Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2, 3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and related compounds was replaced with some N5-substituted glutamines. Antifolates (4A-S) were effectively prepared by coupling pyrrolo[2,3-d]pyrimidine carboxylic acids (11a, b) with some properly protected N5-substituted glutamine derivatives (10A-S), which were prepared by coupling Boc-Glu-OMe (7) with various amines (8A-S) using a suitable condensing reagent, followed by hydrolysis. The inhibitory effects of the resulting products on dihydrofolate reductase (DHFR), thymidylate synthetase (TS) and the growth of murine fibrosarcoma Meth A cells in culture were examined. All N5-substituted glutamine analogs (4A-S) inhibited DHFR much more strongly than TNP-351 and some analogs exhibited the same potent growth inhibition of Meth A cells as TNP-351. Some typical analogs (4Bb, 4Db, 4F, 4Oa) were also examined for inhibitory effects on the growth of methotrexate (MTX)-resistant human CCRF-CEM cells in culture and for in vivo antitumor activities against murine leukemia and solid tumors. MTX-resistant cells, with a defect in transport and decreased polyglutamylation activity, showed little cross resistance to the analog (4Oa) having a tetrazole moiety as a substituent of glutamine, which exhibited potent antitumor activities. These results demonstrate that the antifolate analogs (4) with N5-substituted glutamine in place of glutamic acid are novel potent DHFR inhibitors with activity against MTX-resistant tumors. The potent antitumor activity of these analogs (4) may result from their effective uptake via reduced folate carrier in combination with their potent inhibition of DHFR.
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PMID:Non-glutamate type pyrrolo[2,3-d]pyrimidine antifolates. II. Synthesis and antitumor activity of N5-substituted glutamine analogs. 879 69

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 microM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 microM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuire et al., Adv. Exptl. Med. Biol. 163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (K(ii) = 0.9 microM; K(is) = 1.1 microM) and glutamic acid (K(ii) = 1.0 microM; K(is) = 5.2 microM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (K(ii) = 3.4 microM; K(is) = 0.35 microM), since the major effect is on its K(m). Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 microM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 microM; MTX, 48 microM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideout et al. (Int. J. Cancer 61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.
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PMID:Potent inhibition of human folylpolyglutamate synthetase by suramin. 891 44

F2-MTX'A is an L1210 leukemia cell line with a functional detect in the reduced folate carrier and high level expression of folate receptor beta. The pH dependence of methotrexate (MTX) influx by folate receptor beta in F2-MTX'A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by folate receptor beta had a pH optimum of 6.5, whereas influx mediated by the reduced folate carrier showed a pH optimum of 7.5. Increased folate receptor beta-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTX'A cells proceeded by another mechanism. This transport route became active at pH < 7.5, operated optimally at pH 6.0 to 6.5, and, unlike folate receptor beta-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a Ki of 5.3 microM and a Vmax of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a Ki of 148 microM, and had similar relative affinities for folic acid, leucovorin, and 5 methyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTX'A cells, optimal at low pH and distinct from the reduced folate carrier or the folate receptor.
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PMID:pH dependence of methotrexate transport by the reduced folate carrier and the folate receptor in L1210 leukemia cells. Further evidence for a third route mediated at low pH. 903 55

Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTXrA line, which lacks a functional endogenous carrier to obtain the MTXrA-R16 clonal derivative. Influx was increased approximately 9-fold in MTXrA-R16 cells without a change in Km. The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX]i, when [MTX]e was 1 microM. The concentrative advantage for RFC1 (the ratio of [MTX]i in MTXrA-R16 to L1210 cells) increased from 1.8 at 0.1 microM MTX to 3.8 at an [MTX]e level of 30 microM. Augmented transport in MTXrA-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a approximately 50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX]i and not the much larger change in influx at low [MTX]e levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.
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PMID:Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells. 926 Nov 28

Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 microM) and was completely inhibited by co-exposure to either methotrexate or the thymidylate synthase inhibitor, ZD1694. There was no significant intracellular F-MTX accumulation over this period in a K562 subline (K500E) with a documented defect (approximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was re-established in K500E cells transfected with a cDNA to human RFC, establishing a role for RFC in the cellular uptake of this compound. High levels of intracellular labelling were detected in all cell lines after prolonged (24 h) F-MTX incubations, however F-MTX accumulation at this time was not inhibited by ZD1694. F-MTX uptake by RFC was also detected in leukaemic blasts from children with acute lymphoblastic leukaemia and could be blocked with ZD1694. In leukaemic blasts with a documented defect in MTX uptake, F-MTX accumulation was abolished in almost all the cells. These results display the power of confocal microscopy for directly visualizing RFC-mediated anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long drug exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low levels of RFC transport in leukaemic blasts from patients undergoing chemotherapy with methotrexate.
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PMID:Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells. 931 Feb 38

Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.
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PMID:Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells. 938 86

The resistance to folate-based antifolates is associated with impaired function of the reduced folate carrier (RFC), one of the major routes of folate transport into cancer cells. To clarify the importance of RFC functions in the antifolate resistance, we have examined the expression of RFC1 and its phenotype as a folate transporter in human leukemia cell lines resistant to various antifolates. MOLT-3 cells resistant to ZD9331 (a thymidylate synthase (TS) inhibitor that utilizes the RFC for cell entry) (MOLT-3/ZD9331) showed decreased expression of RFC1 concomitant with diminished cellular uptake of [3H]methotrexate (MTX). K562 cells resistant to raltitrexed (ZD1694, another TS inhibitor that utilizes the RFC for cell entry) (K562/ ZD1694 x C) scarcely expressed RFC1, which is in accordance with the impaired uptake of folate analogs and the high degree of resistance to ZD1694 and MTX. On the other hand, no apparent decrease of RFCI1 expression was found in transport-deficient MTX-resistant MOLT-3 cells (MOLT-3/MTX10000) though its phenotype showed defective transport of MTX or ZD1694. In these cell lines with impaired RFC function, [3H]leucovorin (LV) uptake was only moderately decreased as compared to [3H]MTX or [3H]ZD1694 uptake. These cells grew with a minimal retardation in folate-free medium supplemented with 10 nM LV, suggesting that these cell lines with impaired RFC function had enough folate transporters to transport LV. In contrast to downregulation of RFC, the much greater uptake of [3H]MTX was observed in the MOLT-3/trimetrexate (TMQ)800-MTX10000 in parallel with increased RFC1 expression. These cell lines with the altered expression of RFC1 may serve as models useful for investigating the regulation of RFC1 expression and for understanding the molecular mechanism(s) behind the transport-mediated antifolate resistance.
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PMID:Variable expression of RFC1 in human leukemia cell lines resistant to antifolates. 950 Feb 2

CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 microM to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type Km value for MTX transport but a 31-fold and 9-fold lower Km values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G --> A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 microM folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.
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PMID:A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. 980 75

Chemical mutagenesis with N-methyl-N-nitrosourea was employed to study the pattern of mutations in the reduced folate carrier (RFC1) that results in transport-related methotrexate resistance and to identify amino acid residues that are critical to carrier structure and/or function. Thirty-four methotrexate transport-defective L1210 leukemia cell lines were isolated with folic acid as the sole folate source under antifolate selective pressure. The RFC1 mRNA levels were comparable with, or not substantially decreased, in most of these cell lines relative to wild-type L1210 cells. The molecular basis for the transport defects was investigated by sequencing multiple RFC1 cDNA clones isolated from these mutants by reverse transcription-polymerase chain reaction, which encompassed the entire coding region. The mutations identified were further confirmed either by direct sequencing or, when applicable, by restriction analysis of total reverse transcription-polymerase chain reaction products. The majority of mutations (21) led to single amino acid substitutions that were in, or near, 9 of 12 predicted transmembrane domains, with the highest frequencies in the first, fifth, and eighth. There were no mutations in the sixth, ninth, and twelfth transmembrane domains. Glycine, serine, and arginine were the most frequently mutated residues. These data suggest that several transmembrane domains, rather than the amino- and carboxyl-termini, and the large intracellular loop between the sixth and seventh transmembrane domains play key roles as sites for RFC1 inactivation because of single point mutations. This panel of mutated cell lines offers an important resource for studies on RFC1 structure-function and for the evaluation of transport-related cross-resistance patterns with new-generation antifolate inhibitors of tetrahydrofolate cofactor-dependent enzymes.
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PMID:Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis. 1038 85

We investigated the expression of the folylpolyglutamate synthetase (FPGS) gene at the mRNA level in MOLT-3 and K562 human leukemia cell lines sensitive, or made resistant, to methotrexate (MTX) and/or trimetrexate (TMQ), or raltitrexed (ZD1694). Northern blot analysis demonstrated approximately 3-fold higher FPGS mRNA expression in K562 cells than that in MOLT-3 cells, being consistent with graded polyglutamation capacities of these cell lines. A slight increase in the expression of the FPGS gene was observed in the TMQ-resistant MOLT-3 cells (MOLT-3/TMQ800); moreover, sequential development of MTX resistance in the TMQ-resistant cells (MOLT-3/TMQ800-MTX10,000) resulted in a further enhancement of FPGS mRNA expression despite of decreased polyglutamation capacity in this subline. Another MTX-resistant subline with impaired reduced folate carrier (MOLT-3/MTX10,000) also showed overexpression of FPGS mRNA. Conversely, both raltitrexed-resistant sublines (MOLT-3/ZD1694 x C and K562/ZD1694 x C) displayed a moderately decreased expression of FPGS mRNA. These findings did not correspond to the virtual absence of ZD1694 polyglutamates inside the former cells nor to possibly intact polyglutamation capacity in the latter cells. These results indicate that FPGS mRNA expression may predict cellular ability to produce polyglutamate metabolites of antifolate drugs in the sensitive cells, but does not necessarily reflect FPGS function at the enzyme level in the antifolate-resistant tumor cells.
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PMID:Variable expression of the folylpolyglutamate synthetase gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant, to various antifolate drugs. 1050 18


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