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)

Effects of lipophilic thymidylate synthase (TS) inhibitor AG337 on human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (MTX) resistance were assayed using continuous or intermittent drug exposure. During 120-h continuous exposure, HNSCC cell lines A253 and FaDu are equally MTX sensitive (EC50 equals approximately 15nM); AG337 is less potent (EC50 approximately equals 1 microM). A253 is intrinsically resistant to 24-h intermittent MTX exposure (EC50 equals approximately 17 microM; FaDu, EC50 equals approximately 0.3 microM); both HNSCC cell lines are resistant to 24-h AG337 exposure (EC50 >100 microM). CCRF-CEM shows MTX (EC50 =14 nM) and AG337 (EC50 equals approximately 0.6 microM) sensitivity in continuous exposure similar to HNSCC; however, AG337 retains potency against CCRF-CEM cells in intermittent exposure (24-h, EC50 equals approximately 2 microM; 6-h, EC50 equals approximately 48 microM). The reduced folate leucovorin (LV) at > or = 0.1 microM fully protects from growth inhibition by continuous MTX exposure, but growth inhibition by AG337 is reversed only slightly by < or = 100 microM LV. Thymidine fully protects A253 and FaDu against growth inhibition by AG337, while hypoxanthine alone is without effect, suggesting inhibition is TS-specific. CCRF-CEM sublines with acquired MTX-resistance resulting from DHFR overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG337 in continuous exposure (all EC50 =0.4 microM). These data indicate that AG337 may be useful in therapy of tumors that have acquired resistance to MTX by most common mechanisms.
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PMID:Characterization of the effect of AG337, a novel lipophilic thymidylate synthase inhibitor, on human head and neck and human leukemia cell lines. 1056 35

The mechanism of action of an antifolate may be investigated using a variety of experimental methods. These include experiments in a cell culture setting to observe possible protection against drug effects afforded by the end products of metabolic pathways, assessing the activity of purified target enzymes in the presence of the antifolate, and, finally, the measurement of drug effects on intracellular folate and nucleoside triphosphate pools. The current discussion is focused on studies using CCRF-CEM leukemia cells that were designed to compare and contrast mechanisms of action of the antifolates methotrexate, which is primarily a dihydrofolate reductase inhibitor, raltitrexed, a thymidylate synthase inhibitor, LY309887, a glycinamide ribonucleotide formyltransferase inhibitor, and MTA (multitargeted antifolate), which is a novel antifolate antimetabolite. The results of these studies support the hypothesis that MTA affects multiple enzymatic targets and has a distinct mechanism of action from methotrexate, raltitrexed, and LY309887.
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PMID:Cellular pharmacology of MTA: a correlation of MTA-induced cellular toxicity and in vitro enzyme inhibition with its effect on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells. 1059 55

Prior studies have indicated that MTA requires intracellular polyglutamation for optimal cytotoxic effect and that these polyglutamates potently inhibit several key enzymes of folate metabolism, including thymidylate synthase (TS), dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). In the present studies, we have investigated the mechanistic basis for resistance to MTA in several human tumor cell lines. The cell lines were developed for resistance by the gradual exposure to stepwise (fivefold) increases in the concentration of MTA over a 5-month period. The degree of resistance was 140-fold for GC3 colon carcinoma, 117-fold for HCT-8 ileocecal carcinoma, and 729-fold for CCRF-CEM leukemia cells adapted to 2 micromol/L MTA. The lines had strong cross-resistance (>3,200-fold) to raltitrexed. Only modest resistance was noted for methotrexate and the GARFT inhibitor, LY309887. The cytotoxicity of MTA in wild-type cells was only partially alleviated by thymidine addition (5 micromol/L) and complete protection required the addition of both hypoxanthine (100 micromol/L) and thymidine. In contrast, thymidine alone totally lacked protective activity in the MTA-resistant lines. The cells either demonstrated a GARFT-like reversal pattern (complete protection by hypoxanthine) for GC3MTA or a dihydrofolate reductase-like reversal pattern (complete protection by the combination of hypoxanthine and thymidine) for HCT-8MTA and CCRF-CEM(MTA) cells. Cellular resistance was multifactorial and stable on removal of selective pressure. Only GC3MTA cells showed increased TS activity (approximately 40-fold). Accumulations of 3H-MTA at 24 hours in CCRF-CEM(MTA), HCT-8MTA, and GC3MTA cells were 2%, 6%, and 46% of wild-type values, respectively. We also evaluated the cytotoxic activity of MTA in MCF-7 breast carcinoma and H630 colon carcinoma cells selected for resistance to raltitrexed and 5-fluorouracil, respectively, via TS amplification (provided by Dr P.G. Johnston, Belfast, Ireland). These cells demonstrated more than 200-fold less resistance to MTA compared with raltitrexed and MTA-induced cytotoxicity was prevented by hypoxanthine. These studies suggest that in addition to TS modulation, secondary targets emerge during the development of MTA resistance.
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PMID:Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs. 1059 58

The cellular pharmacology of methotrexate (MTX) is complex, involving the inhibition of both de novo thymidylate and purine biosynthesis. Measurement of MTX-induced inhibition of de novo thymidylate and purine biosynthesis may allow optimisation of MTX therapy, and the aim of this study was to develop an assay to measure the activity of both pathways in the same cell sample, and so determine the effects of MTX treatment. In situ thymidylate synthase (EC 2.1.1.45) activity was measured by the release of 3H2O from [5'-3H]deoxyuridine and de novo purine synthesis by the incorporation of [14C]formate into adenine and guanine. Incubation of human leukaemia CCRF-CEM cells for 22 hr with 50 nM MTX resulted in approximately 90% inhibition of in situ thymidylate synthase activity, relative to control untreated cells, and after exposure to 1000 nM MTX activity could not be detected. In contrast, de novo purine synthesis, measured in the same sample, was not inhibited by exposure to 50 nM MTX, although activity was again completely abolished by exposure to 1000 nM MTX. To demonstrate the utility of the assay, lymphoblasts isolated from a child with acute lymphoblastic leukaemia (ALL) were also incubated for 22 hr with 1000 nM MTX. Both in situ thymidylate synthase activity and de novo purine synthesis were significantly inhibited, by 70% and 60% respectively, relative to the activity in untreated cells.
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PMID:Development of a whole cell assay to measure methotrexate-induced inhibition of thymidylate synthase and de novo purine synthesis in leukaemia cells. 1064 39

The novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung (A549, COR L23) and two breast (MCF7, T47D) tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC50 values (continuous exposure three population doublings) were: A549-640 nM, COR L23-28 nM, MCF7-52 nM and T47D-46 nM. Thymidine (1 microM) completely prevented growth inhibition at the MTA IC50 in all cell lines. At 10 x IC50, growth inhibition was only partially reversed by thymidine (< or = 10 microM); both thymidine and hypoxanthine (30 microM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 microM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by > or = 89% by 1 microM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone.
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PMID:Dipyridamole potentiates the in vitro activity of MTA (LY231514) by inhibition of thymidine transport. 1073 67

Treatment failure in childhood acute lymphoblastic leukaemia (ALL) might be associated with methotrexate (MTX) resistance. Little is known about MTX resistance in relapsed ALL. In this study, we determined ex vivo MTX resistance in precursor-B ALL at relapse (rALL) and determined possible defects in MTX membrane transport and polyglutamylation. Using the in situ thymidylate synthase inhibition assay, 21 rALL samples were threefold more MTX resistant than 63 initial precursor-B ALL samples, both after short-term and after continuous MTX exposure (P < or = 0.01). [3H]-MTX membrane transport did not differ between eight rALL and 25 precursor-B ALL samples. Incubation for 24 h with 1 microM [3H]-MTX resulted in a trend towards a lower accumulation of MTX in 20 relapsed than in 83 initial samples of precursor-B ALL samples (906 vs. 1364 pmol/109 cells; P = 0.07). Accumulation of long-chain MTX polyglutamates (MTX-Glu4-6) did not differ between relapsed and newly diagnosed samples (746 and 889 pmol/109 cells; P = 0.1). Activities of the enzymes involved in polyglutamylation (folylpolyglutamate synthetase and folylpolyglutamate hydrolase) did not differ between rALL and untreated c/pre-B-ALL. This study demonstrates that leukaemic cells of children with relapsed precursor-B ALL are relatively MTX resistant, but that this MTX resistance is not associated with major impairments in MTX uptake or polyglutamylation.
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PMID:Methotrexate resistance in relapsed childhood acute lymphoblastic leukaemia. 1088 14

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.
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PMID:Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. 1089 64

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.
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PMID:5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity. 1110 56

In this study, the downstream effects of thymidylate synthase (TS) inhibition in L1210 (p53 mutant) and HL60 (p53 null) leukaemia cells were investigated. TS inhibition was induced by the specific TS inhibitor Thymitaq. Within 24 h, TS inhibition resulted in S-phase cell cycle arrest in both cell lines and subsequent apoptotic cell death as characterized by nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. A biphasic hyper/hypopolarization of the mitochondrial membrane potential (delta psi m) was also observed. The mitochondrial permeability transition inhibitor, cyclosporin A, increased the baseline level of delta psi m in L1210 cells. However, along with bongkrekic acid, it did not influence the changes in delta psi m induced by TS inhibition in either cell line. In both cell lines the broad spectrum caspase inhibitor, zVAD.fmk as a single agent, induced a significant downward shift in the baseline of delta psi m. However, only in HL60 cells was this accompanied by a slight increase in cytotoxicity. In L1210 cells zVAD.fmk inhibited DNA fragmentation induced by Thymitaq but did not influence other cell cycle events (S-phase arrest) or the biphasic mitochondrial alterations, indicating caspase involvement downstream but not upstream of the mitochondria following TS inhibition. In HL60 cells, zVAD.fmk reduced the hyperpolarization of delta psi m observed with Thymitaq alone and failed to inhibit the increase in the sub-G1 population induced by Thymitaq. Moreover, zVAD.fmk significantly increased the cell death response of these cells following TS inhibition. In conclusion, cell death induced by TS inhibition is mediated via the apoptotic pathway which clearly involves biphasic alterations in delta psi m. In L1210 cells, but not in HL60 cells, caspases function as the final executioner of apoptosis.
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PMID:Thymidylate synthase inhibition induces S-phase arrest, biphasic mitochondrial alterations and caspase-dependent apoptosis in leukaemia cells. 1113 61

A phase I study of nolatrexed, administered as a continuous 5 day intravenous infusion every 28 days, has been undertaken for children with advanced malignancy. 16 patients were treated at 3 dose levels; 420, 640 and 768 mg/m(2)24 h(-1). 8 patients were evaluable for toxicity. In the 6 patients treated at 768 mg/m(2)24 h(-1), dose-limiting oral mucositis and myelosuppression were observed. Plasma nolatrexed concentrations and systemic exposure, measured in 14 patients, were dose related, with mean AUC values of 36 mg(-1)ml(-1)min(-1), 50 mg ml(-1)min(-1)and 80 mg ml(-1)min(-1)at the 3 dose levels studied. Whereas no toxicity was encountered if the nolatrexed AUC was <45 mg ml(-1)min(-1), Grade 3 or 4 toxicity was observed with AUC values of >60 mg ml(-1)min(-1). Elevated plasma deoxyuridine levels, measured as a surrogate marker of thymidylate synthase inhibition, were seen at all of the dose levels studied. One patient with a spinal primitive neuroectodermal tumour had stable disease for 11 cycles of therapy, and in two patients with acute lymphoblastic leukaemia a short-lived 50% reduction in peripheral lymphoblast counts was observed. Nolatrexed can be safely administered to children with cancer, and there is evidence of therapeutic activity as well as antiproliferative toxicity. Phase II studies of nolatrexed in children at the maximum tolerated dose of 640 mg/m(2)24 h(-1)are warranted.
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PMID:A phase I study of nolatrexed dihydrochloride in children with advanced cancer. A United Kingdom Children's Cancer Study Group Investigation. 1113 6


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