Characteristics of transport of 4-amino antifolates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate

B. T. Hill, B. D. Bailey, I. David Goldman, I. D. Goldman

Research output: Contribution to journalArticle

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Abstract

In a line of L5178Y lymphoblasts resistant to methotrexate (MTX) by virtue of a permeability defect, there is a marked decrease in the membrane transport of MTX, aminopterin, and 5-methyltetrahydrofolate. Influx is depressed by 93, 92, and 96%, respectively, as compared to MTX-sensitive cells when the extracellular levels are 1 μM. This is compatible with a common defect in a high-affinity transport carrier mechanism shared by these three folate compounds. Conversely, however, adsorption of MTX to the surface of resistant cells is slightly increased. Uptake of 3H following exposure of these cells to [3H]folic acid is comparable in both resistant and sensitive lines, supporting the concept that influx of folic acid is mediated largely by a process independent of the MTX-tetrahydrofolate cofactor carrier. In addition to the quantitative differences in the rates of transport of 4-amino antifolates and 5-methyltetrahydrofolate, there are important differences in the characteristics of transport of these substances in these two cell lines. The Q (27-37°)'s for MTX influx in MTX-sensitive and -resistant cells are 6.4 and 1.4, respectively. While influx is markedly inhibited by p-chloromercuriphenylsulfonic acid and 5-formyltetrahydrofolate in MTX-sensitive cells, only a small component of influx in resistant cells (~ 25%) is inhibited by these agents. Transstimulation of MTX influx can be demonstrated in both cell lines but to a much lesser extent in resistant L5178Y cells. Sodium azide stimulates MTX influx in sensitive cells but has no effect on influx in resistant cells. However, this metabolic poison augments net uptake of MTX in both cell lines. Net accumulation of exchangeable MTX is markedly reduced in resistant cells, but differences in influx and net transport between resistant and sensitive cells decrease as extracellular MTX is increased, indicating the utility of high extracellular MTX levels in generating free intracellular drug as an approach to overcoming the permeability defect. These data suggest that the bulk of transport of antifolates and 5-methyltetrahydrofolate in resistant cells occurs by a process distinct from the MTX-tetrahydrofolate cofactor carrier system. The MTX-tetrahydrofolate cofactor transport carriers in MTX-resistant cells have undergone a marked reduction in affinity for their substrate, or else the major portion of these carriers have been deleted or are not operational. The observation that azide enhances net MTX-uptake inresistant as well as sensitive L5178Y cells along with findings in other studies that the net uptake of folic acid is enhanced by azide raises the possiility that the effects of metabolic inhibitors on net transport of MTX and folic acid may be mediated, at least in part, by inhibition of a common exit process.

Original languageEnglish (US)
Pages (from-to)2440-2446
Number of pages7
JournalCancer Research
Volume39
Issue number7 I
StatePublished - 1979
Externally publishedYes

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Folic Acid Antagonists
Folic Acid
Methotrexate
Azides
Cell Line
Permeability
Aminopterin

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Characteristics of transport of 4-amino antifolates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate. / Hill, B. T.; Bailey, B. D.; Goldman, I. David; Goldman, I. D.

In: Cancer Research, Vol. 39, No. 7 I, 1979, p. 2440-2446.

Research output: Contribution to journalArticle

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title = "Characteristics of transport of 4-amino antifolates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate",
abstract = "In a line of L5178Y lymphoblasts resistant to methotrexate (MTX) by virtue of a permeability defect, there is a marked decrease in the membrane transport of MTX, aminopterin, and 5-methyltetrahydrofolate. Influx is depressed by 93, 92, and 96{\%}, respectively, as compared to MTX-sensitive cells when the extracellular levels are 1 μM. This is compatible with a common defect in a high-affinity transport carrier mechanism shared by these three folate compounds. Conversely, however, adsorption of MTX to the surface of resistant cells is slightly increased. Uptake of 3H following exposure of these cells to [3H]folic acid is comparable in both resistant and sensitive lines, supporting the concept that influx of folic acid is mediated largely by a process independent of the MTX-tetrahydrofolate cofactor carrier. In addition to the quantitative differences in the rates of transport of 4-amino antifolates and 5-methyltetrahydrofolate, there are important differences in the characteristics of transport of these substances in these two cell lines. The Q (27-37°)'s for MTX influx in MTX-sensitive and -resistant cells are 6.4 and 1.4, respectively. While influx is markedly inhibited by p-chloromercuriphenylsulfonic acid and 5-formyltetrahydrofolate in MTX-sensitive cells, only a small component of influx in resistant cells (~ 25{\%}) is inhibited by these agents. Transstimulation of MTX influx can be demonstrated in both cell lines but to a much lesser extent in resistant L5178Y cells. Sodium azide stimulates MTX influx in sensitive cells but has no effect on influx in resistant cells. However, this metabolic poison augments net uptake of MTX in both cell lines. Net accumulation of exchangeable MTX is markedly reduced in resistant cells, but differences in influx and net transport between resistant and sensitive cells decrease as extracellular MTX is increased, indicating the utility of high extracellular MTX levels in generating free intracellular drug as an approach to overcoming the permeability defect. These data suggest that the bulk of transport of antifolates and 5-methyltetrahydrofolate in resistant cells occurs by a process distinct from the MTX-tetrahydrofolate cofactor carrier system. The MTX-tetrahydrofolate cofactor transport carriers in MTX-resistant cells have undergone a marked reduction in affinity for their substrate, or else the major portion of these carriers have been deleted or are not operational. The observation that azide enhances net MTX-uptake inresistant as well as sensitive L5178Y cells along with findings in other studies that the net uptake of folic acid is enhanced by azide raises the possiility that the effects of metabolic inhibitors on net transport of MTX and folic acid may be mediated, at least in part, by inhibition of a common exit process.",
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N2 - In a line of L5178Y lymphoblasts resistant to methotrexate (MTX) by virtue of a permeability defect, there is a marked decrease in the membrane transport of MTX, aminopterin, and 5-methyltetrahydrofolate. Influx is depressed by 93, 92, and 96%, respectively, as compared to MTX-sensitive cells when the extracellular levels are 1 μM. This is compatible with a common defect in a high-affinity transport carrier mechanism shared by these three folate compounds. Conversely, however, adsorption of MTX to the surface of resistant cells is slightly increased. Uptake of 3H following exposure of these cells to [3H]folic acid is comparable in both resistant and sensitive lines, supporting the concept that influx of folic acid is mediated largely by a process independent of the MTX-tetrahydrofolate cofactor carrier. In addition to the quantitative differences in the rates of transport of 4-amino antifolates and 5-methyltetrahydrofolate, there are important differences in the characteristics of transport of these substances in these two cell lines. The Q (27-37°)'s for MTX influx in MTX-sensitive and -resistant cells are 6.4 and 1.4, respectively. While influx is markedly inhibited by p-chloromercuriphenylsulfonic acid and 5-formyltetrahydrofolate in MTX-sensitive cells, only a small component of influx in resistant cells (~ 25%) is inhibited by these agents. Transstimulation of MTX influx can be demonstrated in both cell lines but to a much lesser extent in resistant L5178Y cells. Sodium azide stimulates MTX influx in sensitive cells but has no effect on influx in resistant cells. However, this metabolic poison augments net uptake of MTX in both cell lines. Net accumulation of exchangeable MTX is markedly reduced in resistant cells, but differences in influx and net transport between resistant and sensitive cells decrease as extracellular MTX is increased, indicating the utility of high extracellular MTX levels in generating free intracellular drug as an approach to overcoming the permeability defect. These data suggest that the bulk of transport of antifolates and 5-methyltetrahydrofolate in resistant cells occurs by a process distinct from the MTX-tetrahydrofolate cofactor carrier system. The MTX-tetrahydrofolate cofactor transport carriers in MTX-resistant cells have undergone a marked reduction in affinity for their substrate, or else the major portion of these carriers have been deleted or are not operational. The observation that azide enhances net MTX-uptake inresistant as well as sensitive L5178Y cells along with findings in other studies that the net uptake of folic acid is enhanced by azide raises the possiility that the effects of metabolic inhibitors on net transport of MTX and folic acid may be mediated, at least in part, by inhibition of a common exit process.

AB - In a line of L5178Y lymphoblasts resistant to methotrexate (MTX) by virtue of a permeability defect, there is a marked decrease in the membrane transport of MTX, aminopterin, and 5-methyltetrahydrofolate. Influx is depressed by 93, 92, and 96%, respectively, as compared to MTX-sensitive cells when the extracellular levels are 1 μM. This is compatible with a common defect in a high-affinity transport carrier mechanism shared by these three folate compounds. Conversely, however, adsorption of MTX to the surface of resistant cells is slightly increased. Uptake of 3H following exposure of these cells to [3H]folic acid is comparable in both resistant and sensitive lines, supporting the concept that influx of folic acid is mediated largely by a process independent of the MTX-tetrahydrofolate cofactor carrier. In addition to the quantitative differences in the rates of transport of 4-amino antifolates and 5-methyltetrahydrofolate, there are important differences in the characteristics of transport of these substances in these two cell lines. The Q (27-37°)'s for MTX influx in MTX-sensitive and -resistant cells are 6.4 and 1.4, respectively. While influx is markedly inhibited by p-chloromercuriphenylsulfonic acid and 5-formyltetrahydrofolate in MTX-sensitive cells, only a small component of influx in resistant cells (~ 25%) is inhibited by these agents. Transstimulation of MTX influx can be demonstrated in both cell lines but to a much lesser extent in resistant L5178Y cells. Sodium azide stimulates MTX influx in sensitive cells but has no effect on influx in resistant cells. However, this metabolic poison augments net uptake of MTX in both cell lines. Net accumulation of exchangeable MTX is markedly reduced in resistant cells, but differences in influx and net transport between resistant and sensitive cells decrease as extracellular MTX is increased, indicating the utility of high extracellular MTX levels in generating free intracellular drug as an approach to overcoming the permeability defect. These data suggest that the bulk of transport of antifolates and 5-methyltetrahydrofolate in resistant cells occurs by a process distinct from the MTX-tetrahydrofolate cofactor carrier system. The MTX-tetrahydrofolate cofactor transport carriers in MTX-resistant cells have undergone a marked reduction in affinity for their substrate, or else the major portion of these carriers have been deleted or are not operational. The observation that azide enhances net MTX-uptake inresistant as well as sensitive L5178Y cells along with findings in other studies that the net uptake of folic acid is enhanced by azide raises the possiility that the effects of metabolic inhibitors on net transport of MTX and folic acid may be mediated, at least in part, by inhibition of a common exit process.

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