Membrane protein changes in an L1210 leukemia cell line with a translocation defect in the methotrexate-tetrahydrofolate cofactor transport carrier

Abstract

We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.