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. 1995 Sep 12;92(19):8861–8865. doi: 10.1073/pnas.92.19.8861

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

Y Pommier 1, G Kohlhagen 1, K W Kohn 1, F Leteurtre 1, M C Wani 1, M E Wall 1
PMCID: PMC41067  PMID: 7568032

Abstract

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bonven B. J., Gocke E., Westergaard O. A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell. 1985 Jun;41(2):541–551. doi: 10.1016/s0092-8674(85)80027-1. [DOI] [PubMed] [Google Scholar]
  2. Capranico G., Kohn K. W., Pommier Y. Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin. Nucleic Acids Res. 1990 Nov 25;18(22):6611–6619. doi: 10.1093/nar/18.22.6611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Capranico G., Palumbo M., Tinelli S., Mabilia M., Pozzan A., Zunino F. Conformational drug determinants of the sequence specificity of drug-stimulated topoisomerase II DNA cleavage. J Mol Biol. 1994 Jan 28;235(4):1218–1230. doi: 10.1006/jmbi.1994.1075. [DOI] [PubMed] [Google Scholar]
  4. Chen A. Y., Liu L. F. DNA topoisomerases: essential enzymes and lethal targets. Annu Rev Pharmacol Toxicol. 1994;34:191–218. doi: 10.1146/annurev.pa.34.040194.001203. [DOI] [PubMed] [Google Scholar]
  5. Covey J. M., Jaxel C., Kohn K. W., Pommier Y. Protein-linked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I. Cancer Res. 1989 Sep 15;49(18):5016–5022. [PubMed] [Google Scholar]
  6. Crow R. T., Crothers D. M. Structural modifications of camptothecin and effects on topoisomerase I inhibition. J Med Chem. 1992 Oct 30;35(22):4160–4164. doi: 10.1021/jm00100a022. [DOI] [PubMed] [Google Scholar]
  7. Fossé P., René B., Le Bret M., Paoletti C., Saucier J. M. Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative. Nucleic Acids Res. 1991 Jun 11;19(11):2861–2868. doi: 10.1093/nar/19.11.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Freudenreich C. H., Kreuzer K. N. Localization of an aminoacridine antitumor agent in a type II topoisomerase-DNA complex. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11007–11011. doi: 10.1073/pnas.91.23.11007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujimori A., Harker W. G., Kohlhagen G., Hoki Y., Pommier Y. Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin. Cancer Res. 1995 Mar 15;55(6):1339–1346. [PubMed] [Google Scholar]
  10. Fukada M. Action of camptothecin and its derivatives on deoxyribonucleic acid. Biochem Pharmacol. 1985 Apr 15;34(8):1225–1230. doi: 10.1016/0006-2952(85)90499-x. [DOI] [PubMed] [Google Scholar]
  11. Hertzberg R. P., Busby R. W., Caranfa M. J., Holden K. G., Johnson R. K., Hecht S. M., Kingsbury W. D. Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site. J Biol Chem. 1990 Nov 5;265(31):19287–19295. [PubMed] [Google Scholar]
  12. Hertzberg R. P., Caranfa M. J., Hecht S. M. On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. Biochemistry. 1989 May 30;28(11):4629–4638. doi: 10.1021/bi00437a018. [DOI] [PubMed] [Google Scholar]
  13. Hertzberg R. P., Caranfa M. J., Holden K. G., Jakas D. R., Gallagher G., Mattern M. R., Mong S. M., Bartus J. O., Johnson R. K., Kingsbury W. D. Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity. J Med Chem. 1989 Mar;32(3):715–720. doi: 10.1021/jm00123a038. [DOI] [PubMed] [Google Scholar]
  14. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
  15. Hsiang Y. H., Liu L. F., Wall M. E., Wani M. C., Nicholas A. W., Manikumar G., Kirschenbaum S., Silber R., Potmesil M. DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res. 1989 Aug 15;49(16):4385–4389. [PubMed] [Google Scholar]
  16. Jaxel C., Capranico G., Kerrigan D., Kohn K. W., Pommier Y. Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin. J Biol Chem. 1991 Oct 25;266(30):20418–20423. [PubMed] [Google Scholar]
  17. Jaxel C., Kohn K. W., Pommier Y. Topoisomerase I interaction with SV40 DNA in the presence and absence of camptothecin. Nucleic Acids Res. 1988 Dec 9;16(23):11157–11170. doi: 10.1093/nar/16.23.11157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jaxel C., Kohn K. W., Wani M. C., Wall M. E., Pommier Y. Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity. Cancer Res. 1989 Mar 15;49(6):1465–1469. [PubMed] [Google Scholar]
  19. Leteurtre F., Fesen M., Kohlhagen G., Kohn K. W., Pommier Y. Specific interaction of camptothecin, a topoisomerase I inhibitor, with guanine residues of DNA detected by photoactivation at 365 nm. Biochemistry. 1993 Aug 31;32(34):8955–8962. doi: 10.1021/bi00085a029. [DOI] [PubMed] [Google Scholar]
  20. Leteurtre F., Fujimori A., Tanizawa A., Chhabra A., Mazumder A., Kohlhagen G., Nakano H., Pommier Y. Saintopin, a dual inhibitor of DNA topoisomerases I and II, as a probe for drug-enzyme interactions. J Biol Chem. 1994 Nov 18;269(46):28702–28707. [PubMed] [Google Scholar]
  21. Luzzio M. J., Besterman J. M., Emerson D. L., Evans M. G., Lackey K., Leitner P. L., McIntyre G., Morton B., Myers P. L., Peel M. Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I. J Med Chem. 1995 Feb 3;38(3):395–401. doi: 10.1021/jm00003a001. [DOI] [PubMed] [Google Scholar]
  22. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  23. Pommier Y., Capranico G., Orr A., Kohn K. W. Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide. Nucleic Acids Res. 1991 Nov 11;19(21):5973–5980. doi: 10.1093/nar/19.21.5973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pommier Y., Leteurtre F., Fesen M. R., Fujimori A., Bertrand R., Solary E., Kohlhagen G., Kohn K. W. Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors. Cancer Invest. 1994;12(5):530–542. doi: 10.3109/07357909409021413. [DOI] [PubMed] [Google Scholar]
  25. Porter S. E., Champoux J. J. The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I. Nucleic Acids Res. 1989 Nov 11;17(21):8521–8532. doi: 10.1093/nar/17.21.8521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Potmesil M. Camptothecins: from bench research to hospital wards. Cancer Res. 1994 Mar 15;54(6):1431–1439. [PubMed] [Google Scholar]
  27. Sawada S., Nokata K., Furuta T., Yokokura T., Miyasaka T. Chemical modification of an antitumor alkaloid camptothecin: synthesis and antitumor activity of 7-C-substituted camptothecins. Chem Pharm Bull (Tokyo) 1991 Oct;39(10):2574–2580. doi: 10.1248/cpb.39.2574. [DOI] [PubMed] [Google Scholar]
  28. Slichenmyer W. J., Rowinsky E. K., Donehower R. C., Kaufmann S. H. The current status of camptothecin analogues as antitumor agents. J Natl Cancer Inst. 1993 Feb 17;85(4):271–291. doi: 10.1093/jnci/85.4.271. [DOI] [PubMed] [Google Scholar]
  29. Svejstrup J. Q., Christiansen K., Gromova I. I., Andersen A. H., Westergaard O. New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site. J Mol Biol. 1991 Dec 5;222(3):669–678. doi: 10.1016/0022-2836(91)90503-x. [DOI] [PubMed] [Google Scholar]
  30. Tanizawa A., Beitrand R., Kohlhagen G., Tabuchi A., Jenkins J., Pommier Y. Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance. J Biol Chem. 1993 Dec 5;268(34):25463–25468. [PubMed] [Google Scholar]
  31. Tanizawa A., Fujimori A., Fujimori Y., Pommier Y. Comparison of topoisomerase I inhibition, DNA damage, and cytotoxicity of camptothecin derivatives presently in clinical trials. J Natl Cancer Inst. 1994 Jun 1;86(11):836–842. doi: 10.1093/jnci/86.11.836. [DOI] [PubMed] [Google Scholar]
  32. Tanizawa A., Kohn K. W., Kohlhagen G., Leteurtre F., Pommier Y. Differential stabilization of eukaryotic DNA topoisomerase I cleavable complexes by camptothecin derivatives. Biochemistry. 1995 May 30;34(21):7200–7206. doi: 10.1021/bi00021a035. [DOI] [PubMed] [Google Scholar]
  33. Tanizawa A., Kohn K. W., Pommier Y. Induction of cleavage in topoisomerase I c-DNA by topoisomerase I enzymes from calf thymus and wheat germ in the presence and absence of camptothecin. Nucleic Acids Res. 1993 Nov 11;21(22):5157–5166. doi: 10.1093/nar/21.22.5157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang J. C. DNA topoisomerases: why so many? J Biol Chem. 1991 Apr 15;266(11):6659–6662. [PubMed] [Google Scholar]

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