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Updated: Jul 13, 2026

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System
08:48

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Published on: December 21, 2016

Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes

D A Haber, R T Schimke

    Cell
    |November 1, 1981
    PubMed

    Insights

    Mouse fibroblasts develop methotrexate resistance through gene amplification and mutation. Variant cells with altered dihydrofolate reductase genes on extrachromosomal elements are selected for, demonstrating gene amplification and chromosome dynamics.

    Area of Science:

    • Molecular biology
    • Genetics
    • Cell biology

    Background:

    • Methotrexate (MTX) resistance in cancer therapy is a significant challenge.
    • Dihydrofolate reductase (DHFR) is a key enzyme in folate metabolism and a target for MTX.
    • Gene amplification and mutation are known mechanisms of drug resistance.

    Purpose of the Study:

    • To investigate the genetic mechanisms of methotrexate resistance in 3T6 mouse fibroblasts.
    • To characterize the emergence and stability of drug-resistant cell populations.
    • To elucidate the role of extrachromosomal elements in acquired drug resistance.

    Main Methods:

    • Progressive selection of 3T6 mouse fibroblasts in increasing MTX concentrations.
    • Fluorescence-activated cell sorting (FACS) to analyze DHFR gene copy number and expression.
    • Analysis of gene mutation and extrachromosomal DNA (double-minute chromosomes).

    Main Results:

    • Initial MTX resistance mediated by amplification of the normal DHFR gene.
    • Emergence of cells with altered DHFR genes (reduced MTX affinity) at higher MTX concentrations.
    • Variant DHFR genes reside on double-minute chromosomes, which are lost upon removal of MTX selection.
    • Autonomous replication and selective advantage of cells with minimal extrachromosomal elements.

    Conclusions:

    • Acquired MTX resistance involves both gene amplification and mutation of the DHFR gene.
    • Double-minute chromosomes play a crucial role in the amplification and maintenance of drug resistance.
    • The study provides insights into the dynamics of extrachromosomal elements and gene amplification in response to selective pressure.

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