Methotrexate resistance in an L1210 cell line resulting from increased dihydrofolate reductase, decreased thymidylate synthetase activity, and normal membrane transport. Computer simulations based on network thermodynamics

J. C. White, I. David Goldman

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36 Citations (Scopus)

Abstract

The biochemical factors determining resistance to the antifolate, methotrexate, were quantitatively evaluated for a methotrexate-resistant L1210 cell line. The concentration needed for 50% inhibition of [3H]deoxyuridine incorporation into DNA was increased 60-fold, as compared to a sensitive cell line. Properties of the membrane transport system for methotrexate were unchanged. Dihydrofolate reductase levels were elevated 16-fold. Computer simulations based on the principles of network thermodynamics predicted that this single alteration would necessitate only a 16-fold increase in free intracellular methotrexate to achieve the same degree of inhibition. Further biochemical examination of the resistant cell type revealed a 4-fold decrease in thymidylate synthetase activity. Computer simulations which included this information agreed closely with the experimental data. In sensitive cells a small fraction of the cell's dihydrofolate reductase can meet the requirement for reduced folate and some free methotrexate is needed for inhibition of DNA synthesis. In cell lines with increased dihydrofolate reductase activity or with a decreased rate of oxidation of tetrahydrofolate cofactor in the synthesis of thymidylate, an even smaller fraction of total reductase activity is sufficient and more free methotrexate is needed to inhibit a larger fraction of the target enzyme to make reduction of dihydrofolate rate-limiting for DNA synthesis. Properties of the membrane transport system for methotrexate, however, may prevent the accumulation of free intracellular methotrexate to levels needed for complete inhibition in some resistant tumor cell types, despite very high extracellular concentrations. Simulations which included consideration of methotrexate pharmacokinetics predicted that following a high dose of methotrexate, cells of the resistant phenotype would recover normal DNA synthesis days earlier than sensitive cells. Further, shortly after methotrexate administration in vivo, the rate of methotrexate exchange across the cell membrane is so fast compared to the rate of change of the extracellular methotrexate level that intracellular methotrexate is always at steady state with extracellular drug, and the net rate of methotrexate loss from the cell is determined not by the transport system but by the rate of decline of the extracellular drug level.

Original languageEnglish (US)
Pages (from-to)5722-5727
Number of pages6
JournalJournal of Biological Chemistry
Volume256
Issue number11
StatePublished - 1981
Externally publishedYes

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Thymidylate Synthase
Tetrahydrofolate Dehydrogenase
Thermodynamics
Methotrexate
Computer Simulation
Cells
Membranes
Cell Line
Computer simulation
DNA
Folic Acid Antagonists
Deoxyuridine
Pharmacokinetics
R Factors
Cell membranes
Folic Acid
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Methotrexate resistance in an L1210 cell line resulting from increased dihydrofolate reductase, decreased thymidylate synthetase activity, and normal membrane transport. Computer simulations based on network thermodynamics",
abstract = "The biochemical factors determining resistance to the antifolate, methotrexate, were quantitatively evaluated for a methotrexate-resistant L1210 cell line. The concentration needed for 50{\%} inhibition of [3H]deoxyuridine incorporation into DNA was increased 60-fold, as compared to a sensitive cell line. Properties of the membrane transport system for methotrexate were unchanged. Dihydrofolate reductase levels were elevated 16-fold. Computer simulations based on the principles of network thermodynamics predicted that this single alteration would necessitate only a 16-fold increase in free intracellular methotrexate to achieve the same degree of inhibition. Further biochemical examination of the resistant cell type revealed a 4-fold decrease in thymidylate synthetase activity. Computer simulations which included this information agreed closely with the experimental data. In sensitive cells a small fraction of the cell's dihydrofolate reductase can meet the requirement for reduced folate and some free methotrexate is needed for inhibition of DNA synthesis. In cell lines with increased dihydrofolate reductase activity or with a decreased rate of oxidation of tetrahydrofolate cofactor in the synthesis of thymidylate, an even smaller fraction of total reductase activity is sufficient and more free methotrexate is needed to inhibit a larger fraction of the target enzyme to make reduction of dihydrofolate rate-limiting for DNA synthesis. Properties of the membrane transport system for methotrexate, however, may prevent the accumulation of free intracellular methotrexate to levels needed for complete inhibition in some resistant tumor cell types, despite very high extracellular concentrations. Simulations which included consideration of methotrexate pharmacokinetics predicted that following a high dose of methotrexate, cells of the resistant phenotype would recover normal DNA synthesis days earlier than sensitive cells. Further, shortly after methotrexate administration in vivo, the rate of methotrexate exchange across the cell membrane is so fast compared to the rate of change of the extracellular methotrexate level that intracellular methotrexate is always at steady state with extracellular drug, and the net rate of methotrexate loss from the cell is determined not by the transport system but by the rate of decline of the extracellular drug level.",
author = "White, {J. C.} and Goldman, {I. David}",
year = "1981",
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N2 - The biochemical factors determining resistance to the antifolate, methotrexate, were quantitatively evaluated for a methotrexate-resistant L1210 cell line. The concentration needed for 50% inhibition of [3H]deoxyuridine incorporation into DNA was increased 60-fold, as compared to a sensitive cell line. Properties of the membrane transport system for methotrexate were unchanged. Dihydrofolate reductase levels were elevated 16-fold. Computer simulations based on the principles of network thermodynamics predicted that this single alteration would necessitate only a 16-fold increase in free intracellular methotrexate to achieve the same degree of inhibition. Further biochemical examination of the resistant cell type revealed a 4-fold decrease in thymidylate synthetase activity. Computer simulations which included this information agreed closely with the experimental data. In sensitive cells a small fraction of the cell's dihydrofolate reductase can meet the requirement for reduced folate and some free methotrexate is needed for inhibition of DNA synthesis. In cell lines with increased dihydrofolate reductase activity or with a decreased rate of oxidation of tetrahydrofolate cofactor in the synthesis of thymidylate, an even smaller fraction of total reductase activity is sufficient and more free methotrexate is needed to inhibit a larger fraction of the target enzyme to make reduction of dihydrofolate rate-limiting for DNA synthesis. Properties of the membrane transport system for methotrexate, however, may prevent the accumulation of free intracellular methotrexate to levels needed for complete inhibition in some resistant tumor cell types, despite very high extracellular concentrations. Simulations which included consideration of methotrexate pharmacokinetics predicted that following a high dose of methotrexate, cells of the resistant phenotype would recover normal DNA synthesis days earlier than sensitive cells. Further, shortly after methotrexate administration in vivo, the rate of methotrexate exchange across the cell membrane is so fast compared to the rate of change of the extracellular methotrexate level that intracellular methotrexate is always at steady state with extracellular drug, and the net rate of methotrexate loss from the cell is determined not by the transport system but by the rate of decline of the extracellular drug level.

AB - The biochemical factors determining resistance to the antifolate, methotrexate, were quantitatively evaluated for a methotrexate-resistant L1210 cell line. The concentration needed for 50% inhibition of [3H]deoxyuridine incorporation into DNA was increased 60-fold, as compared to a sensitive cell line. Properties of the membrane transport system for methotrexate were unchanged. Dihydrofolate reductase levels were elevated 16-fold. Computer simulations based on the principles of network thermodynamics predicted that this single alteration would necessitate only a 16-fold increase in free intracellular methotrexate to achieve the same degree of inhibition. Further biochemical examination of the resistant cell type revealed a 4-fold decrease in thymidylate synthetase activity. Computer simulations which included this information agreed closely with the experimental data. In sensitive cells a small fraction of the cell's dihydrofolate reductase can meet the requirement for reduced folate and some free methotrexate is needed for inhibition of DNA synthesis. In cell lines with increased dihydrofolate reductase activity or with a decreased rate of oxidation of tetrahydrofolate cofactor in the synthesis of thymidylate, an even smaller fraction of total reductase activity is sufficient and more free methotrexate is needed to inhibit a larger fraction of the target enzyme to make reduction of dihydrofolate rate-limiting for DNA synthesis. Properties of the membrane transport system for methotrexate, however, may prevent the accumulation of free intracellular methotrexate to levels needed for complete inhibition in some resistant tumor cell types, despite very high extracellular concentrations. Simulations which included consideration of methotrexate pharmacokinetics predicted that following a high dose of methotrexate, cells of the resistant phenotype would recover normal DNA synthesis days earlier than sensitive cells. Further, shortly after methotrexate administration in vivo, the rate of methotrexate exchange across the cell membrane is so fast compared to the rate of change of the extracellular methotrexate level that intracellular methotrexate is always at steady state with extracellular drug, and the net rate of methotrexate loss from the cell is determined not by the transport system but by the rate of decline of the extracellular drug level.

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