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In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically activate non-toxic prodrugs in tumour tissue. The A33 cognate antigen is a promising target for immunotherapy of gastrointestinal cancers. We have explored A33-based ADEPT with carboxypeptidase A (CPA) and the prodrug, methotrexate-phenylalanine (MTX-Phe). In A33-positive SW1222 cells, the toxicity of MTX-Phe was about 3 logarithms lower compared to MTX. Preincubation with a huA33 antibody-CPA conjugate (huA33-CPA), but not with an isotypic control conjugate, rendered MTX-Phe equally toxic to MTX. No toxicity was observed in mice receiving MTX-Phe in 8-fold the LD50 of MTX. Nude mice bearing A33-positive SW1222 colon carcinoma xenografts were injected intravenously (IV) with 125I-labeled huA33-CPA. The conjugate localised to the tumour with a maximum from 6-24 h. Pre-treating these mice with excess A33 substantially reduced subsequent conjugate uptake, demonstrating immunologic specificity of tumour-uptake. Total tumour uptake and ratios of tumour over blood or normal tissues, however, were lower than with unconjugated A33. This may explain in part why no significant tumour responses were observed in xenografted mice. In summary, our results demonstrate in principle the feasibility of A33-based enzyme targeting, but they call for small recombinant antibody-enzyme constructs to facilitate tumour penetration and clearance from the bloodstream.

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a K(m) of 41 microM and a V(max) of 337 pmol.mg protein(-1).min(-1) was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model.

The Protein Data Bank (PDB) has been processed to extract a screening protein library (sc-PDB) of 2148 entries. A knowledge-based detection algorithm has been applied to 18,000 PDB files to find regular expressions corresponding to either protein, ions, co-factors, solvent, or ligand atoms. The sc-PDB database comprises high-resolution X-ray structures of proteins for which (i) a well-defined active site exists, (ii) the bound-ligand is a small molecular weight molecule. The database has been screened by an inverse docking tool derived from the GOLD program to recover the known target of four unrelated ligands. Both the database and the inverse screening procedures are accurate enough to rank the true target of the four investigated ligands among the top 1% scorers, with 70-100 fold enrichment with respect to random screening. Applying the proposed screening procedure to a small-sized generic ligand was much less accurate suggesting that inverse screening shall be reserved to rather selective compounds., (Copyright 2004 Wiley-Liss, Inc.)

Although heat shock protein Hsp72 confers resistance to oxidative injury, the mechanisms are unknown. These studies demonstrate that Hsp72 protects dihydrofolate reductase (DHFR) against injury caused by the thiol oxidant monochloramine (NH(2)Cl). When exposed to NH(2)Cl, DHFR catalytic activity is impaired and SDS-PAGE migration retarded. These may be blocked by prior addition of Hsp72 or the folate analog methotrexate. Methotrexate binding to DHFR is diminished by oxidant treatment, preventable by prior Hsp72 incubation. Hsp72 also protects DHFR in IEC-18 cells following oxidant exposure. Hsp72 co-immunoprecipitates with DHFR, especially after partial oxidation. The DHFR-Hsp72 interaction is modulated by cofactor/substrate binding for both Hsp72 (ATP) and DHFR (methotrexate). Thiol oxidation of DHFR increases susceptibility for tryptic proteolysis. Preincubation of DHFR with Hsp72 prevents the NH(2)Cl-induced sensitivity to proteolysis. Thus, Hsp72 binds DHFR through enhanced protein-chaperone interactions upon oxidant exposure, a process that may protect against irreversible modification of DHFR catalytic and structural integrity.

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

We present here a novel approach to induce protein dimerization in living cells through covalent labeling of fusion proteins with ligands which are interacting with other proteins. The covalent labeling is based on the reaction of the DNA repair protein O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine coupled to an appropriate ligand. Using methotrexate as a ligand, it is shown that the approach can be used to control transcription in the yeast Saccharomyces cerevisiae. The specificity of the labeling reaction and its irreversible nature should allow the approach to become a valuable tool to study and control protein dimerization in vivo.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit the renal tubular secretion of methotrexate. However, the relative contribution of the active S- and inactive R-enantiomers is unknown. This study examined the effect of racemic ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney (IPK). Nineteen kidneys were divided between a control and three treatment groups. Controls were perfused with methotrexate alone (25 micrograms mL-1, n = 5) over three 30-min periods. Treatment groups were perfused with methotrexate (25 micrograms m-1) for the first period, followed by a second period of methotrexate (25 micrograms mL-1) plus R- (n = 5), S- (n = 5) or RS-ketoprofen (n = 4) at 25 micrograms mL-1, and a third period of methotrexate (25 micrograms mL-1) plus R-, S- or RS-ketoprofen (50 micrograms mL-1). Perfusate and urine were collected over 10-min intervals. Methotrexate was measured by HPLC and its binding in perfusate by ultrafiltration. The clearance ratio (CR) for methotrexate was obtained by dividing the renal clearance by the product of its fraction unbound and the glomerular filtration rate. During control experiments, there was no significant change in the CR over 90 min. R-, S- and RS-ketoprofen at 50 micrograms mL-1 reduced the CR of methotrexate significantly, but there was no difference between the three groups. While the enantiomers of ketoprofen reduced the renal excretion of methotrexate, the interaction was not enantioselective.

The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH(-) gradient or an inwardly directed H(+)gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.

Folates are polyglutamylated in most organisms, and in cancer cells the polyglutamylation of folates and of the antifolate methotrexate (MTX) is an important determinant of MTX susceptibility. The folylpolyglutamate synthetase (FPGS) responsible for polyglutamylation of folates was recently characterized in the parasite Leishmania. We show here that MTX is polyglutamylated in Leishmania tarentolae and that triglutamates are the predominant form. The glutamate chain length of MTX increases significantly in Leishmania cells transfected with the FPGS gene and decreases in cells with one FPGS allele disrupted. Modulation in the expression of the FPGS gene also has a profound effect on MTX susceptibility and this effect was found to be dependent on the folate concentration of the medium. In the folate-rich medium SDM-79, overexpression of FPGS will confer MTX resistance while in M-199 medium, which has much less folates, FPGS transfectants are more sensitive to MTX. Cells with one allele of FPGS disrupted are more resistant to MTX in low folate medium. The modulation of FPGS expression affects both the short-term and long-term accumulation of folate and MTX, showing a marked decrease in accumulation in the FPGS haploid mutant. This differential accumulation was mediated by decreased influx of the drug into the cell. Finally, the analysis of MTX-resistant Leishmania mutants indicated that the presence of shorter glutamate chains on MTX is correlated with MTX resistance.

Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were approximately 1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing approximately 400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

Intrinsic or acquired resistance to cisplatin in cancer cells remains a major obstacle to successful chemotherapy. The clinically relevant genetic and molecular mechanisms of resistance have not yet been identified. Cisplatin-resistant (CP-r) human KB epidermoid carcinoma cell lines (HeLa) resistant to varying levels of cisplatin after single and multiple selection steps are cross-resistant to other platinum compounds and to methotrexate. Intraspecies hybrids of the sensitive and KB CP-r cells were fused with HeLa D98(OR) CP-s, hypoxanthine-aminopterin-thymidine (HAT) sensitive, ouabain resistant, to determine whether cisplatin resistance is dominant or recessive. Cell-cell hybridization between the sensitive cells and single-step or two-step KB CP-r cells both indicated codominance of cisplatin resistance compared to hybrids between sensitive cell lines (D98(OR)xKB). The hybrids between sensitive cell lines (D98xKB) and a single-step CP-r KB cell line (D98xKB-CP.5) also were cross-resistant to carboplatin and methotrexate. In addition, the relatively slower growth rate of CP-r cells appears to be dominant. In the two-step CP-r KB cell line, KB-CP1, resistance is no more dominant than in the single-step CP-r KB cell line, KB-CP.5, suggesting that one of the two steps of resistance in KB-CP1 may not be dominant. These dominance data suggest that it might be possible to identify one or more genes responsible for cisplatin resistance by gene transfer from a resistant cell line to a sensitive cell line., (Copyright 2003 Wiley-Liss, Inc.)

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.

The cell-permeable dihydrofolate reductase inhibitor methotrexate was covalently linked to a ligand for the protein FKBP to create a bifunctional molecule called MTXSLF. The covalent tether between the two ligands was designed to be prohibitively short, so that unfavorable protein-protein interactions between DHFR and FKBP preclude formation of a trimeric complex. In vitro and in vivo experiments demonstrate that MTXSLF is an effective inhibitor of human DHFR, but that efficacy is decreased in the presence of human FKBP due to the high concentration of FKBP and its tight affinity for MTXSLF. MTXSLF also inhibits Plasmodium falciparum DHFR in vitro, but a low concentration of the weaker binding Plasmodium FKBP has no effect on the inhibitory potency of MTXSLF in vivo. These studies illustrate a potentially general strategy for modulating the biological activity of synthetic molecules that depends on the ligand-binding properties of a nontarget protein.

Acute, subacute and chronic neurotoxicity have been observed after the administration of high-dose and/or intrathecal methotrexate (MTX). Acute toxicity is usually transient without permanent damage. Subacute and chronic toxicity are associated with changes in the brain and/or the spinal cord which may be progressive and even lead to coma and death in severe cases. It is believed that MTX can induce direct toxic effects to the CNS by damaging the neuronal tissue. Moreover, MTX interferes with the metabolic pathways of folates, excitatory amino acids, homocysteine, S-adenosylmethionine/S-adenosylhomocysteine, adenosine and biopterins, inducing biochemical alterations which have been associated with neurotoxic symptoms. It has been suggested that acute toxicity is partly mediated by adenosine, whereas homocysteine, S-adenosylmethionine/S-adenosylhomocysteine, excitatory amino acids and biopterins may play an important role in the development of subacute and chronic toxicity. A better understanding of the pathogenesis of MTX neurotoxicity would offer the possibility of developing new therapeutic strategies for its treatment or prevention., (Copyright 2003 S. Karger AG, Basel)

Methotrexate (MTX) has transformed the outlook for children with juvenile idiopathic arthritis (JIA). Most of the evidence from uncontrolled clinical trials suggests that MTX is an effective agent for treating active JIA. Data from controlled clinical trials suggests that MTX has statistically significant effects on patient centred disability measures in JIA patients with active arthritis. Although we would like a much larger study directed evidence base for our use of the drug, the studies that have been done are sound and have been followed by a change in clinical expectations and advice that speak of qualitative evidence from clinical practice, confirming the scientifically acquired data. Randomised controlled multicentre trials using sufficient numbers of patients, including functional assessment and quality of life measures, are needed to confirm the long term efficacy and safety of MTX in JIA.

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.

Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.

Methotrexate (MTX) remains the most commonly used disease modifying antirheumatic drug in rheumatoid arthritis (RA) because of its cost and experience in its use, despite the availability of new treatments such as leflunomide and the biological agents. However, a significant number of patients with RA either do not benefit from the drug or are unable to tolerate it. Pharmacogenetic approaches may help optimise treatment with MTX, and also other agents, in RA.

The addition of methotrexate to treatment protocols in children with acute lymphoblastic leukemia has been found beneficial in preventing central nervous system relapse. However, MTX itself may be associated with neurologic morbidities, the most significant of which is leukoencephalopathy. The present study describes the clinical spectrum of leukoencephalopathy, which ranges from a subclinical disease manifested only radiologically to a progressive, devastating encephalopathy. The interaction of MTX with other components of the treatment protocol is discussed, as is the effect of leucovorin. A summary is presented of the metabolic pathways that may be involved in the development of MTX toxicity. Researchers are still seeking a biochemical marker to aid in the determination of the amount of MTX that may be safely administered.

The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C(4) (LTC(4)), 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys(332), His(335), and Asp(336), predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys(332) mutants had lost the ability to transport LTC(4), and GSH transport was reduced; whereas E(2)17betaG, estrone 3-sulfate, and MTX transport were unaffected. E(2)17betaG transport was not inhibited by LTC(4) and could not be photolabeled with [(3)H]LTC(4), indicating that the MRP1-Lys(332) mutants no longer bound this substrate. Substitutions of MRP1-His(335) also selectively diminished LTC(4) transport and photolabeling but to a lesser extent. Kinetic analyses showed that V(max) (LTC(4)) of these mutants was decreased but K(m) was unchanged. In contrast to the selective loss of LTC(4) transport in the Lys(332) and His(335) mutants, the MRP1-Asp(336) mutants no longer transported LTC(4), E(2)17betaG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys(332), His(335), and Asp(336) of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys(332) and His(335) in determining substrate specificity and of Asp(336) in overall transport activity suggests that such interactions are critical for the binding and transport of LTC(4) and other substrates of MRP1.

Background: The purpose of this study was to determine if a correlation exists between clinical outcome and accumulation and polyglutamation of methotrexate by lymphoblasts in vitro in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL)., Patients and Methods: The amount of accumulated methotrexate and of long-chain methotrexate polyglutamates (MTXPG(3-7)) by lymphoblasts was determined in 52 children newly diagnosed with BCP-ALL after incubation with 1 micromol/L [(3)H]MTX for 24 hours in vitro. All patients then received intensive multiagent chemotherapy that used divided-dose oral methotrexate during consolidation and intensive continuation and standard oral weekly methotrexate during maintenance., Results: Eight patients had a bone marrow relapse at a median of 40.4 months (range 18.5-48.3 months) after diagnosis. The median follow-up for the remaining 44 patients is 69.0 months (range 22-92.8 months). There was no significant difference in the amount of accumulated methotrexate (1450.0 +/- 896.3 vs. 640 +/- 472.5 pmol/10 cells) or of accumulated MTXPG (1450.0 +/- 919.4 vs. 617.4 +/- 482.7 pmol/10(9) cells) (median +/- semi-interquartile ranges) between patients who relapsed and those who remained in continuous complete remission. The estimated 5-year event-free survival rate for patients whose lymphoblasts accumulated more than 500 pmol MTXPG(3-7)/10(9) cells was 80.0% +/- 7.3% versus 90.5% +/- 6.4% for those whose lymphoblasts accumulated less than 500 pmol MTXPG(3-7)/10(9) cells., Conclusions: In the context of effective prolonged divided-dose oral methotrexate-based therapy in the treatment of BCP-ALL, methotrexate accumulation and polyglutamation no longer seem to have prognostic significance.

Methotrexate (MTX), an antineoplastic agent, demonstrates phototoxicity. The mechanism of damage to biomacromolecules induced by photoirradiated MTX was examined using 32P-labeled DNA fragments obtained from a human gene. Photoirradiated MTX caused DNA cleavage specifically at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA only when the DNA fragments were treated with piperidine, which suggests that DNA cleavage was caused by base modification with little or no strand breakage. With denatured single-stranded DNA the damage occurred at most guanine residues. The amount of formation of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo), an oxidative product of 2'-deoxyguanosine, in double-stranded DNA exceeded that in single-stranded DNA. These results suggest that photoirradiated MTX participates in 8-oxodGuo formation at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA through electron transfer, and then 8-oxodGuo undergoes further oxidation into piperidine-labile products. Fluorescence measurement, high-pressure liquid chromatography and mass spectrometry have demonstrated that photoexcited MTX is hydrolyzed into 2,4-diamino-6-(hydroxymethyl)pteridine (DHP). DNA damage induced by DHP was observed in a similar manner as was the damage induced by MTX. The extent of DNA damage and the formation of 8-oxodGuo by DHP were much larger than those induced by MTX. The kinetic analysis, based on the time course of DNA oxidation by photoirradiated MTX, suggests that DNA damage is caused by photoexcited DHP rather than by photoexcited MTX. In conclusion, photoexcited MTX undergoes hydrolysis through intramolecular electron transfer, resulting in the formation of DHP, which exhibits a phototoxic effect caused by oxidation of biomacromolecules through photoinduced electron transfer.

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Control of gene expression using small molecules is a powerful research tool and has clinical utility in the context of regulated gene therapy. Use of chemical inducers of dimerization, or dimerizers, for this purpose has several advantages, including tight regulation, modularity to facilitate iterative improvements, and assembly from human proteins to minimize immune responses in clinical applications. Recent developments include the use of the rapamycin-based dimerizer system to regulate the expression of endogenous genes, the generation of new chemical dimerizers based on FK506, dexamethasone and methotrexate, and progress towards the clinical use of adeno-associated virus and adenovirus vectors regulated by rapamycin analogs.

The aim of this methodological study was to present and exemplify a system-approach-based technology in modeling the formation of the metabolite from the parent drug. To represent this process, the parent-metabolite dynamic system was defined in such a way that the concentration-time profile of the parent drug was considered the input, while the concentration-time profile of the metabolite the output, of this system. The system-approach-based modeling technology was used to determine the model of the parent-metabolite dynamic system and to obtain the model-based estimates of the rate of metabolite formation, the rate of metabolic ratio and the mean time of metabolite formation. The technology was applied to concentration data for methotrexate and 7-hydroxymethotrexate in patients with psoriasis after a single oral methotrexate dose. Third-order linear models were selected as optimal to approximate the parent-metabolite dynamic systems representing the formation of 7-hydroxymethotrexate from methotrexate of these patients. The model-based estimates of the metabolic ratios ranged from 0.53 to 0.95. The model-based estimates of the mean times of formation of 7-hydroxymethotrexate from methotrexate ranged from 9.13 to 25.13 h. The model-based estimates of the rates of formation of 7-hydroxymethotrexate from methotrexate reached peak values (ranging from 0.03 to 0.11 h-1) in the time interval 1.5-4.5 h after administration of methotraxate. This study does not only introduce the method that may be useful in gaining insight into metabolite formation, but also presents a new example of the methodological, conceptual and computational uniformity of the system-approach-based technology in modeling various biomedical systems.

Small-molecule three-hybrid systems show promise as an in vivo alternative to affinity chromatography for detecting small-molecule-protein interactions. While several three-hybrid systems have been reported, little has been done to characterize these systems and, in particular, to test the assumption that the protein-small-molecule interaction can be varied without disrupting the transcription read-out. Recently we reported a dexamethasone-methotrexate chemical inducer of dimerization (CID) for use in the yeast three-hybrid system, based on the well-studied ligand-receptor pairs dexamethasone (Dex)-glucocorticoid receptor (GR) and methotrexate (Mtx)-dihydrofolate reductase (DHFR). Here we describe our first efforts to characterize this system, by focusing on a comparison of the activity of a bacterial and a mammalian DHFR as a test case of the influence of the ligand-receptor pair on the transcription read-out. By using a lacZ reporter gene, the activity of several GR and DHFR protein chimeras with different orientations and linker sequences and Dex-Mtx CIDs with different chemical linkers have been compared. In addition, Western analyses and in vivo biochemical assays have been carried out to confirm the integrity of the GR and DHFR protein chimeras. The transcription read-out is found to be much more sensitive to the structure of the protein chimeras than the CID. The most surprising result is that the levels of transcription activation are consistently higher with the bacterial than the mammalian DHFR, despite the fact that both proteins bind Mtx with an inhibition constant (K(I)) in the low pM range. These results set the stage for understanding three-hybrid systems at the biochemical level so that they can be used to detect ligand-receptor pairs with a range of structures and dissociation constants.

Background: Cooperative interactions between natural killer (NK) cells and macrophages occur normally during the course of the early immune response to bacterial, protozoal, and viral pathogens, with each cellular compartment providing the other with critical stimulatory factors. We conducted the present study to determine whether NK cells contribute to the dysregulated immune response that accompanies septic shock., Methods: An analysis of the lethality of Escherichia coli CP9 was conducted in mice that had been depleted of NK cells via the injection of an anti-asialo GM1 antibody and in CD epsilon transgenic mice that are deficient in both NK cells and T cells. The 2 groups of mice were analyzed for serum levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta as well as activation of NFkappaB and STAT1, 2 proinflammatory transcription factors., Results: NK cell-depleted and NK cell-deficient mice exhibited 80% survival in the face of an intraperitoneal bacterial challenge, whereas control mice all died within 12 hours. Serum levels of proinflammatory cytokines were markedly reduced in NK-depleted mice. NF kappaB and STAT1 activation were also reduced. NK-depleted mice exhibited less inflammation within multiple organs on histologic analysis., Conclusions: These results show that NK cells may contribute to the lethality of bacterial infections via effects on cytokine production.

Methotrexate (MTX) is an antifolate that is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL) and a number of other malignant and nonmalignant diseases. Within cells, MTX is metabolized to more active methotrexate polyglutamates (MTXPG), and these polyglutamates are subsequently cleaved in lysosomes by gamma-glutamyl hydrolase (GGH). GGH is reported to act as either an endopeptidase or an exopeptidase, exhibiting species differences in these functions. To better define the in vivo functions of GGH in human leukemia cells, we characterized GGH activity with different MTXPG substrates (MTX with three to five glutamates) in human T- and B-lineage leukemia cell lines, and in primary leukemia cells from newly diagnosed patients with ALL. Parameters estimated from fitting a series of hypothetical mathematical models to the data revealed that the experimental data were best fit by a model where GGH simultaneously cleaved multiple glutamyl residues, with highest activity at cleaving the outermost or two outermost residues from a polyglutamate chain. The model also revealed that GGH has a higher affinity for longer chain polyglutamates. Together, these findings provide new insights to the intracellular disposition of MTX in human ALL cells, and provides a mechanism-based model for characterizing differences among patients and genetic subtypes of ALL.

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [3H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.

Background: 5-fluorouracil remains the standard therapy for patients with advanced/metastatic colorectal cancer. Pre-clinical studies have demonstrated the biological modulation of 5-fluorouracil by methotrexate and leucovorin. This phase II study was initiated to determine the activity and toxicity of sequential methotrexate--leucovorin and 5-fluorouracil chemotherapy in patients with advanced colorectal cancer., Methods: Ninety-seven patients with metastatic colorectal cancer were enrolled onto the study. Methotrexate--30 mg/m2 was administered every 6 hours for 6 doses followed by a 2 hour infusion of LV--500 mg/m2. Midway through the leucovorin infusion, patients received 5-fluorouracil--600 mg/m2. This constituted a cycle of therapy and was repeated every 2 weeks until progression., Results: The median age was 64 yrs (34-84) and the Eastern Cooperative Group Oncology performance score was 0 in 37%, 1 in 55% and 2 in 8% of patients. Partial and complete responses were seen in 31% of patients with a median duration of response of 6.4 months. The overall median survival was 13.0 months. The estimated 1-year survival was 53.7%. Grade III and IV toxic effects were modest and included mucositis, nausea and vomiting., Conclusions: This phase II study supports previously reported data demonstrating the modest clinical benefit of 5-FU modulation utilizing methotrexate and leucovorin in patients with metastatic colorectal cancer. Ongoing studies evaluating 5-fluorouracil modulation with more novel agents (Irinotecan and/or oxaliplatin) are in progress and may prove encouraging.

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.

Purpose: To evaluate the methotrexate (MTX)-exposed swine brain, functional magnetic resonance imaging (MRI), including perfusion, diffusion, and blood-oxygen-level-dependent (BOLD) contrast imaging, was used., Material and Methods: Juvenile pigs received either 2 x 5 g/m(2), or 5 x 2 g/m(2) MTX intravenously within one month. MRI was performed (sedative: propofol) before (14-17 kg, N = 6) and after (21-27 kg, N = 4) the MTX exposure. Also, age-matched controls (22-27 kg, N = 4) were imaged., Results: After the MTX exposure, reduced (from 2%-4% to 0%-1%) or negative (-2% to -3%) BOLD responses were detected; apparent diffusion coefficient (ADC) or relative perfusion values did not change., Conclusion: This study suggests that MTX-related changes in the brain may be detected as changes in flow-metabolism coupling as reduced or negative response (for somatosensory activation) in the BOLD contrast MRI. The contrast agent perfusion MRI, without absolute quantification, may not show global damage in brain perfusion related to the MTX exposure in the swine model used. ADC (in one direction) may not indicate MTX-related changes in the brain., (Copyright 2002 Wiley-Liss, Inc.)

Osteopenia is a complicating problem that may occur during and after treatment for childhood malignancy. Clinical studies suggest that chemotherapeutic agents directly affect osteoblasts in vivo. Since combinations of agents are used for treatment, we individually investigated the chemosensitivity of human osteoblast-like cells to 11 of the chemotherapeutic agents used. The relative chemosensitivity of osteoblast-like cells representing different stages of cell differentiation was also examined. Cell numbers were evaluated following culture of an established human osteoblast-like cell line (MG63) for 3 days with clinically relevant concentrations of the chemotherapeutic agents. The chemosensitivity of MG63 cells was compared to that of a human osteoprogenitor cell line (HCC1) and primary osteoblast-like (HOB) cells derived from pediatric bone. Cell numbers were reduced by all agents in all cell types, although there was a varied response between agents at equimolar concentrations. In MG63 cells the lowest concentration of agent significantly reducing cell numbers varied between agents, for example, methotrexate (10(-7) M), vincristine (10(-9) M), and etoposide (10(-7) M) (all P <0.01). The less differentiated osteoblast phenotypes were significantly more chemosensitive at equimolar concentrations of methotrexate, vincristine, asparaginase, and dexamethasone than more differentiated phenotypes (all P <0.01). Furthermore, four agents significantly increased alkaline phosphatase (AP) activity in HOB cells. We conclude that individual chemotherapeutic agents added to osteoblast cell cultures reduce cell numbers, with osteoblast precursor cells being preferentially depleted. These results suggest that most of the agents may contribute to osteopenia in childhood malignancy by direct effects on cell numbers.

Here we report the synthesis and characterization of a lipophilic phosphatidylcholine containing the omega-3 fatty acid docosahexaenoic acid (DHA) and the cytotoxic drug methotrexate (MTX). This novel phospholipid combines the fatty acid's and the drug's anticancer activities in a molecule amenable to a liposome bilayer for safe, simultaneous delivery of the two agents. Two phosphatidylcholines were synthesized, from 1-stearoyl or 1-docosahexaenoyl, 2-hydroxy-sn-glycero-3-phosphocholine, to contain MTX in the sn-2 position and either stearic acid or DHA in the sn-1 position. The products contain fatty acid, MTX and phosphorus (1:1:1), and the MTX was released by phospholipase A(2), consistent with the proposed phospholipid structure. The predominant product linked MTX to the glycerol moiety through MTX's gamma-carboxyl group. Liposomes composed of 1-stearoyl, 2-oleoyl phosphatidylcholine plus 1-stearoyl, 2-oleoyl phosphatidylethanolamine and various concentrations of the novel phospholipids caused dose-dependent inhibition of murine leukemia cell proliferation in culture. The DHA- and MTX-containing phosphatidylcholine was more effective than that containing stearic acid, and DHA appeared to synergize with MTX when they were added as free agents or covalently linked in the phospholipid. These data show the feasibility of synthesizing, and the inhibitory activity of phosphatidylcholine with DHA in the sn-1 position and MTX in the sn-2 position, and suggest the compound's potential use in cancer chemotherapy.

P-glycoprotein is a product of the multidrug resistance (MDR-1) gene. In non-Hodgkin's lymphoma, less than 20% of untreated de novo lymphomas express MDR-1 compared with approximately 50% after failure of chemotherapy. We wished to study the expression of MDR-1 in AIDS-related non-Hodgkin's lymphoma (AIDS-NHL). Tissue biopsies from 50 patients with newly diagnosed AIDS-NHL were studied by immunohistochemical analysis using C494, a monoclonal antibody specific for the MDR-1 isoform of P-gp. MDR-1 expression was correlated with patient demographics, lymphoma characteristics, response to chemotherapy, and survival. Forty-six males and four females with a median age of 38 years (range 26-63) were studied. A prior AIDS-defining opportunistic infection was reported in 35 patients (70%). The median CD4+ lymphocyte count was 69/mm(3) (range 0-920). Thirty-two patients (63%) had received prior anti-HIV therapy, including a protease inhibitor in five (10%). Pathologic types consisted of diffuse large cell in 13 (26%), immunoblastic in 13 (26%), small non-cleaved in 22 (44%), and high grade not otherwise specified in two (4%). The majority of patients (76%) had stage III/IV disease. Pre-treatment lymphoma tissues from 33 patients (66%) stained positively for MDR-1. MDR-1 positive patients had a significantly lower complete remission rate compared to MDR-1 negative patients (33 versus 65%, P=0.042). Duration of complete response was significantly longer in MDR-1 negative patients compared with MDR-1 positive patients (not reached versus 9.9 months, P=0.003). Strategies to overcome MDR-1 expression may be important for initial treatment in patients with AIDS-NHL.

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.

To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex alpha/beta motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways., (Copyright 2002 Academic Press.)

Periconceptional use of folic acid reduces the risk of neural tube defects considerably. In Switzerland, implementation of these findings could be improved through fortification of a staple food with folic acid. The present paper reviews possible hazards associated with high intake of folic acid in the general population. Among the potential safety issues are interaction between folic acid and zinc, interaction between folic acid and drugs (phenytoin, methotrexate etc.) and hypersensitivity to folic acid. Of main concern are adverse effects of folic acid in cobalamin deficiency. Solutions are discussed.

Purpose: Multidrug resistance-associated protein 3 (MRP3) was initially cloned as a hepatic transporter induced under cholestatic/ hyperbilirubinemic conditions. In the present study, transport property of human MRP3 (hMRP3) was compared with that of rat MRP3 (rMRP3)., Methods: Adenosine 5' triphosphate (ATP)-dependent uptake of several organic anions into the membrane vesicles isolated from the Sf9 cells expressing hMRP3 and rMRP3 was measured by rapid filtration technique., Results: ATP-dependent uptake of glucuronide conjugates, glutathione conjugates. and [3H]methotrexate (MTX) was stimulated by infection of cDNAs for hMRP3 and rMRP3. The mean (+/- SE) Km values for the uptake of 17beta estradiol 17beta-D-glucuronide ([3H]E(2)17 betaG) by hMRP3 and rMRP3 were 42.9 +/- 4.3 microM and 33.4 +/- 2.2 microM, respectively. Although the Ki values of glucuronides on the uptake of E217betaG were similar in humans and rats, hMRP3 exhibited higher Ki values toward MTX. In addition, although glycocholate and taurolithocholate 3-sulfate (TLC-S) were transported by both hMRP3 and rMRP3, taurocholate was only transported to a significant degree by rMRP3. Moreover, the inhibitory effect of taurocholate and glycocholate on the transport of E(2)17beta3G was much more potent in rMRP3 compared to hMRP3., Conclusion: Collectively, the substrate specificity of hMRP3 resembles that of rMRP3 although differences were observed, particularly in bile acid transport.

Uptake of methotrexate into the LNCaP human prostate cancer cells was linear for the first 60 min. The initial rate of methotrexate uptake was highest at extracellular pH 4.5 and decreased markedly until pH 7.0 to 8.0. Transport of methotrexate into LNCaP cells showed two components, one saturable -K(m) = 0.13 +/- 0.06 microM and V(max) = 1.20 +/- 0.16 pmol x 45 min(-1) x mg(-1) protein at low concentrations and the other apparently not saturable up to 10 microM. Uptake of methotrexate was inhibited by structural analogs with the K(i) values being 6.53, 12.4, and 85.6 microM for 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and folic acid, respectively. Uptake of methotrexate into LNCaP cells was not inhibited by the energy poisons in contrast to methotrexate uptake into PC-3 prostate cancer cells. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions and by the organic anions probenecid and DIDS, suggesting that methotrexate may be transported by an anion-exchange mechanism. These results show that methotrexate is transported into LNCaP prostate cancer cells by a carrier-mediated process.

The establishment of pregnancy in the human decisively depends on the competence of the early trophoblast to interact during implantation with (1). the uterine epithelium and subsequently (2). with the endometrial stroma and blood vessels. In the interaction with uterine epithelium cell-to-cell adhesion appears to be a critical element, involving initially (and astonishingly) apical cell poles of both epithelia. The subsequent invasion of the stroma includes both adhesive interactions with and degradation of extracellular matrix. How these different processes are regulated in detail remains largely unknown. While the invasiveness of the trophoblast is known to be regulated in local and temporal terms it has remained unclear so far whether trophoblast adhesiveness to cells and/or matrix is subject to a coupled regulation or whether both properties involve different, maybe sequentially effective, control mechanisms. It is also not known how the regulation of these activities is related to the differentiation pathways leading to the formation of noninvasive villous trophoblast serving endocrine as well as nutritive functions. This communication reviews experiments using normal cytotrophoblast cells isolated from first trimester or term placentae as well as malignant trophoblast (choriocarcinoma) cells treated with a panel of compounds known to modulate cell differentiation [retinoic acid, methotrexate, dibutyryl-cAMP, phorbol-(12-myristoyl-13-acetyl)-diester]. Parameters indicative of trophoblast differentiation [in particular chorionic gonadotrophin (hCG) secretion] as well as adhesion to uterine epithelial cells and invasion into extracellular matrix in vitro were monitored. While expression of differentiation parameters was increased by all drug treatments, adhesion to uterine epithelial cells in vitro was reduced. Modulation of invasiveness, however, followed a different pattern: while it was reduced in normal trophoblast cells it was even increased in choriocarcinoma cells with various substances. The response of cells with respect to production of extracellular matrix proteins or matrix-degrading proteinases showed a complex pattern that again lacked a stringent correlation with hCG production and adhesion, and in addition also with invasive behavior. These results suggest that adhesiveness of trophoblast to uterine epithelial cells and invasiveness into the uterine stroma (extracellular matrix) are subject to different control mechanisms. They support the view that trophoblast-endometrium interactions involve a cascade of various adhesion and migration processes whose cellular and molecular basis is complex but accessible to experimental investigation using a variety of available in vitro systems., (Copyright 2002 S. Karger AG, Basel)

Objective: It has been reported that several kinds of tumors express increased insulin receptor and the molecules of insulin can be internalized in cells and may thence enter into the nuclei mediated by insulin receptor. In this study, we investigated the receptor binding characteristics of insulin-MTX for the possibility of using insulin as a carrier for carcinoma targeted therapy by receptor mediation., Methods: MTX(methotrexate) was covalently linked to insulin directly. The insulin-MTX conjugate was purified by polyacrylamine agarose gel electrophoresis and analysed by high performance liquid chromatography and SDS- polyacrylamine agarose gel electrophoresis. Histologically confirmed human hepatocellular carcinoma specimens were obtained from patients at surgery and immediately frozen under -80 degrees C. Cell membrane fractions were isolated by sucrose density gradient centrifugation. Competitive displacement of 125I-insulin with insulin and insulin-MTX binding to insulin receptor were carried out and the values of IC50 and Ki were calculated so as to observe the characteristics of insulin-MTX binding to insulin receptor., Results: Insulin-MTX competed as effectively as insulin with 125I-insulin for insulin receptor. The values of IC50 and Ki for insulin-MTX were 93.82 +/- 19.32 nmol/L and 91.88 +/- 16.86 nmol/L respectively, while the values of IC50 and Ki for insulin were 5.01 +/- 1.24 nmol/L and 4.85 +/- 1.12 nmol/L respectively., Conclusion: Insulin-MTX could bind with insulin receptor with high affinity. The result demonstrates us that there is a possibility of using insulin as a carrier for carcinoma targeted therapy by receptor mediation.

Intestinal absorption of folates has been characterized as a facilitative process with a low pH optimum. Studies with intestinal epithelial cells have suggested that this activity is mediated by the reduced folate carrier (RFC1). In this paper, we report on folate transport characteristics in an immortalized rat IEC-6 cell line that was found to exhibit the predominant influx activity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs and high folic acid uptake only at the low pH. For the two clones with the highest level of transport activity, relative MTX influx at the two pHs was reversed. Moreover, the low pH MTX influx activity ([MTX](e) = 0.5 microM) was markedly inhibited by 20 microM folic acid while influx at neutral pH was not. Furthermore, in the presence and absence of glucose at low pH, MTX and folic acid influx activity was inhibited by azide, while MTX influx at pH 7.4 was stimulated by azide in the absence of glucose but was unchanged in the presence of glucose and azide. This was contrasted with the results of transfection of the same RFC1 construct into an L1210 murine leukemia cell line bearing a nonfunctional endogenous carrier. In this case, the activity expressed was only at pH 7.4. These data indicate that RFC1 can exhibit two distinct types of folate transport activities in intestinal cells that must depend on tissue-specific modulators.

The molecular basis of methotrexate resistance was studied in human MDA-MB-231 breast cancer cells, which are inherently defective in methotrexate uptake and lack expression of the reduced folate carrier (RFC). Transfection of MDA-MB-231 cells with RFC cDNA restored methotrexate uptake and increased methotrexate sensitivity by approximately 50-fold. A CpG island in the promoter region of RFC was found to be methylated in MDA-MB-231 cells, but was unmethylated in RFC expressing, methotrexate-sensitive MCF-7 breast cancer cells. Chromatin immunoprecipitation with antibodies against acetylated histones H3 and H4 showed that the RFC promoter was enriched for acetylated histones on expressed, unmethylated alleles only. Treatment of MDA-MB-231 cells with 5-aza-2'-deoxycytidine restored RFC expression but also led to increased methotrexate efflux and did not reverse methotrexate resistance. This suggests that 5-aza-2'-deoxycytidine up-regulates both methotrexate uptake and some methotrexate-resistance mechanism(s). Reverse transcription-polymerase chain reaction analysis showed increased expression levels of several ATP-dependent efflux pumps in response to 5-aza-2'-deoxycytidine treatment, including P-glycoprotein and members of the multidrug resistance-associated protein family. Up-regulation of P-glycoprotein in response to 5-aza-2'-deoxycytidine was associated with demethylation of a CpG island in the MDR1 promoter, whereas the mechanism(s) for 5-aza-2'-deoxycytidine-induced up-regulation of multidrug resistance-associated proteins is probably indirect. Dipyridamole inhibited methotrexate efflux and reversed methotrexate resistance in 5-aza-2'-deoxycytidine-treated MDA-MB-231 cells.

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Transport of methotrexate (MTX) into human prostatic PC-3 cells was studied. Uptake of MTX vs concentration was saturable at pH 7.4 in cells grown in normal medium and in cells incubated for 24 h in folate-free medium (Km = 3.24 and 4.84 microM, respectively (P > 0.05, n = 3) and Vmax = 0.64 and 0.92 nmol x min(-1) x 10(-9) cells, respectively (P < 0.05, n = 3)). In contrast, uptake at pH 4.5 showed both a saturable component (Km = 1.03 microM, Vmax = 0.42 nmol x min(-1) x 10(-9) cells) and a nonsaturable, linear component. Uptake was inhibited by the structural analogs 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and folic acid (K(i) = 6.8, 10.9, and 89.6 microM, respectively). Uptake was inhibited by increasing concentrations of chloride ion, suggesting that MTX transport in PC-3 cells may be via an anion-exchange mechanism. Uptake was significantly decreased by high concentrations of sodium cyanide and sodium arsenate but not by sodium azide. Uptake was inhibited by the sulfhydryl inhibitor p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Uptake of MTX was independent of sodium ions in the medium. It is concluded that PC-3 human prostate cancer cells have a carrier-mediated system for the uptake of MTX and other folates., (Copyright 2001 Academic Press.)