Measurement of folylpolyglutamate synthetase in mammalian tissues

Moran, Richard G.; Colman, Paul D.

doi:10.1016/0003-2697(84)90174-x

Cited by 40 publications

(25 citation statements)

References 18 publications

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“…These findings indicate increased FPGS mRNA as a basis for greater FPGS activity and MTXPG accumulation in the B-lineage NALM6 cells, providing a mechanism for our previous in vivo finding of greater increases in FPGS activity in B-lineage blasts after MTX treatment (19). Differences in FPGS activity are known to exist among normal tissues, with FPGS activity highest in liver and low or undetectable in brain, muscle, kidney, and circulating erythrocytes, granulocytes, and lymphocytes (34,35). FPGS activity has also been linked to cell proliferation, as a 4-fold increase in FPGS mRNA occurs after phytohemagglutinin stimulation of human lymphocytes (36).…”

Section: Discussionmentioning

confidence: 99%

Differences in Folylpolyglutamate Synthetase and Dihydrofolate Reductase Expression in Human B-Lineage versus T-Lineage Leukemic Lymphoblasts: Mechanisms for Lineage Differences in Methotrexate Polyglutamylation and Cytotoxicity

et al. 1997

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SUMMARYCellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) ( p Ͻ 0.01) and was characterized by a saturable (E max ) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 M), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (E max ) in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4 -6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC 50 ) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.

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Section: Discussionmentioning

confidence: 99%

Differences in Folylpolyglutamate Synthetase and Dihydrofolate Reductase Expression in Human B-Lineage versus T-Lineage Leukemic Lymphoblasts: Mechanisms for Lineage Differences in Methotrexate Polyglutamylation and Cytotoxicity

et al. 1997

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“…Cellular FPGS activity was determined using (6S)-tetrahydrofolate as a substrate (26). For some experiments, FPGS was partially purified as described previously and kinetic experiments were performed using a charcoal-adsorption based assay with either aminopterin or DDATHF as substrates (27). For determination of FPGS and GARFT activities, enzyme assays were performed on a high speed supernatant fraction produced by centrifugation for 30 min at full speed in a Beckman Airfuge (about 200,000 ϫ g) or for 1 h in a Beckman Ti50 rotor at 160,000 ϫ g. ␥-Glutamylcarboxypeptidase (conjugase) activity was measured by the release of glutamic acid from (6R)-DDATHF tetraglutamate, which was tritium-labeled in the second through fourth side chain glutamate moieties.…”

Section: Synthesis Of 3 H-labeled (6r)-ddathf-mentioning

confidence: 99%

Cellular Folates Prevent Polyglutamation of 5,10-Dideazatetrahydrofolate

Tse

Moran

1998

Journal of Biological Chemistry

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Mouse L1210 cell variants were selected for resistance to 5,10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. The drug-resistant phenotype selected was conditional to the folate compound used to support growth: grown on folic acid cells were 400-fold resistant, whereas they were 2.5-fold more sensitive to 5,10-dideazatetrahydrofolate than wild-type L1210 cells when grown on folinic acid. In folic acid-containing media, polyglutamation of 5,10-dideazatetrahydrofolate was markedly reduced, yet folylpolyglutamate synthetase activity was not different from that in parental L1210 cells. Resistance was due to two changes in membrane transport: a minor increase in the K m for 5,10-dideazatetrahydrofolate influx, and a major increase in folic acid transport. Enhanced folic acid transport resulted in an expanded cellular content of folates which blocked polyglutamation of 5,10-dideazatetrahydrofolate.We propose that polyglutamation of 5,10-dideazatetrahydrofolate is limited by feedback inhibition by cellular folates on folylpolyglutamate synthetase, an effect which reflects a mechanism in place to control the level of cellular folates. Although the primary alteration causative of resistance is different from those reported previously, all 5,10-dideazatetrahydrofolate resistance phenotypes result in decreased drug polyglutamation, reflecting the centrality of this reaction to the action of 5,10-dideazatetrahydrofolate.Development of classes of folate antimetabolites inhibitory to target enzymes other than dihydrofolate reductase has offered new therapeutic agents for the treatment of human malignancies, and has also provided new biochemical probes for studying folate metabolism and the linkages between cell proliferation and survival. The prototypical members of three of these classes are (6R)-5,10-dideazatetrahydrofolate ((6R)-DDATHF, lometrexol) 1 (1), the quinazoline-based compound, ZD-1694 (tomudex) (2), and N

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“…Previous studies (4,6) of the levels of FPGS activity in normal mouse tissues had indicated high enzyme activity in murine liver and kidney, with lower levels in spleen, and very low to undetectable levels in several other tissues. All mouse tumors surveyed in those studies expressed substantial levels of FPGS activity (4,6). The hybridization of a near full-length mouse cDNA probe to Northern blots mirrored this pattern (Fig.…”

Section: Fpgs Mrna Levels In Normal and Neoplastic Tissues-mentioning

confidence: 99%

“…Earlier studies (5,6) had shown that FPGS activity was high in cells undergoing rapid proliferation and dropped when cells either committed to differentiation or entered an extended G 0 phase. In addition, enzyme levels have been shown to range from undetectable to rather high among differentiated tissues (4,6), leading to the concept that control of FPGS expression was obeying a tissue-specific regulatory mechanism. The Northern analysis presented here shows a similar pattern.…”

Section: Design Of Fpgs-luciferase Reporter Constructs and Definitionmentioning

confidence: 99%

Transcription of the Human Folylpoly-γ-glutamate Synthetase Gene

Freemantle

Moran

1997

Journal of Biological Chemistry

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In mammals, folylpoly-␥-glutamate synthetase (FPGS) activity is found in any cell undergoing sustained proliferative phases, but this enzyme also displays a tissuespecific pattern of expression in differentiated tissues. It is now reported that the steady state levels of FPGS mRNA in normal and neoplastic cells reflect these patterns, supporting the concept that the control mechanisms underlying this distribution are transcriptional. Folylpoly-␥-glutamate synthetase (FPGS)1 catalyzes the ATPdependent formation of an amide bond between the ␥-carboxyl group of the naturally occurring folates and the amino group of glutamic acid. The addition of glutamic acid moieties to folate compounds allows their intracellular retention and concentration for both the folate cofactors (1-3) as well as for all of the "classical" folate antimetabolites studied to date. As a result of its role in the retention of folate cofactors in the cytosol and mitochondria, FPGS is essential for folate homeostasis and the survival of proliferating mammalian cells. The metabolism of antimetabolites by this enzyme plays a major role in the cytotoxicity, and perhaps the selective cytotoxicity, of several folate antagonists used or under development for the treatment of human cancers.Because any cell that is attempting to proliferate during exposure to classical antifolates is susceptible to cytotoxicity if it expresses FPGS, the tissue distribution of this enzyme and the factors controlling its expression are of critical importance. From early studies on the distribution of enzyme activity, it was known that the levels of FPGS are moderately high in some tumors, in normal gut and bone marrow stem cells, and in the two specialized organs of folate metabolism, liver and kidney, but enzyme levels in other mouse adult tissues were barely detectable (4 -6). Studies relating methotrexate polyglutamylation and FPGS activity in individual childhood leukemias to the therapeutic activity of this drug have suggested that the sensitivity of some of these tumors to antifolate chemotherapy is causally related to the level of expression of FPGS (6 -9). In a recent study of FPGS in human leukemias (9), B-lineage leukemias, which are more responsive to methotrexate, had higher levels of FPGS activity than did T-lineage leukemias. Cells from acute nonlymphoblastic leukemias, which are generally resistant to antifolates, had lower enzyme levels (9). This lineage-specific effect was also seen in cells selected from normal bone marrow; lymphoid progenitor cells (CD10/CD19 ϩ ) had high levels of FPGS activity similar to those seen in blast cells from patients with B-lineage leukemias, whereas normal nonlymphoid progenitors (CD34 ϩ ) had lower levels (9). On the other hand, a decrease in cellular FPGS activity in tumor cells can contribute to or cause both acquired (10, 11) and intrinsic (12) resistance to methotrexate.A previous study in this laboratory (6) concluded that FPGS levels are controlled by at least two mechanisms, one of which is linked to proliferation a...

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Measurement of folylpolyglutamate synthetase in mammalian tissues

Cited by 40 publications

References 18 publications

Differences in Folylpolyglutamate Synthetase and Dihydrofolate Reductase Expression in Human B-Lineage versus T-Lineage Leukemic Lymphoblasts: Mechanisms for Lineage Differences in Methotrexate Polyglutamylation and Cytotoxicity

Differences in Folylpolyglutamate Synthetase and Dihydrofolate Reductase Expression in Human B-Lineage versus T-Lineage Leukemic Lymphoblasts: Mechanisms for Lineage Differences in Methotrexate Polyglutamylation and Cytotoxicity

Cellular Folates Prevent Polyglutamation of 5,10-Dideazatetrahydrofolate

Transcription of the Human Folylpoly-γ-glutamate Synthetase Gene

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