Abstract
Camptothecin (CPT) has heen recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I‐mediated DNA single‐strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), in HL‐60 cells. DNA single‐ strand breaks were detected using alkaline sucrose gradient centrifugation when HL‐60 cells were incubated with 10 μM CPT or 10 μM SN‐38 for 30 min. These DNA single‐strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL‐60 cells with CPT or SN‐38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single‐strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug‐free medium after treatment with CPT or SN‐38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I‐mediated DNA single‐strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL‐60 cell death induced by Topo I inhibitor.
Keywords: Camptothecin, DNA damage, HL‐60
Full Text
The Full Text of this article is available as a PDF (478.7 KB).
REFERENCES
- 1. ) Wall , M. E. , Wani , M. C. , Cook , C. E. , Palmer , K. H. , McPhail , A. T. and Sim , G. A.Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata . J. Am. Chem. Soc. , 88 , 3888 – 3890 ( 1966. ). [Google Scholar]
- 2. ) Gallo , R. C. , Whang‐Peng , J. and Adamson , R. H.Studies on the antitumor activity, mechanism of action, and cell cycle effects of camptothecin . J. Natl. Cancer Inst. , 46 , 789 – 795 ( 1971. ). [PubMed] [Google Scholar]
- 3. ) Gottlieb , J. A. , Guarino , A. M. , Call , J. B. , Oliverio , V. T. and Block , J. B.Preliminary pharmacologic and clinical evaluation of camptothecin sodium (NSC‐100880) . Cancer Chemother. Rep. , 54 , 461 – 470 ( 1970. ). [PubMed] [Google Scholar]
- 4. ) Kunimoto , T. , Nitta , K. , Tanaka , T. , Uehara , N. , Baba , H. , Takeuchi , M. , Yokokura , T. , Sawada , S. , Miyasaka , T. and Mutai , M.Antitumor activity of a new camptothecin derivative, SN‐22, against various murine tumors . J. Pharmacobiodyn. , 10 , 149 – 152 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 5. ) Kaneda , N. , Nagata , H. , Furuta , T. and Yokokura , T.Metabolism and pharmacokinetics of the camptothecin analogue CPT‐11 in the mouse . Cancer Res. , 50 , 1715 – 1720 ( 1990. ). [PubMed] [Google Scholar]
- 6. ) Ohno , R. , Okada , K. , Masaoka , T. , Kuramoto , A. , Arima , T. , Yoshida , Y. , Ariyoshi , H. , Ichimaru , M. , Sakai , Y. , Oguro , M. , Ito , Y. , Morishima , Y. , Yokomaku , S. and Ota , K.An early phase‐II study of CPT‐11: a new derivative of camptothecin, for the treatment of leukemia and lymphoma . J.Clin. Oncol. , 8 , 1907 – 1912 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 7. ) Hsiang , Y‐H. , Hertzberg , R. , Hecht , S. and Liu , L. F.Camptothecin induces protein‐linked DNA breaks via mammalian DNA topoisomerase I . J. Biol. Chem. , 260 , 14873 – 14878 ( 1985. ). [PubMed] [Google Scholar]
- 8. ) Hertzberg , R. P. , Caranfa , M. J. and Hecht , S. M.On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme‐DNA complex . Biochemistry , 28 , 4629 – 638 ( 1989. ). [DOI] [PubMed] [Google Scholar]
- 9. ) Horwitz , M. S. and Horwitz , S. B.Intracelluiar degradation of HeLa and adenovirus type 2 DNA induced by camptothecin . Biochem. Biophys. Res. Commun. , 45 , 723 – 727 ( 1971. ). [DOI] [PubMed] [Google Scholar]
- 10. ) Spataro , A. and Kessel , D.Studies on camptothecin‐ induced degradation and apparent reaggregation of DNA from L1210 cells . Biochem. Biophys. Res. Commun. , 48 , 643 – 648 ( 1972. ). [DOI] [PubMed] [Google Scholar]
- 11. ) Mattern , M. R. , Mong , S‐M. , Bartus , H. F. , Mirabelli , C. K. , Crooke , S. T. and Johnson , R. K.Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells . Cancer Res. , 47 , 1793 – 1798 ( 1987. ). [PubMed] [Google Scholar]
- 12. ) Andoh , T. , Ishii , K. , Suzuki , Y. , Ikegami , Y. , Kusunoki , Y. , Takemoto , Y. and Okada , K.Characterization of a mammalian mutant with a camptothec in‐resistant DNA topoisomerase I . Proc. Natl Acad. Sci. USA , 84 , 5565 – 5569 ( 1987. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. ) Nitiss , J. and Wang , J. C.DNA Topoisomerase‐targeting antitumor drugs can be studied in yeast . Proc. Natl. Acad. Sci. USA , 85 , 7501 – 7505 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. ) Jaxel , C. , Kohn , K. W. , Wani , M. C. , Wall , M. E. and 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. , 49 , 1465 – 1469 ( 1989. ). [PubMed] [Google Scholar]
- 15. ) Kessel , D. , Bosmann , H. B. and Lohr , K.Camptothecin effects on DNA synthesis in murine leukemia cells . Biochim. Biophys. Acta , 269 , 210 – 216 ( 1972. ). [DOI] [PubMed] [Google Scholar]
- 16. ) Heck , M. M. S. , Hittelman , W. N. and Earnshaw , W. C.Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle . Proc. Natl. Acad. Sci. USA , 85 , 1086 – 1090 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. ) Kunkel , L. M. , Smith , K. D. , Boyer , S. H. , Borgaonkar , D. S. , Wachtel , S. S. , Miller , O. J. , RoyBreg , W. , Jones , H. W. , Jr. and Rary , J. M.Analysis of human Y‐ chromosome‐specific reiterated DNA in chromosome variants . Proc. Natl. Acad. Sci. USA , 74 , 1245 – 1249 ( 1977. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. ) Bradford , M. M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding . Anal. Biochem. , 72 , 248 – 254 ( 1976. ). [DOI] [PubMed] [Google Scholar]
- 19. ) Liu , L. F , and Miller , K. G.Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerase from HeLa cell nuclei . Proc. Natl. Acad. Sci. USA , 78 , 3487 – 3491 ( 1981. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. ) Goelz , S. E. , Hamilton , S. R. and Vogelstein , B.Purification of DNA from formaldehyde fixed and paraffin embedded human tissue . Biochem. Biophys. Res. Commun. , 130 , 118 – 126 ( 1985. ). [DOI] [PubMed] [Google Scholar]
- 21. ) Cohen , J. J. and Duke , R. C.Glucocorticoid activation of a calcium dependent endonuclease in thymocyte nuclei leads to cell death . J. Immunol. , 132 , 38 – 42 ( 1984. ). [PubMed] [Google Scholar]
- 22. ) Duke , R. C. , Cherverank , R. and Cohen , J. J.Endogenous endonuclease‐induced DNA fragmentation: an early event in cell‐mediated cytolysis . Proc. Natl. Acad. Sci. USA , 80 , 6361 – 6365 ( 1983. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. ) Wyllie , A. H.Glucocorticoid‐induced thymocyte apoptosis is associated with endogenous endonuclease activation . Nature. , 284 , 555 – 556 ( 1980. ). [DOI] [PubMed] [Google Scholar]
- 24. ) Wielckens , K. and Delfs , T.Glucocorticoid‐induced cell death and poly [adenosine diphosphate (ADP)‐ribosyl ‐ ation: increased toxicity of dexamethasone on mouse S49. 1 Lymphoma cells with the poly (ADP‐ribosyl)ation inhibitor benzamide . Endocrinology , 119 , 2383 – 2392 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 25. ) Kaufmann , S. H.Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note . Cancer Res. , 49 , 5870 – 5878 ( 1989. ). [PubMed] [Google Scholar]
- 26. ) Barry , M. A. , Behnke , C. A. and Eastman , A.Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia . Biochem. Pharmacol. , 40 , 2353 – 2362 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 27. ) Hsiang , Y‐H. , Lihou , M. G. and Liu , L. F.Arrest of replication forks by drug‐stabilized topoisomerase I‐DNA cleavable complexes as a mechanism of cell killing by camptothecin . Cancer Res. , 49 , 5077 – 5082 ( 1989. ). [PubMed] [Google Scholar]
- 28. ) Flieger , D. , Riethmuller , G. and Ziegler‐Heitbrock , H. W. L.Zn++ inhibits both tumor necrosis factor‐mediated DNA fragmentation and cytolysis . Int. J. Cancer. , 44 , 315 – 319 ( 1989. ). [DOI] [PubMed] [Google Scholar]
- 29. ) Shimizu , T. , Kubota , M. , Tanizawa , A. , Sano , H. , Kasai , Y. , Hashimoto , H. , Akiyama , Y. and Mikawa , H.Inhibition of both etoposide‐induced DNA fragmentation and activation of poly (ADP‐ribose) synthesis by zinc ion . Biochem. Biophys. Res. Commun. , 169 , 1172 – 1177 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 30. ) Martin , S. J. , Lennon , S. V. , Bonham , A. M. and Cotter , T. G.Induction of apoptosis (programmed cell death) in human leukemic HL‐60 cells by inhibition of RNA or protein synthesis . J. Immunol. , 145 , 1859 – 1867 ( 1990. ). [PubMed] [Google Scholar]
- 31. ) Nakamura , T.Action mechanism of antileukemic agents with special reference to nucleic acid metabolism of leukemic cells . Acta Hematol. Jpn. , 45 , 1203 – 1218 ( 1982. ). [PubMed] [Google Scholar]
- 32. ) Ueda , T. , Nakamura , T. , Kagawa , D. , Yamamoto , K. , Uchida , M. , Sasada , M. and Uchino , H.Intracellular distribution of N4‐behenoyl‐l‐β‐D‐arabinofuranosylcytosine in blood cells . Gann , 74 , 445 – 451 ( 1983. ). [PubMed] [Google Scholar]