Involvement of DNA topoisomerase II in the selective resistance of a mammalian cell mutant to DNA minor groove ligands: ligand-induced DNA—protein crosslinking and responses to topoisomerase poisons

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Abstract
A mutant murine cell line has previously been reported to be resistant to the AT-specific DNA minor groove ligand 2',5'-bi-1H-benzimidazole, 2',(4-ehoxyphenyl)-5-(4-methyl-1-pipera zinyl), trichloride (Ho33342), due to an enhanced capacity to remove ligand molecules from cellular DNA via a pathway which can be blocked by DNA topoisomerase poisons. We have studied the relationship between ligand resistance and DNA topoisomerase II activity. The cross-sensitivity patterns of the mutant were examined for covalently (anthramycin) and non-covalently (distamycin A) binding minor groove ligands, and DNA intercalating [adriamycin, mitoxantrone and 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA)] and non-intercalating (VP16-213) topoisomerase II poisons. The mutant was cross-resistant to distamycin A alone. The mutant showed no abnormality in: (i) the in vitro decatenation activity of topoisomerase II, (ii) VP16-213 or mAMSA induced protein—DNA cross-linking activities in nuclear extracts, (iii) ‘cleavable complex’ generation (or DNA strand scisson) in intact cells exposed to topoisomerase poisons. Ho33342 and the topoisomerase II inhibitor novobiocin were found to disrupt both the in vitro binding of nudear extracted proteins, from mutant and parental cells, to plasmid DNA and the formation of drug-induced cleavable complexes in vitro. Unexpectedly, Ho33342 induced significant levels of DNA—protein crosslinking in both parental and mutant cells. We conclude that: (i) resistance of the mutant is limited to non-covalently binding minor groove ligands, (ii) Ho33342 can block the trapping of DNA topoisomerase II by enzyme poisons in vitro, (iii) Ho33342 can induce a novel form of DNA—protein cross-link in intact cells, and (iv) the resistance of the mutant is not dependent upon some abnormality in topoisomerase II function.