Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in pl ace of the conventional six-membered alpha-hydroxylactone ring found in cam ptothecin and its tumor active analogues, including topotecan and irinoteca n. The homologation of the lactone E-ring reinforces the stability of the l actone, thus reducing considerably its conversion into a carboxylate form w hich is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenog raft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further eviden ce that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring wit h a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT sti mulate the cleavage by topoisomerase I at T(down arrow)G sites, but in addi tion, hCPT stabilizes cleavage at specific sites containing the sequence AA C(down arrow)G. At low drug concentrations, the cleavage at the Tic sites a nd at the hCPT-specific C(down arrow)G sites is more pronounced and more st able with hCPT than with CPT. The in vitro data were confirmed in cells. Hi gher levels of protein-DNA complexes were detected in P388 leukemia cells t reated with hCPT than those treated with CPT. Immunoblotting experiments re vealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT pr ovided evidence that topoisomerase I is involved in the cytotoxicity of hCP T. Altogether, the results show that the beta-hydroxylactone ring of hCPT p lays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequ ence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complex es.