Ouabain-resistant mutants of mouse and hamster cells in culture

R. M. Baker, D. M. Brunette, R. Mankovitz, L. H. Thompson, G. F. Whitmore, L. Siminovitch, J. E. Till

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Abstract

Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5-6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10-4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5-6 × 10-8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

Original languageEnglish (US)
Pages (from-to)9-21
Number of pages13
JournalCell
Volume1
Issue number1
DOIs
StatePublished - 1974
Externally publishedYes

Fingerprint

Ouabain
Cell culture
Cricetinae
Cell Culture Techniques
Cells
Adenosine Triphosphatases
Clone Cells
Cricetulus
Ovary
Cell membranes
Clone cells
Cell Membrane
Hybrid Cells
Mutagens
Mutation Rate
Growth
Pharmaceutical Preparations
Population

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L., & Till, J. E. (1974). Ouabain-resistant mutants of mouse and hamster cells in culture. Cell, 1(1), 9-21. https://doi.org/10.1016/0092-8674(74)90149-4

Ouabain-resistant mutants of mouse and hamster cells in culture. / Baker, R. M.; Brunette, D. M.; Mankovitz, R.; Thompson, L. H.; Whitmore, G. F.; Siminovitch, L.; Till, J. E.

In: Cell, Vol. 1, No. 1, 1974, p. 9-21.

Research output: Contribution to journalArticle

Baker, RM, Brunette, DM, Mankovitz, R, Thompson, LH, Whitmore, GF, Siminovitch, L & Till, JE 1974, 'Ouabain-resistant mutants of mouse and hamster cells in culture', Cell, vol. 1, no. 1, pp. 9-21. https://doi.org/10.1016/0092-8674(74)90149-4
Baker RM, Brunette DM, Mankovitz R, Thompson LH, Whitmore GF, Siminovitch L et al. Ouabain-resistant mutants of mouse and hamster cells in culture. Cell. 1974;1(1):9-21. https://doi.org/10.1016/0092-8674(74)90149-4
Baker, R. M. ; Brunette, D. M. ; Mankovitz, R. ; Thompson, L. H. ; Whitmore, G. F. ; Siminovitch, L. ; Till, J. E. / Ouabain-resistant mutants of mouse and hamster cells in culture. In: Cell. 1974 ; Vol. 1, No. 1. pp. 9-21.
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abstract = "Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5-6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10-4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5-6 × 10-8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.",
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AU - Baker, R. M.

AU - Brunette, D. M.

AU - Mankovitz, R.

AU - Thompson, L. H.

AU - Whitmore, G. F.

AU - Siminovitch, L.

AU - Till, J. E.

PY - 1974

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AB - Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5-6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10-4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5-6 × 10-8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

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