Effect of cellular ATP depletion on topoisomerase II poisons. Abrogation of cleavable-complex formation by etoposide but not by amsacrine

Topoisomerase (topo) II poisons have been categorized into ATP-independent and -dependent drugs based on in vitro studies. We investigated drug-induce d topoII-DNA complexes in intact cells almost completely depleted of ATP. V irtually no DNA single-strand breaks (SSBs), as measured by alkaline elutio n, were detected in energy-depleted cells treated with the topoII poisons e toposide, teniposide, daunorubicin, doxorubicin, mitoxantrone, or clerocidi n. This inhibition was reversible; subsequent incubation with glucose resto red the level of DNA SSBs, The effect of ATP depletion was specific for top oII, because topoI-mediated cleavable complexes induced by camptothecin wer e unaffected by ATP depletion. Furthermore, etoposide-induced DNA-protein c omplexes and DNA double-strand breaks, as measured by filter elution techni ques, and topoII alpha and -beta trapping, as measured by a band depletion assay, were completely inhibited by energy depletion. Differences in drug t ransport could not explain the effect of ATP depletion. The topoII poison a msacrine (m-AMSA) was unique with respect to ATP dependence. In ATP-deplete d cells, m-AMSA-induced DNA SSBs, DNA double-strand breaks, DNA-protein com plexes, topoII alpha and -beta trapping were only modestly reduced. The acc umulation of m-AMSA was reduced in ATP-depleted cells, which indicates that drug transport could contribute to the modest decrease in m-AMSA-induced c leavable complexes. In conclusion, drug-induced topoII-DNA complexes were c ompletely antagonized in ATP-depleted cells, except in the case of m-AMSA, One possible interpretation is that m-AMSA mainly produces prestrand passag e DNA lesions, whereas the other topoII poisons tested exclusively stabiliz e poststrand passage DNA lesions in intact cells.