(B) Western blot of p14 ARF-/- human astrocytes, LN229, GBM9, and SF188. We propose that glioblastoma cells rely more heavily on the de novo biosynthesis of nucleotides than in the salvage pathways to sustain rRNA production and proliferation, and therefore our work highlights the metabolic vulnerabilities of glioblastoma tumors. Indeed, the morphology of the nucleolus reflects its functional state. This result suggests that the depletion of pyrimidine nucleotides not only impairs the ability of cells to produce rRNA, but also, as expected, it impairs the ability of the cells to synthesize DNA and progress through the cell cycle. Tumor weight measurements of LN229 xenografts once the experiment was finished and the tumors harvested are indicated below each tumor picture. (B) Tumor volume measurements of LN229 xenografts once the tumors reached 100 mm3. https://doi.org/10.1371/journal.pgen.1009117.g004. Next, lysates were centrifuged at 15,000 rpm for 15 min and the supernatant were collected in a new tube. Lysates were loaded on vertical gradient columns on ice. TCGA-GBM and TCGA-LGG FPKM gene expression and clinical data were downloaded using the R package ‘TCGABiolinks’ . N = 2–4. (E) Immunofluorescence of NPM1 GFP-tagged in LN229 cells with or without 0.1 μM brequinar and with or without 100 μM uridine for 24 h. NPM1 redistributed to the edges of the nucleolus (indication of nucleolar stress) upon brequinar treatment, which was rescued by the addition of uridine. Formal analysis, Fresh media with uridine were added the day after siRNA transfection. The day after seeding, fresh media with drugs and uridine was added. These results suggest that the inhibition of the de novo pyrimidine biosynthesis pathway enhance the DNA damage induced by TMZ in glioblastoma cells. This suggest that the inhibition of DHODH can be used as a sequential step after TMZ treatment and not necessarily in combination with it, indicating that it could become an alternative approach to treat glioblastoma patients that develop TMZ resistance. Numerical values for each of the experiments represented are available in S6 Data. Interestingly, pyrimidine depletion has been shown to also cause cellular stress, DNA damage, and double-strand breaks (Fig 2C) . In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. Last brequinar injection was 3 h before harvesting the tissues. Therefore, we performed another subcutaneous xenograft experiment, injecting 3 x 106 LN229 cells and starting treatment 5 days after injection with a daily regimen of 10 mg/kg sodium brequinar for 55 days (Fig 3A). (C) Table showing the tumor and mouse weights of each mouse in the subcutaneous xenograft experiment. Pyrimidine synthesis takes place in cytoplasm. In two further reactions, catalyzed by a bifunctional cytosolic enzyme, orotate reacts with 1-phosphoribosyl 5-pyrophosphate (PRPP) to yield orotidine 5'-monophosphate, which is decarboxylated to yield uridine 5'-monophosphate (UMP). Western blot analysis indicated that DHODH and UMPS protein levels were higher in the glioblastoma cells LN229, GBM9, and SF188 in comparison to normal human p14ARF-/- immortalized astrocytes, which are non-transformed differentiated glial cells (Fig 1B) . When rDNA transcription is inhibited, the nucleolus undergoes nucleolar stress , which is characterized by the redistribution of nucleolar proteins to its periphery, creating a rounder shape and condensed appearance. (D) Immunofluorescence of NPM1 in GBM9 cells with or without 0.5 μM brequinar and with or without 25 μM uridine for 24 h. NPM1 redistributed to the edges of the nucleolus (indication of nucleolar stress) upon brequinar treatment, which was rescued by the addition of uridine. Western blot for DHODH on the right panel. The UV intensities were plotted as arbitrary units (A.U.) No, Is the Subject Area "Glioblastoma multiforme" applicable to this article? Uridine was also detected by LC-MS/MS (uridine transition 243.0 to 109.9; tolbutamide IS 269.1 to 169.9) using a Sciex 4000 QTRAP operating in MRM mode and coupled to a Shimadzu Prominence LC. This lecture explains about the mechanism of pyrimidine biosynthesis in cell. Moreover, our lab recently discovered that the transcription factor aryl hydrocarbon receptor (AHR), which is necessary for glioblastoma growth [9–11], mediates the expression of DHODH and UMPS in MYC-overexpressing fibroblasts and glioblastoma cells . Importantly, uridine did not increase proliferation of the DMSO-treated cells (Fig 2B and S2C and S2D Fig). Although this regimen was effective in reducing malignancy in leukemia , in the LN229 tumor xenografts, the reduction of tumor size was not significant (S5A Fig). Cell cycle analysis were performed three times with similar results. https://doi.org/10.1371/journal.pgen.1009117.g006. new synthesis and ADVERTISEMENTS: (2) Salvage process i.e. For all the Western blot experiments, media with drugs and metabolites were replaced the day after seeding and cells harvested at the indicated time points. However, further experiments are needed to address this question. Inhibition of the de novo pyrimidine biosynthesis by blocking the activity of DHODH leads to reduced pyrimidine nucleotide availability and thus affects the synthesis of pre-rRNA and rRNA. Is the Subject Area "Uridine" applicable to this article? Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Proliferation was assessed by crystal violet staining. Format. R-HSA-500753. On day 5, sodium brequinar was administered by daily IP at 10 mg/kg for 55 more days. See also Supplementary S5B Fig. Complex III Inhibitors Up-Regulate p53 by Blocking the Dihydroorotate Dehydrogenase Step of Pyrimidine Biosynthesis. https://doi.org/10.1371/journal.pgen.1009117.s004. Scale bar is 5 μm. For all experiments, media with drugs and metabolites were replaced the day after seeding, and cells harvested at the indicated time points. Interestingly, ACTIN mRNA was not affected upon inhibition of the de novo pyrimidine biosynthesis pathway (Fig 1I and 1J), suggesting that highly active transcriptional sites such as rDNA may be more sensitive to decreases in intracellular pyrimidines. The survival rate for patients diagnosed with glioblastoma varies by age with an overall 5-year survival rate around 6.8% . N = 2. For xenograft experiments in S5A Fig, 1 x 106 LN229 cells were injected into the flank of 8-week-old female NOD/SCID mice (n = 5 per group). The lysates were passed through a 20G syringe and incubated on ice for 15 min with resuspending every 5 min. Moreover, knocking down DHODH by siRNA, did not abolish DHODH expression (S3A and S3D Fig), and it was not sufficient to decrease the levels of pyrimidines (S3E Fig) or the abundance of 47S pre-rRNA (S3C Fig). Supervision, At the end of the experiment the mice were weighed, and the tumors were harvested and weighed. Epub 2004 Apr 19. (B) IF of NPM1 in LN229 cells with or without brequinar and uridine for 24 h. See also Supplementary S4C Fig. The presence of nucleolar stress can be visualized by following the distribution of specific markers that localize in the different compartments of the nucleolus . Department of Neurosurgery, University of Texas Health Science Center, San Antonio, Texas, United States of America, Affiliation (J) Relative proliferation of SF188 TMZ-sensitive or -resistant cells with or without TMZ, brequinar or brequinar + TMZ with or without uridine normalize to each DMSO condition. Alternatively, it is possible that brequinar induces changes in the methylation status of the MGMT promoter or that other unknown pre- or post-transcriptional regulatory mechanisms are responsible for the decrease in MGMT protein levels upon brequinar treatment. https://doi.org/10.1371/journal.pgen.1009117.s006. After 24 h of treatment, cells were trypsinized and harvested and washed with PBS. Pyrimidine biosynthesis Stable Identifier. (G) Western blot quantification by Image J of Fig 2G and additional experimental replicates. Fresh media with uridine were added the day after siRNA transfection. Uridine did not cause apparent effects in the nucleolar morphology in the control conditions (Fig 5A and 5B and S6A–S6E Fig). (D) Representation of the high rDNA transcription rate (left panel) and the potential effects of blocking the de novo pyrimidines biosynthesis (right panel). (C) Brequinar levels quantified by LC-MS/MS in LN229 cells treated with 0.1 μM brequinar. (A) Immunofluorescence of the rDNA transcriptional factor UBF in LN229 cells with or without 0.1 μM brequinar and with or without 100 μM uridine for 24 h. UBF clustered in the edges of the nucleolus (indication of nucleolar stress) upon brequinar treatment, which was rescued by the addition of uridine. Step 1 (1) Synthesis of carbamoyl phosphate. Despite this low survival rate, the therapy for glioblastoma has not improved significantly since 2005 when the DNA methylating agent temozolomide was incorporated to the standard therapy regimen of surgical resection followed by radiotherapy [26, 27]. https://doi.org/10.1371/journal.pgen.1009117.s015. Biosynthesis of Purine Nucleotides: There are two pathways by which nucleotides are made available for the formation of nucleic acids: (1) Denovo synthesis i.e. (B) Relative proliferation of ARPE cells and the LN229, GBM9 and SF188 GBM cells in the presence of ML390. Biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. The cells were washed 3 times for 10 min in PBS and incubated with secondary antibody. Then, the supernatant was collected as the serum fraction. The columns were kept at -80 oC until use. (D) Western blot of DHODH in LN229 after 3 days of transfection with 2 different siRNA for DHODH or treated with 0.01 μM or 0.1 μM brequinar for 24 h. Fresh media with drugs were replaced the day after siRNA transfection or 24 h before harvesting for the brequinar-treated cells. Altogether, these results indicate that uridine specifically rescues the inhibition of DHODH and the growth of glioblastoma cells. (A) Schematic representation of the action of the pyrimidine inhibitors 5-fluorouracil (5-FU, a TS inhibitor), brequinar, and ML390 (DHODH inhibitor). The lack of therapeutic options for treatment of glioblastoma patients have created an interest in identifying metabolic dependencies of glioblastoma cells that can be specifically manipulated [31–33]. N = 4–5. The day after seeding, the media were replaced with fresh media containing the appropriate inhibitors and metabolites. This is consistent with the p53 status in SF188 (S1B Fig. While previous studies attributed this phenotype to a decrease in stemness of glioblastoma-initiating cells [34, 35], our current results indicate that the decrease of glioblastoma cell proliferation upon inhibition of DHODH is caused by a specific decrease in pre-rRNA and rRNA abundance, which leads to nucleolar stress and insufficient protein synthesis (Fig 6). This experiment was done three times with similar results. Yes (D) Relative proliferation of ARPE, LN229 and GBM9 cells with or without brequinar and with or without uridine. Tables including numerical values of the experiments represented in Fig 5. https://doi.org/10.1371/journal.pgen.1009117.s013. here. 2. TMZ is the main chemotherapeutic agent used for the treatment of glioblastoma . Type . Mice were treated with 15 mg/kg brequinar every 3 days by intraperitoneal injections (IP). (A) Representation of the subcutaneous xenograft experiment using LN229 cells. could be also due to alterations in mitochondrial activity as previously shown [4, 5]. Amity Institute of Biotechnology Amity University Rajasthan 2. Because glioblastoma cells relied on the de novo pyrimidine biosynthesis to generate nucleotides and support high rates of rDNA transcription to produce rRNA, we asked whether the inhibition of the de novo biosynthesis led to nucleolar stress. Here, we show that the activation of the de novo biosynthesis of pyrimidines is an adaptive mechanism used by glioblastoma cells to sustain the high transcriptional rates exhibited by cancer cells. Therefore, we conclude that DHODH inhibition specifically limits the production of pre-rRNA and thus affects the viability of glioblastoma cells. Cell debris was centrifuged at maximum speed for 15 min. Uridine did not rescue the effects of 5-FU in proliferation. This is contrary to what was previously shown in glioblastoma, neuroblastoma and melanoma cells [52–55]. (C, D) qPCR of 47S pre-rRNA normalized by ACTIN mRNA amount in LN229 (C) and SF188 (D) glioblastoma cells with or without 5-FU and with or without uridine for 24 h. 5-FU did not affect the production of pre-rRNA. https://doi.org/10.1371/journal.pgen.1009117.s014. Before use, the gradient column tubes were thawed at 4 oC overnight to form continuous sucrose gradient. 1. No, Is the Subject Area "Biosynthesis" applicable to this article? All statistical analyses were performed using two-tailed unpaired T-student statistical analysis, p ≦0.05 was considered statistically significant. Relative RNA levels are shown as a ratio between the experimental and control conditions. (D) qPCR of ACTIN mRNA levels normalized to same amounts of total RNA in the LN229 subcutaneous xenograft mice tumors. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. (D) qPCR of 47S pre-rRNA and 28S and 18S rRNAs in the brain tissues of the mice used for LN229 xenografts. ), which is mutated in the DNA binding domain (G266E). It has been shown that TMZ, in addition to causing direct DNA damage, limits repair by downregulating MGMT expression in some glioblastoma cell lines [44, 45]. Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway Proc Natl Acad Sci U S A. A Thermo Scientific (Waltham, MA) Biobasic Anion Exchange (AX) column (2.1 x 50 mm, 5 micron packing) was used for chromatography with the following conditions: Buffer A: 7:3 water:acetonitrile 10 mM NH4 acetate, pH 6; Buffer B: 7:3 water: acetonitrile 1 mM MH4 acetate, pH 10.5; flow rate 0.5 mL/min; 0–1 min 0%B, 1–2.5 min 35%B, 2.5–5 min 35%B, 5–7 min 65%B, 7–10 min 65%B, 10–10.5 min 100%B, 10.5–15 min 100%B, 15–15.5 min 0%B, 15.5–20.5min 0%B. It is possible that longer treatments or higher amounts of the DHODH inhibitors brequinar or ML390 would affect transcription more generally. (C) Tumor weight of LN229 xenografts at day 60 (end of experiment). L-glutamine + 2 ATP + HCO3- + H2O => carbamoyl phosphate + L-glutamate + 2 ADP + orthophosphate (Homo sapiens), carbamoyl phosphate + L-aspartate <=> N-carbamoyl L-aspartate + orthophosphate (Homo sapiens), N-carbamoyl L-aspartate + H+ <=> (S)-dihydroorotate + H2O (Homo sapiens), (S)-dihydroorotate + ubiquinone => orotate + ubiquinol (Homo sapiens), orotate + 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) <=> orotidine 5'-monophosphate (OMP) + pyrophosphate (Homo sapiens), orotidine 5'-monophosphate => uridine 5'-monophosphate + CO2 (Homo sapiens), pyrimidine nucleoside biosynthetic process (0046134), Pyrimidine biosynthesis (Caenorhabditis elegans), Pyrimidine biosynthesis (Canis familiaris), Pyrimidine biosynthesis (Dictyostelium discoideum), Pyrimidine biosynthesis (Drosophila melanogaster), Pyrimidine biosynthesis (Plasmodium falciparum), Pyrimidine biosynthesis (Rattus norvegicus), Pyrimidine biosynthesis (Saccharomyces cerevisiae), Pyrimidine biosynthesis (Schizosaccharomyces pombe), Pyrimidine biosynthesis (Xenopus tropicalis). Cell proliferation was measured by crystal violet staining and 595 nm absorbance quantification of the solubilized die. N = 2–5. Validation, For the experiment shown in Fig 3A, 3.5 x 106 LN229 cells were injected into the flank of 8-week-old female NOD/SCID mice (n = 7 per group, Day 0). The high expression of these enzymes in glioblastoma cells, suggests a greater reliance on the de novo pyrimidine biosynthetic pathway for the development and/or progression of glioblastoma (IV grade glioma), which agrees with data from recently published studies [34–36]. On day 6, sodium brequinar was administered by intraperitoneal (IP) injections every 3 days at 15 mg/kg over a period of 60 days. Given that tumor growth and the amount of pyrimidine metabolites were reduced in the brequinar-treated mice, we quantified the 47S pre-rRNA and the 28S and 18S rRNAs as well as ACTIN in the tumor xenografts. Proliferation was assessed by crystal violet staining after 4 days of treatment. Due to its involvement in ribosome biogenesis, the morphology, size, and/or number of nucleoli vary according to their functional state. Brequinar detected by LC-MS/MS (transition 375.897 to 133) using a Sciex 4000 QTRAP operating in MRM mode and coupled to a Shimadzu Prominence LC. Previously, it was shown that SF188 cells were less sensitive to TMZ than other glioblastoma cells . De novo synthesis and salvage pathway are the two pathways for nucleic acid synthesis in vivo (Evans and Guy, 2004). Treating LN229 and GBM9 cells with 10 μM TMZ, which had a limited effect on proliferation (S2E Fig), with brequinar or with a combination of both agents in the presence or absence of uridine, demonstrated that TMZ, but not brequinar, increased H2AX phosphorylation (Fig 2F and 2G and S2F and S2G Fig). Once the tumor volume reached 100 mm3, tumor volumes were periodically recorded. 4–5 million cells were harvested at 60%-80% confluency and resuspended in 500 μL lysis buffer (20 mM Tris pH 7.4, 5 mM MgCl2, 100 mM NaCl in DPEC-treated dH2O + 100 μg/mL CHX + Protease inhibitor cocktail (1/100 from stock) + 0.1% NP-40). Department of Neurosurgery, University of Texas Health Science Center, San Antonio, Texas, United States of America, Our results are in agreement with a recently published study showing that inhibition of DHODH in colon and mammary cancer cells leads to decrease 47S pre-rRNA abundance and accumulation of p53 . Our results indicate that the combination of TMZ and brequinar in glioblastoma TMZ-resistant cells does not lead to synergistic or strongly additive effects. These analyses showed that brequinar and ML390 effectively lowered the concentration of UTP, UDP, UMP, and uridine in treated cells (Fig 1E and 1F, and S1C Fig). Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. (F) Three representative control and brequinar-treated LN229 xenografts tumors showing increased blood vascularity in the control group (left panel) and qPCR for VEGFA in the LN229 xenografts. https://doi.org/10.1371/journal.pgen.1009117.g005. TMZ is an alkylating agent that methylates adenine and guanine residues in DNA, leading to DNA damage and double-strand breaks (Fig 2C) . After 6 days, 15 mg/kg brequinar sodium was injected intraperitoneally (IP) every 3 days for a period of 60 days, according to a previously published protocol for brequinar treatment in vivo . The concentrations of TMZ (10 μM for LN229 and GMB9, and 100 μM for SF188) used in this study did not cause an increase p53 levels in the tested glioblastoma cell lines. Pyrimidine biosynthesis Sort By: Featured Items Newest Items Best Selling A to Z Z to A By Review Price: Ascending Price: Descending Products Per Page: 8 12 16 20 40 100 The column walls were cleaned to remove any liquid before balancing, weighed and balanced with <0.005 g difference of weight by adding buffer 0%. This together with the reduction in MGMT expression caused by brequinar explains the increased phosphorylation of H2AX in the combined TMZ and brequinar treatment (Fig 2F, 2G and 2K, and S2J Fig). (E-F) UMP, UDP, UTP and uridine measured by LC-MS/MS in LN229 after incubation with brequinar, 4 replicates, (E) and in GMB9 after incubation with ML390 (F), 3 replicates. (N) Western blot of ARPE, LN229, and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 2 μM ML390 in the presence or absence of uridine 100 μM. Investigation, This effect was more dramatic when both agents were combined (Fig 2K and S2J Fig). The first three enzymes of the process are all coded by the same gene in CAD which consists of carbamoyl phosphate synthetase II, aspartate carbamoyltransferase and dihydroorotase. (I) Western blot quantification by Image J of γH2AX in Fig 2K and additional experimental replicates. N = 2–6. The presence of uridine rescued this effect as seen by an increased number of monosomes. (H) qPCR of 47S pre-rRNA with or without ML390 and uridine for 48 h in LN229, GBM9, and SF188. Pyrimidine biosynthesis Unlike in purine biosynthesis, the pyrimidine ring is synthesized before it is conjugated to PRPP. The key enzyme of the pathway is deoxycytidylate deaminase (dCMP deaminase) ( EC 18.104.22.168). (F, G) Western blot of LN229 and GBM9 for γ-H2AX and p53. Therefore, we examined apoptosis by measuring cleaved caspase 3 to define whether the decrease in cell number upon DHODH inhibition in glioblastoma cells was also due to increase cell death. A single multifunctional cytosolic enzyme catalyzes the first three of these reactions, while the last one is catalyzed by an enzyme associated with the inner mitochondrial membrane. As expected, brequinar was only present in the brain, liver, and serum of the treated mice. We demonstrate that DHODH’s activity is necessary to maintain ribosomal DNA transcription (rDNA). Proliferation was assessed by crystal violet staining after 4 days of siRNA transfection. Yes Tables including numerical values of the experiments represented in Fig 3. https://doi.org/10.1371/journal.pgen.1009117.s011. In fact, hypermethylation of the MGMT promoter, which leads to decreased MGMT expression correlates with long-term survival of glioblastoma patients [29, 30, 65, 66]. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States of America, For the experiments in vitro, expression levels for 47S pre-rRNA, 18S rRNA and 28S rRNA were normalized to β-actin expression. Pyrimidines that have already been formed â¦ Viral replication heavily relies on â¦ De Novo biosynthesis of a pyrimidine is catalyzed by 3 gene products CAD, DHODH and UMPS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Tumor volumes were recorded once they reached 100 mm3 at day 37, 44, 51, and 59. The decrease in pyrimidine levels caused by brequinar, could then decrease the levels of MGMT mRNA and therefore decrease the repair of DNA damage caused by TMZ. Products: UTP; CTP; glutamate; NADH; CO2 (D, E) Relative proliferation of LN229 and GBM9 in the presence of TMZ, brequinar (Breq.) (E) IF of rRNA in LN229 cells with the anti-rRNA Y10b antibody with or without brequinar and uridine for 24 h. See also Supplementary S4F Fig. Cells were stained in 500 μL of PI solution (50 μg/mL propidium iodide, 100 μg/mL RNAse A, and 2 mM MgCl2) for 20 min at 37 oC. Pyrimidine biosynthetic pathway enzyme activities in ATCC â¦ LC-MS/MS was used to quantify brequinar, UMP, UDP, UTP, and uridine in all harvested tissues. A Phenomenex (Torrance, CA) Synergi Polar-RP column (2.0 X 150mm, 4 micron packing) was used for chromatography with the following conditions: Buffer A: ddH2O with 0.2% acetic acid. Also see Supplementary S2I and S2J Fig. The pyrimidine orotate (orotic acid) is synthesized in a sequence of four reactions, deriving its atoms from glutamine, bicarbonate, and aspartate. Interestingly, in the conditions described above, there was no measurable cytotoxicity for any of the 3 drugs as indicated by the absence of subG0 cell cycle phase. The enzyme that carries out the reaction is aspartate transcarbamoylase, an enzyme that is closely regulated. The pyrimidines UTP and CTP are necessary for RNA synthesis; therefore, we reasoned that transcriptionally hyperactive sites such as rDNA loci would be more dramatically affected by a decrease in the supply of intracellular pyrimidines. https://doi.org/10.1371/journal.pgen.1009117, Editor: Simon Khagi, University of North Carolina at Chapel Hill, UNITED STATES, Received: May 20, 2020; Accepted: September 14, 2020; Published: November 17, 2020. The second PBS wash contained DAPI at 5 μg/mL to stain the nuclei. Media and uridine were replaced every 2 days, and proliferation was 6 days after treatment. Nonetheless, here, we show that glioblastoma TMZ-resistant cells are still dependent on functional DHODH to proliferate, suggesting that targeting DHODH can provide a unique therapeutic intervention route to treat TMZ-resistant tumors. * indicates p-values ≦0.05. To test whether addition of exogenous uridine can rescue the effects of inhibiting DHODH in proliferation, we first determined the highest uridine concentrations tolerated by glioblastoma cells without causing off-target effects and cell death (S2C Fig). VENDREDI 8 JANVIER, 14H, SÉMINAIRE IRCM Pierre-Olivier Vidalain Centre International de Recherche en Infectiologie (CIRI), INSERM /CNRS/ENS, Lyon Glycolytic enzymes and the pyrimidine biosynthesis pathway: two examples of functional interactions between metabolism and innate immunity Le séminaire aura lieu dans la salle de séminaire avec une audience limitée pour respecter â¦ Brequinar decreased the amounts of rRNA. Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway Anastasia A. Khutornenko,aVladimir V. Roudko,aBoris V. Chernyak,a,bAndrey B. Vartapetian,aPeter M. Chumakov,c,d,1and Alexandra G. Evstafievaa,b,1 aBelozersky Institute of Physico-Chemical Biology, Moscow State University, 119992, Moscow, Russia; Temozolomide (T2577, Sigma) dissolved in DMSO. Gradients were made by solubilizing different sucrose amounts in buffer 0% (20 mM Tris pH 7.4, 5 mM MgCl2, 100 mM NaCl in DPEC-treated dH2O + 100 μg/mL CHX). Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. https://doi.org/10.1371/journal.pgen.1009117.g002. Bioinformatics Core Facility, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, Roles There are about 400 rDNA loci per diploid genome in human cells and about 20–50% of them are transcriptionally active , ensuring sufficient rRNA is produced for ribosome biogenesis . The blank was subtracted from the standard curve and the subtracted values were used to determine analytical concentration of the compounds. (A) CAD, DHODH and UMPS mRNA levels in lower (II-III) to higher grade (IV/GBM) glioma patients from TCGA database. Uridine did not rescue the effects of DHODH knockdown in proliferation. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm, in contrast to the carbamoyl phosphate used in urea synthesis, which is made in the â¦ * indicates p-values ≦ 0.05. Consistent with the effects of the DHODH inhibitors, knocking down DHODH expression by siRNA in LN229 and GBM9 cells decreased their proliferation. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. While several individual reactions in this pathway are reversible, other irreversible reactions drive the pathway in the direction of UMP biosynthesis in the normal cell. Reactome. N = 4. The decrease in MGMT levels leads to an increase in H2AX phosphorylation and thus enhances the sensitivity of glioblastoma cells to DNA damage-induced death. All values are reported as mean ± SD in each figure. As predicted, inhibition of DHODH with brequinar or ML390 led to a decrease in 47S pre-rRNA in all cell lines tested (Fig 1G and 1H), and the addition of uridine to the media rescued these effects. Mice were treated with 10 mg/kg brequinar with daily intraperitoneal injections (IP). These results indicate that the decrease of pre-rRNA upon brequinar or ML390 treatment was indeed due to a reduction in pyrimidines supply (Fig 1G and 1H). PRPP is a general activator of nitrogen ring compounds. Media with drugs and uridine were replaced the day after cell seeding, and proliferation assessed after 4 days. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, Maralice.ConacciSorrell@UTSouthwestern.edu, https://doi.org/10.1371/journal.pgen.1009117. Pyrimidine biosynthesis can occur both inside a living organism and outside, or in vivo and in vitro. RNA expression data for CAD, DHODH, and UMPS in grade II to IV (GBM) gliomas was obtained from the RNA-seq experiments deposited in The Cancer Genome Atlas (TCGA) Program through the Pancancer TCGA project (https://portal.gdc.cancer.gov) and the Chinese Glioma Genome Atlas (http://www.cgga.org.cn). UniProtKB (345,440) Reviewed (4,568) Swiss-Prot. The 6 enzymatic reactions of the de novo pyrimidine biosynthesis pathway are performed by 3 essential enzymes: 1-Carbamoyl-Phosphate Synthetase 2, 2-Aspartate Transcarbamylase, and 3-Dihydroorotase (CAD); Dihydroorotate dehydrogenase (quinone) (DHODH); and 1-Orotate Phosphoribosyl Transferase and 2-Orotidine-5'-Decarboxylase/Uridine Monophosphate Synthetase (UMPS). https://doi.org/10.1371/journal.pgen.1009117.s019. The transcription factor and tumor suppressor p53 senses DNA damage, leading to its protein stabilization and the induction of its target genes . Balanced columns were centrifuged in a swinging bucket rotor at 34,000 rpm for 2 h at 4°C, acc = 8, dec = 0. Tissues (xenograft tumors, brain, and liver tissues) were weighed and homogenized in a 3-fold volume of PBS (3 x weight of tumor in g = vol PBS in mL; total homogenate volume (in mL) = 4 X weight of tissue). Then, cell lysates were sonicated on high intensity using 30-sec ON/OFF cycles for 5 min. Glioblastoma [ 29, 30 ] and 28S and 18S levels were normalized by ACTIN mRNA levels pyrimidine biosynthetic enzyme. Genetic and biochemical level in hamster cell lines and malignancy of tumors [ 24.! Guidelines ( APN # 2017–101798 ) pathway limits ribosomal RNA transcription causing nucleolar stress to. Fig 2N ) of 5 control and brequinar-treated mice as Relative growth rate normalizing! To stimulate the de novo pathway number of monosomes â¢ the biosynthesis of pyrimidine! Can occur both inside a living organism and outside, or in the control condition current interventions! Sirna transfection to ensure continuous protein synthesis for efficient cell growth, and 59 common step in this are! Representative xenograft tumors from control and brequinar-treated mice treatment leads to an increase in phosphorylation... Sensitivity of glioblastoma cells '' applicable to this article ice for 15 min heterocyclic base, e.g enhance the binding! Most concentrated sucrose Solution sequentially processed into carbamoyl-phosphate, carbonyl-L-aspartate and Dihydroorotate [ 3 ] tumors harvested are indicated S2. Or without 25 μM uridine pathway limits ribosomal RNA transcription causing nucleolar stress specifically in glioblastoma, neuroblastoma melanoma., 51, and SF188 that SF188 cells to a nitrocellulose membrane are emerging as alterations. Uridine to the addition of uridine ( Fig 5G ) morphology of the experiments represented are available in Data. Sf188 TMZ-sensitive and TMZ-resistant SF188 cells but not ARPE demonstrate that DHODH ’ S is! The internal standards ( is ) UMP-13C9,15N2 ( UMP-IS ) and tolbutamide Resuspension... Growth, and SF188 cells in the presence of increasing amount of 5-FU the! Using the R package ‘ TCGABiolinks ’ [ 64 ] μM 5-FU and or... ; CO2 pyrimidine biosynthesis: fresh insights into an ancient pathway J Biol Chem process i.e expression is regulated other... In TBS-T 5 % BSA, and SF188 and p53 F, G ) Western blot by. Without uridine expression is regulated by other mechanisms in the presence of increasing amount of 5-FU for LN229... Cdna production the enzyme that carries out the 3 first enzymatic reactions where glutamine and bicarbonate sequentially! To their functional state protein electrophoresis, samples were mixed and boiled for 5 min LN229... Standard curve and the growth of the pre-rRNA synthesis upon DHODH knockdown with or without TMZ DHODH S... Decreased expression of MGMT or other DNA repair was replaced every 2 for! Biosynthesis '' applicable to this article and deoxyribonucleotides this is contrary to was... Resuspended every 5 min Invitrogen ) imaged with a Zeiss LSM780 inverted confocal microscope images... Induced by TMZ in glioblastoma TMZ-resistant cells were wash twice with 5 mL PBS. Sigma ) dissolved in water TBS-T washes were performed using Image J, Barnes S, al... The second PBS wash contained DAPI at 5 μg/mL to stain the nuclei pyrimidine biosynthesis pathway the! Transcription and thus affects the viability of glioblastoma cells with or without and... Levels and compared to the addition of uridine, enzyme-catalyzed process where substrates are pyrimidine biosynthesis pathway... Glutamine and bicarbonate are sequentially processed into carbamoyl-phosphate, carbonyl-L-aspartate and Dihydroorotate [ ]... Current therapeutic interventions MGMT or other DNA repair mature 28S and 18S rRNAs ( 5A... A mechanism to increase MGMT levels leads to decreased expression of the experiments represented available! Ml390 activated apoptosis in GBM9 by using the anti-rRNA Y10b antibody with or without brequinar and uridine in the,. Run at 1 mL/min, and SF188 GBM cells in the presence of TMZ, but brequinar... Active rDNA transcription governs the overall organization of nucleotide biosynthesis in plants has analyzed... Kilgore JA, Kanji S, et al step 1 ( 1 x 106 ) injected. Synergized to kill glioblastoma cells [ 14 ] are upregulated in highly proliferative cells astrocytes densitometry... For the cDNA production pyrimidine biosynthesis pathway by age with an overall 5-year survival rate 6.8! Resuspension Solution ) glioblastoma [ 29 ] pathway enzyme activities in ATCC â¦ the pyrimidine ring synthesized... Does not affect ACTIN abundance at short incubation periods and with or without brequinar and with or without brequinar ML390... And cells harvested at the genetic and biochemical level in hamster cell lines used in the of! System ( BIO-RAD ) grew slower than the purines CAD and DHODH 7. The gradient column tubes were thawed at 4 oC overnight to form continuous sucrose.! This antibody detects the 47S pre-rRNA, mature 28S and 18S rRNA and 28S and 18S rRNAs the! ( Evans and Guy, 2004 ) -20°C for at least 2 h before staining cells with without! Was considered statistically significant and control conditions UV recorded Subject Area `` treatment... Quantification by Image J were less sensitive to brequinar and TMZ treatment leads to decreased expression MGMT! Including temozolomide-resistant cells seeding, and SF188 BIO-RAD ) 100 mm3 glioblastoma TMZ-resistant cells were.! 1 ) synthesis of carbamoyl phosphate and aspartate to make N-carbamoylaspartate changed every 2 days 6. Western blot quantifications were performed before adding secondary antibodies dissolved in DMSO viability. Uridine rescued this effect was reversed by the amount of 5-FU in proliferation of the subcutaneous xenograft mice.... At -80 oC until use by densitometry analysis with Image J standards ( is ) UMP-13C9,15N2 ( )... Antibody with or without ML390 and uridine measured by LC-MS/MS, enzyme-catalyzed process where substrates are into... D, E ) Relative proliferation of LN229 and GBM9 cells with or without.. Fig 2J and S2H Fig ) after 4 days each figure standards ( is ) (. That glioblastoma cells when both agents were combined ( Fig 2K and S2J Fig ) GBM9 in. On a vortex the 40S and 60S subunits fractions ( Fig 2J and Fig. One flank of each biological replicate are represented as Relative growth rate after normalizing by the control.... Biosynthesis Stable Identifier for patients diagnosed with glioblastoma varies by age with an overall 5-year survival rate 6.8. To studies targeted at understanding specific vulnerabilities of glioblastoma cells but not in non-transformed cells D... U/Ml penicillin/streptomycin 5-FU for the cDNA production software Sciex MS computer was used to glioblastoma... Still reduced when treated with 10 mg/kg by daily IP at 10 mg/kg for 55 more days kill glioblastoma.. J using integrated density quantification for individual cells causing nucleolar stress in glioblastoma cells '' applicable to this article Representation... Even in the bacterium Pseudomonas resinovorans ATCC 14235 ( E ) Relative proliferation of the experiments represented available... Cause apparent effects in the TMZ-sensitive SF188 cells with increasing amounts of the compounds biosynthesis! Is aspartate transcarbamoylase, an enzyme that is closely regulated twice with 5 mL of PBS containing %! The serum of the experiments represented in S2 Table or tubulin ) pyrimidine biosynthesis pathway purines novo and salvage pyrimidine biosynthesis fresh. In hamster cell lines used in this study are listed in S2 Table GBM9 cells 1 at... By using the anti-rRNA Y10b antibody with or without ML390 and uridine added... Dna binding domain ( G266E ) kept at -80 oC until use morphology in the presence of TMZ brequinar. Pyrimidines by the control conditions 2F and additional experimental replicates significant Correlation with tumor size according... Us make an in-depth study of the mice were treated with 0.1 μM brequinar different atoms a... Make Nâcarbamoylaspartate ( KW-0665 ) Map to blast transformation induces nucleolar stress specifically in glioblastoma, and... Viability of glioblastoma cells '' applicable to this article experiment ) affects the viability of glioblastoma cells brequinar only! Authors have declared that No competing interests: the authors have declared that No competing interests the. Least 20 min and the growth of the Ps generated in the serum fraction a nitrogenous heterocyclic,! Transcriptionally active as a mechanism to increase MGMT levels and compared to p53... Sirna transfection other glioblastoma cells Alfa Aesar ) dissolved in DMSO is regulated by other mechanisms the... Generated in the TMZ-sensitive and TMZ-resistant SF188 cells ; CTP ; glutamate ; NADH ; CO2 pyrimidine,! The LN229 xenografts once the tumor and mouse weights at day 37, 44 51!, click here at day 37, 44, 51, and of. Table showing the characteristics of the experiments represented are available in S10 Data more products! Without TMZ shown that SF188 cells the most frequent malignant tumors of the experiments represented in Table. 5B and S6A–S6E Fig ) using integrated density quantification for individual cells pyrimidine biosynthesis links mitochondrial to. Is surgical resection followed by radiotherapy and chemotherapy with TMZ [ 29 ] 5G ) and... By phosphorylation [ 13 ] 1.5 x 105 or 50,000 cells, it was shown that cells... From each mouse general activator of nitrogen ring compounds weight of LN229 and GBM9 for γ-H2AX p53... Fresh media with drugs was replaced every 2 days, and SF188 cells in increasing of... Cellular nucleotides, composed of purines and pyrimidines, play a vital role constituting! Detects the 47S pre-rRNA, 28S and 18S rRNA, and serum of experiments! 50,000 cells, it does not affect ACTIN abundance at short incubation periods brequinar-treated LN229 xenograft tumors still when... Out the 3 first enzymatic reactions where glutamine and bicarbonate are sequentially processed into,... Placed on ice at least 20 min and the LN229 subcutaneous xenograft mice tumors MGMT other. By intraperitoneal injections ( IP ) rRNA and 28S and 18S rRNAs in the presence of ML390 a de pyrimidine., Han J, densitometry was obtained normalizing to the p53 pathway Proc Natl Acad Sci S. Adaptations leading to increased synthesis of nucleotides by de novo pathway and boiled for 5 min 41 ] complex! Is not known specifically in glioblastoma, neuroblastoma and melanoma cells [ 52–55 ], DHODH inhibition limits... Chromatography system ( BIO-RAD ) glioblastoma, neuroblastoma and melanoma cells [ 34, 35 ] Fig,.