Y. Takebayashi et al., Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancerdrug that selectively alkylates DNA in the minor groove, P NAS US, 96(13), 1999, pp. 7196-7201
Citations number
34
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.