We studied the molecular basis of the up to 46-fold increased accumulation of folates and
methotrexate (MTX) in human
leukemia CEM-7A cells established by gradual deprivation of
leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and
folic acid growth requirements and 23-25-fold
hypersensitivity to MTX and
edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the
reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased
folate/MTX accumulation because RFC functions as a nonconcentrative
anion exchanger. We therefore explored the possibility that
folate efflux activity mediated by members of the multidrug resistance
protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of
MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and
breast cancer resistance
protein were undetectable. In contrast, CEM-7A cells lost 95% of
MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]
folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their
folic acid efflux was blocked (78%) with
probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more
calcein fluorescence. Treatment of parental cells with the
MRP1 efflux inhibitors MK571 and
probenecid resulted in a 60-100% increase in
calcein fluorescence. In contrast, these inhibitors failed to alter the
calcein fluorescence in CEM-7A cells, which markedly lost
MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal
MRP1 expression. These results establish for the first time that
MRP1 is the primary
folate efflux route in CEM
leukemia cells and that the loss of
folate efflux activity is an efficient means of markedly augmenting cellular
folate pools. These findings suggest a functional role for
MRP1 in the maintenance of cellular
folate homeostasis.