Skip to main content
SpringerLink
Log in

Induction of Mitochondrial Mutations in Human Cells by Methotrexate

  • Chapter
Genetic Consequences of Nucleotide Pool Imbalance

Part of the book series: Basic Life Sciences ((BLSC,volume 31))

Abstract

Inhibition of dihydrofolate reductase by the folate analog, methotrexate (MTX) results in a depletion of tetrahydrofolate dependent one carbon transfer reactions in amino acid and nucleic acid biosynthesis. When human cells (either HeLa or normal skin fibroblasts) are exposed to MTX in a defined medium containing dialyzed fetal calf serum, essential and non-essential amino acids, and purine source, the thymidylate pools alone are depleted. Under these conditions exposure to 10-6 M MTX induces mitochondrial mutagenesis, measured as an increase in the frequency of chloramphenicol resistant (CAPR) colonies, without altering the rate of nuclear mutation monitored by determining the frequency of 6-thioguanine resistance (TGr). The occurrence of CAPR mutations is time, and MTX concentration dependent and the frequency of CAPR can be decreased quantitatively by adding thymidine to the culture medium. This mitochondrial specific mutagenesis can also be achieved using the thymidylate synthetase inhibitor, 5-fluorodeoxyuridine further implicating thymidylate pools as the mediator of this effect. During the course of exposure to 10-6 M MTX the thymidine kinase deficient HeLa BU25 cell line exhibits a progressive depletion and degradation of mitochondrial DNA suggesting that the mutagenesis and DNA degradation represent portions of a progressive process. The basis for the selective sensitivity of the mitochondrial genome to thymidylate depletion mutagenesis may be the consequence of its differences from the nuclear genome in mechanisms of DNA replication or repair.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. C. T. M. Anderson and E. C. Friedberg, The presence of nuclear and mitochondrial uracil-DNA-glycohydrolase in extracts of human KB cells, Nucleic Acid Res., 8: 875 - 888 (1980).

    Article  PubMed  CAS  Google Scholar 

  2. J. R. Andrews, in: “Radiotherapy,” pp. 395 - 416, University Park Press, Baltimore (1978).

    Google Scholar 

  3. B. J. Barclay and J. G. Little, Genetic damage during thymidylate starvation in Saccharomyces cerevisiae, Molec. Gen. Genet., 160: 33 - 40 (1978).

    CAS  Google Scholar 

  4. H. Blanc, C. T. Wright, M. J. Bibb, D. C. Wallace, and D. A. Clayton, Mitochondrial DNA of chloramphenicol-resistant mouse cells contains a single nucleotide change in the region encoding the 3’-end of the large ribosomal RNA, Proc. Natl. Acad. Sci., U.S.A., 78: 3789 - 3793 (1981).

    Article  PubMed  CAS  Google Scholar 

  5. D. Bogenhagen and D. A. Clayton, The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells, J. Biol. Chem., 249: 7991 - 7995 (1974).

    PubMed  CAS  Google Scholar 

  6. J. Borsa and G. F. Whitmore, Cell killing studies on the mode of action of methotrexate on L-cells in vivo, Cancer Research, 29: 737 - 744 (1969).

    PubMed  CAS  Google Scholar 

  7. G. G. Carmichael and G. K. McMaster, The analysis of nucleic acids in gels using glyoxal and acridine orange, in: “Methods in Enzymology”, Vol. 65, pp. 380 - 391, Academic Press, New York (1980).

    Google Scholar 

  8. R. Carroll, J. Ash, P. Vogt, and J. Singer, Reversion of transformed glycolysis to normal by inhibition of protein synthesis in rat kidney cells infected with temperature sensitive mutant of Rous sarcoma virus, Proc. Natl. Acad. Sci., U.S.A., 75: 5015 - 5019 (1978).

    Article  PubMed  CAS  Google Scholar 

  9. B. Demple and S. Linn, DNA N-glycosylases and UV repair, Nature (London), 287: 203 - 208 (1980).

    Article  CAS  Google Scholar 

  10. L. Dimnik, Mutagenesis of the mitochondrial genome by methotrexate, M.Sc. Thesis, University of Calgary (1982).

    Google Scholar 

  11. L. Dimnik, R. B. Church, and D. I. Hoar, Induction of mitochondrial mutants by methotrexate, submitted for publication (1983).

    Google Scholar 

  12. M. Goulian, B. Bleile, and B. Y. Tseng, Methotrexate-induced misincorporation of uracil into DNA, Proc. Natl. Acad. Sci., U.S.A., 77: 1956 - 1960 (1980).

    Article  PubMed  CAS  Google Scholar 

  13. T. C. Hall and G. A. Gudouskas, Chemical pharmacology and biochemical interactions of currently useful anticancer drugs, Meth. Can. Res., 17: 313 - 349 (1979).

    CAS  Google Scholar 

  14. K. R. Harrap, B. T. Hill, M. E. Furness, and L. I. Hart, Sites of action of amethopterin: Intrinsic and acquired drug resistance, Ann. N.Y. Acad. Sci., 186: 312 - 324 (1971).

    Article  PubMed  CAS  Google Scholar 

  15. D. I. Hoar and P. Sargent, Chemical mutagen sensitivity in Ataxia telangiectasia, Nature (London), 261: 590 - 592 (1976).

    Article  CAS  Google Scholar 

  16. H. A. Ingraham, B. Y. Tseng, and M. Goulian, Mechanism for exclusion of 5-fluorouracil from DNA, Cancer Research, 80: 998 - 1001 (1980).

    Google Scholar 

  17. H. A. Ingraham, B. Y. Tseng, and M. Goulian, Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine, Molec. Pharmacol., 21: 211 - 216 (1982).

    CAS  Google Scholar 

  18. S. Kearsey and I. W. Craig, Altered ribosomal RNA genes in mitochondria from mammalian cells with chloroamphenical resistance, Nature, 290: 607 - 608 (1981).

    Article  PubMed  CAS  Google Scholar 

  19. S. Kit, D. R. Dubbs, and P. M. Frearson, HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity, J. Cancer, 1: 19 - 30 (1966).

    CAS  Google Scholar 

  20. S. Kit, W.-C. Leung, and L. A. Kaplan, Distinct molecular form of thymidine kinase in mitochondria of normal and BudR-resistant HeLa cells, Eur. J. Biochem., 39: 43 - 48 (1973).

    Article  PubMed  CAS  Google Scholar 

  21. T. A. Kunkel and L. A. Loeb, Fidelity of mammalian DNA polymerases, Science, 211: 765 - 767 (1981).

    Article  Google Scholar 

  22. B. A. Kunz, B. J. Barclay, J. C. Game, J. G. Little, and R. H. Haynes, Induction of mitotic recombination in yeast by starvation for thymidine nucleotides, Proc. Natl. Acad. Sci., U.S.A., 77: 6057 - 6061 (1980).

    Article  PubMed  CAS  Google Scholar 

  23. B. A. Kunz and R. H. Haynes, DNA repair and the genetic effects of thymidylate stress in yeast, Mutation Research, 93: 353 - 375 (1982).

    Article  CAS  Google Scholar 

  24. J. Littlefield, Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants, Since, 145: 709 - 710 (1964).

    Article  CAS  Google Scholar 

  25. H. Mahler, Biogenetic autonomy of mitochondria, C.R.C. Crit. Rev. Biochem., 1: 381 - 460 (1973).

    Article  CAS  Google Scholar 

  26. V. McKusick, Medelian inheritance in Man, John Hopkins Press (1979).

    Google Scholar 

  27. J. A. Montgomery, Synthetic chemicals, in: “Methods in Cancer Research: Cancer Drug Development,” Part A (V. T. DeVita, Jr., and H. Busch, eds.), pp. 3 - 25, Academic Press, New York (1979).

    Google Scholar 

  28. G. Pontecorvo, Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment, Somatic Cell Genet., 1: 377 - 400 (1975).

    Article  Google Scholar 

  29. J. W. Shay, Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts, Proc. Natl. Acad. Sci., U.S.A., 74: 2461 - 2464 (1977).

    Article  PubMed  CAS  Google Scholar 

  30. E. M. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 89: 503 - 517 (1975).

    Article  Google Scholar 

  31. C. M. Spolsky and J. M. Eisenstadt, Chloramphenicol-resistant mutants of human HeLa cells, FEBS Letters, 25: 319 - 324 (1972).

    Article  PubMed  CAS  Google Scholar 

  32. E.-M. Suolinna, D. R. Long, and E. Racher, Quercetin, and artifical regulation of the high aerobic glycolysis of tumor cells, J. Natl. Cancer Inst., 53: 1515 - 1519 (1974).

    PubMed  CAS  Google Scholar 

  33. D. C. Wallace, C. L. Bunn, and J. M. Eisenstadt, Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells, J. Cell Biol., 67: 174 - 199 (1975).

    Article  PubMed  CAS  Google Scholar 

  34. D. C. Wallace, N. A. Oliver, H. Blanc, and C. W. Adams, A system to study human mitochondrial genetics: Application to chloramphenicol resistance, in: “Mitochondrial Genetics” (P. Slonimski, et al., eds.), pp. 105 - 116, Cold Spring Harbor Laboratories, New York (1982).

    Google Scholar 

  35. R. Waters and E. Moustacchi, The fate of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae on various postirradiation treatments and its influence on survival and cytoplasmic “petite” induction, in: “Molecular Mechanisms for DNA Repair” (P. C. Hanawalt and R. B. Setlow, eds.), pp. 556 - 565, Plenum Press, New York (1975).

    Google Scholar 

  36. A. Wiseman and G. Attardi, Reversible ten fold reduction in mitochondrial DNA content of human cells treated with ethidium bromide, Mol. Gen. Genet., 167: 51 - 63 (1978).

    CAS  Google Scholar 

  37. A. Wiseman and G. Attardi, Cytoplasmically inherited mutations of human cell line resulting in deficient mitochondrial protein synthesis, Somatic Cell Genet., 5: 241 - 262 (1980).

    Article  Google Scholar 

  38. E. A. Wurtz, B. B. Sears, D. K. Rabert, H. S. Sheperd, N. W. Gillham, and J. E. Boynton, A specific increase in chloroplast gene mutations following growth of Chlamydomonas in 5-fluoro-deoxyuridine, Molec. Gen. Genet., 170: 235 - 242 (1979).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Medical Biochemistry and Pediatrics, The University of Calgary, Calgary, Alberta, Canada

    David I. Hoar & Leo S. Dimnik

Authors
  1. David I. Hoar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leo S. Dimnik
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA

    Frederick J. de Serres

Rights and permissions

Reprints and permissions

Copyright information

© 1985 Plenum Press, New York

About this chapter

Cite this chapter

Hoar, D.I., Dimnik, L.S. (1985). Induction of Mitochondrial Mutations in Human Cells by Methotrexate. In: de Serres, F.J. (eds) Genetic Consequences of Nucleotide Pool Imbalance. Basic Life Sciences, vol 31. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-2449-2_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4613-2449-2_16

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4612-9488-7

  • Online ISBN: 978-1-4613-2449-2

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation