Different Antifolate-resistant L1210 Cell Variants with either Increased or Decreased Folylpolyglutamate Synthetase Gene Expression at the Level of mRNA Transcription

Roy, Krishnendu; Mitsudo, Kenji; Sirlin, Sonia; Shane, Barry; Sirotnak, Francis M.

doi:10.1074/jbc.270.45.26918

Cited by 19 publications

(14 citation statements)

References 32 publications

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“…Only Galpin et al 56 observed such a relationship in 11 samples obtained from six patients before and after MTX treatment, which may be due to potential upregulation by MTX of FPGS within leukemic blasts. 40 Also in subtypes of cell lines with induced resistance, a relation was found, 35,57,58 but not in a panel of human CEM leukemia cell lines. 59,60 Post-translational modifications may explain this discrepancy since mRNA levels correlated with protein expression, but not with activity of FPGS.…”

Section: Discussionmentioning

confidence: 96%

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

et al. 2000

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Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of ␤-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three-and four-fold higher, respectively; P Ͻ 0.001), while FPGS expression was lower (three-fold, P ‫؍‬ 0.006) compared to common/preB-ALL. The ratio of (DHFR ؋ FPGH)/(RFC ؋ FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P Ͻ 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P ‫؍‬ 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (fourfold higher; P ‫؍‬ 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r ‫؍‬ 0.80; P Ͻ 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance. Leukemia (2000) 14, 2166-2175.

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Section: Discussionmentioning

confidence: 96%

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

et al. 2000

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“…The human FPGS peptide utilized was deduced from the cDNA nucleotide sequence (23) and linked to keyhole limpet hemocyanin (35). The amino acid sequence of this peptide and its use as an antigen for antibody production has already been described (19). During blotting, the anti-rabbit horseradish peroxidase-IgG conjugate (Sigma) at a 1:3000 dilution was used as the secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

“…Derivation of a Murine FPGS cDNA Probe-A murine FPGS cDNA (ZAP-L1210/R83-1) 77.3% homologous to a human FPGS cDNA was obtained by hybridization screening (19) of an L1210 cell cDNA library in gt11 (Stratagene, La Jolla, CA) using the human cDNA, pTZ18U (25), as a probe. This gt11 cDNA construct incorporates a 2.247-kilobase pair insert ligated at the XhoI polylinker site including 3Ј-and 5Ј-untranslated region sequences of 460 and 26 base pairs respectively, and an open reading frame of 1761 base pairs that codes for a putative mitochondrial leader peptide as well as the enzyme protein.…”

Section: Methodsmentioning

confidence: 99%

“…1 This process is not only important to the conservation and efficient utilization by proliferating cells of folate coenzymes (12)(13)(14)(15)(16)(17)(18) but is responsible, as well, for converting folate analogues (9 -18) to longer chain polyglutamate anabolites that are more retentive and more effective inhibitors of folate-dependent enzymatic targets. Subsequently, examples have been reported from our laboratory (4,19) and elsewhere (20,21) of tumor cell variants with acquired resistance to methotrexate and other folate analogues that exhibit lower levels of FPGS activity when compared with parental cells and are cross-resistant to most other classical folate analogues. In one of our own studies with the L1210 leukemia, resistant variants (L1210/EDX) were independently selected (4) during therapy of tumor-bearing mice with edatrexate (EDX).…”

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confidence: 99%

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Posttranscriptionally Mediated Decreases in Folylpolyglutamate Synthetase Gene Expression in Some Folate Analogue-resistant Variants of the L1210 Cell

Roy

Egan

Sirlin

et al. 1997

Journal of Biological Chemistry

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L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Biochemical alterations associated with acquired resistance of tumor cells to classical folate analogues are highly diverse (1-5). This phenotypic diversity most likely reflects the common occurrence of multiple genomic alterations among clonal variants selected for resistance to these agents either in vitro or in vivo. As a consequence, acquired resistance to methotrexate and newer folate analogues under development will remain (6 -8) a major limitation to their effective clinical utility. One of the determinants of cytotoxicity shared (9 -11) by most folate analogues, whether targeted to dihydrofolate reductase or folatedependent biosynthetic enzymes, is the process of intracellular polyglutamylation mediated by the enzyme folylpolyglutamate synthetase (FPGS).1 This process is not only important to the conservation and efficient utilization by proliferating cells of folate coenzymes (12-18) but is responsible, as well, for converting folate analogues (9 -18) to longer chain polyglutamate anabolites that are more retentive and more effective inhibitors of folate-dependent enzymatic targets. Subsequently, examples have been reported from our laboratory (4, 19) and elsewhere (20, 21) of tumor cell variants with acquired resistance to methotrexate and other folate analogues that exhibit lower levels of FPGS activity when compared with parental cells and are cross-resistant to most other classical folate analogues. In one of our own studies with the L1210 leukemia, resistant variants (L1210/EDX) were independently selected (4) during therapy of tumor-bearing mice with edatrexate (EDX). Some of these variants exhibited levels of FPGS activity that were substantially lower than in parental cells. In the current studies, we address the underlying basis for these alterations in FPGS activity in these resistant variants at the level of the FPGS protein elaborated by these cells as well as cognate gene expression. Our results appear to define a novel mechanism underlying these modifications in FPGS activity in these variants that may have broad significance in regard to the regulation of FPGS gene expression and for therapy with classical folate analogues. A preliminary report of some of these findings has already been presented (22) in abstract form. EXPERIMENTAL PROCEDURESCells and Culture Conditions-Methods for the isolation of variants of the L1210 cell used in these studies were described in detail earlier (4). These variants were independently isolated from ascite fluid in the peritoneal cavity of mice following treatment with EDX. ...

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“…FPGS enzyme activity was not detectable in human pe-ripheral blood lymphocytes (6), but both enzyme activity and FPGS mRNA increased when the cells were stimulated to proliferate with phytohaemagglutinin (17). Conversely, in a cell line selected for resistance to the second generation dihydrofolate reductase inhibitor edatrexate, the decreased FPGS levels causative of resistance were demonstrated to be due to a decrease in the rate of transcription of the FPGS gene (18). Hence, the limited information available implicates the level of transcription of this gene as the major determinant of cellular FPGS activity.…”

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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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Different Antifolate-resistant L1210 Cell Variants with either Increased or Decreased Folylpolyglutamate Synthetase Gene Expression at the Level of mRNA Transcription

Cited by 19 publications

References 32 publications

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Posttranscriptionally Mediated Decreases in Folylpolyglutamate Synthetase Gene Expression in Some Folate Analogue-resistant Variants of the L1210 Cell

Transcription of the Human Folylpoly-γ-glutamate Synthetase Gene

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