FORMATION OF TOPOISOMERASE II-ALPHA COMPLEXES WITH NASCENT DNA IS RELATED TO VM-26-INDUCED CYTOTOXICITY

Several clinically active anticancer drugs an known to interfere with DNA topoisomerase II activity. However, the importance of the individu al alpha (170 kDa) and beta (180 kDa) isozymes as targets of topoisome rase II-active drugs is not clear. To address this question, human CCR F-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified b y immunoprecipitation with an anti-bromodeoxyuridine antibody. The top oisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The alpha isozyme form ed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the beta isozyme was only bound to bulk DNA. At m oderately cytotoxic concentrations, VM-26 enhanced the binding of topo isomerase II alpha to nascent DNA at least 5.2-fold compared to bulk D NA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxi c concentrations of VM-26, topoisomerase II alpha complex formation wi th nascent DNA was decreased at least 5.5-fold compared to bulk DNA. D rug-induced binding of topoisomerase II beta with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these result s indicate that the formation of VM-26 stabilized complexes of topoiso merase II alpha with nascent DNA are critical to the development of cy totoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide ind irect evidence that topoisomerase II alpha is involved in DNA replicat ion.