Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression

Abstract

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.