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Selection for methotrexate resistance in mammalian cells bearing a Drosophila dihydrofolate reductase transgene

Methotrexate resistance in transgenic mammalian cells

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Abstract

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.

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Abbreviations

AP:

Aminopterin

CHO:

Chinese hamster ovary

DHFR:

Dihydrofolate reductase

MTX:

Methotrexate

PMA:

Pyromethamine

TMP:

Trimethoprim

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Acknowledgements

Natural Sciences and Engineering Research Council of Canada (NSERC) is acknowledged for scholarship support to J.G.A. and grant support to V.K.W. We acknowledge Dr. W. Flintoff for the CHONULL cell line, the Protein Discovery and Function group for the flow cytometer, and Dr. S. Cole for cell culture facilities.

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  1. Joslynn G. Affleck

    Present address: Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada

Authors and Affiliations

  1. Department of Biology, Queen’s University, Biosciences Complex, Kingston, ON, K7L 3N6, Canada

    Shaun M. Nowickyj & Virginia K. Walker

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  1. Joslynn G. Affleck
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  2. Shaun M. Nowickyj
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  3. Virginia K. Walker
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Correspondence to Virginia K. Walker.

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Affleck, J.G., Nowickyj, S.M. & Walker, V.K. Selection for methotrexate resistance in mammalian cells bearing a Drosophila dihydrofolate reductase transgene. Cell Biol Toxicol 26, 117–126 (2010). https://doi.org/10.1007/s10565-009-9122-1

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  • DOI: https://doi.org/10.1007/s10565-009-9122-1

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