Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks.
Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which 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 xenograft 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 evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence 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 complexes.
Bailly
C
C
INSERM U-524, Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, France. bailly@lille.inserm.fr
Lansiaux
A
A
Dassonneville
L
L
Demarquay
D
D
Coulomb
H
H
Huchet
M
M
Lavergne
O
O
Bigg
D C
DC
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D007941:Leukemia P388; D051379:Mice; D013329:Structure-Activity Relationship; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; C120029:homocamptothecin; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin