Impact of Overexpression of the Reduced Folate Carrier (RFC1), an Anion Exchanger, on Concentrative Transport in Murine L1210 Leukemia Cells

Abstract

Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTXrA line, which lacks a functional endogenous carrier to obtain the MTXrA-R16 clonal derivative. Influx was increased approximately 9-fold in MTXrA-R16 cells without a change in Km. The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX]i, when [MTX]e was 1 microM. The concentrative advantage for RFC1 (the ratio of [MTX]i in MTXrA-R16 to L1210 cells) increased from 1.8 at 0.1 microM MTX to 3.8 at an [MTX]e level of 30 microM. Augmented transport in MTXrA-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a approximately 50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX]i and not the much larger change in influx at low [MTX]e levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase

“…In addition to their conserved tandem DH and PH domains, Dbl RhoGEFs contain a diverse array of conserved functional domains, including intermolecular interaction and catalytic domains. The excess of RhoGEFs compared to Rho (Zhao et al, 1997). In contrast, the bombesin receptor couples to both PDZRhoGEF and LARG, while none of the three receptors appears to couple to p115 RhoGEF.…”

Resistance to antifolates

“…Under these conditions, overexpression of RFC will increase the steady-state level as the carrier becomes the dominant transport pathway. When MTX influx was increased ninefold in L1210 murine leukemia cells by transfection of RFC, the efflux rate constant increased by a factor of five, resulting in only a twofold increase in the free drug level achieved (Zhao et al, 1997). In contrast, in MTX r -ZR-75 breast cancer cells, transfection of RFC increased steady-state free drug by a factor Bhalf that of the increase in influx (Sharif et al, 1998).…”

“…MRP K t 's are at least one to two orders of magnitude higher. This means that as the drug level is increased, the influx mechanism saturates long before the export system; and this results in a progressive decline in the ratio of free intracellular to extracellular drug (Goldman et al, 1968;Zhao et al, 1997). When the primary resistance mechanism is a decrease in affinity of a target enzyme for drug (i.e.…”

“…Owing to the high membrane potential in tumor cells and the divalent nature of the folate molecule, transmembrane chemical gradients achieved by RFC are small in leukemic and other cells (Goldman et al, 1968;Zhao et al, 1997;Sharif et al, 1998). Another factor in limiting concentrative transport is the activity of exporters that pump monoglutamyl folates and antifolates out of cells (see below).…”

A Reduced Folate Carrier Mutation Produces Substrate-dependent Alterations in Carrier Mobility in Murine Leukemia Cells and Methotrexate Resistance with Conservation of Growth in 5-Formyltetrahydrofolate

“…RNA (20 g) was fractionated by electrophoresis on 1.0% formaldehyde-agarose gels. Transfer and hybridization were performed as described previously (27). Transcripts were quantitated by PhosphorImager analysis of the hybridization signals and normalized to ␤-actin.…”