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Molecular Pharmacology

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Research ArticleArticle

Multiple Mechanisms of Resistance to Polyglutamatable and Lipophilic Antifolates in Mammalian Cells: Role of Increased Folylpolyglutamylation, Expanded Folate Pools, and Intralysosomal Drug Sequestration

Gerrit Jansen, Haim Barr, Ietje Kathmann, Marlene A. Bunni, David G. Priest, Paul Noordhuis, Godefridus J. Peters and Yehuda G. Assaraf
Molecular Pharmacology April 1999, 55 (4) 761-769;
Gerrit Jansen
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Haim Barr
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Ietje Kathmann
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Marlene A. Bunni
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David G. Priest
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Paul Noordhuis
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Godefridus J. Peters
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Yehuda G. Assaraf
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Abstract

Chinese hamster ovary PyrR100 cells display more than 1000-fold resistance to pyrimethamine (Pyr), a lipophilic antifolate inhibitor of dihydrofolate reductase. PyrR100 cells had wild-type DHFR activity, lost folate exporter activity, and had a 4-fold increased activity of a low pH folic acid transporter. Here we report on the marked alterations identified in PyrR100cells compared with parental cells: 1) ∼100-fold decreased folic acid growth requirement; 2) a 25-fold higher glucose growth requirement in Pyr-containing medium; 3) a 2.5- to 4.1-fold increase in folylpolyglutamate synthetase activity; 4) a 3-fold increase in the accumulation of [3H]folic acid and a 3-fold expansion of the intracellular folate pools; 5) a 4-fold increase in the activity of the lysosomal marker β-hexoseaminidase, suggesting an increased lysosome number/PyrR100 cell; and 6) a small reduction in the steady-state accumulation of [3H]Pyr and no evidence of catabolism or modification of cellular [3H]Pyr. Consequently, PyrR100 cells were markedly resistant to the lipophilic antifolates trimetrexate (40-fold) and AG377 (30-fold) and to the polyglutamatable antifolates 5,10-Dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) (26-fold) and AG2034 (14-fold). Resistance to these drugs was reversed in PyrR100 cells transferred into folate-depleted medium. In conclusion, these multiple resistance factors collectively result in a prominent increase in folate accumulation, an expansion of the intracellular folylpolyglutamate pool, and abolishment of the cytotoxic activity of polyglutamatable and lipophilic antifolates. The role of increased lysosome number per cell in sequestration of hydrophobic weak base drugs such as Pyr is also discussed as a novel mechanism of drug resistance.

Footnotes

  • Send reprint requests to: Dr. Yehuda G. Assaraf, Ph.D., Department of Biology, The Technion–Israel Institute of Technology, Haifa 32000, Israel. E-mailassaraf{at}tx.technion.ac.il

  • This study was supported by grants from the Dutch Cancer Society (NKB-VU-96-1260) (G.J.) and Chemotech Technologies Ltd. (Y.G.A.).

  • Abbreviations:
    MTX
    methotrexate
    RFC
    reduced folate carrier
    TMQ
    trimetrexate
    Pyr
    pyrimethamine
    HF
    high folate
    LF
    low folate
    CHO
    Chinese hamster ovary
    FPGS
    folylpolyglutamate synthetase
    DHFR
    dihydrofolate reductase
    TS
    thymidylate synthase
    GARFT
    glycinamide ribonucleotide formyltransferase
    THF
    tetrahydrofolate
    DHF
    dihydrofolate
    LV
    leucovorin/5-formyltetrahydrofolate
    10-CHOTHF
    10-formyltetrahydrofolate
    DDATHF
    5-10-Dideaza-5,6,7,8-tetrahydrofolic acid
    • Received September 11, 1998.
    • Accepted January 4, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 55 (4)
Molecular Pharmacology
Vol. 55, Issue 4
1 Apr 1999
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Research ArticleArticle

Multiple Mechanisms of Resistance to Polyglutamatable and Lipophilic Antifolates in Mammalian Cells: Role of Increased Folylpolyglutamylation, Expanded Folate Pools, and Intralysosomal Drug Sequestration

Gerrit Jansen, Haim Barr, Ietje Kathmann, Marlene A. Bunni, David G. Priest, Paul Noordhuis, Godefridus J. Peters and Yehuda G. Assaraf
Molecular Pharmacology April 1, 1999, 55 (4) 761-769;

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Research ArticleArticle

Multiple Mechanisms of Resistance to Polyglutamatable and Lipophilic Antifolates in Mammalian Cells: Role of Increased Folylpolyglutamylation, Expanded Folate Pools, and Intralysosomal Drug Sequestration

Gerrit Jansen, Haim Barr, Ietje Kathmann, Marlene A. Bunni, David G. Priest, Paul Noordhuis, Godefridus J. Peters and Yehuda G. Assaraf
Molecular Pharmacology April 1, 1999, 55 (4) 761-769;
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