homocamptothecin and Leukemia-P388

Homocamptothecin (hCPT), which differs from camptothecin (CPT) by the presence of an additional methylene group in the E-ring, was evaluated in CPT-resistant cell lines. Topoisomerase I (top1)-deficient leukemia P388/CPT45 cells were highly resistant to hCPT, which demonstrates that top1 is the primary target of hCPT. Three CPT-resistant cell lines with top1 point mutations (Chinese hamster lung fibroblast DC3F/C10, human prostate carcinoma DU-145/RC1, and human leukemia CEM/C2) and their top1 enzymes were cross-resistant to hCPT. The antiproliferative activity of hCPT was greater than that of CPT in both parental and CPT-resistant cell lines, particularly in the prostate cell lines. The top1 cleavage complexes formed in the presence of hCPT appear to be more stable than those induced by CPT. Together, these data indicate that hCPT is a specific top1 inhibitor, which shares a common binding site with CPT in the topl-DNA cleavage complexes. Because of its potency, hCPT might overcome resistance to CPT in some cancer cells.

Topics: Animals; Antineoplastic Agents; Camptothecin; Cricetinae; DNA Topoisomerases, Type I; DNA, Neoplasm; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Enzyme Stability; Humans; Inhibitory Concentration 50; Leukemia P388; Male; Mice; Point Mutation; Prostatic Neoplasms; Topoisomerase I Inhibitors; Tumor Cells, Cultured

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.

Topics: Animals; Antineoplastic Agents; Camptothecin; Cell Survival; DNA Topoisomerases, Type I; DNA, Superhelical; Drug Resistance, Neoplasm; Enzyme Inhibitors; Leukemia P388; Mice; Structure-Activity Relationship; Topoisomerase I Inhibitors; Tumor Cells, Cultured