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 n m 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased V max for [ 3H]-MTX influx as compared to parental cells. The values for [ 3H]-MTX influx K m, efflux t 1 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°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.