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Results for “Reduced Folate Carrier Protein”

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PubMed2003

Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport.

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed1991

Synthesis and biological evaluation of a fluorescent analogue of folic acid.

A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗

PubMed2004

Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate.

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.

Animals↗

PubMed2001

Carrier-mediated hepatobiliary transport of a novel antifolate, N-[4-[(2,4-dianninopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl-L-homoglutamic acid, in rats.

The hepatic uptake and biliary excretion of a novel methotrexate derivative, N-[4-[(2,4-diamminopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl-L-homoglutamic acid (MX-68), were examined in rats in vitro using isolated hepatocytes and bile canalicular membrane vesicles (CMVs), respectively. The uptake of MX-68 by isolated rat hepatocytes showed a saturable component (Km = 2.15 microM and Vmax = 2.34 pmol/min/mg of protein) and was inhibited by ATP-depletors and anionic compounds such as taurocholate and probenecid. [3H]MX-68 uptake was also inhibited by folate analogs such as methotrexate and 5CH3-tetrahydrofolate, but the effect of these compounds was slightly less than that of unlabeled MX-68. On replacing Na+ with choline, MX-68 uptake remained unchanged, whereas the methotrexate uptake was reduced. Uptake of MX-68 increased as the extracellular pH fell from 7.5 to 5.5. These results suggest that MX-68 is taken up via active transport systems. The uptake of MX-68 by CMVs prepared from normal rats exhibited clear ATP dependence, whereas ATP had only a minimal effect on the uptake by CMVs from Eisai-hyperbilirubinemic rats with a hereditary deficiency in canalicular multispecific organic anion transporter (cMOAT). The initial uptake rate of ATP-dependent MX-68 transport showed saturation with kinetic parameters similar to those of methotrexate. MX-68 inhibited the ATP-dependent transport of 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, the inhibition constant (162 microM) being comparable with the Km of ATP-dependent MX-68 transport. These results suggest that the biliary excretion of MX-68 via the bile canalicular membrane is mediated mainly by cMOAT. In conclusion, active transport systems are involved in membrane penetration of MX-68 both at sinusoidal and canalicular sides in the liver, the latter being mainly involved with methotrexate (MTX) whereas the former differs partially from that for MTX.

2-Aminoadipic Acid↗

PubMed2004

Antisense oligonucleotides targeted to the human alpha folate receptor inhibit breast cancer cell growth and sensitize the cells to doxorubicin treatment.

Folates are essential for cell survival and are required for numerous biochemical processes. The human alpha isoform folate receptor (alphahFR) has a very high affinity for folic acid and is considered an essential component in the cellular accumulation of folates and folate analogues used in chemotherapy. The expression of alphahFR is not detected inmost normal tissues. In contrast, high levels of the expression of alphahFR have been reported in a variety of cancer cells. The significance of alphahFR overexpression in malignant tissues has not been elucidated, but it is possible that it promotes cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. The purpose of the present study was to evaluate alphahFR as a potential target for the treatment of breast cancer. Initial studies were done in nasopharyngeal carcinoma (KB) cells, which express high levels of alphahFR. In KB cells, antisense oligodeoxyribonucleotides (ODN) complementary to the alphahFR gene sequences were found to reduce newly synthesized alphahFR protein up to 60%. To examine the effect of alphahFR antisense ODNs in a panel of cultured human breast cancer cell lines, we used a tumor cell-targeted, transferrin-liposome-mediated delivery system. The data show that alphahFR antisense ODNs induced a dose-dependent decrease in cell survival. Finally, we determined that alphahFR antisense ODNs sensitized MDA-MB-435 breast cancer cells by 5-fold to treatment with doxorubicin. The data support the application of alphahFR antisense ODNs as a potential anticancer agent in combination with doxorubicin.

Antibiotics, Antineoplastic↗

PubMed1995

Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias.

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

Animals↗

PubMed1995

Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro.

Receptors for the vitamin folic acid are frequently overexpressed on epithelial cancer cells. To examine whether this overexpression might be exploited to specifically deliver liposome-encapsulated drug molecules in vitro, folate-targeted liposomes were prepared by incorporating 0.1 mol% of a folate-polyethyleneglycol-distearoylphatidylethanolamine (folate-PEG-DSPE) construct into the lipid bilayer, and were loaded with doxorubicin (DOX), an anti-cancer drug. Uptake of folate-PEG-liposomal DOX by KB cells was 45-fold higher than that of non-targeted liposomal DOX, and 1.6-times higher than that of free DOX, while the cytotoxicity was 86 and 2.7-times higher, respectively. Folate-targeting is fully compatible with PEG-coating of the liposomes, since incorporation of 4 mol% PEG2000-DSPE does not reduce the uptake or cytotoxicity of folate-PEG-liposomal DOX. Uptake of folate-PEG-liposomes was inhibited by 1 mM free folic acid but was unaffected by physiological concentrations of folate. In HeLa/WI38 co-cultures, folate-PEG-liposomes encapsulating calcein, a fluorescent dye, were found to be almost exclusively internalized by the HeLa cells which overexpress the folate receptors. We suggest that folate targeting constitutes a possible mechanism for improving the specificity of PEG-coated liposomes for cancer cells.

Biological Transport↗

PubMed1997

Fumonisin B1-induced sphingolipid depletion inhibits vitamin uptake via the glycosylphosphatidylinositol-anchored folate receptor.

The folate receptor, like many glycosylphosphatidylinositol-anchored proteins, is found associated with membrane domains that are insoluble in Triton X-100 at low temperature and that are enriched in cholesterol and sphingolipids. Depletion of cellular cholesterol has been shown to inhibit vitamin uptake by this receptor (Chang, W. -J., Rothberg, K. G., Kamen, B. A., and Anderson, R. G. W. (1993) J. Cell Biol. 118, 63-69), suggesting that these domains regulate this process. In this study, the importance of sphingolipids for folate receptor function was investigated in Caco-2 cells using fumonisin B1, a mycotoxin that inhibits the biosynthesis of these lipids. The folate receptor-mediated transport of 5-methyltetrahydrofolate was almost completely blocked in cells in which sphingolipids had been reduced by approximately 40%. This inhibition was dependent on the concentration and duration of the treatment with the mycotoxin and was mediated by the sphingolipid decrease. Neither receptor-mediated nor facilitative transport was inhibited by fumonisin B1 treatment, indicating that the effect of sphingolipid depletion was specific for folate receptor-mediated vitamin uptake. A concurrent loss in the total amount of folate binding capacity in the cells was seen as sphingolipids were depleted, suggesting a causal relationship between folate receptor number and vitamin uptake. These findings suggest that dietary exposure to fumonisin B1 could adversely affect folate uptake and potentially compromise cellular processes dependent on this vitamin. Furthermore, because folate deficiency causes neural tube defects, some birth defects unexplained by other known risk factors may be caused by exposure to fumonisin B1.

Biological Transport↗

PubMed2006

Dietary folate and APC mutations in sporadic colorectal cancer.

Folate deficiency has been associated with colorectal cancer risk and may be involved in colorectal carcinogenesis through increased chromosome instability, gene mutations, and aberrant DNA methylation. Within the Netherlands Cohort Study on diet and cancer, we investigated the associations between dietary folate intake and colorectal cancer risk with (APC(+)) and without (APC(-)) truncating APC mutations, accounting for hMLH1 expression and K-ras mutations. In total, 528 cases and 4200 subcohort members were available for data analyses of the study cohort (n = 120,852) from a follow-up period between 2.3 and 7.3 y after baseline. Adjusted gender-specific incidence rate ratios (RR) over tertiles of folate intake were calculated in case-cohort analyses for colon and rectal cancer. Although relatively high folate intake was not associated with overall colorectal cancer risk, it reduced the risk of APC(-)colon tumors in men (RR 0.58, 95% CI 0.32-1.05, P(trend) = 0.06 for the highest vs. lowest tertile of folate intake). In contrast, it was positively associated with APC(+) colon tumors in men (highest vs. lowest tertile: RR 2.77, 95% CI 1.29-5.95, P(trend) = 0.008) and was even stronger when the lack of hMLH1 expression and K-ras mutations were excluded (RR 3.99, 95% CI 1.43-11.14, P(trend) = 0.007). Such positive associations were not observed among women; nor was folate intake associated with rectal cancer when APC mutation status was taken into account. Relatively high folate consumption reduced the risk of APC(-) colon tumors, but folate intake was positively associated with APC(+) colon tumors among men. These opposite results may indicate that folate enhances colorectal carcinogenesis through a distinct APC mutated pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

New drugs for the treatment of advanced-stage diffuse large cell lymphomas.

The rapid pace of discovering new signaling pathways that influence the growth and survival of different types of cancer cells has produced a daunting array of potentially new, "drugable" targets for the treatment of cancer. This has been particularly true for the lymphomas. The empiric observation that many kinds of "novel targeted" drugs appear to exhibit relatively selective patterns of activity is testament to the concept that the lymphomas represent an incredibly diverse group of human cancers. Targeting one pathway in a subtype of lymphoma may not be universally effective against all subtypes of lymphoma, even closely related ones. These observations suggest that pharmacologically driven "target identification" plays an important role in the continued development of new therapeutic agents, and underscores the need for more research into the biologic basis for different subtypes of lymphoma. Over the past 10 years, there has been an explosion of new drugs making their way through preclinical laboratories and early clinical studies. These experiences have taught us some frustrating lessons. Sometimes, the strongest biologic rationale is associated with the poorest clinical results. Sometimes, where no rationale exists, golden therapeutic opportunities emerge. In either case, both experiences typically end up teaching us something about the disease we didn't historically appreciate. In this review, we will take a close look at some of the more intriguing targets in aggressive large B-cell lymphomas, all of which now have small molecules that can profoundly affect their activity, activity we hope will lead to new treatment opportunities. We will focus on the biologic rationale for targeting novel pathways regulated by Bcl-6, Bcl-2, BLysS, and APRIL for example. In addition, we will review how new concepts in structural biology and chemical design can help produce new generation compounds with novel activity, as is the case for pralatrexate and targeting the reduced folate carrier-type-1. Clearly, given the breadth and depth of information emerging on these and other relevant biologic pathways, we are limited to discussing only a few illustrative examples, which should in no way detract from the importance of other critical signaling and survival pathways now being exploited in the treatment of lymphoma.

Antibodies, Monoclonal↗

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↗

PubMed1996

Cellular transport of CI-980.

CI-980, originally synthesized as a potential folate antagonist, is a tubulin-binding mitotic inhibitor currently in pediatric phase I and adult phase II clinical trials. Because of its extensive tissue distribution in animals and its favorable activity against multidrug resistant (MDR)-cells compared with other mitotic inhibitors, such as vincristine, we examined the membrane transport properties of CI-980. CI-980 accumulated rapidly in L1210 and CHO/K1 cells, reaching intracellular levels 40- and 8-fold higher, respectively, than those in the extracellular medium. Efflux was also quite rapid, but a small fraction of drug remained associated with the cells in drug-free medium. The uptake of CI-980 was not temperature or energy dependent, nor was it saturable up to an extracellular concentration of 100 microM. Inhibitors of nucleoside transport had no effect on CI-980 uptake. A cell line deficient in the transport of reduced folate was not resistant to CI-980, nor did it exhibit reduced CI-980 uptake. A 100-fold excess of the R-enantiomer inhibited CI-980 uptake by only 50%. These results are consistent with a model of CI-980 uptake involving passive diffusion followed by significant but largely reversible binding to intracellular or membrane components.

Animals↗

PubMed1991

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed2004

In vivo efficacy of folate-targeted lipid-protamine-DNA (LPD-PEG-Folate) complexes in an immunocompetent syngeneic model for breast adenocarcinoma.

Gene therapy utilizing lipid-based delivery systems holds tremendous promise for the treatment of cancer. However, due to the potential adverse inflammatory and/or immune effects upon systemic administration, treatments thus far have been predominantly limited to intratumoral or regional treatment. Previous studies from our group have demonstrated the antitumor efficacy of systemically administered, folate-targeted, lipid-protamine-DNA complexes (LPD-PEG-Folate) against breast cancer using an immunodeficient xenogenic murine model. In the current study, the antitumor efficacy of LPD-PEG-Folate in a syngeneic, immune competent, murine model of breast cancer was examined. In this model, the potential inflammatory or immune responses and their effects on systemic delivery can be addressed. The 410.4 murine breast adenocarcinoma cell line was initially evaluated in vitro for its interactions with LPD-PEG-Folate and control LPD-PEG formulations. Utilizing fluorescently labeled formulations and fluorescence-activated cell sorting (FACS) analysis, a 1.6-fold enhancement of binding and internalization of LPD-PEG-Folate over LPD-PEG formulations was observed, suggestive of specific receptor interaction. Increased binding was manifested as 5-26-fold increases in luciferase gene expression in 410.4 cell transfection when comparing LPD-PEG-Folate to LPD-PEG. Moreover, in vivo treatment of 410.4 breast tumors in BALB/c mice with i.v. injected LPD-PEG-Folate delivering the HSV-1 thymidine kinase (TK) gene, in combination with gancyclovir treatment, resulted in a significant reduction in mean tumor volume (260.1 mm3) compared to the LPD-PEG-TK (914.1 mm3), as well as the vehicle (749.7 mm3) and untreated (825.3 mm3) control groups (day 25, P<.019). In addition to a reduced tumor volume, LPD-PEG-Folate-TK treatment also increased median survival from 25 days in the nontargeted LPD-PEG-TK groups to 31 days (P=.0011), which correlated with the termination of treatment. Together, these results demonstrate that in the context of a fully functional immune system, LPD-PEG-Folate-TK treatment possesses significant specific antitumor efficacy and the potential for further preclinical development.

Adenocarcinoma↗

PubMed1988

Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling.

Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗

PubMed1996

Isolation and characterization of a folate receptor-directed metalloprotease from human placenta.

Glycosyl-phosphatidylinositol-anchored hydrophobic placental folate receptors (PFRs), which have an important functional role in maternal-to-fetal transplacental folate transport, can be converted to soluble hydrophilic forms by a placental metalloprotease. Using a Triton X-114 temperature-induced phase separation assay to monitor enzyme-mediated conversion of radiolabeled hydrophobic PFR into hydrophilic PFR, a metalloenzyme was isolated to apparent homogeneity from Triton X-114-solubilized human placenta using concanavalin A-Sepharose and reverse-phase high performance liquid chromatography (HPLC) as major purification steps. The purified hydrophobic enzyme eluted as a single protein peak on reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis revealed a single 63,000 M(r) species, which was reduced to 58,000 M(r) following deglycosylation, findings comparable with amino acid analysis (M(r) approximately 59,000). The metalloenzyme was activated by Mg2+, Zn2+, Mn2+, and Ca2+, optimally at physiologic pH; it also exhibited EDTA-sensitive endoproteolytic cleavage of [3H]leucine-labeled full-length nascent PFR polypeptide generated in vitro in the absence of microsomes. Rabbit polyclonal anti-metalloprotease antiserum specifically immunoprecipitated 125I-metalloprotease and recognized cross-reacting moieties on plasma membranes of normal human hematopoietic progenitor cells and human cervical carcinoma cells, both of which also express FR.

Amino Acids↗

PubMed2003

Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies.

The alpha-folate receptor (FR) is selectively overexpressed in 90% of nonmucinous ovarian carcinomas, whereas no expression is detectable in normal ovarian surface epithelium (OSE). Indirect evidence suggests that FR expression is associated with tumor progression and affects cell proliferation. To evaluate better the role of FR, we developed an approach based on intracellular expression of single-chain (sc) antibodies (intrabody) to downmodulate membrane expression of FR in ovary cancer cells. IGROV-1 and SKOV3 ovarian carcinoma cell lines were transfected with an anti-FR intrabody. Transfectants and parental cells were tested for FR, integrins and anti-FR intrabody expression by fluorescence-activated cell sorting (FACS), reverse transcription and polymerase chain reaction (RT-PCR) and/or immunoblotting. Cell growth characteristics and adhesion properties were evaluated in liquid, semisolid and organotypic cultures. The anti-FR scFv inhibited FR expression from 60 to 99%. At physiological concentrations of folate, proliferation varied directly as a function of FR expression. FR downmodulation was accompanied by reduced colony-forming ability in soft agar, morphological change of the cells, significant enhanced adhesion to laminin or Matrigel, a two- to three-fold increase in alpha6beta4 integrin expression, and a marked reduction in laminin production. In three-dimensional organotypic cultures, anti-FR intrabody-transfected IGROV1 cells grew as a single-ordered layer, reminiscent of normal OSE growth in vivo. In conclusion, the anti-FR intrabody reverses the transformed phenotype in ovary cancer cells and may provide an efficient means to inhibit selectively the growth of these cells.

Antibodies, Monoclonal↗

PubMed1995

Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.

Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation.

Animals↗