RESUMEN
The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, R26L, R42L, and K332L, there was little or no change in the influx K(t) value for MTX or influx K(i) value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was most prominent for K404L, which had 11- and 4-fold increases in influx K(i) for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.
Asunto(s)
Proteínas Portadoras/metabolismo , Antagonistas del Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Proteínas de Transporte de Membrana , Metotrexato/metabolismo , Animales , Aniones/farmacología , Arginina/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Secuencia Conservada , Análisis Mutacional de ADN , Lisina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutagénesis , Proteína Portadora de Folato Reducido , TransfecciónRESUMEN
MTA (LY231514) is an antifolate that targets multiple folate-dependent enzymes. In this report, MTA transport was characterized in wild-type L1210 cells and variants with impaired membrane transport or polyglutamation. MTA influx via the reduced folate carrier was somewhat faster (approximately 30%) than that for methotrexate (MTX). Unlike MTX, MTA was rapidly polyglutamated in L1210 cells; hence, a folylpoly-gamma-glutamate synthetase-deficient L1210 variant was used to assess net transport and efflux properties. The MTA transmembrane gradient for exchangeable drug was 2.5 times greater than the MTX gradient, attributable primarily to an efflux rate constant 40% that of MTX. No MTA was bound to dihydrofolate reductase. When grown with folic acid, MTX-resistant L1210 variants with mutations in the reduced folate carrier demonstrated cross-resistance to MTA, markedly reduced MTA accumulation, and only a slightly decreased intracellular folate cofactor pool as compared to L1210 cells. However, when 5-formyltetrahydrofolate was the growth substrate, these MTX-resistant cells were less resistant or negligibly resistant to MTA, accumulated more MTA, and had a lower folate pool as compared to L1210 cells. MTA activity and the intracellular folate pool in L1210 cells were inversely related. These data indicate that MTA polyglutamation in L1210 cells is favored by both the generation of high intracellular drug levels and high MTA affinity for FPGS relative to MTX. Cells resistant to MTX because of impaired transport may retain appreciable sensitivity to MTA because of a concurrent reduction in tetrahydrofolate cofactor transport resulting in cellular folate depletion, which diminishes endogenous folate suppression of MTA polyglutamation.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Antagonistas del Ácido Fólico/farmacocinética , Glutamatos/farmacocinética , Guanina/análogos & derivados , Guanina/farmacocinética , Proteínas de Transporte de Membrana , Metotrexato/farmacocinética , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , División Celular/fisiología , Resistencia a Antineoplásicos , Ácido Fólico/metabolismo , Leucovorina/metabolismo , Leucovorina/farmacología , Leucemia L1210/enzimología , Leucemia L1210/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Pemetrexed , Péptido Sintasas/metabolismo , Ácido Poliglutámico/biosíntesis , Prostaglandinas A/farmacologíaRESUMEN
A murine leukemia cell line was identified that is highly resistant to methotrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dideazatetrahydrofolate (DDATHF). A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in drug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell line markedly increased DDATHF influx (32x) but only modestly increased influx of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively). The growth inhibition or growth requirements for these folates fell by factors of 18, 2, and 4, respectively, in the transfectant. Preservation of DDATHF influx in cells with V104M RFC1 resulted in even greater preservation (60%) of the exchangeable drug level. Another major element in the preservation of DDATHF activity was the impact of the mutated carrier on cellular folate pools. For folic acid, folate pools were essentially unchanged but DDATHF polyglutamate levels decreased in lines that express the V104M carrier. However, with 5-formyltetrahydrofolate as the growth source, there was a marked decrease in folate pools in the lines carrying the mutated carrier, and DDATHF polyglutamate levels were unchanged. Hence, DDATHF activity was preserved in cells with V104M RFC1 due to (a) relative conservation of DDATHF transport, and (b) depletion of cellular THF cofactors with diminishing folate cofactor competition at folylpolyglutamate synthetase and possibly glycinamide ribonucleotide formyltransferase. Hence, resistance to one antifolate, in this case MTX, because of a loss of RFC1 transport activity need not exclude the subsequent utility of another antifolate that uses the same carrier.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Antagonistas del Ácido Fólico/farmacología , Leucemia L1210/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Metotrexato/farmacología , Tetrahidrofolatos/farmacología , Aminoácidos/análisis , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Transporte Biológico , Proteínas Portadoras/genética , ADN Complementario/genética , Resistencia a Antineoplásicos , Antagonistas del Ácido Fólico/farmacocinética , Leucovorina/farmacología , Leucemia L1210/tratamiento farmacológico , Proteínas de la Membrana/genética , Metotrexato/farmacocinética , Ratones , Tetrahidrofolatos/farmacocinética , TransfecciónRESUMEN
Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A1 (PGA1) on processes that govern the transport of folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the reduced folate carrier, RFC1, and ATP-dependent exporters. PGA1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a Ki of approximately 21 microM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity. In contrast, the effect of PGA1 on folic acid transport was small (approximately 20% inhibition of total influx), consistent with the observation that the major portion of folic acid transport in CHO cells is mediated by a low pH mechanism distinct from RFC1. PGA1 was also a potent inhibitor of the ATP-driven efflux of both MTX and folic acid. At a concentration of 7 microM PGA1, the efflux rate constants for these folates were depressed by approximately 70 and approximately 50%, respectively. The net effects of PGA1 on the bidirectional folate fluxes translated into marked alterations in net transport. The addition of 7 microM PGA1 to cells at steady state with 1 microM MTX produced a rapid onset of net uptake and the achievement of an approximately 3-fold increase in the steady-state free MTX level as compared with untreated CHO cells. The addition of 7 microM PGA1 to cells at steady state with 1 microM folic acid produced an approximately 5-fold increase in the free folate level. These studies establish PGA1 as a potent inhibitor of both the reduced folate carrier and ATP-driven folate exporter(s). The noncompetitive nature of the inhibition of RFC1 is unique among anionic compounds, which are usually competitive inhibitors of the carrier.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Ácido Fólico/metabolismo , Prostaglandinas A/farmacología , Receptores de Superficie Celular , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células CHO , Membrana Celular/metabolismo , Cricetinae , Receptores de Folato Anclados a GPI , Antagonistas del Ácido Fólico/farmacocinética , Metotrexato/farmacocinéticaRESUMEN
Previous studies described a Chinese hamster ovary cell line, PyrR100, resistant to lipid-soluble antifolates due to the loss of an energy-coupled folate exporter resulting in a marked increase in intracellular folate cofactor accumulation. There was, in addition, an unexplained increase in folic acid influx in PyrR100 cells which is shown in this paper to be mediated by a transporter with a low pH optimum. The pH profile for folic acid influx in parental Chinese hamster ovary AA8 cells indicated peak activity at pH 6; this was increased >3-fold in PyrR100 cells. In contrast, methotrexate (MTX) influx in AA8 cells showed two peaks of comparable activities at pH 6 and 7.5; in PyrR100 cells, the component at pH 6 was increased 2-fold. Folic acid was a potent inhibitor of [3H]MTX or [3H]folic acid influx (1 microM) via the low pH route with IC50 values of approximately 1 microM. Prostaglandin A1 was a potent inhibitor of [3H]MTX influx via the reduced folate carrier 1 at pH 7.5 with only a small inhibitory effect on the low pH route. The addition of 10 microM folic acid to PyrR100 cells resulted in a MTX influx pH profile identical to that of AA8 cells, consistent with suppression of the low pH route. In contrast, addition of 25 microM prostaglandin A1 to PyrR100 cells resulted in a MTX influx pH profile comparable to that of folic acid, consistent with the loss of the reduced folate carrier-mediated component. Inhibition ( approximately 70%) of [3H]folic acid influx by approximately 10 microM unlabeled folic acid at pH 7.5 indicated that the low pH transporter accounts for the majority of folic acid transport at physiological pH. This study demonstrates the functional importance of a low pH folate transporter that is increased when enhanced folic acid entry into cells is required as an adaptive response to antifolate selective pressure. This may represent a mechanism of resistance to new antifolate inhibitors of folate cofactor-dependent enzymes in which cytotoxic activity is limited by expanded cellular folate pools.