Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Dec;87(24):9883–9887. doi: 10.1073/pnas.87.24.9883

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

J Thomale 1, N H Huh 1, P Nehls 1, G Eberle 1, M F Rajewsky 1
PMCID: PMC55278  PMID: 2263639

Abstract

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.

Full text

PDF
9883

Selected References

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

  1. Adamkiewicz J., Eberle G., Huh N., Nehls P., Rajewsky M. F. Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies. Environ Health Perspect. 1985 Oct;62:49–55. doi: 10.1289/ehp.856249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aquilina G., Zijno A., Moscufo N., Dogliotti E., Bignami M. Tolerance to methylnitrosourea-induced DNA damage is associated with 6-thioguanine resistance in CHO cells. Carcinogenesis. 1989 Jul;10(7):1219–1223. doi: 10.1093/carcin/10.7.1219. [DOI] [PubMed] [Google Scholar]
  3. Backer J. M., Boerzig M., Weinstein I. B. When do carcinogen-treated 10T1/2 cells acquire the commitment to form transformed foci? Nature. 1982 Sep 30;299(5882):458–460. doi: 10.1038/299458a0. [DOI] [PubMed] [Google Scholar]
  4. Bedell M. A., Lewis J. G., Billings K. C., Swenberg J. A. Cell specificity in hepatocarcinogenesis: preferential accumulation of O6-methylguanine in target cell DNA during continuous exposure to rats to 1,2-dimethylhydrazine. Cancer Res. 1982 Aug;42(8):3079–3083. [PubMed] [Google Scholar]
  5. Beranek D. T., Weis C. C., Swenson D. H. A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis. 1980 Jul;1(7):595–606. doi: 10.1093/carcin/1.7.595. [DOI] [PubMed] [Google Scholar]
  6. Bogden J. M., Eastman A., Bresnick E. A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA. Nucleic Acids Res. 1981 Jul 10;9(13):3089–3103. doi: 10.1093/nar/9.13.3089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Day R. S., 3rd, Ziolkowski C. H., Scudiero D. A., Meyer S. A., Lubiniecki A. S., Girardi A. J., Galloway S. M., Bynum G. D. Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains. Nature. 1980 Dec 25;288(5792):724–727. doi: 10.1038/288724a0. [DOI] [PubMed] [Google Scholar]
  8. Deilhaug T., Myrnes B., Aune T., Krokan H., Haugen A. Differential capacities for DNA repair in Clara cells, alveolar type II cells and macrophages of rabbit lung. Carcinogenesis. 1985 Apr;6(4):661–663. doi: 10.1093/carcin/6.4.661. [DOI] [PubMed] [Google Scholar]
  9. Demple B., Sedgwick B., Robins P., Totty N., Waterfield M. D., Lindahl T. Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(9):2688–2692. doi: 10.1073/pnas.82.9.2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doniger J., Day R. S., DiPaolo J. A. Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents. Proc Natl Acad Sci U S A. 1985 Jan;82(2):421–425. doi: 10.1073/pnas.82.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Druckrey H., Ivanković S., Preussmann R. Teratogenic and carcinogenic effects in the offspring after single injection of ethylnitrosourea to pregnant rats. Nature. 1966 Jun 25;210(5043):1378–1379. doi: 10.1038/2101378a0. [DOI] [PubMed] [Google Scholar]
  12. Ellison K. S., Dogliotti E., Connors T. D., Basu A. K., Essigmann J. M. Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8620–8624. doi: 10.1073/pnas.86.22.8620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Erickson L. C., Laurent G., Sharkey N. A., Kohn K. W. DNA cross-linking and monoadduct repair in nitrosourea-treated human tumour cells. Nature. 1980 Dec 25;288(5792):727–729. doi: 10.1038/288727a0. [DOI] [PubMed] [Google Scholar]
  14. Fong A. T., Rasmussen R. E. DNA ethylation in hamster tissues during subchronic diethylnitrosamine administration and in the hamster trachea after acute diethylnitrosamine administration. Carcinogenesis. 1986 Sep;7(9):1457–1461. doi: 10.1093/carcin/7.9.1457. [DOI] [PubMed] [Google Scholar]
  15. Gerson S. L., Trey J. E., Miller K., Berger N. A. Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues. Carcinogenesis. 1986 May;7(5):745–749. doi: 10.1093/carcin/7.5.745. [DOI] [PubMed] [Google Scholar]
  16. Gibson N. W., Zlotogorski C., Erickson L. C. Specific DNA repair mechanisms may protect some human tumor cells from DNA interstrand crosslinking by chloroethylnitrosoureas but not from crosslinking by other anti-tumor alkylating agents. Carcinogenesis. 1985 Mar;6(3):445–450. doi: 10.1093/carcin/6.3.445. [DOI] [PubMed] [Google Scholar]
  17. Goth R., Rajewsky M. F. Persistence of O6-ethylguanine in rat-brain DNA: correlation with nervous system-specific carcinogenesis by ethylnitrosourea. Proc Natl Acad Sci U S A. 1974 Mar;71(3):639–643. doi: 10.1073/pnas.71.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gräslund A., Jernström B. DNA-carcinogen interaction: covalent DNA-adducts of benzo(a)pyrene 7,8-dihydrodiol 9,10-epoxides studied by biochemical and biophysical techniques. Q Rev Biophys. 1989 Feb;22(1):1–37. [PubMed] [Google Scholar]
  19. Hayakawa H., Koike G., Sekiguchi M. Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA. J Mol Biol. 1990 Jun 20;213(4):739–747. doi: 10.1016/S0022-2836(05)80260-8. [DOI] [PubMed] [Google Scholar]
  20. Huh N. H., Rajewsky M. F. Enzymatic elimination of O6-ethylguanine from the DNA of ethylnitrosourea-exposed normal and malignant rat brain cells grown under cell culture versus in vivo conditions. Int J Cancer. 1988 May 15;41(5):762–766. doi: 10.1002/ijc.2910410521. [DOI] [PubMed] [Google Scholar]
  21. Huh N., Rajewsky M. F. Enzymatic elimination of O6-ethylguanine and stability of O4-ethylthymine in the DNA of malignant neural cell lines exposed to N-ethyl-N-nitrosourea in culture. Carcinogenesis. 1986 Mar;7(3):435–439. doi: 10.1093/carcin/7.3.435. [DOI] [PubMed] [Google Scholar]
  22. Karran P., Lindahl T., Griffin B. Adaptive response to alkylating agents involves alteration in situ of O6-methylguanine residues in DNA. Nature. 1979 Jul 5;280(5717):76–77. doi: 10.1038/280076a0. [DOI] [PubMed] [Google Scholar]
  23. Karran P., Stephenson C., Macpherson P., Cairns-Smith S., Priestley A. Coregulation of the human O6-methylguanine-DNA methyltransferase with two unrelated genes that are closely linked. Cancer Res. 1990 Mar 1;50(5):1532–1537. [PubMed] [Google Scholar]
  24. Kodama K. I., Nakabeppu Y., Sekiguchi M. Cloning and expression of the Bacillus subtilis methyltransferase gene in Escherichia coli ada- cells. Mutat Res. 1989 Sep;218(2):153–163. doi: 10.1016/0921-8777(89)90022-0. [DOI] [PubMed] [Google Scholar]
  25. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  26. Lindahl T., Sedgwick B., Sekiguchi M., Nakabeppu Y. Regulation and expression of the adaptive response to alkylating agents. Annu Rev Biochem. 1988;57:133–157. doi: 10.1146/annurev.bi.57.070188.001025. [DOI] [PubMed] [Google Scholar]
  27. Morimoto K., Tanaka A., Yamaha T., Takahashi A. Alkylation of DNA in F344 rat thymus following administration of 1-n-propyl-1-nitrosourea in vivo and comparison of O6-alkylguanine DNA alkyltransferase activity in thymus from F344 and Long-Evans rats in vitro. Carcinogenesis. 1988 May;9(5):853–856. doi: 10.1093/carcin/9.5.853. [DOI] [PubMed] [Google Scholar]
  28. Morten J. E., Margison G. P. Increased O6-alkylguanine alkyltransferase activity in Chinese hamster V79 cells following selection with chloroethylating agents. Carcinogenesis. 1988 Jan;9(1):45–49. doi: 10.1093/carcin/9.1.45. [DOI] [PubMed] [Google Scholar]
  29. Myrnes B., Giercksky K. E., Krokan H. Interindividual variation in the activity of O6-methyl guanine-DNA methyltransferase and uracil-DNA glycosylase in human organs. Carcinogenesis. 1983 Dec;4(12):1565–1568. doi: 10.1093/carcin/4.12.1565. [DOI] [PubMed] [Google Scholar]
  30. Müller R., Rajewsky M. F. Immunological quantification by high-affinity antibodies of O6-ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea. Cancer Res. 1980 Mar;40(3):887–896. [PubMed] [Google Scholar]
  31. Nehls P., Adamkiewicz J., Rajewsky M. F. Immuno-slot-blot: a highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA. J Cancer Res Clin Oncol. 1984;108(1):23–29. doi: 10.1007/BF00390969. [DOI] [PubMed] [Google Scholar]
  32. Nehls P., Rajewsky M. F. Monoclonal antibody-based immunoassay for the determination of cellular enzymatic activity for repair of specific carcinogen-DNA adducts (O6-alkylguanine). Carcinogenesis. 1990 Jan;11(1):81–87. doi: 10.1093/carcin/11.1.81. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Potter P. M., Wilkinson M. C., Fitton J., Carr F. J., Brennand J., Cooper D. P., Margison G. P. Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli. Nucleic Acids Res. 1987 Nov 25;15(22):9177–9193. doi: 10.1093/nar/15.22.9177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Quade K. Transformation of mammalian cells by avian myelocytomatosis virus and avian erythroblastosis virus. Virology. 1979 Oct 30;98(2):461–465. doi: 10.1016/0042-6822(79)90569-5. [DOI] [PubMed] [Google Scholar]
  36. Rydberg B., Spurr N., Karran P. cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase. cDNA expression in Escherichia coli and gene expression in human cells. J Biol Chem. 1990 Jun 5;265(16):9563–9569. [PubMed] [Google Scholar]
  37. Samson L., Cairns J. A new pathway for DNA repair in Escherichia coli. Nature. 1977 May 19;267(5608):281–283. doi: 10.1038/267281a0. [DOI] [PubMed] [Google Scholar]
  38. Satoh M. S., Huh N. H., Horie Y., Thomale J., Rajewsky M. F., Kuroki T. Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine. Jpn J Cancer Res. 1987 Oct;78(10):1094–1099. [PubMed] [Google Scholar]
  39. Singer B., Bodell W. J., Cleaver J. E., Thomas G. H., Rajewsky M. F., Thon W. Oxygens in DNA are main targets for ethylnitrosourea in normal and xeroderma pigmentosum fibroblasts and fetal rat brain cells. Nature. 1978 Nov 2;276(5683):85–88. doi: 10.1038/276085a0. [DOI] [PubMed] [Google Scholar]
  40. Sklar R., Strauss B. Removal of O6-methylguanine from DNA of normal and xeroderma pigmentosum-derived lymphoblastoid lines. Nature. 1981 Jan 29;289(5796):417–420. doi: 10.1038/289417a0. [DOI] [PubMed] [Google Scholar]
  41. Sukumar S., Notario V., Martin-Zanca D., Barbacid M. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature. 1983 Dec 15;306(5944):658–661. doi: 10.1038/306658a0. [DOI] [PubMed] [Google Scholar]
  42. Swenberg J. A., Dyroff M. C., Bedell M. A., Popp J. A., Huh N., Kirstein U., Rajewsky M. F. O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1692–1695. doi: 10.1073/pnas.81.6.1692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tano K., Shiota S., Collier J., Foote R. S., Mitra S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc Natl Acad Sci U S A. 1990 Jan;87(2):686–690. doi: 10.1073/pnas.87.2.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wiestler O., Kleihues P., Pegg A. E. O6-alkylguanine-DNA alkyltransferase activity in human brain and brain tumors. Carcinogenesis. 1984 Jan;5(1):121–124. doi: 10.1093/carcin/5.1.121. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. de Kok A. J., van Zeeland A. A., Simons J. W., Den Engelse L. Genetic and molecular mechanisms of the in vitro transformation of Syrian hamster embryo cells by the carcinogen N-ethyl-N-nitrosourea II. Correlation of morphological transformation, enhanced fibrinolytic activity, gene mutations, chromosomal alterations and lethality to specific carcinogen-induced DNA lesions. Carcinogenesis. 1985 Nov;6(11):1571–1576. doi: 10.1093/carcin/6.11.1571. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES