The “induction” of leukocyte and erythrocyte dihydrofolate reductase by methotrexate

Bertino, Joseph R.; Cashmore, Arlene R.; Fink, Mary Lynn; Calabresi, Paul; Lefkowitz, Eliot J

doi:10.1002/cpt196566763

Cited by 45 publications

(18 citation statements)

References 10 publications

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“…Following this initial report, several in vivo and in vitro studies have shown that DHFR protein levels rapidly increase, or are "induced" in response to MTX treatment, associated with MTX bound to the protein (5)(6)(7)(8). The increase in DHFR protein levels seen upon exposure to MTX was unaffected by the transcriptional inhibitor actinomycin D but was blocked by the translational inhibitor cycloheximide indicating that this increase was not due to increased transcription (7).…”

mentioning

confidence: 99%

Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Skacel

Menon

Mishra

et al. 2005

Journal of Biological Chemistry

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Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.

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mentioning

confidence: 99%

Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Skacel

Menon

Mishra

et al. 2005

Journal of Biological Chemistry

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“…[3',5',9-3H]MTX-Glu, (20 (17). After pelleting cellular debris by centrifugation at 10,000 g for 5 min, the remaining cell lysate was immediately applied to a 0.8 X 2.5-cm DEAE-Sephacell minicolumn equilibrated with KH2PO4 0.15 M, pH 6.2, at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…That the expanding drug-bound fraction represented DHFR was demonstrated by a corresponding increase of the enzyme activity measured by a spectrophotometric assay after drug removal from DHFR by alkaline dialysis. Increases in DHFR levels have been described previously during MTX therapy and were initially thought to result from enzyme stabilization by MTX (20), although more recent data suggest that MTX stimulates DHFR synthesis at the ribosomal level by an as yet undefined mechanism (21).…”

Section: Reversibility Of the Binding Of Mtx And Mtxpg To Dhfrmentioning

confidence: 97%

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

Jolivet¹,

Chabner²

1983

J. Clin. Invest.

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A B S T R A C T Methotrexate (MTX-Glul) exerts its antitumor effects through its potent inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for maintaining the cellular pool of reduced folates. Since the drug-enzyme complex (bound drug) is slowly dissociable, an excess of drug (unbound or free drug) above that required to bind all enzyme sites is required in order to compete with substrate for sites made available by enzyme-drug dissociation. We have examined the role of the polyglutamyl metabolites of MTX-Glul containing two to five glutamyl (MTX-Glu2-5) groups in gamma peptide linkage in maintaining an intracellular pool of free drug and in forming slowly dissociable complexes with DHFR. During 24-h incubations of ZR-75-B human breast cancer cells with 2 uM MTX-Glu,, we observed the progressive formation of derivatives with two to five glutamyl groups, which rapidly replaced the parent compound on enzyme binding sites and represented 85% of both unbound and bound intracellular drug at the end of incubation. When cells were then placed in drug-free medium, the rates of disappearance of drug and metabolites from the intracellular bound and free fractions decreased with increasing glutamyl chain length. Over 90% of both bound and free MTX-Glul left the cells within 1 h, >90% of MTX-glu2 left within 6 h, and >90% of MTX-Glu3 left the bound and free fractions within 24 h. In contrast, free MTX-Glu4 fell by only 63% and bound by only 23% affter 24 h, while free MTX-Glu5 increased by 52% after 6 h in drug-free tively, while -Glu3, -Glu4, and -Glu5 had t½ of 102, 108, and 120 min. These experiments demonstrated that the longer chain polyglutamates have prolonged intracellular retention and can be dissociated less readily than MTX-Glu2 from DHFR, properties likely to make them more efficient DHFR inhibitors than the parent drug and of potential importance in extending the duration of drug action in tumor cells.

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“…Bertino and co-workers (32) were the first to demonstrate that acute rises in intracellular MTX binding capacity could be a determinant ofdrug resistance in patients with acute leukemia. More recently it has been shown that in vivo resistance after MTX treatment may be due to DHFR gene amplification and elevated target enzyme levels (33-37).…”

Section: Introductionmentioning

confidence: 99%

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

Curt¹,

Jolivet²,

Carney³

et al. 1985

J. Clin. Invest.

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We hate characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with <0.1 M&M MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 ;M) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 M&M) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 MM drug. Two cell lines with resistance to 10 MM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 MM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTIX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.

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The “induction” of leukocyte and erythrocyte dihydrofolate reductase by methotrexate

Cited by 45 publications

References 10 publications

Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

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