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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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.[1]


  1. 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. Adv. Enzyme Regul. (1992) [Pubmed]
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