Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines.
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.
Freisheim
J H
JH
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008.
Ratnam
M
M
McAlinden
T P
TP
Prasad
K M
KM
Williams
F E
FE
Westerhof
G R
GR
Schornagel
J H
JH
Jansen
G
G
D000345:Affinity Labels; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013936:Thymidine / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011687:Purines; D011956:Receptors, Cell Surface; D005492:Folic Acid; D013936:Thymidine; D008727:Methotrexate