Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.

McCloskey, Diane E.; McGuire, John J.; Russell, Cynthia A.; Rowan, Brian G.; Bertino, Joseph R.; Pizzorno, Giuseppe; Mini, Enrico

doi:10.1016/s0021-9258(18)38101-8

Cited by 154 publications

(90 citation statements)

References 19 publications

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“…1 and 10). The importance of FPGS for C1 metabolism in bacteria, yeast, and plant and mammalian cells (30,(59)(60)(61)(62) is wellestablished, but most studies in mammalian systems have been on cancer cells with the ultimate aim being cancer therapeutics (63)(64)(65)(66)(67)(68). For example, Kim et al (32) used RNA interference to knock down FPGS activity in breast cancer cells (32,35) and found that FPGS modulation altered global DNA methylation and expression of several genes involved in important biologic pathways.…”

Section: Discussionmentioning

confidence: 99%

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells

et al. 2019

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Folates are vital cofactors for the regeneration of S-adenosyl methionine, which is the methyl source for DNA methylation, protein methylation, and other aspects of one-carbon (C1) metabolism. Thus, folates are critical for establishing and preserving epigenetic programming. Folypolyglutamate synthetase (FPGS) is known to play a crucial role in the maintenance of intracellular folate levels. Therefore, any modulation in FPGS is expected to alter DNA methylation and numerous other metabolic pathways. To explore the role of polyglutamylation of folate, we eliminated both isoforms of FPGS in human cells (293T), producing FPGS knockout (FPGS ko ) cells. The elimination of FPGS significantly decreased cell proliferation, with a major effect on oxidative phosphorylation and a lesser effect on glycolysis. We found a substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGS ko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.-

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Section: Discussionmentioning

confidence: 99%

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells

et al. 2019

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“…To compare AS tools, we used RNA-seq datasets from two human T-cell acute lymphoblastic leukemia (T-ALL), CCRF-CEM (ATCC, Manassas, VA) and its subclone CEM/R30dm (provided by Dr. J. McGuire [ 26 ]). The cell line CEM/R30dm has been made resistant to MTX by repeated exposure to MTX and has been shown to harbor specific splice variants in FPGS, an enzyme critically involved in metabolism and intracellular retention of MTX [ 27 – 29 ]. Loss of FPGS function, e.g.…”

Section: Methodsmentioning

confidence: 99%

“…due to AS, is implicated in MTX resistance [ 13 , 30 ] . The choice for this cell line model for the simulation experiments was based on the proven clinical and pathological relevance of the validated AS event implicated in (MTX) therapy resistance [ 13 , 22 , 26 , 29 , 30 ]. These cell lines provide a practical case for laboratory researchers providing perspective on tool applicability and output variation in a controlled setting.…”

Section: Methodsmentioning

confidence: 99%

Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

et al. 2021

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Background Computational tools analyzing RNA-sequencing data have boosted alternative splicing research by identifying and assessing differentially spliced genes. However, common alternative splicing analysis tools differ substantially in their statistical analyses and general performance. This report compares the computational performance (CPU utilization and RAM usage) of three event-level splicing tools; rMATS, MISO, and SUPPA2. Additionally, concordance between tool outputs was investigated. Results Log-linear relations were found between job times and dataset size in all splicing tools and all virtual machine (VM) configurations. MISO had the highest job times for all analyses, irrespective of VM size, while MISO analyses also exceeded maximum CPU utilization on all VM sizes. rMATS and SUPPA2 load averages were relatively low in both size and replicate comparisons, not nearing maximum CPU utilization in the VM simulating the lowest computational power (D2 VM). RAM usage in rMATS and SUPPA2 did not exceed 20% of maximum RAM in both size and replicate comparisons while MISO reached maximum RAM usage in D2 VM analyses for input size. Correlation coefficients of differential splicing analyses showed high correlation (β > 80%) between different tool outputs with the exception of comparisons of retained intron (RI) events between rMATS/MISO and rMATS/SUPPA2 (β < 60%). Conclusions Prior to RNA-seq analyses, users should consider job time, amount of replicates and splice event type of interest to determine the optimal alternative splicing tool. In general, rMATS is superior to both MISO and SUPPA2 in computational performance. Analysis outputs show high concordance between tools, with the exception of RI events.

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“…The IC 50 value of each drug was calculated from an analysis of growth inhibition. To evaluate the proliferative activity of each cell line, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 h-tetrazolium-5-carboxanilide (XTT) assays were performed according to the manufacturer's instructions (Roche, Indianapolis, IN, USA) [10,[14][15][16][17][18]. Spheroid (3D) cell viability of Col-Tgel encapsulated cells was also assessed using XTT assays.…”

Section: Growth Inhibition Assaymentioning

confidence: 99%

“…The resistance mechanisms associated with intracellular folate metabolism are thought to be related to deficiencies in FPGS activity [17,18,23] and/or increased DHFR expression [24][25][26][27]. FPGS catalyzes the formation of polyglutamate chains, yielding active PDX polyglutamates.…”

Section: Expression Levels Of Fpgs and Dhfrmentioning

confidence: 99%

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

et al. 2021

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Background Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. Methods To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. Results Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. Conclusions The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

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Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.

Cited by 154 publications

References 19 publications

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells

Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

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