Increase of dihydrofolate reductase activity in cultured mammalian cells after exposure to methotrexate.

Hillcoat, Brian L.; Swett, V.; Bertino, Joseph R.

doi:10.1073/pnas.58.4.1632

Cited by 64 publications

(20 citation statements)

References 11 publications

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“…The observation that DHFR levels may rise as a result of treatment with MTX in cancer patients was first made 2 decades ago (17,18). It has been shown that this "induction" phenomenon is consistent with the binding of MTX to the enzyme, which is thereby protected from proteolytic degradation (17,19), rather than reflecting net overproduction. The increased levels of free enzyme measured may be related to the assay conditions-the dilution and a higher pH than occurs intracellularly-making dissociation of the enzymeMTX complex more likely (17).…”

Section: Discussionmentioning

confidence: 99%

Increase of dihydrofolate reductase in peripheral blood lymphocytes of rheumatoid arthritis patients treated with low‐dose oral methotrexate

Rodenhuis¹,

Kremer²,

Bertino³

1987

Arthritis & Rheumatism

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Because of a previously observed plateau of clinical response after long‐term methotrexate (MTX) therapy for rheumatoid arthritis (RA), we investigated whether such treatment might lead to acquired resistance to the drug. We studied the activity of dihydrofolate reductase (DHFR) (the target enzyme of MTX) in peripheral blood mononuclear cells of 11 RA patients who had been treated with MTX for a median of 43 months. The enzyme levels were markedly increased compared with levels found in the cells of 6 RA patients treated with other slow‐acting drugs. Quantitative dot‐blot analysis of DNA from 7 of these patients showed no evidence of DHFR gene amplification. No correlation was observed between increased levels of DHFR and either response to therapy or to the weekly MTX dosage. Phytohemagglutinin‐stimulated peripheral blood lymphocytes from 6 patients with increased levels of DHFR showed no evidence of MTX resistance in vitro. The increased DHFR levels may result from binding of MTX to the enzyme, which may block the normal degradation pathways; they do not appear to be a marker of impending drug resistance.

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Section: Discussionmentioning

confidence: 99%

Increase of dihydrofolate reductase in peripheral blood lymphocytes of rheumatoid arthritis patients treated with low‐dose oral methotrexate

Rodenhuis¹,

Kremer²,

Bertino³

1987

Arthritis & Rheumatism

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“…The initial response of cells to methotrexate exposure is to upregulate DHFR protein level. This upregulation is thought to be mediated at the translational level (12)(13)(14); likely due to a conformational change of the DHFR mRNA complex (11,15). At the posttranslational level recent evidence suggests that DHFR is subject to both monoubiquitination and sumoylation (16,17).…”

mentioning

confidence: 99%

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

McEntee

Minguzzi

O’Brien

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K m for NADPH is similar for both enzymes but DHFRL1 has a higher K m for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.

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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). Additional evidence for a translational role in the up-regulation response came from studies showing that DHFR mRNA levels remain the same in the presence or absence of MTX (9,10).…”

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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Increase of dihydrofolate reductase activity in cultured mammalian cells after exposure to methotrexate.

Cited by 64 publications

References 11 publications

Increase of dihydrofolate reductase in peripheral blood lymphocytes of rheumatoid arthritis patients treated with low‐dose oral methotrexate

Increase of dihydrofolate reductase in peripheral blood lymphocytes of rheumatoid arthritis patients treated with low‐dose oral methotrexate

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

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

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