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Somatic Cell and Molecular Genetics

, Volume 12, Issue 1, pp 21–31 | Cite as

Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts

  • Kamal D. Mehta
  • Radhey S. Gupta
Article

Abstract

Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fomr) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10−6. Cross-resistance studies with these mutants show that the Fomr mutants exhibit increased resistance to all adenosine analogs (N- and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fomr mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N7-(Δ2-isopentenyl) formycin A for similar inhibition of [3H]adenosine phosphorylation. These results indicate that AK from the Fomr mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fomr mutants may thus be directly affecting the structural gene for AK.

Keywords

Adenosine Cell Extract Chinese Hamster Ovary Cell Parental Cell Inosine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Literature cited

  1. 1.
    Bloch, A. (1975).Ann. N.Y. Acad. Sci. 255:576–595.PubMedGoogle Scholar
  2. 2.
    Suhadolnik, R.J. (1979).Prog. Nucleic Acid Res. Mol. Biol. 22:195–289.Google Scholar
  3. 3.
    Suhadolnik, R.J. (1970).Nucleoside Antibiotics, Wiley-Interscience, New York.Google Scholar
  4. 4.
    Bennett, L.L., Jr., Schnebli, H.P., Vail, M.H., Allan, P.W., and Montgomery, J.A. (1966).Mol. Pharmacol. 2:432–443.PubMedGoogle Scholar
  5. 5.
    McBurney, M.W., and Whitmore, G.F. (1974).J. Cell. Physiol. 85:87–100.Google Scholar
  6. 6.
    Chan, T., Creagan, R.P., and Reardon, M.P. (1978).Somat. Cell Genet. 4:1–12.PubMedGoogle Scholar
  7. 7.
    Gupta, R.S., and Siminovitch, L. (1978).Somat. Cell Genet. 4:715–735.PubMedGoogle Scholar
  8. 8.
    Rabin, M.S., and Gottesman, M.M. (1979).Somat. Cell Genet. 5:571–584.PubMedGoogle Scholar
  9. 9.
    Gupta, R.S., and Singh, B. (1983).Mutat. Res. 113:441–454.PubMedGoogle Scholar
  10. 10.
    Plagemann, P.G.W., and Wohlhueter, R.M. (1983).J. Cell. Physiol. 116:236–246.PubMedGoogle Scholar
  11. 11.
    Mehta, K.D., and Gupta, R.S. (1983).Mol. Cell. Biol. 3:1468–1477.PubMedGoogle Scholar
  12. 12.
    Thacker, J. (1980).Mutat. Res. 74:37–59.PubMedGoogle Scholar
  13. 13.
    Kao, F.T., and Puck, T.T. (1967).Genetics 55:513–524.PubMedGoogle Scholar
  14. 14.
    Gupta, R.S., Chan, D.H.Y., and Siminovitch, L. (1978).Cell 14:1007–1013.PubMedGoogle Scholar
  15. 15.
    Robins, M.J., and Trip, E.M. (1973).Biochemistry 12:2179–2187.PubMedGoogle Scholar
  16. 16.
    Chan, T.S., Ishii, K., Long, C., and Green, H. (1973).J. Cell. Physiol. 81:315–322.PubMedGoogle Scholar
  17. 17.
    Gupta, R.S. (1984). InHandbook of Mutagenicity Test Procedures, 2nd ed. (eds.) Kilbey, B.J., Legator, M., Nichols, W., and Ramel, C. (Elsevier Science Publishers, New York), pp. 291–319.Google Scholar
  18. 18.
    Dix, D.E., Lehman, C.P., Jakabowski, A., Moyer, J.D., and Handschumacher, R.E. (1979).Cancer Res. 39:4458–4490.Google Scholar
  19. 19.
    Saunders, P.P., Kuttan, R., Lai, M.M., and Robins, R.K. (1983).Mol. Pharmacol. 23:535–539.Google Scholar
  20. 20.
    Chasin, L.A. (1974).Cell 2:37–42.PubMedGoogle Scholar
  21. 21.
    Caskey, C.T., and Kruh, G.D. (1979).Cell 16:1–10.PubMedGoogle Scholar
  22. 22.
    Littlefield, J.W. (1964).Science 145:709–710.PubMedGoogle Scholar
  23. 23.
    Norwood, T.H., Zeigler, C.J., and Martin, G.M. (1976).Somat. Cell Genet. 2:263–270.PubMedGoogle Scholar
  24. 24.
    Mehta, K.D., and Gupta, R.S. (1985).Biochem. Biophys. Res. Commun. 130:910–917.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1986

Authors and Affiliations

  • Kamal D. Mehta
    • 1
  • Radhey S. Gupta
    • 1
  1. 1.Department of BiochemistryMcMaster UniversityHamiltonCanada

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