Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancerdrug that selectively alkylates DNA in the minor groove

Ecteinascidin 743 (Et743, National Service Center 648766) is a potent antit umor agent from the Caribbean tunicate Ecteinascidia turbinata, Although Et 743 is presently in clinical trials for human cancers, the mechanisms of an titumor activity of Et743 hale not been elucidated. Et743 can alkylate sele ctively guanine NZ from the DNA minor groove, and this alkylation is revers ed by DNA denaturation, Thus, Et743 differs from other DNA alkylating agent s presently in the clinic (by both its biochemical activities and its profi le of antitumor activity in preclinical models), In this study, we investig ated cellular proteins that can bind to DNA. alkylated by Et743. By using a n oligonucleotide containing high-affinity Et743 binding sites and nuclear extracts from human leukemia CEM cells, we purified a 100-kDa protein as a cellular target of Et743 and identified it as topoisomerase I (top 1), Puri fied top1 was then tested and found to produce cleavage complexes in the pr esence of Et743, whereas topoisomerase Il had no effect. DNA alkylation was essential for the formation of top1-mediated cleavage complexes by Et743. and the distribution of the drug-induced top1 sites was different for Et743 and camptothecin. top1-DNA complexes were also detected in Et743-treated C EM cells by using cesium chloride gradient centrifugation followed by top1 immunoblotting. These data indicate that DNA minor groove alkylation by Et7 43 induces top1-mediated protein-linked DNA breaks and that top1 is a targe t for Et743 in vitro and in vivo.