Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines).

A wild-type Datura innoxia cell line (Px4) was used to select methotrexate-resistant cells through a stepwise procedure. Two independently selected cell lines, MTX161 and MTX132, were stable and shown to be 5 to 15 times more resistant to methotrexate than wild type. These methotrexate-resistant cells were similar to the wild-type cells in levels and kinetic properties of dihydrofolate reductase, the sensitivity of dihydrofolate reductase to methotrexate, the binding of [3H]methotrexate to soluble proteins, and the formation of methotrexate polyglutamate derivatives. High performance liquid chromatographic analyses indicated that methotrexate polyglutamylation is only slight and may not be significant in the toxicity of methotrexate to Datura cells. The uptake of methotrexate was also investigated in the wild-type and resistant cells. The Px4 cells exhibited a linear uptake that lasted for 1 to 7 h. The uptake was saturable, pH and energy dependent, and had a Km of 65.6 nM and a Vmax of 12.5 nmol h-1g-1 fresh weight. Neither MTX161 nor MTX132 exhibited the sustained uptake of methotrexate shown by the Px4 cells. As a result, there were greatly reduced concentrations of intracellular methotrexate in resistant cells. Resistant cell lines had 2- to 3-fold higher Km values for methotrexate uptake compared with Px4 cells. It is proposed that these cells became resistant as a result of a stable change in the membrane transport system for methotrexate.

Folylpolyglutamate synthesis in Neurospora crassa: primary structure of the folylpolyglutamate synthetase gene and elucidation of the met-6 mutation.

In Neurospora crassa, the met-6+ gene encodes folylpoly-gamma-glutamate synthetase (FPGS) which catalyzes the formation of polyglutamate forms of folate. Methionine auxotrophy of the Neurospora crassa met-6 mutant is related to a lesion affecting this enzyme. Functional complementation of the mutant strain was achieved by introducing copies of the wild-type met-6+ gene into mutant spheroplasts. The complementing sequences were found to be contained on a 3.5 kb EcoRI-BamHI restriction fragment. The nucleotide sequence of the met-6+ gene was determined and an open reading frame of 1587 bp was identified, interrupted by two introns. This open reading frame contained several AUG codons but translation beginning from either of the first two would theoretically produce a protein of appropriate size and with similarity to five other FPGS proteins. Northern blot analyses of met-6+ transcripts revealed a 2.0 kb product. The position of the transcription stop site and an intron were identified by sequencing partial cDNA clones which were truncated at the 5' end. DNA sequence analysis of the met-6 mutant allele revealed a T to C transition which would result in replacement of a highly conserved serine with a proline.

Folylpolyglutamate synthesis in Neurospora crassa: transformation of polyglutamate-deficient mutants.

The methionine auxotrophy of Neurospora crassa met-6 and mac mutants is related to an inability to synthesize long-chained folylpolyglutamates. Both of these lesions affect folylpolyglutamate synthetase activity, but it is not clear whether these mutations occur in different genes or in functional domains of the same gene. To address this question, copies of the met-6+ gene have been introduced into both mutants using plasmid and cosmid vectors. Transformation to prototrophy was achieved in both mutants. The ability of these mutant and transformant strains to synthesize folylpolyglutamates was assessed by HPLC analysis of folate cleavage products. Mycelial extracts of the wild type revealed a folate pool dominated by folylhexaglutamates. These folates were also detected in the transformants but were lacking in both mutants. In the latter strains, the conjugated folates were mainly di- and triglutamates. When incubated for 24 hr with [14C]p-aminobenzoate, transformant and wild type cultures synthesized long chain folates, ca60-80% of these being hexaglutamyl derivatives. In contrast, the labelled folates of mac and met-6 were mainly mono- and diglutamyl derivatives, respectively. Polyglutamate synthesis was also studied in vitro by partial purification and characterization of mycelial folylpolyglutamate synthetase protein. Mycelial extracts of the wild type and transformant cultures utilized 5,10-methylenetetrahydrofolate monoglutamate and its diglutamate as substrates in this synthetase reaction. In contrast, extracts of met-6 and mac mycelia utilized only one of these folate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora.

The possible association of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) in Neurospora crassa (FGSC 853, wild type) has been examined using mycelial extracts prepared and fractionated in the presence of protease inhibitors. DHFS and FPGS were assayed by following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. Both of these activities were predominately cytosolic in mycelia that were harvested 24 hr after spore inoculation of defined minimal medium. Relatively small amounts of total mycelial DHFS activity were associated with mitochondrial fractions isolated by differential centrifugation. In contrast, ca 20% of the mycelial FPGS activity was mitochondrial. Treatment of the mitochondrial fractions with Triton X-100 suggested that these activities were not latent under the assay conditions employed. Separate peaks of DHFS and FPGS activity were observed when (NH4)2SO4-fractionated protein was desalted and chromatographed on columns of either Mono Q HR, DEAE-cellulose, heparin agarose, Matrex Green A or Reactive Green 5. Gel filtration indicated average Mr values of 52 and 66 x 10(3) for DHFS and FPGS protein, respectively. Dihydrofolate synthetase protein was purified over 1000-fold by a protocol that included chromatography on DEAE-cellulose, DEAE-Sephacel, heparin agarose and Matrex Green A. The isolated protein lacked ability to glutamyl conjugate 5,10-methylene tetrahydrofolate. SDS-polyacrylamide gel electrophoresis of the Matrex Green A peak fractions revealed a major protein band of average Mr 52 x 10(3) whose concentration appeared to parallel DHFS activity. FPGS protein (average Mr 66 x 10(3)), which lacked ability to glutamyl conjugate dihydropteroate, was recovered by a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)