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)

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.
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PMID:Role of thymidylate synthetase activity in development of methotrexate cytotoxicity. 16 May 58

Biochemical and biological studies have been carried out with 2-desamino-2-methylaminopterin (dmAMT), which inhibits tumor cell growth in culture but is only a weak inhibitor of dihydrofolate reductase (DHFR). Since it was possible that the species responsible for growth inhibition are polyglutamylated metabolites, the di-, tri-, and tetraglutamates of dmAMT were synthesized and tested as inhibitors of purified recombinant human DHFR, murine L1210 leukemia thymidylate synthase (TS), chicken liver glycinamide ribonucleotide formyltransferase (GARFT), and murine L1210 leukemia aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT). The compounds with three and four gamma-glutamyl residues were found to bind two orders of magnitude better than dmAMT itself to DHFR, TS, and AICARFT, with 50% inhibitory concentration values in the 200 to 300 nM range against all three enzymes. In contrast, at a concentration of 10 microM, dmAMT polyglutamates had no appreciable effect on GARFT activity. These findings support the hypothesis that dmAMT requires intracellular polyglutamylation for activity and indicate that replacement of the 2-amino group by 2-methyl is as acceptable a structural modification in antifolates targeted against DHFR as it is in antifolates targeted against TS. In growth assays against methotrexate (MTX)-sensitive H35 rat hepatoma cells and MTX-resistant H35 sublines with a transport defect, dmAMT was highly cross-resistant with MTX, but not with the TS inhibitors N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-ox-oquinazolin-6-yl)-N- methylamino]thenoyl)-L-glutamic acid, implicating DHFR rather than TS as the principal target for dmAMT polyglutamates in intact cells. On the other hand, an H35 subline resistant to 2'-deoxy-5-fluorouridine by virtue of increased TS activity was highly cross-resistant to N10-propargyl-5,8-dideazafolic acid and not cross-resistant to MTX, but showed partial cross-resistance to dmAMT. Both thymidine and hypoxanthine were required to protect H35 cells treated with concentrations of dmAMT and MTX that inhibited growth by greater than 90% relative to unprotected controls. In contrast, N10-propargyl-5,8-dideazafolic acid and N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)-N-methylamino] thenoyl)- L-glutamic acid required only thymidine for protection. Like MTX, therefore, dmAMT appears to inhibit purine as well as pyrimidine de novo synthesis, and its effect on cell growth probably reflects the ability of dmAMT polyglutamates to not only block dihydrofolate reduction but also interfere with other steps of folate metabolism, either directly or indirectly via alteration of reduced folate pools.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical and biological studies on 2-desamino-2-methylaminopterin, an antifolate the polyglutamates of which are more potent than the monoglutamate against three key enzymes of folate metabolism. 131 37

Previous studies from this laboratory demonstrated that marked suppression of thymidylate synthase activity is required to slow the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate when dihydrofolate reductase is blocked by an antifolate. This finding is due to the high catalytic activity of thymidylate synthase within cells in comparison to the tetrahydrofolate cofactor pool size. In the present study, we assessed the rate of resumption of thymidylate synthase catalytic activity in terms of [3H]deoxyuridine incorporation into DNA and dihydrofolate generation from tetrahydrofolate cofactors following exposure of cells to fluorodeoxyuridine. Log phase L1210 leukemia cells, incubated with fluorodeoxyuridine to abolish thymidylate synthase catalytic activity, were suspended into drug-free medium. Resumption of [3H]deoxyuridine incorporation into DNA was negligible; by 4 hr enzyme activity was still inhibited by approximately 98%. However, this was sufficient to interconvert all available tetrahydrofolate cofactors to dihydrofolate (T1/2 approximately 2 hr) when dihydrofolate reductase was inhibited by the lipophilic antifolate trimetrexate. Interconversion of tetrahydrofolate cofactors to dihydrofolate correlated with a decline, then cessation, of purine synthesis as measured by the incorporation of [14C]formate into purine bases. These data suggest that an earlier than previously expected depletion of tetrahydrofolate cofactors with consequent inhibition of purine and other folate-dependent synthetic processes is likely to occur when antifolates are administered after a fluoropyrimidine.
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PMID:Interconversion of tetrahydrofolate cofactors to dihydrofolate induced by trimetrexate after suppression of thymidylate synthase by fluorodeoxyuridine in L1210 leukemia cells. 138 49

The inhibitory effects of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline antifolate and a potent thymidylate synthase inhibitor, were evaluated in human leukemia cell lines resistant to methotrexate (MTX) and trimetrexate (TMQ). MTX-resistant MOLT-3 cell lines, MOLT-3/MTX200 and MOLT-3/MTX10,000, were cross-resistant to CB3717; however, the degree of resistance was only tenfold for both cell lines, and increased dihydrofolate reductase activity in MOLT-3/MTX10,000 had little influence on the degree of CB3717 resistance. The MOLT-3 cell line made resistant to TMQ, MOLT-3/TMQ200, was as sensitive to CB3717 as the parent line. The cell growth inhibitory effect of CB3717 on MOLT-3 was reversed by the addition of thymidine. Leucovorin also partially reversed CB3717-induced growth inhibition. Cellular uptake of MTX and 5-methyl-tetrahydrofolate was hindered by the presence of a high concentration of CB3717, whereas TMQ uptake was not influenced by CB3717. CB3717 appears to enter the cells not only through reduced folate transport system, but by other route(s). CB3717 does not share the transport pathway with TMQ. Our observations that MTX-resistant cells with increased dihydrofolate reductase are not more resistant than cells without increased enzyme activity, and that TMQ-resistant cells are not cross-resistant to CB3717, may have clinical relevance.
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PMID:N10-propargyl-5,8-dideazafolic acid (CB3717): inhibitory effects on human leukemia cell lines resistant to methotrexate or trimetrexate. 143 41

Metabolic effects and mode of cytotoxicity of 5-deazaacyclotetrahydrofolate (5-DACTHF, BW543U76), a glycineamide ribonucleotide transformylase inhibitor, were studied in MOLT-4 cells, a human T-cell leukemia line. 5-DACTHF inhibits purine synthesis with 50% inhibitory concentration values of 0.5 microM and 0.08 microM following 6- or 24-h exposure to drug, respectively. At 6 h, adenine nucleotide synthesis is preferentially inhibited over guanine nucleotide synthesis. A similar effect was observed with another glycineamide ribonucleotide transformylase inhibitor, 5,10-dideazatetrahydrofolate. GTP was depleted to 40% of control and ATP to 10% of control by 5 microM 5-DACTHF. After a transitory increase, UTP and CTP were depleted to 30% of control. Deoxynucleotides were also depleted by the drug; dCTP was depleted to the greatest extent, followed by dATP, dTTP, and dGTP, respectively. MOLT-4 cell growth was inhibited by 5-DACTHF with a 50% inhibitory concentration of 0.066 microM. Complete reversal was effected by hypoxanthine, and there was no reversal by thymidine. The drug was cytotoxic to MOLT-4 cells in the range 0.25 to 5.0 microM, but a minimum of 48 h was required for trypan blue-staining dead cells to appear. The rate and extent of kill with the thymidylate synthase inhibitor 2-methyl-10-propargyl-5,8-dideazafolate was greater than with 5-DACTHF, which indicates that kill by inhibition of thymidylate synthase is more effective than that by inhibition of purine synthesis. Electron microscopy of MOLT-4 cells exposed to 5-DACTHF showed electron-dense mitochondria and nuclear changes reminiscent of apoptosis. These morphological changes were accompanied by the appearance of DNA strand breaks at approximately 180-base pair intervals (internucleosomal breaks). Concomitant proteolysis of nuclear proteins poly(ADP-ribose) polymerase and lamin B was observed.
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PMID:Metabolic effects and kill of human T-cell leukemia by 5-deazaacyclotetrahydrofolate, a specific inhibitor of glycineamide ribonucleotide transformylase. 151 46

The synthesis of a novel series of gamma-substituted folic acid analogues, pteroyl-S-alkyl-DL-homocysteine (RS)-sulfoximines, and the corresponding S-methylhomocysteine sulfone is described. Side reactions of the sulfoximine groups of the amino acid ester reactants were considered. The correct structures of the isolated target compounds were confirmed by NMR and FAB/MS excluding other alternatives. The replacement of the gamma-COOH of the glutamate moiety of folic acid with S-alkylsulfoximine groups or S-methylsulfone did not affect the substrate activity of the vitamin for dihydrofolate reductase. The resulting tetrahydrofolate analogues could serve as cofactors for the thymidylate synthase cycle of murine leukemia L1210 cells in situ. The analogues inhibited the growth of these cells in culture with 2 orders of magnitude lower IC50 values [(2-4) x 10(-4) M] than the parent folic acid.
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PMID:Synthesis and biological activity of novel folic acid analogues: pteroyl-S-alkylhomocysteine sulfoximines. 156 Apr 36

The thymidylate synthase (TS) inhibitor ICI D1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-S-glutamic acid) is a structural analogue of the substrate N5,N10-methylenetetrahydrofolate (5,10-CH2FH4) and is currently under clinical evaluation as a treatment for cancer. The compound is shown here to be a mixed non-competitive inhibitor of TS from murine leukemia (L1210) cells when 5,10-CH2FH4 is varied. This result suggests formation of an inactive complex between TS, 5,10-CH2FH4 and the inhibitor. Thus, binding to only one of the two active sites on the TS homodimer may be sufficient to prevent catalysis fully. Treatment of L1210 cells with ICI D1694 is known to cause intracellular accumulation of the tetraglutamate derivative which is shown here to have a 60-fold higher affinity for TS. The IC50 for inhibition of L1210 cell growth is below the Ki value of ICI D1694 for L1210 TS but above that of the tetraglutamate. The formation of polyglutamates and concentration of drug inside cells, therefore, seem to be responsible for biological activity.
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PMID:Kinetic characteristics of ICI D1694: a quinazoline antifolate which inhibits thymidylate synthase. 159 89

Hydroxyurea is a potent inhibitor of the enzyme ribonucleotide reductase. Due to its effects on cellular deoxyribonucleotide pools, hydroxyurea can modulate the activity of several pyrimidine and purine antimetabolites. As an inhibitor of DNA repair, it can potentially interact with DNA-damaging agents such as alkylating agents or inhibitors of topoisomerase II. Both cytokinetic and biochemical interactions occur between hydroxyurea and cytarabine (ara-C), which account for their synergistic cytotoxicity. Inhibition of ribonucleotide reductase by hydroxyurea depletes cellular deoxycytidine triphosphate pools, thereby enhancing ara-C uptake and phosphorylation to ara-C triphosphate. In a phase II clinical trial, the combination of hydroxyurea and ara-C produced a 43% response rate in patients with refractory malignant lymphoma. Studies in murine leukemia models have demonstrated therapeutic synergy when hydroxyurea is combined with fluoropyrimidines. High levels of deoxyuridine monophosphate that have been associated with resistance to 5-fluorouracil can be suppressed by hydroxyurea, leading to greater inhibition of thymidylate synthase. Despite the strong biochemical rationale for the use of hydroxyurea and 5-fluorouracil in combination, few clinical trials have been conducted thus far. Antimetabolites and topoisomerase II inhibitors have also been shown to be synergistic in vitro. Hydroxyurea has been shown to enhance the formation of DNA strand breaks produced by amsacrine and to produce synergistic cytotoxicity with etoposide. A phase I clinical trial of these drugs has demonstrated bone marrow suppression to be the major toxicity of the combination. In summary, hydroxyurea has been shown to undergo cytokinetic and biochemical interactions with a number of established antitumor agents. Clinical trials of hydroxyurea in combination with these agents have identified doses and schedules of administration that produce acceptable levels of clinical toxicity and appear feasible for further testing.
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PMID:Laboratory and clinical studies of biochemical modulation by hydroxyurea. 164 59

Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells. 182 99

Following exposure of L1210 leukemia cells to antifolates, tetrahydrofolate-dependent purine and pyrimidine biosyntheses are blocked despite the presence of the major portion of tetrahydrofolate cofactors. Previous studies from this laboratory demonstrated that this cannot be due to direct inhibition of thymidylate synthase by dihydrofolate polyglutamates or other endogenous folates and suggested that this phenomenon is due to compartmentation of tetrahydrofolate cofactors unavailable for interconversion and/or oxidation when dihydrofolate reductase activity is abolished by antifolates. The present paper evaluates the possibility that tetrahydrofolate cofactors in subcellular organelles, in particular, mitochondria, are unavailable for oxidation by thymidylate synthase. Particulate and cytosolic fractions were obtained from L1210 cells following homogenization and differential centrifugation. The crude mitochondrial fraction contained 20.1% of the total folate pool and included 5-formyltetrahydrofolate, 10-formyltetrahydrofolate and tetrahydrofolate in proportions similar to intact cells. The cytosolic fraction had an increased proportion of tetrahydrofolate and decreased proportions of 5-formyl- and 10-formyltetrahydrofolate relative to intact cells or the particulate fraction. Exposure of cells to 10 microM trimetrexate for 30 min produced approximately 45% interconversion of tetrahydrofolate cofactors to dihydrofolate in the cytosolic fraction, a level much greater than that observed in whole cell extracts (25-30%), but had no effect on folate pools in the crude mitochondrial fraction. These data indicate that subcellular compartmentation accounts, in part, for the failure to oxidize tetrahydrofolate cofactors to dihydrofolate in the presence of antifolate levels that abolish dihydrofolate reductase activity.
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PMID:Compartmentation of intracellular folates. Failure to interconvert tetrahydrofolate cofactors to dihydrofolate in mitochondria of L1210 leukemia cells treated with trimetrexate. 183 61


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