Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines

J H Freisheim, M Ratnam, T P McAlinden, K M Prasad, F E Williams, G R Westerhof, J H Schornagel, G Jansen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

A variant line (CEM-7A) "overproducing" the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.

Original languageEnglish
Pages (from-to)17-31
Number of pages15
JournalAdvances in enzyme regulation
Volume32
Publication statusPublished - 1992

Cite this

Freisheim, J. H., Ratnam, M., McAlinden, T. P., Prasad, K. M., Williams, F. E., Westerhof, G. R., ... Jansen, G. (1992). Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. Advances in enzyme regulation, 32, 17-31.
Freisheim, J H ; Ratnam, M ; McAlinden, T P ; Prasad, K M ; Williams, F E ; Westerhof, G R ; Schornagel, J H ; Jansen, G. / Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. In: Advances in enzyme regulation. 1992 ; Vol. 32. pp. 17-31.
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title = "Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines",
abstract = "A variant line (CEM-7A) {"}overproducing{"} the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.",
keywords = "Affinity Labels, Biological Transport, Active/drug effects, Carrier Proteins/metabolism, Folate Receptors, GPI-Anchored, Folic Acid/analogs & derivatives, Humans, Leukemia/metabolism, Methotrexate/analogs & derivatives, Purines/pharmacology, Receptors, Cell Surface/metabolism, Thymidine/pharmacology, Tumor Cells, Cultured/metabolism",
author = "Freisheim, {J H} and M Ratnam and McAlinden, {T P} and Prasad, {K M} and Williams, {F E} and Westerhof, {G R} and Schornagel, {J H} and G Jansen",
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language = "English",
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journal = "Advances in enzyme regulation",
issn = "0065-2571",
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Freisheim, JH, Ratnam, M, McAlinden, TP, Prasad, KM, Williams, FE, Westerhof, GR, Schornagel, JH & Jansen, G 1992, 'Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines' Advances in enzyme regulation, vol. 32, pp. 17-31.

Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. / Freisheim, J H; Ratnam, M; McAlinden, T P; Prasad, K M; Williams, F E; Westerhof, G R; Schornagel, J H; Jansen, G.

In: Advances in enzyme regulation, Vol. 32, 1992, p. 17-31.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines

AU - Freisheim, J H

AU - Ratnam, M

AU - McAlinden, T P

AU - Prasad, K M

AU - Williams, F E

AU - Westerhof, G R

AU - Schornagel, J H

AU - Jansen, G

PY - 1992

Y1 - 1992

N2 - A variant line (CEM-7A) "overproducing" the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.

AB - A variant line (CEM-7A) "overproducing" the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.

KW - Affinity Labels

KW - Biological Transport, Active/drug effects

KW - Carrier Proteins/metabolism

KW - Folate Receptors, GPI-Anchored

KW - Folic Acid/analogs & derivatives

KW - Humans

KW - Leukemia/metabolism

KW - Methotrexate/analogs & derivatives

KW - Purines/pharmacology

KW - Receptors, Cell Surface/metabolism

KW - Thymidine/pharmacology

KW - Tumor Cells, Cultured/metabolism

M3 - Article

VL - 32

SP - 17

EP - 31

JO - Advances in enzyme regulation

JF - Advances in enzyme regulation

SN - 0065-2571

ER -

Freisheim JH, Ratnam M, McAlinden TP, Prasad KM, Williams FE, Westerhof GR et al. Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. Advances in enzyme regulation. 1992;32:17-31.