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

Abstract

Ecteinascidin 743 (Et743, National Service Center 648766) is a potent antitumor agent from the Caribbean tunicate Ecteinascidia turbinata. Although Et743 is presently in clinical trials for human cancers, the mechanisms of antitumor activity of Et743 have not been elucidated. Et743 can alkylate selectively guanine N2 from the DNA minor groove, and this alkylation is reversed by DNA denaturation. Thus, Et743 differs from other DNA alkylating agents presently in the clinic (by both its biochemical activities and its profile of antitumor activity in preclinical models). In this study, we investigated cellular proteins that can bind to DNA alkylated by Et743. By using an 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 (top1). Purified top1 was then tested and found to produce cleavage complexes in the presence of Et743, whereas topoisomerase II 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 CEM cells by using cesium chloride gradient centrifugation followed by top1 immunoblotting. These data indicate that DNA minor groove alkylation by Et743 induces top1-mediated protein-linked DNA breaks and that top1 is a target for Et743 in vitro and in vivo.

Interaction of Human Nuclear Topoisomerase I with Guanosine Quartet-forming and Guanosine-rich Single-stranded DNA and RNA Oligonucleotides

“…DNA containing partially singlestranded regions and extra-helical segments (bulges) were shown to be cleaved by the enzyme (26,43,44). Top1 also cleaves DNA sites with local structure distortion including oxidative base modifications (36), base mismatches and abasic sites (45), UV photoproducts (46), base methylation (47), intercalation (48), and minor groove alkylation (49,50). Top1 has also been found to cleave three-stranded flap regions and promote their DNA recombination (30,31).…”

New agents in cancer clinical trials

“…Other compounds that a ect minor groove DNA binding such as distamycins are known to be reactive in AT-rich regions suggesting an overall gene regulation impairment by such agents (Broggini et al, 1989). Through DNA minor groove alkylation, ET-743 also induces topoisomerase I mediated DNA strand breaks (Takebayashi et al, 1999). Additionally, ET-743 also appears to indirectly disorganize microtubule formation (Garcia-Rocha et al, 1996).…”

Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation

“…97 ͉ no. 12 ͉ 6775-6779 bule disruption (13), and target topoisomerase I (14,15), its mechanism of action in vivo is unknown. A recent study showed that ET-743 interfered with the interaction of minor-groove-binding proteins, particularly NF-Y, with their cognate DNA elements in vitro (16).…”