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Depletion of O6-alkylguanine-DNA alkyltransferase activity in mammalian tissues and human tumor xenografts in nude mice by treatment with O6-methylguanine

Cancer Chemotherapy and Pharmacology volume 25, pages 103–108 (1989)Cite this article

Summary

We have previously shown that exposure of cells in culture to O6-methylguanine significantly reduces their level of the repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), thus rendering cells more sensitive to the cytotoxic effects of chemotherapeutic chloroethylating agents. Experiments were carried out in mice to determine whether the AGT content of tissues and tumors could be reduced by in vivo treatment with O6-methylguanine. There was a dose-dependent decrease in AGT activity in liver tissues of CD-1 mice to 24% of basal levels after four hourly intraperitoneal injections of O6-methylguanine (110 mg/kg). Although the decline in AGT activity in the liver was reversible, the activity remained at 75% of basal levels for up to 25 h after the final injection. The effect of O6-methylguanine treatment on AGT activity was measured in mouse tissues as well as human colonic carcinoma tumors (HT29 and BE) grown in Swiss athymic nude mice. The activity in the liver, kidney, and spleen of these mice decreased to 33%–35% of control levels, whereas the activity in HT29 tumors was likewise diminished to 25% of control levels after four hourly injections of O6-methylguanine (100 mg/kg). There was no enhancement of the tumoricidal effectiveness of chloroethylating agents on the HT29 tumor after O6-methylguanine treatment, probably due to a disproportionately higher level of AGT in human tissue than in murine tissue. However, these studies suggest that O6-methylguanine can be given in vivo to examine the role of the AGT protein in protecting against the toxic and carcinogenic effects of alkylating agents.

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Abbreviations

AGT:

O6-alkylguanine-DNA alkyltransferase

MeCCNU:

1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea

Clomesone:

2-chloroethyl(methylsulfonyl) methanesulfonate

HPLC:

high-pressure liquid chromatography

References

  1. Aida T, Cheitlin RA, Bodell WJ (1987) Inhibition of O6-alkylguanine-DNA-alkyltransferase activity potentiates cytotoxicity and induction of SCEs in human glioma cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea. Carcinogenesis 8: 1219–1223

    Google Scholar 

  2. Anderson T, DeVita VT, Young RC (1976) BCNU (NSC 409962) in the treatment of advanced Hodgkin's disease: its role in remission induction and maintenance. Cancer Treat Rep 60: 761–767

    Google Scholar 

  3. Balsiger RW, Montgomery JA (1960) Synthesis of potential anticancer agents: XXV. Preparation of 6-alkoxy-2-aminopurines. J Org Chem 25: 1573–1574

    Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72: 248–254

    Google Scholar 

  5. Brent TP (1984) Suppression of cross-link formation in chloroethylnitrosourea-treated DNA by an activity in extracts of human leukemic lymphoblasts. Cancer Res 44: 1887–1892

    Google Scholar 

  6. Dempke W, Nehls P, Wandl U, Soll D, Schmidt CG, Osieka R (1987) Increased cytotoxicity of 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea by pretreatment with O6-methylguanine in resistant but not in sensitive human melanoma cells. J Cancer Res Clin Oncol 113: 387–391

    Google Scholar 

  7. Dolan ME, Corsico CD, Pegg AE (1985) Exposure of HeLa cells to O6-alkylguanines increases sensitivity to the cytotoxic effects of alkylating agents. Biochem Biophys Res Commun 132: 178–185

    Google Scholar 

  8. Dolan ME, Morimoto K, Pegg AE (1985) Reduction of O6-alkylguanine-DNA-alkyltransferase activity in HeLa cells treated with O6-alkylguanines. Cancer Res 45: 6413–6417

    Google Scholar 

  9. Dolan ME, Young GS, Pegg AE (1986) Effect of O6-alkylguanine pretreatment on the sensitivity of human colon tumor cells to the cytotoxic effects of chloroethylating agents. Cancer Res 46: 4500–4504

    Google Scholar 

  10. Dolan ME, Pegg AE, Hora NK, Erickson LC (1988) Effect of O6-methylguanine on DNA interstrand cross-link formation by chloroethylnitrosoureas and 2-chloroethyl(methylsulfonyl)methanesulfonate. Cancer Res 48: 3603–3606

    Google Scholar 

  11. Dolan ME, Scicchitano D, Pegg AE (1988) Use of oligodeoxynucleotides containing O6-alkylguanine for the assay of O6-alkylguanine-DNA-alkyltransferase activity. Cancer Res 48: 1184–1188

    Google Scholar 

  12. Domoradzki J, Pegg AE, Dolan ME, Maher VM, McCormick JJ (1984) Correlation between O6-methylguanine-DNA-methyltransferase activity and resistance of human cells to the cytotoxic and mutagenic effect ofN-methyl-N′-nitro-N-nitrosoguanidine. Carcinogenesis 5: 1641–1647

    Google Scholar 

  13. Erickson LC, Osieka R, Kohn KW (1978) Differential repair of 1-(2-chloroethyl)3-(4-methyl-cyclohexyl)-1-nitrosourea-induced DNA damage in two human colon tumor cell lines. Cancer Res 38: 802–808

    Google Scholar 

  14. Erickson LC, Bradley MO, Ducore JM, Ewig RA, Kohn KW (1980) DNA cross-linking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas. Proc Natl Acad Sci USA 77: 467–471

    Google Scholar 

  15. Erickson LC, Micetich K, Fisher R (1988) Preclinical and clinical experience with drug combinations designed to inhibit DNA repair enzymes. In: Woolley P, Tew K (eds) Mechanisms of drug resistance in neoplastic cells, vol IX. Bristol Myers Cancer Symposium. Academic, New York, pp 173–182

    Google Scholar 

  16. Ewig RA, Kohn KW (1978) DNA-protein cross-linking and DNA interstrand cross-linking by haloethyl-nitrosoureas in L1210 cells. Cancer Res 38: 3197–3203

    Google Scholar 

  17. Gerson SL, Trey JE, Miller K (1988) Potentiation of nitrosourea cytotoxicity in human leukemic cells by inactivation of O6-alkylguanine-DNA alkyltransferase. Cancer Res 48: 1521–1527

    Google Scholar 

  18. Grafstrom RC, Pegg AE, Trump BF, Harris CC (1984) O6-Alkylguanine-DNA alkyltransferase activity in normal human tissues and cells. Cancer Res 44: 2855–2857

    Google Scholar 

  19. Graves RJ, Li BFL, Swann PF (1987) Repair of synthetic oligonucleotides containing O6-methylguanine, O6-ethylguanine and O4-methylthymine, by O6-alkylguanine-DNA-alkyltransferase. In: Bartsch H, O'Neill IK, Schulte-Hermann R (eds) Relevance ofN-nitroso compounds to human cancer: exposures and mechanisms (IARC Scientific Publication 84). Oxford Press, Oxford pp 41–43

    Google Scholar 

  20. Karran P, Williams SA (1985) The cytotoxic and mutagenic effects of alkylating agents on human lymphoid cells are caused by different DNA lesions. Carcinogenesis 6: 789–792

    Google Scholar 

  21. Kohn KW (1977) Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas. Cancer Res 37: 1450–1454

    Google Scholar 

  22. Labarca C, Paigen K (1980) A simple, rapid and sensitive DNA procedure. Anal Biochem 102: 344–352

    Google Scholar 

  23. Likhachev A, Anisimov V, Zhukovskaya N, Petrov A (1988) Inactivation of O6-alkylguanine-DNA alkyltransferase in the rat liver due to an intraperitoneal administration of methylated DNA. Carcinogenesis 9: 1139–1142

    Google Scholar 

  24. Marcus-Segura CJ (1988) Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal Biochem 172: 289–295

    Google Scholar 

  25. Mehta JR, Ludlum DB, Renard A, Verly W (1981) Repair of O6-ethylguanine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group to an acceptor protein. Proc Natl Acad Sci USA 78: 6766–6770

    Google Scholar 

  26. Morimoto K, Dolan ME, Scicchitano D, Pegg AE (1985) Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver andE. coli. Carcinogenesis 6: 1027–1031

    Google Scholar 

  27. Osieka R, Houchens DP, Goldin A, Johnson RK (1977) Chemotherapy of human colon cancer xenografts in athymic nude mice. Cancer 40: 2640–2650

    Google Scholar 

  28. Pegg AE (1983) Alkylation and subsequent repair of DNA after exposure to dimethylnitrosamine and related carcinogens. Rev Biochem Toxicol 5: 83–133

    Google Scholar 

  29. Pegg AE, Roberfroid M, Bahr C von, Foote RS, Mitra S, Bresil H, Likachev A, Montesano R (1982) Removal of O6-methylguanine from DNA by human liver fractions. Proc Natl Acad Sci USA 79: 5162–5165

    Google Scholar 

  30. Pegg AE, Wiest L, Foote RS, Mitra S, Perry W (1983) Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem 258: 2327–2333

    Google Scholar 

  31. Pegg AE, Scicchitano D, Dolan ME (1984) Comparison of the rates of repair of O6-alkylguanines in DNA by rat liver and bacterial O6-alkylguanine-DNA alkyltransferase. Cancer Res 44: 3806–3811

    Google Scholar 

  32. Robins P, Harris AL, Goldsmith I, Lindahl T (1983) Cross-linking of DNA induced by chloroethylnitrosourea is prevented by O6-methylguanine-DNA methyltransferase. Nucleic Acids Res 11: 7743–7758

    Google Scholar 

  33. Scicchitano D, Jones RA, Kuzmich S, Gaffney B, Lasko DD, Essigmann JM, Pegg AE (1986) Repair of oligodeoxy-nucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase. Carcinogenesis 7: 1383–1386

    Google Scholar 

  34. Scudiero DA, Meyer SA, Clatterbuck BE, Mattern MR, Ziolkowski CHJ, Day RS (1984) Sensitivity of human cell strains having different abilities to repair O6-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas. Cancer Res 44: 2467–2474

    Google Scholar 

  35. Stein CA, Cohen JS (1988) Oligodeoxynucleotides as inhibitors of gene expression: review. Cancer Res 48: 2659–2668

    Google Scholar 

  36. Thielmann HW, Edler L, Müller N, Eisenbrand G (1987) 6-Methylguanine and 6-methylguanosine inhibit colony forming ability in malignant xeroderma pigmentosum cell line but not in other xeroderma pigmentosum and normal human fibroblast strains after treatment with 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea. J Cancer Res Clin Oncol 113: 67–72

    Google Scholar 

  37. Tong WP, Kirk MC, Ludlum DB (1982) Formation of the cross-link 1[N3-deoxycytidyl],2-N1-deoxyguanosinyl]-ethane in DNA treated withN,N1-bis(2-chloroethyl)-N-nitrosourea. Cancer Res 42: 3102–3105

    Google Scholar 

  38. Weinkam RJ, Lin H-S (1982) Chloroethylnitrosourea cancer chemotherapeutic agents. Adv Pharmacol Chemother 19: 1–33

    Google Scholar 

  39. Wu RS, Hurst-Calderone S, Kohn KW (1987) Measurement of O6-alkylguanine-DNA alkyltransferase activity in human cell and tumor tissues by restriction endonuclease inhibition. Cancer Res 47: 6229–6235

    Google Scholar 

  40. Yarosh DB (1985) The role of O6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mut Res 145: 1–16

    Google Scholar 

  41. Yarosh DB, Hurst-Calderone S, Babich MA, Day RS (1986) Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base. Cancer Res 46: 1663–1668

    Google Scholar 

  42. Zlotogorski C, Erickson LC (1984) Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts. Carcinogenesis 5: 83–87

    Google Scholar 

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Author information

Affiliations

  1. Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, 17033, Hershey, Pennsylvania, USA

    M. Eileen Dolan, Gregory L. Larkin & Anthony E. Pegg

  2. Divisions of Endocrinology and Urology and Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, 17033, Hershey, Pennsylvania, USA

    Hugh F. English

Authors
  1. M. Eileen Dolan
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  2. Gregory L. Larkin
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  3. Hugh F. English
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  4. Anthony E. Pegg
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Dolan, M.E., Larkin, G.L., English, H.F. et al. Depletion of O6-alkylguanine-DNA alkyltransferase activity in mammalian tissues and human tumor xenografts in nude mice by treatment with O6-methylguanine. Cancer Chemother. Pharmacol. 25, 103–108 (1989). https://doi.org/10.1007/BF00692348

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  • DOI: https://doi.org/10.1007/BF00692348

Keywords

  • Nude Mouse
  • Control Level
  • Tumor Xenograft
  • Mammalian Tissue
  • Mouse Tissue