXN(4) cells, as well as topoisomerase II alpha purifie d 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 a s wild-type activity. Thus, the resistance of MXN(4) cells to mitoxant rone involves the expression of a topoisomerase II alpha with altered DNA cleavage activity. The hypersensitivity of this cell line to plati num analogs is due to an apparent increased uptake of these drugs whic h results in augmented DNA interstrand crosslinking.