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. 2005 Aug 19;95(5):454–458. doi: 10.1111/j.1349-7006.2004.tb03231.x

DNA intrastrand cross‐link at the 5′‐GA‐3′ sequence formed by busulfan and its role in the cytotoxic effect

Takuya Iwamoto 1,2, Yusuke Hiraku 1, Shinji Oikawa 1, Hideki Mizutani 1,2, Michio Kojima 2, Shosuke Kawanishi 1,✉
PMCID: PMC11158704  PMID: 15132775

Abstract

Busulfan (1,4‐butanediol dimethanesulfonate) has been used widely for the treatment of patients with chronic myelogenous leukemia. Busulfan is bifunctional and thus may effectively induce DNA damage, which may play an important role in the cytotoxicity. In this study, we compared the cytotoxicity of bifunctional busulfan with that of monofunctional ethyl methanesulfonate (EMS) in human promyelocytic leukemia HL‐60 cells. Busulfan showed a significant inhibitory effect on cell growth, whereas the cells grew in the presence of EMS. To clarify the mechanism of cytotoxicity of busulfan, we investigated DNA damage induced by busulfan using 32P‐5′‐end‐labeled DNA fragments obtained from the human p16 tumor suppressor gene. Busulfan induced DNA damage dose‐dependently, whereas EMS caused little DNA damage. DNA‐sequencing experiments using piperidine and 3‐methyladenine DNA glycosylase indicated that busulfan caused double‐base lesions mainly at 5′‐GA‐3′and, to a lesser extent, at 5′‐GG‐3’sequences. Time of flight mass spectrometry confirmed that busulfan forms an intrastrand cross‐link at the 5′‐GA‐3’sequence, in addition to mono‐alkylation. The mechanism and the role of cross‐linking at the 5′‐GA‐3’sequence are discussed in relation to the cytotoxicity induced by busulfan.

References

  • 1. Kohn KW. Beyond DNA cross‐linking: history and prospects of DNA‐targeted cancer treatment‐fifteenth Bruce F. Cain Memorial Award Lecture. Cancer Res 1996; 56: 5533–46. [PubMed] [Google Scholar]
  • 2. Hurley LH. DNA and its associated processes as targets for cancer therapy. Nat Rev Cancer 2002; 2: 188–200. [DOI] [PubMed] [Google Scholar]
  • 3. Newbold RF, Warren W, Medcalf AS, Amos J. Mutagenicity of carcinogenic methylating agents is associated with a specific DNA modification. Nature 1980; 283: 596–9. [DOI] [PubMed] [Google Scholar]
  • 4. Lawley PD, Phillips DH. DNA adducts from chemotherapeutic agents. Mutat Res 1996; 355: 13–40. [DOI] [PubMed] [Google Scholar]
  • 5. Brookes P, Lawley PD. Reaction of mustard gas with nucleic acids in vitro and in vivo. Biochem J 1960; 77: 478–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Kohn KW, Hartley JA, Mattes WB. Mechanisms of DNA sequence selective alkylation of guanine‐N7 positions by nitrogen mustards. Nucleic Acids Res 1987; 15: 10531–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Buggia I, Locatelli F, Regazzi MB, Zecca, M. Busulfan. Ann Pharmacother 1994; 28: 1055–62. [DOI] [PubMed] [Google Scholar]
  • 8. Westerhof GR, Ploemacher RE, Boudewijn A, Blokland I, Dillingh JH, McGown AT, Hadfield JA, Dawson MJ, Down JD. Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor‐type chimerism in murine bone marrow transplant recipients. Cancer Res 2000; 60: 5470–8. [PubMed] [Google Scholar]
  • 9. Brookes P, Lawley PD. The reactions of mono‐ and difunctional alkylating agents with nucleic acids. Biochem J 1960; 80: 496–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Tong WP, Ludlum DB. Crosslinking of DNA by busulfan. Formation of diguanyl derivatives. Biochim Biophys Acta 1980; 608: 174–81. [DOI] [PubMed] [Google Scholar]
  • 11. Ponti M, Souhami RL, Fox BW, Hartley JA. DNA interstrand crosslinking and sequence selectivity of dimethanesulphonates. Br J Cancer 1991; 63: 743–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Serrano M, Hannon GJ, Beach DA. A new regulatory motif in cell‐cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993; 366: 704–7. [DOI] [PubMed] [Google Scholar]
  • 13. Oikawa S, Murakami K, Kawanishi S. Oxidative damage to cellular and isolated DNA by homocysteine: implications for carcinogenesis. Oncogene 2003; 22: 3530–8. [DOI] [PubMed] [Google Scholar]
  • 14. Oikawa S, Kawanishi S. Detection of DNA damage and analysis of its site‐specificity. In: Taniguchi N, Gutteridge JMC, editors. Experimental protocols for reactive oxygen and nitrogen species. New York : Oxford University Press; 2000. p. 229–35. [Google Scholar]
  • 15. Maxam AM, Gilbert W. Sequencing end‐labeled DNA with base‐specific chemical cleavages. Methods Enzymol 1980; 65: 499–560. [DOI] [PubMed] [Google Scholar]
  • 16. Hemminki K, Ludlum DB. Covalent modification of DNA by antineoplastic agents. J Natl Cancer Inst 1984; 73: 1021–8. [PubMed] [Google Scholar]
  • 17. Seker H, Bertram B, Burkle A, Kaina B, Pohl J, Koepsell H, Wiesser M. Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent. Br J Cancer 2000; 82: 629–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Osborne MR, Wilman DE, Lawley PD. Alkylation of DNA by the nitrogen mustard bis(2‐chloroethyl)methylamine. Chem Res Toxicol 1995; 8: 316–20. [DOI] [PubMed] [Google Scholar]
  • 19. Bedford P, Fox BW. DNA‐DNA interstrand crosslinking by dimethanesulphonic acid esters. Correlation with cytotoxicity and antitumour activity in the Yoshida lymphosarcoma model and relationship to chain length. Biochem Pharmacol 1983; 32: 2297–301. [DOI] [PubMed] [Google Scholar]
  • 20. Dosanjh MK, Roy R, Mitra S, Singer B. 1,N6‐Ethenoadenine is preferred over 3‐methyladenine as substrate by a cloned human N‐methylpurine‐DNA glycosylase (3‐methyladenine‐DNA glycosylase). Biochemistry 1994; 33: 1624–8. [DOI] [PubMed] [Google Scholar]
  • 21. Mattes WB, Lee CS, Laval J, O'Connor TR. Excision of DNA adducts of nitrogen mustards by bacterial and mammalian 3‐methyladenine‐DNA glycosylases. Carcinogenesis 1996; 17: 643–8. [DOI] [PubMed] [Google Scholar]
  • 22. Roy R, Kennel SJ, Mitra S. Distinct substrate preference of human and mouse N‐methylpurine‐DNA glycosylases. Carcinogenesis 1996; 17: 2177–82. [DOI] [PubMed] [Google Scholar]
  • 23. Saparbaev M, Laval J. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc Natl Acad Sci USA 1994; 91: 5873–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Saparbaev M, Kleibl K, Laval J. Escherichia coli, Saccharomyces cerevisiae, rat and human 3‐methyladenine DNA glycosylases repair 1,N6‐ethenoadenine when present in DNA. Nucleic Acids Res 1995; 23: 3750–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Bouziane M, Miao F, Ye N, Holmquist G, Chyzak G, O'Connor TR. Repair of DNA alkylation damage. Acta Biochim Pol 1998; 45: 191–202. [PubMed] [Google Scholar]
  • 26. Westerhof GR, Down JD, Blokland I, Wood M, Boudewijn A, Watson AJ, McGown AT, Ploemacher RE, Margison GP. O6‐Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells. Exp Hematol 2001; 29: 633–8. [DOI] [PubMed] [Google Scholar]
  • 27. Je KH, Son JK, O'Connor TR, Lee CS. Hepsulfam induced DNA adducts and its excision repair by bacterial and mammalian 3‐methyladenine DNA glycosylases. Mol Cells 1998; 8: 691–7. [PubMed] [Google Scholar]
  • 28. Shiner AC, Newbold RF, Cooper CS. Morphological transformation of immortalized hamster dermal fibroblasts following treatment with simple alkylating carcinogens. Carcinogenesis 1988; 9: 1701–9. [DOI] [PubMed] [Google Scholar]
  • 29. Tada‐Oikawa S, Oikawa S, Kawanishi M, Yamada M, Kawanishi S. Generation of hydrogen peroxide precedes loss of mitochondrial membrane potential during DNA alkylation‐induced apoptosis. FEBS Lett 1999; 442: 65–9. [DOI] [PubMed] [Google Scholar]

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