Pteridines (Nov 1998)
Inhibition of Mycelial Growth by Methotrexate in Neurospora crassa Wild Type and Mutants Deficient in Folylpolyglutamate Synthase
Abstract
In mammalian cells, folylpolyglutamate synthase (FPGS) catalyzes the polyglutamylation of methotrexate (MTX), a reaction that significantly enhances the cellular retention and cytotoxicity of this antifolate. In contrast, MTX is a poor substrate for the cytosolic FPGS of Neurospora crassa. The present study has therefore examined the effect of MTX on growth of N. crassa wild type (FGSC 853 ) and two mutants (met-6, FGSC 1330 and mac, FGSC 3609) that have lesions affecting FPGS expression. Mycelial dry weights after growth in MTX-supplemented media, suggested that met-6 and mac were more sensitive to the antifolate than the wild type (WT). MTX concentrations resulting in 50% inhibition of growth (ICso values) were 5.5 11M, 6.0 11M and 87.5 11M for met-6, mac, and WT, respectively. When MTX treatment was followed by transfer to 50 11M folinic acid-supplemented media, growth of both mutants was enhanced by ca. 20% while that of WT increased by ca. 8%. [3H]-MTX pulse-chase experiments demonstrated that all three strains had limited or no ability to form polyglutamates (MTXGlun ) of the antifolate. In WT cultures, supplied with 1 I-tM [3H]-MTX for 24 hr and then grown in MTX-free media for another 24 hr, over 95% of the recovered label was in MTX; MTXGlu2 and MTXGlu3 accounting for only 2% and 1% respectively. MTXGlun derivatives were not detected in mac but low levels of MTXGlu2 were generated by met-6. In all three strains, the level of expression of dihydrofolate reductase (DHFR) was similar. DHFR was purified to apparent homogeneity (21.6 kDa) from extracts of each strain using a protocol of ammonium sulfate fractionation, gel filtration and Matrex Green A chromatography. It is concluded that in Neurospora, MTX polyglutamylation is not a major factor in the cytotoxicity of this antifolate.
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