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Related Experiment Videos

Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake

F M Williams1, R C Murray, T M Underhill

  • 1Department of Microbiology and Immunology, University of Western Ontario, London.

The Journal of Biological Chemistry
|February 25, 1994
PubMed
Summary

Researchers identified a new gene clone that restores methotrexate uptake in defective Chinese hamster ovary cells. This finding advances understanding of folate transport and drug sensitivity mechanisms.

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ON THE EVOLUTION OF HANDLING TIME.

Evolution; international journal of organic evolution·2017

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Methotrexate (MTX) is a crucial chemotherapy drug that relies on cellular uptake mechanisms.
  • Mutations affecting MTX transport can lead to drug resistance, posing a challenge in cancer treatment.
  • Identifying the specific transporters involved in MTX uptake is vital for understanding drug efficacy.

Purpose of the Study:

  • To isolate and characterize a cDNA clone responsible for methotrexate uptake in Chinese hamster ovary (CHO) cells.
  • To investigate the functional role of the identified clone in restoring MTX binding and transport in mutant cells.
  • To determine the potential identity of the encoded protein and its involvement in folate transport pathways.

Main Methods:

  • Screening of a CHO cell cDNA expression library using a methotrexate uptake-deficient mutant.

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  • Transfection of mutant cells with the isolated clone.
  • Assessment of methotrexate binding and transport in transfected cells.
  • Analysis of the clone's sequence, open reading frame, and predicted protein characteristics (size, hydrophobicity, secondary structure).
  • Northern blot analysis to detect mRNA expression levels in wild-type and mutant cell lines.

    • Main Results:

    • A cDNA clone was isolated that complements mutant cells defective in methotrexate uptake.
    • Transfection restored methotrexate binding and transport, re-sensitizing cells to the drug's cytotoxic effects.
    • The clone encodes a putative 58 kDa protein with high hydrophobicity and beta-sheet structure.
    • An mRNA of 2.5 kilobases was detected in wild-type and one mutant cell line, but absent in another mutant line.
    • The data suggest the clone encodes the reduced folate transporter or an associated auxiliary protein.

    Conclusions:

    • This study reports the first cDNA encoding a function, beyond folate-binding protein, involved in methotrexate transport.
    • The identified clone plays a critical role in mediating methotrexate uptake and sensitivity in CHO cells.
    • This discovery provides a new target for understanding and potentially overcoming drug resistance in cancer therapy.