Disease relevance of FPGS

• Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS [1].
• 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 [1].
• The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar [2].
• CONCLUSION: Our results suggest that normal-appearing colonic mucosa adjacent to primary colon cancer can show altered gene expression levels of FPGS that may have bearing on the development of aggressive metastatic behavior of the tumor and on tumor-specific survival [3].
• MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX [4].

High impact information on FPGS

• In culture, enhanced antifolate sensitivity was also seen in other stably transfected rodent and human glioma cell lines, including one with high pre-existing FPGS activity, and in canine and human glioblastoma cell lines transduced with a vector bearing FPGS cDNA [5].
• Subcutaneous 9L/FPGS tumors responded as well to methotrexate given every third day as 9L tumors did to daily treatment [5].
• CONCLUSIONS: FPGS gene delivery enhances the antifolate sensitivity of several glioma cell lines and merits further evaluation as a therapeutic strategy [5].
• METHODS: 9L rat gliosarcoma cells were stably transfected with a human FPGS complementary DNA (cDNA), producing 9L/FPGS cells [5].
• 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 [1].

Chemical compound and disease context of FPGS

• Low expression of reduced folate carrier-1 and folylpolyglutamate synthase correlates with lack of a deleted in colorectal carcinoma mRNA splice variant in normal-appearing mucosa of colorectal carcinoma patients [6].
• 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 [7].
• In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC [8].
• AIM: We investigated the effects of FPGS modulation on the chemosensitivity of colon cancer cells to 5-FU and MTX [9].
• Aminopterin has higher activity with FPGS than does methotrexate, which partially explains its greater toxicity [10].

Biological context of FPGS

• The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns [11].
• A single FPGS gene was located to chromosome region 9q34 [11].
• However, the human FPGS upstream promoter is infrequently used, and transcripts from this promoter include sequences homologous with only one of the upstream exons found in the mouse [12].
• We conclude that transcription of the FPGS gene in CEM cells involves transactivation events over a limited upstream DNA sequence and that the FPGS promoter used in proliferating human leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 sites to initiate transcription [13].
• The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584) [11].

Anatomical context of FPGS

• An additional region of the minimal promoter, situated between the two translational start codons of the FPGS gene, was bound by protein(s) from HeLa cell nuclear extracts [13].
• A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands ( approximately 60 kDa) present in each subcellular fraction [2].
• Univariate and multivariate analyses showed that the FPGS gene expression level in mucosa, but not in tumor, was a prognostic parameter independent of the clinicopathological factors with regard to survival [3].
• Normal circulating human lymphocytes (peripheral blood mononuclear cells) also expressed mRNA amplified from full-length FPGS and the four exon 1 splice variants, although no detectable FPGS activity was found [14].
• Total FPGS mRNA expression showed tissue-specificity, and higher levels were observed in human fetal tissues compared with adult tissues [14].

Associations of FPGS with chemical compounds

• The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells [11].
• Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity [2].
• 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 [15].
• It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors [16].
• This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate [17].
• AuxB1 cells harboring these same cytosolic variant allozymes displayed significant increases in the EC(50) for folic acid and in the IC(50) values for both methotrexate and pemetrexed relative to the WT Cyt form of FPGS [18].

Other interactions of FPGS

• These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts [15].
• Tomudex belongs to a class of compounds that use the reduced-folate carrier (RFC) for uptake into cells and which are excellent substrates for folylpolyglutamate synthetase (FPGS) [19].
• Compound 13 is a poor inhibitor of purified DHFR and TS, and both 13 and 14 are poor inhibitors of the growth of CCRF-CEM human leukemia cells in culture, indicating that single carbon bridged compounds in these series though conducive to FPGS substrate activity were not potent inhibitors [20].
• CONCLUSION: FPGS mRNA expression is an independent predictive factor associated with poor response to methotrexate therapy in RA patients [21].
• FPGS mRNA expression was not associated with age, sex, disease duration, white blood cell count, erythrocyte sedimentation rate, C-reactive protein (CRP), number of swollen joints, number of painful joints, and combined therapy with other disease-modifying antirheumatic drugs (DMARDs) or additional corticosteroids [21].

Analytical, diagnostic and therapeutic context of FPGS

• Folypoly-gamma-glutamate synthetase (FPGS) is essential for the cytotoxicity of "classical" antifolates and their efficacy in cancer chemotherapy [14].
• Using Western immunoblotting, we show that FPGS protein is expressed in these peripheral blood mononuclear cells; thus, we propose that posttranslational modification(s) is required for expression of a functional FPGS protein in human lymphohematopoietic cells [14].
• METHODS: We determined the expression of FPGS mRNA using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in 141 patients with RA [21].
• METHODS: Quantification of RFC-1 and FPGS expression in mucosa of 53 CRC patients was performed using real-time PCR whereas DCC splicing variants were detected by automated capillary gel electrophoresis [6].
• The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting [22].

References