Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1999 Mar;79(9-10):1332–1339. doi: 10.1038/sj.bjc.6690215

Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2-chloroethyl-1-nitrosourea in mice

M C Bibby 1, M J Thompson 1,2, J A Rafferty 3, G P Margison 3, R S McElhinney 4
PMCID: PMC2362701  PMID: 10188873

Abstract

Previous studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O6-alkylguanine-DNA-alkyltransferase (ATase) depletion by the pseudosubstrate O6-benzylguanine (BG) on the anti-tumour activity and normal tissue toxicity in mice of three such molecular combinations, in comparison with BCNU. When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. © 1999 Cancer Research Campaign

Keywords: O6-benzylguanine, nitrosoureas, anti-tumour, tissue toxicity, mouse

Full Text

The Full Text of this article is available as a PDF (384.9 KB).

Selected References

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

  1. Baer J. C., Freeman A. A., Newlands E. S., Watson A. J., Rafferty J. A., Margison G. P. Depletion of O6-alkylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumour cells. Br J Cancer. 1993 Jun;67(6):1299–1302. doi: 10.1038/bjc.1993.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bibby M. C., Double J. A., Wahed I. A., Hirbawi N., Baker T. G. The logistics of broader pre-clinical evaluation of potential anti-cancer agents with reference to anti-tumour activity and toxicity of mitozolomide. Br J Cancer. 1988 Aug;58(2):139–143. doi: 10.1038/bjc.1988.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brennand J., Margison G. P. Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6292–6296. doi: 10.1073/pnas.83.17.6292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brent T. P., von Wronski M. A., Edwards C. C., Bromley M., Margison G. P., Rafferty J. A., Pegram C. N., Bigner D. D. Identification of nitrosourea-resistant human rhabdomyosarcomas by in situ immunostaining of O6-methylguanine-DNA methyltransferase. Oncol Res. 1993;5(2):83–86. [PubMed] [Google Scholar]
  6. Bronstein S. M., Hooth M. J., Swenberg J. A., Skopek T. R. Modulation of ethylnitrosourea-induced toxicity and mutagenicity in human cells by O6-benzylguanine. Cancer Res. 1992 Jul 15;52(14):3851–3856. [PubMed] [Google Scholar]
  7. Chinnasamy N., Rafferty J. A., Hickson I., Ashby J., Tinwell H., Margison G. P., Dexter T. M., Fairbairn L. J. O6-benzylguanine potentiates the in vivo toxicity and clastogenicity of temozolomide and BCNU in mouse bone marrow. Blood. 1997 Mar 1;89(5):1566–1573. [PubMed] [Google Scholar]
  8. Crone T. M., Pegg A. E. A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine. Cancer Res. 1993 Oct 15;53(20):4750–4753. [PubMed] [Google Scholar]
  9. Dolan M. E., Chae M. Y., Pegg A. E., Mullen J. H., Friedman H. S., Moschel R. C. Metabolism of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase. Cancer Res. 1994 Oct 1;54(19):5123–5130. [PubMed] [Google Scholar]
  10. Dolan M. E., Corsico C. D., Pegg A. E. Exposure of HeLa cells to 0(6)-alkylguanines increases sensitivity to the cytotoxic effects of alkylating agents. Biochem Biophys Res Commun. 1985 Oct 15;132(1):178–185. doi: 10.1016/0006-291x(85)91004-6. [DOI] [PubMed] [Google Scholar]
  11. Dolan M. E., Moschel R. C., Pegg A. E. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5368–5372. doi: 10.1073/pnas.87.14.5368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dolan M. E., Pegg A. E. O6-benzylguanine and its role in chemotherapy. Clin Cancer Res. 1997 Jun;3(6):837–847. [PubMed] [Google Scholar]
  13. Dolan M. E., Stine L., Mitchell R. B., Moschel R. C., Pegg A. E. Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea. Cancer Commun. 1990;2(11):371–377. doi: 10.3727/095535490820873985. [DOI] [PubMed] [Google Scholar]
  14. Down J. D., Boudewijn A., Dillingh J. H., Fox B. W., Ploemacher R. E. Relationships between ablation of distinct haematopoietic cell subsets and the development of donor bone marrow engraftment following recipient pretreatment with different alkylating drugs. Br J Cancer. 1994 Oct;70(4):611–616. doi: 10.1038/bjc.1994.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dumenco L. L., Allay E., Norton K., Gerson S. L. The prevention of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA alkyltransferase. Science. 1993 Jan 8;259(5092):219–222. doi: 10.1126/science.8421782. [DOI] [PubMed] [Google Scholar]
  16. Fairbairn L. J., Watson A. J., Rafferty J. A., Elder R. H., Margison G. P. O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide. Exp Hematol. 1995 Feb;23(2):112–116. [PubMed] [Google Scholar]
  17. Gerson S. L., Trey J. E., Miller K. Potentiation of nitrosourea cytotoxicity in human leukemic cells by inactivation of O6-alkylguanine-DNA alkyltransferase. Cancer Res. 1988 Mar 15;48(6):1521–1527. [PubMed] [Google Scholar]
  18. Hendriks H. R., Pizao P. E., Berger D. P., Kooistra K. L., Bibby M. C., Boven E., Dreef-van der Meulen H. C., Henrar R. E., Fiebig H. H., Double J. A. EO9: a novel bioreductive alkylating indoloquinone with preferential solid tumour activity and lack of bone marrow toxicity in preclinical models. Eur J Cancer. 1993;29A(6):897–906. doi: 10.1016/s0959-8049(05)80434-4. [DOI] [PubMed] [Google Scholar]
  19. Hendriks H. R., Pizao P. E., Berger D. P., Kooistra K. L., Bibby M. C., Boven E., Dreef-van der Meulen H. C., Henrar R. E., Fiebig H. H., Double J. A. EO9: a novel bioreductive alkylating indoloquinone with preferential solid tumour activity and lack of bone marrow toxicity in preclinical models. Eur J Cancer. 1993;29A(6):897–906. doi: 10.1016/s0959-8049(05)80434-4. [DOI] [PubMed] [Google Scholar]
  20. Hickson I., Fairbairn L. J., Chinnasamy N., Dexter T. M., Margison G. P., Rafferty J. A. Protection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA alkyltransferase. Gene Ther. 1996 Oct;3(10):868–877. [PubMed] [Google Scholar]
  21. Jelinek J., Kleibl K., Dexter T. M., Margison G. P. Transfection of murine multi-potent haemopoietic stem cells with an E. coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents. Carcinogenesis. 1988 Jan;9(1):81–87. doi: 10.1093/carcin/9.1.81. [DOI] [PubMed] [Google Scholar]
  22. Jelínek J., Fairbairn L. J., Dexter T. M., Rafferty J. A., Stocking C., Ostertag W., Margison G. P. Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase. Blood. 1996 Mar 1;87(5):1957–1961. [PubMed] [Google Scholar]
  23. Kurpad S. N., Dolan M. E., McLendon R. E., Archer G. E., Moschel R. C., Pegg A. E., Bigner D. D., Friedman H. S. Intraarterial O6-benzylguanine enables the specific therapy of nitrosourea-resistant intracranial human glioma xenografts in athymic rats with 1,3-bis(2-chloroethyl)-1-nitrosourea. Cancer Chemother Pharmacol. 1997;39(4):307–316. doi: 10.1007/s002800050577. [DOI] [PubMed] [Google Scholar]
  24. Lee S. M., Thatcher N., Crowther D., Margison G. P. In vivo depletion of O6-alkylguanine-DNA-alkyltransferase in lymphocytes and melanoma of patients treated with CB 10-277, a new DTIC analogue. Cancer Chemother Pharmacol. 1992;31(3):240–246. doi: 10.1007/BF00685554. [DOI] [PubMed] [Google Scholar]
  25. Lee S. M., Thatcher N., Margison G. P. O6-alkylguanine-DNA alkyltransferase depletion and regeneration in human peripheral lymphocytes following dacarbazine and fotemustine. Cancer Res. 1991 Jan 15;51(2):619–623. [PubMed] [Google Scholar]
  26. Loadman P. M., Matthew A. M., McCormick J. E., McElhinney R. S., Bibby M. C. Pharmacokinetics and anti-tumour activity of B.4152, a reactive molecular combination of 5-fluorouracil and N-(2-chloroethyl)-N-nitrosourea, and its uracil analogue. Anticancer Drug Des. 1996 Mar;11(2):117–128. [PubMed] [Google Scholar]
  27. Loktionova N. A., Pegg A. E. Point mutations in human O6-alkylguanine-DNA alkyltransferase prevent the sensitization by O6-benzylguanine to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea. Cancer Res. 1996 Apr 1;56(7):1578–1583. [PubMed] [Google Scholar]
  28. Lord B. I., Marshall E., Woolford L. B., Hunter M. G. BB-10010/MIP-1 alpha in vivo maintains haemopoietic recovery following repeated cycles of sublethal irradiation. Br J Cancer. 1996 Oct;74(7):1017–1022. doi: 10.1038/bjc.1996.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matthew A. M., Phillips R. M., Loadman P. M., Bibby M. C. Pre-clinical evaluation of a novel chloroethylating agent, Clomesone. Br J Cancer. 1993 Mar;67(3):441–446. doi: 10.1038/bjc.1993.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McElhinney R. S., McCormick J. E., Bibby M. C., Double J. A., Atassi G., Dumont P., Pratesi G., Radacic M. Nucleoside analogues: 7. Effect on colon, breast and lung tumours in mice of 5-fluorouracil/nitrosourea molecular combinations incorporating alkoxy and oxidized sulphur functions. Anticancer Drug Des. 1989 Mar;3(4):255–269. [PubMed] [Google Scholar]
  31. McElhinney R. S., McCormick J. E., Bibby M. C., Double J. A., Atassi G., Dumont P., Pratesi G., Radacic M. Nucleoside analogues: 8. Some isomers of B.3839, the original 5-fluorouracil/nitrosourea molecular combination, and their effect on colon, breast and lung tumours in mice. Anticancer Drug Des. 1989 Jun;4(1):1–20. [PubMed] [Google Scholar]
  32. Mitchell R. B., Moschel R. C., Dolan M. E. Effect of O6-benzylguanine on the sensitivity of human tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea and on DNA interstrand cross-link formation. Cancer Res. 1992 Mar 1;52(5):1171–1175. [PubMed] [Google Scholar]
  33. Moritz T., Mackay W., Glassner B. J., Williams D. A., Samson L. Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo. Cancer Res. 1995 Jun 15;55(12):2608–2614. [PubMed] [Google Scholar]
  34. Nakatsuru Y., Matsukuma S., Nemoto N., Sugano H., Sekiguchi M., Ishikawa T. O6-methylguanine-DNA methyltransferase protects against nitrosamine-induced hepatocarcinogenesis. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6468–6472. doi: 10.1073/pnas.90.14.6468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Naundorf H., Rewasowa E. C., Fichtner I., Büttner B., Becker M., Görlich M. Characterization of two human mammary carcinomas, MT-1 and MT-3, suitable for in vivo testing of ether lipids and their derivatives. Breast Cancer Res Treat. 1992;23(1-2):87–95. doi: 10.1007/BF01831480. [DOI] [PubMed] [Google Scholar]
  36. Newlands E. S., Blackledge G. R., Slack J. A., Rustin G. J., Smith D. B., Stuart N. S., Quarterman C. P., Hoffman R., Stevens M. F., Brampton M. H. Phase I trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856). Br J Cancer. 1992 Feb;65(2):287–291. doi: 10.1038/bjc.1992.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nusbaum N. J., Abraham T. Combination antiretroviral chemotherapy: a potential strategy in AIDS-related malignancy. Anticancer Drugs. 1996 Jan;7(1):109–114. [PubMed] [Google Scholar]
  38. O'Reilly S. M., Newlands E. S., Glaser M. G., Brampton M., Rice-Edwards J. M., Illingworth R. D., Richards P. G., Kennard C., Colquhoun I. R., Lewis P. Temozolomide: a new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours. Eur J Cancer. 1993;29A(7):940–942. doi: 10.1016/s0959-8049(05)80198-4. [DOI] [PubMed] [Google Scholar]
  39. Patchen M. L. Amifostine plus granulocyte colony-stimulating factor therapy enhances recovery from supralethal radiation exposures: preclinical experience in animals models. Eur J Cancer. 1995;31A Suppl 1:S17–S21. doi: 10.1016/0959-8049(95)00147-b. [DOI] [PubMed] [Google Scholar]
  40. Pegg A. E., Boosalis M., Samson L., Moschel R. C., Byers T. L., Swenn K., Dolan M. E. Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry. 1993 Nov 16;32(45):11998–12006. doi: 10.1021/bi00096a009. [DOI] [PubMed] [Google Scholar]
  41. Pegg A. E., Byers T. L. Repair of DNA containing O6-alkylguanine. FASEB J. 1992 Mar;6(6):2302–2310. doi: 10.1096/fasebj.6.6.1544541. [DOI] [PubMed] [Google Scholar]
  42. Pegg A. E., Dolan M. E. Properties and assay of mammalian O6-alkylguanine-DNA alkyltransferase. Pharmacol Ther. 1987;34(2):167–179. doi: 10.1016/0163-7258(87)90010-6. [DOI] [PubMed] [Google Scholar]
  43. Pegg A. E. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 1990 Oct 1;50(19):6119–6129. [PubMed] [Google Scholar]
  44. Rafferty J. A., Hickson I., Chinnasamy N., Lashford L. S., Margison G. P., Dexter T. M., Fairbairn L. J. Chemoprotection of normal tissues by transfer of drug resistance genes. Cancer Metastasis Rev. 1996 Sep;15(3):365–383. doi: 10.1007/BF00046348. [DOI] [PubMed] [Google Scholar]
  45. Reese J. S., Koç O. N., Lee K. M., Liu L., Allay J. A., Phillips W. P., Jr, Gerson S. L. Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to O6-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14088–14093. doi: 10.1073/pnas.93.24.14088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Siemann D. W., Beyers K. L. In vivo therapeutic potential of combination thiol depletion and alkylating chemotherapy. Br J Cancer. 1993 Dec;68(6):1071–1079. doi: 10.1038/bjc.1993.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. TILL J. E., McCULLOCH E. A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res. 1961 Feb;14:213–222. [PubMed] [Google Scholar]
  48. Thompson M. J., Abdhul-Rahmann S., Baker T. G., Bibby M. C. Potentiation of testicular cytotoxicity by the alkyltransferase inhibitor O6 benzylguanine and the 5-fluorouracil/N-(2-chloroethyl)-N-nitrosourea molecular combination, B.4152. Reprod Toxicol. 1996 Jan-Feb;10(1):71–77. doi: 10.1016/0890-6238(95)02020-9. [DOI] [PubMed] [Google Scholar]
  49. Walker M. C., Masters J. R., Margison G. P. O6-alkylguanine-DNA-alkyltransferase activity and nitrosourea sensitivity in human cancer cell lines. Br J Cancer. 1992 Nov;66(5):840–843. doi: 10.1038/bjc.1992.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wedge S. R., Newlands E. S. O6-benzylguanine enhances the sensitivity of a glioma xenograft with low O6-alkylguanine-DNA alkyltransferase activity to temozolomide and BCNU. Br J Cancer. 1996 May;73(9):1049–1052. doi: 10.1038/bjc.1996.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wedge S. R., Porteus J. K., May B. L., Newlands E. S. Potentiation of temozolomide and BCNU cytotoxicity by O(6)-benzylguanine: a comparative study in vitro. Br J Cancer. 1996 Feb;73(4):482–490. doi: 10.1038/bjc.1996.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yarosh D. B. The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mutat Res. 1985 Jan-Mar;145(1-2):1–16. doi: 10.1016/0167-8817(85)90034-3. [DOI] [PubMed] [Google Scholar]
  53. von Hofe E., Fairbairn L., Margison G. P. Relationship between O6-alkylguanine-DNA alkyltransferase activity and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation, transformation, and cytotoxicity in C3H/10T1/2 cells expressing exogenous alkyltransferase genes. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11199–11203. doi: 10.1073/pnas.89.23.11199. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES