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Lookup NU author(s): Gordon Taylor, Professor Herbie Newell
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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. Copyright (C) 2000 Elsevier Science Inc.
Author(s): Barnes M, Taylor G, Newell D
Publication type: Article
Publication status: Published
Journal: Biochemical Pharmacology
Year: 2000
Volume: 59
Issue: 4
Pages: 321-328
ISSN (print): 0006-2952
ISSN (electronic): 1873-2968
URL: http://dx.doi.org/10.1016/S0006-2952(99)00320-2
DOI: 10.1016/S0006-2952(99)00320-2
PubMed id: 10644039
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