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Biomedical subjects

B Shane

Publications and source records attributed to B Shane.

At least 37 records · Page 2Linked to original sources
PubMed1995

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)

Chromatography, Ion Exchange↗
PubMed1995

Evolution of drug resistance in CCRF-CEM human leukemia cells selected by intermittent methotrexate exposure.

A clinically relevant mechanism of acquired methotrexate (MTX) resistance is decreased MTX polyglutamate (MTXGn) synthesis (McCloskey et al. J. Biol. Chem. 266:6181, 1991) secondary to deficient folylpolyglutamate synthetase (FPGS) activity. Earlier studies showed that this mechanism resulted after intermittent MTX treatment, but did not define its evolution in populations or its clonal frequency of occurrence and heterogeneity. We thus studied evolution of resistance in CCRF-CEM human leukemia cell populations and clones after repeated treatment with 30 microM MTX for 24 h. In populations, MTX resistance was detectable after 1 treatment and increased in degree as total cycles increased to 7. Defective MTXGn synthesis in populations was the only resistance mechanism detected, and FPGS activity in extracts decreased with treatment cycle. After 1 treatment, defective MTXGn synthesis was the major (27/48 clones) form of resistance; 18 clones were sensitive, while 1 clone with a DHFR-related change, no clones with decreased MTX uptake, and 2 complex phenotypes were observed. Sporadic clones with DHFR-mediated resistance appeared up to cycle 4, but defective MTXGn synthesis remained the major resistance mechanism. The degree of clonal resistance tended to increase with treatment cycle, but 1 clone in cycle 2 was similar to the clones from cycle 7. No change in FPGS gene copy number or restriction pattern, or FPGS mRNA level or size (2.3 Kb) was detected in populations. Decreased FPGS activity must result from decreased translation, increased protein turnover, or a point mutation affecting catalysis.

Antimetabolites, Antineoplastic↗
PubMed1994

Expression of Escherichia coli folylpolyglutamate synthetase in the Chinese hamster ovary cell mitochondrion.

Chinese hamster ovary (CHO) cell transfectants expressing Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity solely in their cytosol lack mitochondrial folylpolyglutamates and are auxotrophic for glycine. Addition of a mammalian mitochondrial leader sequence targeted E. coli FPGS to the mitochondria of these cells. Mitochondrial expression of FPGS restored mitochondrial folylpolyglutamate pools and overcame the glycine requirement. Pteroyltriglutamates functioned as effectively as the longer glutamate chain length folates found in wild type CHO cells in the metabolic cycle of glycine synthesis provided they were located in the mitochondria. Although folypolyglutamates cannot enter the mitochondria, mitochondrial folylpolyglutamates can be released without prior hydrolysis and CHO transfectants expressing E. coli FPGS activity solely in the mitochondria possessed normal cytosolic folylpolyglutamate pools. The proportion of cellular folate in the mitochondrion is governed by competition between mitochondrial and cytosolic FPGS activities.

Amino Acid Sequence↗
PubMed1994

Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazaacyclotetrahydrofolate, an anti-purine drug.

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF), an anti-purine agent that requires conversion to polyglutamate forms to be a potent inhibitor of its target enzymes. The sensitivity of cells continuously exposed to DATHF was not influenced by FPGS activity. After short term exposure to DATHF, cells expressing higher levels of human FPGS were more sensitive to the drug while cells expressing low levels were resistant. Cells expressing E. coli FPGS solely in the cytosol were resistant to DATHF and accumulated only low levels of the drug. Expression of E. coli FPGS in the mitochondria of these cells restored drug sensitivity and cytosolic drug accumulation. DATHF is extensively metabolized in the mitochondria of mammalian cells and, despite an inability to enter the mitochondria, folylpolyglutamates and DATHF polyglutamates formed in the mitochondria are released into the cytosol without prior hydrolysis. DATHF was not solely an inhibitor of de novo purine biosynthesis but also inhibited glycine synthesis in the cell.

Animals↗
PubMed1994

Quantitative analysis of folylpolyglutamate synthetase gene expression in tumor tissues by the polymerase chain reaction: marked variation of expression among leukemia patients.

Evidence from previous in vitro studies indicates that the enzyme folylpolyglutamate synthetase (FPGS) may be an important determinant of the antitumor activity of antifolate drugs that are substrates for this enzyme. To facilitate investigations regarding the association between FPGS content of tumor tissues and the sensitivity of tumors to antifolates, we developed a polymerase chain reaction (PCR)-based gene expression quantitation assay for measuring relative amounts of FPGS mRNA in tumor tissue specimens. From the known sequence of the human gene, FPGS-specific PCR primers were chosen that flanked a 263-base segment of the FPGS gene. The PCR carried out with these primers was linear over at least a three orders of magnitude range of starting cDNA concentration. The amount of cDNA required per assay corresponded to the quantity of RNA contained in nanogram to microgram amounts of tissue, depending on the level of gene expression. In CHO AUXB1 (FPGS) cell lines transfected with human DNA and expressing different levels of human FPGS, FPGS gene expression measured by this assay was linear with the FPGS enzyme activity in the cells. In human head and neck cell lines, which contained naturally varying levels of FPGS enzyme activity, FPGS gene expressions were also linearly proportional to FPGS enzyme content as measured both by activity in cell-free extracts and by intracellular methotrexate polyglutamate formation. Among leukemic cells from 11 acute lymphocytic leukemia and acute myelogenous leukemia patients, FPGS expression varied by over 500-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

Regulation of folate and one-carbon metabolism in mammalian cells. I. Folate metabolism in Chinese hamster ovary cells expressing Escherichia coli or human folylpoly-gamma-glutamate synthetase activity.

Chinese hamster ovary (CHO) cell transfectants expressing various levels of human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity and possessing different folylpolyglutamate chain length distributions have been developed as models for folate and antifolate metabolism. The synthesis of pteroyltriglutamate was sufficient for normal cellular retention of folate and also overcame the phenotypic requirement for purines and thymidine of AUXB1, a CHO cell mutant lacking FPGS activity and lacking folylpolyglutamates. Only low levels of FPGS are required to enable cellular metabolism of folates to forms that are retained by mammalian cells. The higher levels found in mammalian cells are required for the synthesis of the long chain polyglutamate derivatives characteristic of mammalian cells. At low medium folate concentrations, folate accumulation by transfectants expressing human FPGS was not responsive to FPGS levels as the limiting step in metabolism was beyond the triglutamate, the chain length required for retention. The rate-limiting step in folate metabolism in cells expressing the E. coli enzyme was the conversion of diglutamate to triglutamate, and, at low FPGS levels, the E. coli enzyme was about 50-fold less effective than the human FPGS in enabling cellular folate accumulation. These data suggest that cellular accumulation of any folate analog whose mono- or diglutamate derivative is a poor substrate for FPGS would be very responsive to the level of FPGS activity.

Animals↗
PubMed1993

Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism.

The effect of folylpoly-gamma-glutamate synthetase (FPGS) levels on folate accumulation was investigated in Chinese hamster ovary cells expressing various levels of human and Escherichia coli FPGS activity. At low medium folate concentrations, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. Essentially all transported folate was metabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity. As medium folate concentration increased through the physiological to the pharmacological range, cellular folate accumulation became proportional to FPGS activity and the chain length of intracellular folates decreased. At high folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cell. Pteroyltriglutamates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosynthesis but were unable to support glycine and methionine synthesis. Transfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supporting glycine synthesis, and transfectants expressing higher levels of FPGS were able to grow in the absence of methionine. Growth in the absence of methionine required high (nonphysiological) intracellular folate levels and longer chain length polyglutamates.

Animals↗
PubMed1993

Regulation of folate and one-carbon metabolism in mammalian cells. III. Role of mitochondrial folylpoly-gamma-glutamate synthetase.

Wild-type Chinese hamster ovary (CHO) cells and CHO cell transfectants expressing human folylpoly-gamma-glutamate synthetase (FPGS) activity contain mitochondrial FPGS activity of higher specific activity than the cytosolic isozyme. Expression of mitochondrial FPGS activity is required for folate accumulation by mitochondria. The mitochondrial folate pool in CHO cells is not in equilibrium with the cytosolic pool and contains folylpolyglutamates of longer glutamate chain length than cytosolic folates. The inability of AUX-coli, a CHO cell expressing high levels of Escherichia coli FPGS activity and containing pteroyltriglutamate, to support glycine synthesis is due to a lack of mitochondrial FPGS activity. AUX-coli cells lack mitochondrial folate despite containing high levels of cytosolic folate.

Animals↗
PubMed1993

Regulation of folate and one-carbon metabolism in mammalian cells. IV. Role of folylpoly-gamma-glutamate synthetase in methotrexate metabolism and cytotoxicity.

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of methotrexate (MTX). CHO cells expressing human FPGS metabolized MTX to polyglutamates characteristic of human cells. Cellular MTX accumulation and metabolism to polyglutamates were dependent on the level of FPGS activity and were unaffected by putative gamma-glutamyl hydrolase inhibitors. The sensitivity of cells continuously exposed to MTX was not influenced by FPGS activity. After short term exposure to MTX, cells expressing higher levels of FPGS were more sensitive to the drug. MTX was not transported into the mitochondria and MTX treatment had no effect on preexisting mitochondrial folates while cytosolic folates were converted to oxidized forms. Mitochondrial folate accumulation was significantly impaired by MTX treatment, suggesting that the mitochondrial folate transport system is specific for reduced folates.

Animals↗
PubMed1993

Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization.

Human cDNAs for cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) were cloned by functional complementation of an Escherichia coli glyA mutant with a human cDNA library. The cDNA for the cytosolic enzyme encodes a 483-residue protein of M(r) 53,020. The cDNA for the mitochondrial enzyme encodes a mature protein of 474 residues of M(r) 52,400. The deduced protein sequences share a high degree of sequence identity to each other (63%), and the individual isozymes are highly homologous to the analogous rabbit liver cytosolic (92% identity) and mitochondrial (97% identity) SHMT isozymes (Martini, F., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1987) J. Biol. Chem. 262, 5499-5509; Martini, F., Maras, B., Tanci, P., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1989) J. Biol. Chem. 264, 8509-8519). SHMT is a highly conserved protein with the human isozymes retaining about 43% sequence identity with the E. coli protein. The human cytosolic and mitochondrial SHMT genes were localized to chromosome regions 17p11.2 and 12q13, respectively. The high degree of nucleotide sequence identity between the two isozymes, and the presence of keratin genes in both chromosomal regions, is consistent with these regions of chromosome 12 and 17 arising by a duplication event.

Amino Acid Sequence↗
PubMed1993

Accumulation of 5-methyltetrahydrofolic acid and folylpolyglutamate synthetase expression by mitogen stimulated human lymphocytes.

The accumulation of 5-methyl[3H]tetrahydrofolic acid (5CH3[3H]FH4) by phytohaemagglutinin stimulated lymphocytes (PHA-L) cultured in folate free media was investigated to determine the mechanism of uptake of 5CH3FH4 and the requirement of the cells for this vitamin as assessed by monitoring de novo thymidine synthesis. When grown in 20 nM 5CH3[3H]FH4 PHA-L accumulate radiolabel at a rate of 0.04 pmol/h/10(6) cells. This doubles the endogenous folate pool of unstimulated cells (0.6 +/- 0.16 pmol/10(6) cells) in about 15 h. Uptake proceeded via a saturable process, independent of a high affinity folate receptor as assessed by ligand binding and by Northern and Western blot analysis. However, transport was blocked by probenecid, which is consistent with an anion carrier mechanism. Unstimulated cells lacked folypolyglutamate synthetase (FPGS) activity and did not express significant amounts of FPGS mRNA. After 48 h of mitogen stimulation there was a 4-10-fold increase in FPGS mRNA and folypolyglutamate formation (Glu > or = 5) was essentially simultaneous with 5CH3[3H]FH4 transport. Increasing extracellular folate to 2 microM only increased intracellular folate 8-fold, but the length of the folylpolyglutamates decreased. The increased folate did not increase de novo thymidine synthesis compared to cells grown in physiological folate. We conclude that mitogen stimulation activates the process(es) for folate accumulation, especially FPGS, and that physiological uptake (0.04 pmol/h/10(6) cells) is adequate for meeting the cells' need for the vitamin.

Cells, Cultured↗
PubMed1992

Inhibition of hog liver folylpolyglutamate synthetase by 5-substituted 5,8-dideaza analogues of folic acid bearing a terminal L-ornithine residue.

Five new N alpha-(5,8-dideazapteroyl)-L-ornithines have been prepared using multistep synthetic sequences. These include N alpha-[5-(trifluoromethyl)-5,8-dideazapteroyl]-L-ornithine, 3, as well as N alpha-[5-(trifluoromethyl)-5,8-dideazaisopteroyl]-L-ornithine, 4, and its 5-fluoro and 5-chloro analogues. Both of the compounds containing a 5-(trifluoromethyl) group (3 and 4) were found to be excellent inhibitors of homogeneous hog liver folylpolyglutamate synthetase, having Ki values in the same range as N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, 2, (approximately 10 nM). However, the bridge-reversed isomer of 2 was 60-fold less inhibitory than 2.

Animals↗
PubMed1992

Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria.

Amino Acid Sequence↗