Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

Separation of bisdioxopiperazine- and vanadate resistance in topoisomerase II

Bisdioxopiperazines are inhibitors of topoisomerase II trapping this protein as a closed clamp on DNA with concomitant inhibition of its ATPase activity. Here, we analyse the effects of N-terminal mutations identified in bisdioxopiperazine-resistant cells on ATP hydrolysis by this enzyme. We present data consistent with bisdioxopiperazine resistance arising by two different mechanisms; one involving reduced stability of the N-terminal clamp (the N-gate) and one involving reduced affinity for bisdioxopiperazines. Vanadate is a general inhibitor of type P ATPases and has recently been demonstrated to lock topoisomerase II as a salt-stable closed clamp on DNA analogous to the bisdioxopiperazines. We show that a R162K mutation in human topoisomerase II alpha renders this enzyme highly resistant towards vanadate while having little effect on bisdioxopiperazine sensitivity. The implications of these findings for the mechanism of action of bisdioxopiperazines versus vanadate with topoisomerase II are discussed.     

N-terminal and core-domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.