Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes.

D. W. Fry, D. A. Gewirtz, J. C. Yalowich, I. David Goldman

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5 Citations (Scopus)

Abstract

The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.

Original languageEnglish (US)
Pages (from-to)215-234
Number of pages20
JournalAdvances in Experimental Medicine and Biology
Volume163
StatePublished - 1983
Externally publishedYes

Fingerprint

Ehrlich Tumor Carcinoma
Methotrexate
Rats
Tumors
Hepatocytes
Cells
Derivatives
Folic Acid
Oxidoreductases
Polyglutamic Acid
Probenecid
methotrexate polyglutamate
Vincristine
Metabolites
Glutamine
Pteroylpolyglutamic Acids
Sulfobromophthalein
Bucladesine
Xanthine
Deoxycholic Acid

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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title = "Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes.",
abstract = "The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.",
author = "Fry, {D. W.} and Gewirtz, {D. A.} and Yalowich, {J. C.} and Goldman, {I. David}",
year = "1983",
language = "English (US)",
volume = "163",
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T1 - Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes.

AU - Fry, D. W.

AU - Gewirtz, D. A.

AU - Yalowich, J. C.

AU - Goldman, I. David

PY - 1983

Y1 - 1983

N2 - The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.

AB - The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.

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