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
. 1993 Jul 15;90(14):6468–6472. doi: 10.1073/pnas.90.14.6468

O6-methylguanine-DNA methyltransferase protects against nitrosamine-induced hepatocarcinogenesis.

Y Nakatsuru 1, S Matsukuma 1, N Nemoto 1, H Sugano 1, M Sekiguchi 1, T Ishikawa 1
PMCID: PMC46953  PMID: 8341657

Abstract

We previously generated transgenic C3H/HeN mice by introducing the Escherichia coli O6-methylguanine-DNA methyltransferase (MGMT, DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC2.1.1.63) gene, ada, attached to the Chinese hamster metallothionein I gene promoter. One transgenic mouse line expressing both ada-specific mRNA and Ada protein could be propagated over many generations in a homozygous state with respect to the integrated DNA. Liver extracts from transgenic homozygous mice have consistently demonstrated about 3 times the control activity of normal mice. Furthermore, in the transgenic homozygotes treated with ZnSO4, activity is increased to 6-8 times the normal level in mice and is equivalent to that for man. To examine whether these increased levels of MGMT activity can actually decrease the susceptibility of animals to N-nitroso compounds, we studied liver carcinogenesis in our transgenic mice expressing high amounts of MGMT. Groups of transgenic and nontransgenic mice, each comprising about 200 suckling animals (14 +/- 1 days old), were divided each into eight subgroups, providing paired groups of transgenic and nontransgenic mice. They received an i.p. injection of ZnSO4 to induce MGMT, and 10 hr thereafter were given an i.p. injection of either dimethylnitrosamine or diethylnitrosamine. Liver tumor development was quantitatively assessed at 7-11 months. Here, we report statistically significant reduction of tumor formation in transgenic mice of four of the six paired groups that received treatment. The remaining two demonstrated results in line with dose dependence. Therefore, our data indicate that MGMT can indeed protect animals from low-dose exposure to environmental alkylating carcinogens.

Full text

PDF
6471

Images in this article

6470Fig. 2
on p.6470

Selected References

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

  1. Belinsky S. A., Foley J. F., White C. M., Anderson M. W., Maronpot R. R. Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 1990 Jun 15;50(12):3772–3780. [PubMed] [Google Scholar]
  2. Brent T. P., Dolan M. E., Fraenkel-Conrat H., Hall J., Karran P., Laval L., Margison G. P., Montesano R., Pegg A. E., Potter P. M. Repair of O-alkylpyrimidines in mammalian cells: a present consensus. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1759–1762. doi: 10.1073/pnas.85.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bronstein S. M., Skopek T. R., Swenberg J. A. Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells. Cancer Res. 1992 Apr 1;52(7):2008–2011. [PubMed] [Google Scholar]
  4. Cleaver J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652–656. doi: 10.1038/218652a0. [DOI] [PubMed] [Google Scholar]
  5. Drinkwater N. R., Ginsler J. J. Genetic control of hepatocarcinogenesis in C57BL/6J and C3H/HeJ inbred mice. Carcinogenesis. 1986 Oct;7(10):1701–1707. doi: 10.1093/carcin/7.10.1701. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Dumenco L. L., Arce C., Norton K., Yun J., Wagner T., Gerson S. L. Enhanced repair of O6-methylguanine DNA adducts in the liver of transgenic mice expressing the ada gene. Cancer Res. 1991 Jul 1;51(13):3391–3398. [PubMed] [Google Scholar]
  8. Fan C. Y., Potter P. M., Rafferty J., Watson A. J., Cawkwell L., Searle P. F., O'Connor P. J., Margison G. P. Expression of a human O6-alkylguanine-DNA-alkyltransferase cDNA in human cells and transgenic mice. Nucleic Acids Res. 1990 Oct 11;18(19):5723–5727. doi: 10.1093/nar/18.19.5723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foote R. S., Mitra S., Pal B. C. Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichia coli. Biochem Biophys Res Commun. 1980 Nov 28;97(2):654–659. doi: 10.1016/0006-291x(80)90314-9. [DOI] [PubMed] [Google Scholar]
  10. Frei J. V., Swenson D. H., Warren W., Lawley P. D. Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography. Biochem J. 1978 Sep 15;174(3):1031–1044. doi: 10.1042/bj1741031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Hall J., Brésil H., Montesano R. O6-Alkylguanine DNA alkyltransferase activity in monkey, human and rat liver. Carcinogenesis. 1985 Feb;6(2):209–211. doi: 10.1093/carcin/6.2.209. [DOI] [PubMed] [Google Scholar]
  14. Klaunig J. E., Weghorst C. M., Pereira M. A. Effect of phenobarbital on diethylnitrosamine and dimethylnitrosamine induced hepatocellular tumors in male B6C3F1 mice. Cancer Lett. 1988 Sep-Oct;42(1-2):133–139. doi: 10.1016/0304-3835(88)90250-9. [DOI] [PubMed] [Google Scholar]
  15. Lim I. K., Dumenco L. L., Yun J., Donovan C., Warman B., Gorodetzkaya N., Wagner T. E., Clapp D. W., Hanson R. W., Gerson S. L. High level, regulated expression of the chimeric P-enolpyruvate carboxykinase (GTP)-bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene in transgenic mice. Cancer Res. 1990 Mar 15;50(6):1701–1708. [PubMed] [Google Scholar]
  16. Lindahl T. DNA repair enzymes. Annu Rev Biochem. 1982;51:61–87. doi: 10.1146/annurev.bi.51.070182.000425. [DOI] [PubMed] [Google Scholar]
  17. Matsukuma S., Nakatsuru Y., Nakagawa K., Utakoji T., Sugano H., Kataoka H., Sekiguchi M., Ishikawa T. Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene. Mutat Res. 1989 Nov;218(3):197–206. doi: 10.1016/0921-8777(89)90004-9. [DOI] [PubMed] [Google Scholar]
  18. Nakabeppu Y., Kondo H., Kawabata S., Iwanaga S., Sekiguchi M. Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase. J Biol Chem. 1985 Jun 25;260(12):7281–7288. [PubMed] [Google Scholar]
  19. Nakatsuru Y., Aoki K., Ishikawa T. Age and strain dependence of O6-methylguanine DNA methyltransferase activity in mice. Mutat Res. 1989 Jan;219(1):51–56. doi: 10.1016/0921-8734(89)90040-4. [DOI] [PubMed] [Google Scholar]
  20. Nakatsuru Y., Matsukuma S., Sekiguchi M., Ishikawa T. Characterization of O6-methylguanine-DNA methyltransferase in transgenic mice introduced with the E. coli ada gene. Mutat Res. 1991 May;254(3):225–230. doi: 10.1016/0921-8777(91)90060-3. [DOI] [PubMed] [Google Scholar]
  21. Nakatsuru Y., Nemoto N., Nakagawa K., Masahito P., Ishikawa T. O6-methylguanine DNA methyltransferase activity in liver from various fish species. Carcinogenesis. 1987 Aug;8(8):1123–1127. doi: 10.1093/carcin/8.8.1123. [DOI] [PubMed] [Google Scholar]
  22. Olsson M., Lindahl T. Repair of alkylated DNA in Escherichia coli. Methyl group transfer from O6-methylguanine to a protein cysteine residue. J Biol Chem. 1980 Nov 25;255(22):10569–10571. [PubMed] [Google Scholar]
  23. Pegg A. E., Wiest L., Foote R. S., Mitra S., Perry W. Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem. 1983 Feb 25;258(4):2327–2333. [PubMed] [Google Scholar]
  24. Peterson L. A., Hecht S. S. O6-methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenesis in A/J mouse lung. Cancer Res. 1991 Oct 15;51(20):5557–5564. [PubMed] [Google Scholar]
  25. Saffhill R., Margison G. P., O'Connor P. J. Mechanisms of carcinogenesis induced by alkylating agents. Biochim Biophys Acta. 1985 Dec 17;823(2):111–145. doi: 10.1016/0304-419x(85)90009-5. [DOI] [PubMed] [Google Scholar]
  26. Shiraishi A., Sakumi K., Nakatsu Y., Hayakawa H., Sekiguchi M. Isolation and characterization of cDNA and genomic sequences for mouse O6-methylguanine-DNA methyltransferase. Carcinogenesis. 1992 Feb;13(2):289–296. doi: 10.1093/carcin/13.2.289. [DOI] [PubMed] [Google Scholar]
  27. Snow E. T., Foote R. S., Mitra S. Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication. J Biol Chem. 1984 Jul 10;259(13):8095–8100. [PubMed] [Google Scholar]
  28. Tanaka K., Miura N., Satokata I., Miyamoto I., Yoshida M. C., Satoh Y., Kondo S., Yasui A., Okayama H., Okada Y. Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain. Nature. 1990 Nov 1;348(6296):73–76. doi: 10.1038/348073a0. [DOI] [PubMed] [Google Scholar]
  29. Thomale J., Huh N. H., Nehls P., Eberle G., Rajewsky M. F. Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9883–9887. doi: 10.1073/pnas.87.24.9883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Waldstein E. A., Cao E. H., Bender M. A., Setlow R. B. Abilities of extracts of human lymphocytes to remove O6-methylguanine from DNA. Mutat Res. 1982 Aug;95(2-3):405–416. doi: 10.1016/0027-5107(82)90274-3. [DOI] [PubMed] [Google Scholar]
  31. Xiao W., Derfler B., Chen J., Samson L. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene. EMBO J. 1991 Aug;10(8):2179–2186. doi: 10.1002/j.1460-2075.1991.tb07753.x. [DOI] [PMC free article] [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