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Biomedical subjects

I David Goldman

Publications and source records attributed to I David Goldman.

At least 19 recordsLinked to original sources
PubMed2006

Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption.

Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.

Animals↗
PubMed2006

The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line.

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro

Amino Acid Substitution↗
PubMed2006

The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity.

Pemetrexed is a new-generation antifolate inhibitor of thymidylate synthase (TS) and a weaker inhibitor of glycinamide ribonucleotide transformylase (GARFT) required for de novo purine synthesis. Methylthioadenosine phosphorylase (MTAP) salvages purines by releasing adenine from methylthioadenosine and is often deleted in mesothelioma. The current study addresses the effect of MTAP on pemetrexed activity using a highly potent transition state inhibitor of MTAP, MT-DADMe-Immucillin A (ImmA; K(i) = 86 pmol/L) in the MTAP(+) NCI-H28 and MTAP(-) NCI-H2052 mesothelioma cell lines. Based on selective nucleoside protection, TS was found to be the primary pemetrexed target in both cell lines with GARFT inhibition requiring 20- to 30-fold higher pemetrexed concentrations. ImmA had no effect on pemetrexed activity but, when thymidine was added, the pemetrexed IC(50) decreased by a factor of approximately 3 in MTAP(+) H28 cells with no effect in MTAP(-) H2052 cells. Conversely, the transfection of MTAP into H2052 cells increased the pemetrexed IC(50) by nearly 3-fold but only in the presence of thymidine; this was reversed by ImmA. An MTAP-specific short interfering RNA produced a 2-fold decrease in pemetrexed IC(50) in MTAP(+) HeLa cells in the presence of thymidine. These data indicate that suppression of constitutive MTAP has no effect on pemetrexed activity when the primary target is TS. There is a modest salutary effect when the pemetrexed target is GARFT alone.

Cell Line, Tumor↗
PubMed2005

The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells.

Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632(R) subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70%, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx K(t) for folic acid and MTX at pH 5.5 in PT632(R) cells was 0.36 and 1.5 microM, respectively. The affinity of the low pH transporter in PT632(R) cells was highest for pemetrexed (K(i)=140 nM), very low for PT632 (K(i)=77 microM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60% resulting in a 40% decrease in MTX influx at pH 7.4 but only a very small (5%) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.

Biological Transport↗
PubMed2005

Loss of reduced folate carrier function and folate depletion result in enhanced pemetrexed inhibition of purine synthesis.

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.

Animals↗
PubMed2004

Antifolate resistance in a HeLa cell line associated with impaired transport independent of the reduced folate carrier.

Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low pH optimum in human solid tumor cell lines. In HeLa cells, this low pH activity has high affinity for pemetrexed [PMX (Alimta)] and is reduced folate carrier (RFC)-independent because it is not diminished in a RFC-null subline (R5). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH. In the current study, a R5 subline, R1, was selected under MTX selective pressure at a modest reduction in pH. There was markedly decreased MTX and PMX transport at both pH 5.5 and pH 7.4. When MTX was removed, there was a slow return of transport activity, and when MTX was added back, there was loss of transport at both pH values within 8 weeks. In R1 cells, there was a marked decrease in accumulation of PMX, MTX, and folic acid along with a decrease in growth inhibition by these and other antifolates that require a facilitative process to gain entry into cells. These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contributes to antifolate activity (in particular, to PMX activity) and can be diminished by antifolate selective pressure and (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process.

Antimetabolites, Antineoplastic↗
PubMed2004

Lack of impact of the loss of constitutive folate receptor alpha expression, achieved by RNA Interference, on the activity of the new generation antifolate pemetrexed in HeLa cells.

Pemetrexed [PMX (Alimta)] is a new generation antifolate with activity in a variety of solid tumors. It is an excellent substrate for most folate transporters, notably the reduced folate carrier (RFC) and folate receptor (FR)-alpha. The role of FR-alpha in PMX pharmacological activity is uncertain. Whereas high-level expression may enhance the activity of this agent, it is not clear what role constitutive levels of this transporter contribute to PMX activity. In this study, constitutive levels of FR-alpha expression were abolished by small interfering RNA-induced silencing in HeLa cells and RFC-null HeLa R5 cells as confirmed by Northern blotting, immunohistochemistry, and cell surface binding. PMX growth inhibition was unchanged in HeLa and R5 cells in the absence of FR-alpha expression. Loss of FR-alpha expression did not decrease net accumulation of PMX in either wild-type or RFC-null HeLa cells. Likewise, folate pools in wild-type HeLa cells were not decreased by FR-alpha gene silencing and were negligibly affected in the RFC-null R5 subline grown with 5-formyltetrahydrofolate. FR-alpha surface binding in HeLa cells was shown to be greater than that in a variety of other human solid tumor cell lines. Hence, constitutively expressed FR-alpha in HeLa cells does not contribute to PMX activity in the presence or absence of RFC function. This is likely the case in many human solid tumor cell lines.

Apoptosis↗
PubMed2004

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function.

Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln(420). A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser(135) was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC.

Animals↗
PubMed2004

Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, approximately 45 nmol/L) than methotrexate (MTX; Kt, approximately 1 micromol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were approximately 400-600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 micromol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 micromol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (approximately 1.2 micromol/L) or the Ki for folic acid (approximately 130 micromol/L) or PT523 ( approximately 0.2 micromol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl- (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

Antimetabolites, Antineoplastic↗
PubMed2004

Selective preservation of pemetrexed pharmacological activity in HeLa cells lacking the reduced folate carrier: association with the presence of a secondary transport pathway.

A methotrexate (MTX)-resistant HeLa subline (R5), developed in this laboratory, with impaired transport due to a genomic deletion of the reduced folate carrier (RFC) was only 2-fold resistant to pemetrexed (PMX), but 200- and 400-fold resistant to raltitrexed (ZD1694) and N(alpha)-(-4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-1-ornithine (PT523), respectively, compared with parental HeLa cells when grown with 2 microM folic acid. When folic acid was replaced with the more physiological 25 nM 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX but still 40- and 200-fold resistant to ZD1694 and PT523, respectively. Sensitivity to PT523 and PMX could be completely restored, and sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells, indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (Kt = 12 microM) with much lower affinities for ZD1694, MTX, and PT523 (Kis of approximately 90, 100, and 250 microM, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nM 5-formyl-tetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity in RFC-null R5 cells is due, in part, to partial preservation of transport by secondary process with a higher affinity for PMX than the other antifolates evaluated.

Antimetabolites, Antineoplastic↗
PubMed2004

A prominent low-pH methotrexate transport activity in human solid tumors: contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier.

Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 micro M) at pH 7.4, the IC(50) for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC(50)s were identical at this pH when the medium folate was 25 nM 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.

Antimetabolites, Antineoplastic↗
PubMed2003

Resistance to antifolates.

The antifolates were the first class of antimetabolites to enter the clinics more than 50 years ago. Over the following decades, a full understanding of their mechanisms of action and chemotherapeutic potential evolved along with the mechanisms by which cells develop resistance to these drugs. These principals served as a basis for the subsequent exploration and understanding of the mechanisms of resistance to a variety of diverse antineoplastics with different cellular targets. This section describes the bases for intrinsic and acquired antifolate resistance within the context of the current understanding of the mechanisms of actions and cytotoxic determinants of these agents. This encompasses impaired drug transport into cells, augmented drug export, impaired activation of antifolates through polyglutamylation, augmented hydrolysis of antifolate polyglutamates, increased expression and mutation of target enzymes, and the augmentation of cellular tetrahydrofolate-cofactor pools in cells. This chapter also describes how these insights are being utilized to develop gene therapy approaches to protect normal bone marrow progenitor cells as a strategy to improve the efficacy of bone marrow transplantation. Finally, clinical studies are reviewed that correlate the cellular pharmacology of methotrexate with the clinical outcome in children with neoplastic diseases treated with this antifolate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

Residues 45 and 404 in the murine reduced folate carrier may interact to alter carrier binding and mobility.

The reduced folate carrier (RFC), a facilitative transporter, plays a major role in the delivery of reduced folates and antifolates into cells. Previous studies indicated that mutations of E45K in the first transmembrane domain (TMD), and K404L in the 11th TMD, produce selective and opposite alterations in binding of natural folate substrates to murine RFC. The former mutation is frequently associated with antifolate resistance. The current study was designed to determine whether there might be an interaction between these sites by comparing the transport properties of RFC-null cell lines stably transfected with K404E, E45K, or E45K/K404E carriers. These studies demonstrated that: (1) All mutant carriers were inserted into the plasma membrane. (2) In the K404E mutant, the influx K(t)'s for 5-formyltetrahydrofolate and 5-methyltetrahydrofolate were markedly increased, and to a much smaller extent folic acid, as compared to L1210 cells. However, with introduction of a second E45K mutation the influx K(t) for these folates reverted to those of the E45K cells which retained wild-type binding for 5-methyltetrahydrofolate and enhanced binding of 5-formyltetrahydrofolate and folic acid. (3) The influx V(max) of the E45K mutant was markedly reduced. Introduction of the second K404E mutation doubled this parameter and the ratio of V(max) to K(t) for 5-formytetrahydrofolate was restored to approximately 50% that of the wild-type carrier consistent with a substantial increase in function. (4) Chloride inhibits wild-type RFC but the E45K mutant requires chloride for activity. The K404E mutant is also suppressed by chloride but introduction of the K404E mutation decreased the chloride-dependence of E45K. The results suggest that there is an interaction between the E45 and K404 residues in the first and 11th TMDs, respectively, but that the E45 residue appears to be the more dominant determinant of binding and anion sensitivity.

Amino Acid Substitution↗
PubMed2003

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line.

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.

Animals↗
PubMed2003

Antifolate resistance associated with loss of MRP1 expression and function in Chinese hamster ovary cells with markedly impaired export of folate and cholate.

Export of folates from a Chinese hamster ovary PyrR100 cell line is markedly impaired, resulting in expansion of cellular folate pools and high-level antifolate resistance. We now report that MRP1 expression is absent in PyrR100 cells along with a marked decrease in MRP5 expression with 3-fold cross-resistance to thiopurines. PyrR100 and wild-type cells had comparable low levels of MRP2 expression; both lacked the breast cancer resistance protein. PyrR100 cells showed a 4-fold decrease in cholate (an MRP substrate) efflux with a 6-fold increase in cellular cholate accumulation compared with wild-type cells. Prostaglandin A1 increased cholate accumulation in wild-type cells to levels comparable with PyrR100 cells. Calcein (an MRP1 substrate) fluorescence increased 5-fold in PyrR100 cells; probenecid increased the intracellular calcein level in wild-type cells to that of PyrR100 cells. Consistent with the loss of MRP1 expression, PyrR100 cells showed modest collateral sensitivity to cholate, etoposide, doxorubicin, and vincristine. Transfection of MRP5 into PyrR100 cells did not alter sensitivity to pyrimethamine or MTX but restored sensitivity to mercaptopurines, indicating that decreased MRP5 expression did not play a role in antifolate resistance. Hence, although MRP-mediated anticancer drug resistance has been associated with gain of function (i.e., overexpression), this is the first report that loss of MRP1 efflux function can expand intracellular folate pools to result in acquired antifolate resistance. The data also suggest that MRP1, and possibly other MRPs that transport folates, can play a role in the maintenance of cellular folate homeostasis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

A novel folate transport activity in human mesothelioma cell lines with high affinity and specificity for the new-generation antifolate, pemetrexed.

Pemetrexed is a novel antifolate effective in the treatment of mesothelioma. Studies were undertaken to characterize the transport of this antifolate in this tumor. We report the presence of a novel, concentrative high-affinity transport activity in three human mesothelioma cell lines, characterized in detail in the NCI-H28 line, with a pemetrexed influx K(t) of 30 nM and V(max) of 10 nmol/g protein/min. This route is highly specific for pemetrexed, with a substrate specificity pattern quite different from that of the reduced folate carrier and folate receptors. In particular, there is an apparent relatively low affinity for other antifolate inhibitors of dihydrofolate-reductase (MTX, aminopterin, PT523) and thymidylate synthase (ZD1694, ZD9331). Besides its impact on the transport of pemetrexed, this high-affinity route may represent another pathway by which physiological folates are transported into human cells.

Antineoplastic Agents↗
PubMed2002

Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function.

The reduced folate carrier (RFC1), a member of the major facilitative superfamily, generates uphill transport of folates into cells through an exchange mechanism with intracellular organic anions. RFC1 has twelve transmembrane domains with N- and C-termini, and the long loop connecting the 6th and 7th transmembrane domains, directed to the cytoplasm. To elucidate the role of the C-terminus and the long cytoplasmic loop in carrier function, mutants with deletion of the entire C-terminus or with progressive deletions of the loop region were constructed and stably transfected into the murine MTX(r)A cell line, which lacks functional RFC1. While expression of the C-terminus-deleted RFC1 protein could not be detected in the cell lysate, the RFC1 mutant lacking 57 of 66 amino acid residues of the long cytoplasmic loop appeared to be inserted into the cytoplasmic membrane but was not functional. In cell lines in which 17 or 31 amino acids were deleted from the carboxyl half of the loop, there was partial preservation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate transport. The loss of 5-formyltetrahydrofolate transport activity in the delta31 and delta17 mutants was due primarily to a decrease in substrate binding to the carrier. Mutants with partially truncated internal loops demonstrated an anion responsiveness similar to that of wild-type RFC1, indicating that this region of the carrier does not contain a site(s) that plays a role in anion exchange. This is the first study to describe the important role of the long cytoplasmic loop in substrate binding and the crucial role of the C-terminus in maintaining stability of RFC1.

Amino Acid Sequence↗