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Related Experiment Videos

Characterisation of methotrexate-resistant clones

M Roy1, S Sengupta, R Ghosh

  • 1Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, Bidhan Nagar, Calcutta, India.

Mutation Research
|February 1, 1993
PubMed
Summary

Methotrexate resistance in V79 cells is linked to dihydrofolate reductase gene amplification, causing increased chromosomal aberrations and sister-chromatid exchange. This study details genetic changes in resistant cell clones.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Methotrexate (MTX) is a chemotherapy drug that inhibits dihydrofolate reductase (DHFR).
  • Drug resistance can arise from gene amplification, leading to increased enzyme levels.
  • Understanding resistance mechanisms is crucial for improving cancer therapy.

Purpose of the Study:

  • To characterize MTX-resistant Chinese hamster V79 cell clones.
  • To investigate the role of DHFR gene amplification in MTX resistance.
  • To assess associated genetic alterations, including aneuploidy and chromosomal aberrations.

Main Methods:

  • Isolation and characterization of MTX-resistant cell clones (M1-M5).
  • Measurement of DHFR activity.
  • Cytogenetic analysis: G-banding for homogeneously staining regions (HSRs) and detection of double minutes.

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  • Assessment of chromosomal aberrations, sister-chromatid exchange (SCE), and mutation frequency at the HGPRT locus.

    • Main Results:

    • MTX-resistant clones exhibited amplified DHFR genes, evidenced by increased DHFR activity, double minutes, and HSRs.
    • Higher MTX concentrations led to clones with significantly elevated DHFR activity (M4, M5).
    • Increased chromosomal aberrations and SCE rates correlated with DHFR activity, while HGPRT mutation frequency remained unchanged.

    Conclusions:

    • DHFR gene amplification is a primary mechanism of MTX resistance in V79 cells.
    • Increased DHFR activity is associated with genomic instability, including aneuploidy and SCE.
    • Specific genetic alterations contribute to drug resistance without affecting all mutation pathways.