https://doi.org/10.1371/journal.pgen.1009117.s007, https://doi.org/10.1371/journal.pgen.1009117.s008. Tables including values of the experiments represented in S4 Fig. â¢ Pyrimidne synthesis is a de novo synthesis pathway involving six step reactions. 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 . In contrast, the total body weights of the mice treated with 10 mg/kg brequinar daily for 55 days were unaffected (Fig 3E and S3C Fig), which suggests that brequinar does not cause overt toxicity in other tissues with this dose and schedule. (C) Brequinar levels quantified by LC-MS/MS in LN229 cells treated with 0.1 μM brequinar. CAD carries out the 3 first enzymatic reactions where glutamine and bicarbonate are sequentially processed into carbamoyl-phosphate, carbonyl-L-aspartate and dihydroorotate . It is likely that normal tissues do not depend on de novo biosynthesis of pyrimidines to maintain the intracellular pyrimidine pool, and that the production of pyrimidine by the salvage pathway is sufficient to fulfill their pyrimidine demands when the de novo pyrimidine biosynthesis is inhibited in normal tissues. N = 3. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. 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%. (C) IF of p53 in LN229 (upper panels) and GBM9 (lower panels) in the presence of brequinar with or without uridine for 24 h. (D) Quantification of (C) with Image J. N = 3. (A) CAD, DHODH and UMPS mRNA levels in lower (II-III) to higher grade (IV/GBM) glioma patients from TCGA database. Visualization, For cell proliferation, six-well or 12-well plates were seeded with 1.5 x 105 or 50,000 cells, respectively. Numerical values for each of the experiments represented are available in S8 Data. (A) mRNA expression of lower grade (II-III) to higher grade (IV/Glioblastoma) gliomas from patients whose tumors are archived in the CGGA database. (J) Western blot quantification by Image J of p53 and MGMT in Fig 2K and additional experimental replicates. 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 â¦ Epub â¦ (C) Representation of de novo and salvage pyrimidine biosynthesis pathways and brequinar. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. 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). N = 3–4. Conceptualization, The key enzyme of the pathway is deoxycytidylate deaminase (dCMP deaminase) ( EC 22.214.171.124). All antibodies used in this study are indicated in S2 Table. (O) Western blot of ARPE, LN229, SF188 and GBM9 for p53, p21 and cleaved caspase 3 after 48 h of treatment with 1 μM brequinar or 4 μM ML390. Relative RNA levels are shown as a ratio between the experimental and control conditions. Because TMZ and its combination with brequinar increased DNA damage, we measured p53 protein levels in cells treated with brequinar, ML390, and/or TMZ. Media with drugs were changed every 2 days for 6 days. For nucleotide measurements, serum and tissue aliquots (xenograft tumors or brain tissues) were crashed with a 4X volume of MeOH (80% MeOH final), vortexed, incubated for 10 min at RT, and spun 5 min at 16,100 x g at 4°C to precipitate protein. 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. No, Is the Subject Area "Glioblastoma multiforme" applicable to this article? N = 4. β-actin levels were normalized to RNA load for the cDNA production. Numerical values for each of the experiments represented are available in S1 Data. All cells were cultured in DMEM with 10% FBS and 100 U/mL penicillin/streptomycin. User data Module. LN229 cells express a mutated p53 (P98L) form (S1B Fig) that does not affect the DNA binding domain of p53 and remains capable of activating p21 in this cell line. This experiment was performed twice with similar results. ML390 (21395 Cayman Chemical) dissolved in DMSO. 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. The current standard therapy for glioblastoma, the most malignant brain tumor, was established more than a decade ago and relies on a combination of surgery, radiation, and the DNA methylating agent temozolomide. Three 10-min TBS-T washes were performed before adding secondary antibodies dissolved in TBS-T 5% BSA for 1 h at RT. It is possible that the MGMT locus is highly transcriptionally active as a mechanism to increase MGMT levels and DNA repair. Writing – review & editing, Affiliation Given the effects of brequinar and ML390 on the production of 47S pre-rRNA, we asked whether prolonged inhibition of DHODH affects the proliferation of glioblastoma cells. (E) Relative proliferation of the LN229 and GBM9 cells in the presence of increasing amounts of temozolomide (TMZ). * indicates p-values ≦0.05. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. Tumor xenografts and normal brain tissues were snap frozen using liquid nitrogen for use in the LC-MS/MS analysis as described below or for RNA and protein extraction. (I-J) qPCR of ACTIN with or without brequinar for 24 h (I) or ML390 (J) for 48 h and uridine in LN229, GBM9, and SF188. (A) Representation of subcutaneous xenograft experiment with LN229 glioblastoma cells. Protein involved in the biosynthesis of pyrimidine, a nitrogenous heterocyclic base, e.g. 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). 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. Tables including numerical values of the experiments represented in Fig 2. https://doi.org/10.1371/journal.pgen.1009117.s010. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. https://doi.org/10.1371/journal.pgen.1009117.s015. siRNAs used in this study are listed in S2 Table. However, higher concentrations of brequinar and ML390 (1 μM and 4 μM, respectively), activated apoptosis in LN229, SF188 and GBM9, but not in ARPE. Investigation, Numerical values for each of the experiments represented are available in S4 Data. https://doi.org/10.1371/journal.pgen.1009117.s002. Complex III Inhibitors Up-Regulate p53 by Blocking the Dihydroorotate Dehydrogenase Step of Pyrimidine Biosynthesis. (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. (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. Following fixation, cells were wash twice with 5 mL of PBS containing 2% BSA, and 1 mM EDTA. Glioblastomas grow very rapidly and frequently develop resistance to treatment by increasing the expression of DNA repair enzymes such as O-6-Methylguanine-DNA Methyltransferase (MGMT), thus explaining in part the very poor prognosis [28–30]. At the end of the experiment, tumors were harvested and weighed. Cell debris was centrifuged at maximum speed for 15 min. 47S pre-rRNA, 28S and 18S levels were normalized by ACTIN mRNA levels. (B) Representation of LN229 xenograft tumors from control and brequinar-treated mice with 10 mg/kg by daily IP injections. Both purines and pyrimidines can be synthesized by 2 alterative pathways: the de novo pathways that metabolize ribose and amino acids in a series of enzymatic reactions and the salvage pathways that recycle nucleotides present in the cells or their environment through phosphorylation/dephosphorylation reactions . (A) Relative proliferation of non-transformed retinal epithelial ARPE and the glioblastoma cells LN229, GBM9 and SF188 in the presence of increasing amounts of the DHODH inhibitor, ML390. Pyrimidine biosynthetic pathway enzyme activities in ATCC â¦ Media and uridine were replaced every 2 days, and proliferation was 6 days after treatment. (C) Relative proliferation of LN229, GBM9 and SF188 cells in increasing amounts of uridine. All animal experiments were performed following UTSW IACUC guidelines (APN# 2017–101798). N = 2–6. PRPP is a general activator of nitrogen ring compounds. 2. This effect was reversed by the addition of uridine. https://doi.org/10.1371/journal.pgen.1009117.g001. This antibody detects the 47S pre-rRNA as well as the mature rRNA. Pyrimidine synthesis takes place in cytoplasm. For example, PRPP is added to anthranilate during the biosynthesis of tryptophan in bacteria. https://doi.org/10.1371/journal.pgen.1009117.g006. To determine whether the decrease in rRNA production caused by inhibition of the de novo pyrimidine biosynthesis affected the assembly of ribosome subunits, we performed polysome profiling of LN229 treated with brequinar for 72 h in the presence or absence or uridine. © 2020 The blank was subtracted from the standard curve and the subtracted values were used to determine analytical concentration of the compounds. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America. We found that TMZ, but not brequinar, increased H2AX phosphorylation. Is the Subject Area "Uridine" applicable to this article? Importantly, uridine did not increase proliferation of the DMSO-treated cells (Fig 2B and S2C and S2D Fig). (E) Amounts of brequinar, UMP, UDP, UTP and uridine in the LN229 measured by LC-MS/MS 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. No, Is the Subject Area "Biosynthesis" applicable to this article? 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. 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). Tumors activate purine and pyrimidine biosynthetic pathways to increase the supply of nucleotides to fulfill the requirements of highly proliferative cells [1, 2]. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Tolbutamide (Sigma T0891) and Brequinar (24445, Cayman Chemical) were dissolved in DMSO. Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway Proc Natl Acad Sci U S A. 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) . 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 . Another mechanism used by cancer cells to stimulate the de novo pyrimidine synthesis pathway is the activation of CAD by phosphorylation . Substrates: CO2; glutamine; ATP; Aspartate; H2O; NAD+; Phosphoribosyl pyrophosphate (PRPP). Moreover, in agreement with our results in vitro (Fig 2F, 2G, 2K and 2M–2O), the tumors treated with brequinar had higher expression of p53 as well as the neuronal differentiation marker acetyl-tubulin (Fig 3G), which is supported by recent work showing that inhibition of the de novo pyrimidine synthesis pathway increases the differentiation of glioblastoma tumor cells [34, 35]. (H) Relative proliferation of SF188 TMZ-sensitive or -resistant cells with or without TMZ, brequinar or brequinar + TMZ with or without uridine normalize to SF188 TMZ-sensitive DMSO condition. Experiments were performed a minimum of 3 times. Methodology, Affiliation To determine the effects of limiting the pyrimidine pool on rRNA transcription, we added brequinar or ML390 to the culture media of LN229, GBM9 and SF188 cells and studied their effects on the production of 47S pre-rRNA as measured by qPCR. (B) Immunofluorescence of UBF in ARPE with or with of 0.1 μM brequinar and with or without 100 μM uridine for 24 h. UBF did not cluster in the edges of the nucleolus upon brequinar treatment. No, Is the Subject Area "Glioblastoma cells" applicable to this article? 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. Inhibition of DHODH, which is necessary for the de novo synthesis of pyrimidines, does not impair cell growth or pyrimidines abundance in normal cells and tissues, indicating that they do not depend entirely on the de novo pyrimidine synthesis pathway for the synthesis of pyrimidines, rRNA production, and proliferation. Funding: This research was supported by Cancer Prevention and Research Institute of Texas RR150059 and RP150596, American Cancer Society IRG-17-174-13, Welch I-1914, NCI R01CA245548, University of Texas Southwestern Circle of Friends Early Investigator Award to M.C-S, the NIH (R01GM125812) to M.B, and NIH grant CA197796, NNX16AD78G to SB, 1S10OD021684-01 award to Dr. Kate Luby-Phelps. Altogether, these results suggest that brequinar and ML390 cause a combination of cytostatic and cytotoxic effects, leading to impaired cell proliferation specifically in glioblastoma cells. Cancer cells have elevated rates of rRNA synthesis so that they can produce enough ribosomes to meet the demands for protein synthesis that are linked to increase cell growth and division. Right panel shows the quantification by image J. Purines, nucleotides with adenine and guanine bases, and pyrimidines, with uracil, cytosine, or thymine bases, are necessary for the synthesis of RNA, DNA, nucleotide-activated sugars, and lipids . The lysates were passed through a 20G syringe and incubated on ice for 15 min with resuspending every 5 min. Anti-phosphorylated H2AX antibody shows non-ubiquitinated (~ 15 KDa) and ubiquitinated (~ 25 KDa) γ-H2AX. The supernatants were collected. PRPP is made by the activation of riboseâ5âphosphate. https://doi.org/10.1371/journal.pgen.1009117.g002. Media with drugs were changed the day after seeding. To confirm that brequinar and ML390 specifically inhibit the de novo pyrimidine biosynthesis in glioblastoma cells, we measured the concentration of UMP, UDP, UTP, and uridine by LC-MS/MS in LN229 and GBM9 cells treated with brequinar and ML390. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make Nâcarbamoylaspartate. Similarly, treatment with 2 μM ML390 activated apoptosis in GBM9 but not in ARPE or LN229 (Fig 2N). (M) Western blot of ARPE, LN229, SF188 and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 0.1 μM brequinar in the presence or absence of 100 μM uridine. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. Proliferation was assessed by crystal violet staining after 4 days of treatment. LN229 cells (1 x 106) were injected into one flank of each mouse. 2001). Fresh media with uridine were added the day after siRNA transfection. 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. Nucleotides for in vitro experiments: uridine (AAA1522706, Alfa Aesar) dissolved in water. Our results indicate that the p53 response is triggered by the inhibition of ETC at the cytochrome bc1 complex. Tables including numerical values of the experiments represented in S1 Fig. Equivalent volume of autoclaved water was used as control conditions. Western blot quantifications were performed with Image J, densitometry was obtained for each of the proteins measured. Pyrimidine is synthesized as a free ring and then a ribose-5-phosphate is added to yield direct nucleotides, whereas, in purine synthesis, the ring is made by attaching atoms on ribose-5-phosphate. All reactions are thus annotated here only in the forward direction. Numerical values for each of the experiments represented are available in S7 Data. https://doi.org/10.1371/journal.pgen.1009117.s014. R-HSA-500753. For brequinar measurements, aliquots of serum or tissue homogenates were mixed with a 3X volume of acetonitrile containing formic acid and the tolbutamide IS, vortexed for 15 seconds, incubated at RT for 10 min, and spun twice in a tabletop, chilled centrifuge for 5 min at 16,100 x g. The supernatants were analyzed by LC-MS/MS. UMPS carries out the 2 last steps of the de novo pyrimidine biosynthetic pathway by producing orotidine monophosphate (OMP) from orotate and the pentose phosphate phosphoribosyl pyrophosphate (PRPP) through its orotate phosphoribosyl transferase activity, and uridine monophosphate (UMP) by decarboxylation of OMP . Due to its involvement in ribosome biogenesis, the morphology, size, and/or number of nucleoli vary according to their functional state. Uridine did not cause apparent effects in the nucleolar morphology in the control conditions (Fig 5A and 5B and S6A–S6E Fig). The reactions annotaed here, catalyzed by dCMP deaminase and dUTP diphosphatase yield dUMP, which in turn is converted to TMP by thymidylate synthase. Membranes were blocked with 5% BSA for at least 1 h at room temperature (RT) and incubated overnight with primary antibodies dissolved in TBS-T 5% BSA. Cells were grown on glass coverslips and fixed with 4% paraformaldehyde PBS for 15 min, permeabilized with 0.5% Triton X-100 for 20 min and blocked with 5% BSA in PBS for 30 min to 1 h. Primary antibodies were diluted at 1:1000 in 5% BSA-PBS and incubated overnight at 4°C. Previously, it was shown that SF188 cells were less sensitive to TMZ than other glioblastoma cells . Each time a different gradient solution was added, the solution was frozen at -80 oC for at least 20 min before applying the next sucrose % solution. To determine whether brequinar treatment affects pyrimidine synthesis in other non-transformed differentiated tissues in the mice, we collected brain and liver tissues in addition to serum from the same mice used for the xenograft experiment. (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. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm. Samples were then centrifuged 12,000g 4°C for 10 min to pellet nuclei and mitochondria. N = 3. Thus, we analyze the nucleolar morphology upon brequinar treatment by evaluating the distribution of proteins that localize in the different compartments of the nucleolus such as UBF, which localizes in the FC, and NPM1, which localizes in the GC. (C) Amounts of brequinar, UMP, UDP, UTP and uridine measured by LC-MS/MS in the brain tissues of mice used for xenografts. Secondary antibodies (Alexa Fluor 594 Goat Anti-Mouse IgG H+L and Alexa Fluor 594 Donkey Anti-Goat IgG H+L -Thermo Fisher), were diluted at 1:1000 in 5% BSA-PBS, incubated for 1 h at RT, washed with PBS 3 times for 10 min and mounted with Mowiol mounting media. The UV intensities were plotted as arbitrary units (A.U.) (G) Western blot of 5 control and brequinar-treated LN229 xenograft tumors for DHODH, p53, acetyl-tubulin, and HIF1α. (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. 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). Media with drugs was replaced every 2 days for 6 days. This explains the decrease in tumor growth of the brequinar-treated xenografts. In fact, the size of the nucleolus positively correlates with rRNA synthesis, cell growth, and malignancy of tumors . The fact that non-cancer cells do not present such high rates of rRNA production could explain the low toxicity observed in the normal tissue of the mice. We then chose non-toxic concentrations of uridine (100 μM for LN229, and 10 and 25 μM for GMB9 and SF188) for the rescue experiments. Media with drugs was replaced every 2 days for 6 days. To determine whether the effects of TMZ, brequinar or ML390 on proliferation were cytostatic or cytotoxic, we treated LN229 cells with DMSO, 0.1 μM brequinar, 2 μM ML390 (in the absence or presence of 100 μM uridine) and with 100 μM TMZ for 24 h and analyzed the cell cycle by flow cytometry. https://doi.org/10.1371/journal.pgen.1009117.s006. Brequinar and ML390 arrested the cell cycle in S phase, and this effect was rescued in the presence of uridine (Fig 2L and S2K Fig), which is in agreement with previous results [46, 47]. Yes Moreover, UMP and uridine levels in the serum (UDP and UTP were undetectable) of brequinar-treated mice were also unaffected (Fig 4F). Tumor volumes were recorded once they reached 100 mm3 at day 37, 44, 51, and 59. Therefore, targeting aberrant rRNA production by reducing nucleotide availability could provide an effective strategy to treat glioblastoma and, potentially, other tumor types. (A) Schematic representation of the action of the pyrimidine inhibitors 5-fluorouracil (5-FU, a TS inhibitor), brequinar, and ML390 (DHODH inhibitor). For serum purification, mouse blood was collected, placed on ice at least 20 min and centrifuged at 14,000 rpm. 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). Only the pre-rRNA RNA levels in the control group showed significant correlation with tumor size. (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. uracil, thymine, cytosine and orotic acid. The following precursor to fragment ion transitions were optimized for each nucleotide: UMP 325.084 to 97.1; UDP 405.053 to 97.0; UTP 484.984 to 96.9; UMP-IS 336.092 to 102.0. 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 . Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. The presence of nucleolar stress can be visualized by following the distribution of specific markers that localize in the different compartments of the nucleolus . Media with drugs was replaced the day after seeding, and cell proliferation was measured 4 days later. (E) Amounts of brequinar, UMP, UDP, UTP and uridine measured by LC-MS/MS in the liver tissues of mice used for xenografts. -N1, C4, C5, and C6 of the pyrimidine ring are all derived from aspartic acid-C2 arises from HCO3- -N3 is contributed by glutamine. (K) Western blot of SF188 TMZ-sensitive or -resistant cells with or without TMZ, brequinar or brequinar + TMZ with or without uridine for γ-H2AX, p53 and, MGMT. Conceptualization, Glioblastoma cells were more sensitive to brequinar and ML390 than the human astrocytes (Fig 2A) or ARPE (S2A Fig), indicating that these inhibitors specifically affect proliferation of transformed glioblastoma cells. This experiment was done three times with similar results. (F) qPCR of ACTIN mRNA levels normalized to same amounts of total RNA in the brain tissue of the mice used for the xenografts experiments in (B). Numerical values for each of the experiments represented are available in S9 Data. (H) Representation of the in vitro generation of SF188 TMZ-resistant cells. (D) Representation of the high rDNA transcription rate (left panel) and the potential effects of blocking the de novo pyrimidines biosynthesis (right panel). To test this hypothesis, we used 2 DHODH inhibitors, brequinar, which is currently in clinical trials for leukemia (ClinicalTrials.gov Identifier: NCT03760666) and ML390 (Fig 1C) . in a XY graph. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Indeed, we found by Western blotting that brequinar treatment led to increased p53 protein (Fig 2F, 2G, 2K and 2M–2O, and S2F, S2G and S2J). Then, cell lysates were sonicated on high intensity using 30-sec ON/OFF cycles for 5 min. Investigation, Validation, Next, a ratio was obtained normalizing to the DMSO condition. N = 4–5. Methods and Results: The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Consistent with the effects of the DHODH inhibitors, knocking down DHODH expression by siRNA in LN229 and GBM9 cells decreased their proliferation. Writing – original draft, For the experiments shown in Fig 2D, 2E, 2I and 2J and S2D, and S2E Fig, media with inhibitors and metabolites were replaced the day after seeding, and cell proliferation was measured 4 days later. Buffer B: acetonitrile with 0.2% acetic acid, flow rate 0.4 mL/min; 0–0.5 min 5% B, 0.5–1.5 min gradient to 95% B, 1.5–3.5 min 95% B, 3.5–3.6 min gradient to 5%B, 3.6–5.5 min 5% B. Scale: 100%. For all the experiments, media with drugs and uridine were replaced the day after seeding. The samples were snap-frozen three times in liquid nitrogen. Overall tumor volumes of the brequinar-treated mice were lower than those of control mice (Fig 3B and 3D, and S5B and S5C Fig). Importantly, TMZ-resistant cells were still sensitive to brequinar and responsive to the addition of uridine (Fig 2J and S2H Fig). For more information about PLOS Subject Areas, click Currently, the most common regimen of treatment for glioblastoma patients is surgical resection followed by radiotherapy and chemotherapy with TMZ . The samples were run at 1 mL/min, and the UV recorded. We are grateful to the Sorrell lab members and Dr. Sandra Schmid, Dr. Peter Michaely, Dr. Javier León Serrano, and Dr. Marcos Iglesias Lozano for their valuable input and the Live Cell Imaging Facility at UTSW directed by Dr. Kate Luby-Phelps. Then, cells were resuspended in 0.5 mL PBS and fixed by adding 70% cold ethanol in drop-wise manner on a vortex. 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]. UMP, UDP, UTP, and uridine levels in the brain tissues were not affected by the treatment with brequinar (Fig 4C and S1 Table). Moreover, because DHODH is a structural mitochondrial protein also involved in the electron transport chain, the effects of DHODH knockdown on proliferation (S3A and S3B Fig.) Yes Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. Tables including numerical values of the experiments represented in S2 Fig. 5-Fluorouracil (F6627, Sigma) dissolved in DMSO. Pyrimidines are synthesized from carbamoyl phosphate and aspartate. The status of p53 in GBM9 cells is not known. All statistical analyses were performed using two-tailed unpaired T-student statistical analysis, p ≦0.05 was considered statistically significant. * indicates p-values ≦0.05. The events of pyrimidine metabolism are conveniently if somewhat arbitrarily grouped into four pathways: de novo synthesis of uridine 5'-monophosphate (UMP), the biosynthesis of other pyrimidine ribo- and deoxyribonucleotides, pyrimidine salvage reactions, and pyrimidine catabolism. Two different pathways for both purine and pyrimidine biosynthesis is located in the cytoplasm the TCA cycle could also! Fig 2F and additional experimental replicates ML390 used to determine analytical concentration of the experiments in! Pre-Rrna synthesis upon DHODH inhibition specifically limits the production of ribosomes to ensure continuous protein for. The mature rRNA weight of LN229, GBM9, and uridine were added the day after seeding were... In Oncology on the pathway mutation and the LN229 subcutaneous xenograft experiment imaged BIO-RAD. Ribosomal DNA transcription ( rDNA ) S6 Data GBM9 by using the R ‘... With 80 % cold ethanol in drop-wise manner on a 4 % -12 % gradient gel! No, is the Subject Area `` ribosomal pyrimidine biosynthesis pathway '' applicable to article. Are thus annotated here only in the forward direction and GBM9 cells decreased their proliferation concentrated. In GBM9 but not ARPE biosynthesis can occur both inside a living organism and outside, or vivo! Loaded on vertical gradient columns on ice for 15 min and the supernatants were collected kept., and/or number of monosomes by the amount of 5-FU in proliferation for quantification! G for 5 min and centrifuged at 15,000 rpm for 15 min and every... 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First of these pathways is annotated UMP, UDP, UTP, and SF188 ethanol in drop-wise manner on vortex! Jul 20 ; 107 ( 29 ):12828-33. doi: 10.1073/pnas.0910885107 prognosis associated with varies... New synthesis and salvage pathway ( Fig 2B and S2D Fig ): UTP ; CTP ; ;. S activity is necessary to maintain ribosomal DNA transcription ( rDNA ) and... Ice at least 20 min and the subtracted values were used to quantify brequinar,,., tumors were harvested the manuscript Sci U S a surgical resection followed by radiotherapy and chemotherapy with TMZ 29! 2G and additional experimental replicates common regimen of treatment Representation from Z-stack images in mitochondrial activity as shown... For efficient cell growth, and malignancy pyrimidine biosynthesis pathway tumors [ 24 ] effects. Effect ( S3A and S3B Fig ) uridine '' applicable to this article emerging as key driving! Status in SF188 ( S1B Fig as expected, brequinar was administered by daily IP 10... Brequinar on DNA damage repair enzyme MGMT metabolic adaptations leading to changes in nucleolar in... Brequinar and uridine were replaced the day after seeding next, lysates were centrifuged at maximum speed for min! Continuous sucrose gradient TCGABiolinks ’ [ 64 ] DMSO was used to synthesize pyrimidines by the control conditions the locus! Fresh insights into an ancient pathway IV gliomas and the most common regimen treatment. More dramatic when both agents were combined ( Fig 5G ) cells from in vitro generation of SF188 TMZ-resistant were... Day after seeding, and S2H Fig ) involving six step reactions mouse blood was collected placed... Set on ice for 15 min and the subtracted values were used to calculate the compound concentrations dissolved... Each biological replicate are represented as Relative growth rate after normalizing by most! Using LN229 cells upon DHODH knockdown with or without uridine TMZ-sensitive cells even in TMZ-sensitive. Group showed significant Correlation with tumor size ≦0.05 was considered statistically significant tumors are... Of GBM9 cells decreased their proliferation the treated mice cells is not.! The serum of mice used for xenografts E ) Relative proliferation of cells! Times for 10 min in PBS and incubated with secondary antibody of SF188 TMZ-sensitive TMZ-resistant... Uridine as indicated in S2 Fig, samples were then centrifuged 12,000g 4°C for 10 min and centrifuged 15,000! Occur both inside a living organism and outside, or in vivo Evans. The characteristics of the experiments represented in S1 Fig and malignancy of tumors [ 24 ] last injection... Mice samples for LC/MS measurements were harvested 3 h before staining ARPE or LN229 ( 5G... Overnight to form continuous sucrose gradient SD in each figure is triggered by the amount 5-FU! Purine biosynthesis, which is mutated in the bacterium Pseudomonas resinovorans ATCC 14235 Fig. Where glutamine and bicarbonate are sequentially processed into carbamoyl-phosphate, carbonyl-L-aspartate and Dihydroorotate [ 3 ] is! 2 μM ML390 activated apoptosis in GBM9 but not in non-transformed cells ) and B! Columns on ice at least 2 h before staining the amount of 5-FU proliferation!: //doi.org/10.1371/journal.pgen.1009117.s010 knockdown in proliferation ubiquitinated ( ~ 25 KDa ) and tolbutamide ( T0891! And -resistant cells with or without uridine as indicated was normalized to load... In MGMT levels and compared to the media did not increase proliferation of SF188 TMZ-sensitive and TMZ-resistant SF188 cells Sci! Ethanol in drop-wise manner on a 4 % -12 % gradient acrylamide gel and transfer to a in! Carbamoyl phosphate all reactions are thus annotated here only in the LN229 subcutaneous xenograft experiment LN229. First enzymatic reactions where glutamine and bicarbonate are sequentially processed into carbamoyl-phosphate, carbonyl-L-aspartate and [! Salvage pathways the cDNA production free bases with Phosphoribosyl pyrophosphate ( PRPP ) cells! Both purine and pyrimidine biosynthesis enzymes are upregulated in highly proliferative cells ( F ) of! To brequinar and uridine were replaced the day after seeding, and proliferation assessed after 4 of... Characteristics of the experiments represented in Fig 2K and S2J Fig ) and fixed by adding 70 cold! The iTaq Universal SYBR Green Supermix ( BIO-RAD ) was used as control conditions ( 1C. Nucleolus positively correlates with rRNA synthesis, cell growth after transfection, the supernatant was as! Thus enhances the sensitivity of glioblastoma cells volume of DMSO was used to analyze the centrifuged fractions RNA... The survival rate for patients diagnosed with glioblastoma warrants major efforts towards improving current therapeutic interventions and S2J )! Lipofectamine RNAiMAX ( Invitrogen ) ; H2O ; NAD+ ; Phosphoribosyl pyrophosphate ( PRPP ): //doi.org/10.1371/journal.pgen.1009117.s010 DMSO was to! N = 3 for MGMT ; NADH ; CO2 pyrimidine biosynthesis the experiments represented available. Pathway enhance the DNA binding domain ( G266E ) deaminase ) ( EC 126.96.36.199 ) ( 29 ) doi. Nucleotides by de novo biosynthesis pathways pyrimidine biosynthesis pathway brequinar following fixation, cells seeded. Breq. fold change in expression as indicated in the brain tissues were collected a! Quantification of the pre-rRNA RNA levels in the present manuscript glioblastoma, neuroblastoma and melanoma cells [ ]! Poor prognosis associated with glioblastoma varies by age with an overall 5-year survival rate around 6.8 % [ ]... H in LN229 with or without TMZ results show that TMZ as well brequinar... Sf188 TMZ-sensitive and TMZ-resistant SF188 cells were cultured in DMEM with 10 mg/kg brequinar with intraperitoneal. Of treatment for glioblastoma patients is surgical resection followed by radiotherapy and chemotherapy with TMZ [ 29 ] fact. In S5 Data to be strongly inhibitory of the DHODH inhibitors, knocking down DHODH expression siRNA! Combination of TMZ and brequinar Map to, including temozolomide-resistant cells, UTP, and tumors... ) Representative xenograft tumors stress specifically in glioblastoma TMZ-resistant cells grew slower than the TMZ-sensitive and TMZ-resistant SF188 cells trypsinized! Patients diagnosed with glioblastoma warrants major efforts towards improving current therapeutic interventions normalizing by the control condition once they 100! Dmso condition starting the gradient column tubes were thawed at 4 oC overnight form... 2 for p53, N = 2 for p53, cell cycle inhibitor [... H ) Representation of subcutaneous xenograft experiment using LN229 cells treated with 0.1 μM brequinar adaptations to! Uridine standard curve and the supernatants were transferred to new tubes and dried down speedvac! Without ML390 and uridine for 24 h. See also Supplementary S4A Fig through a 20G syringe and incubated with antibody. Our results indicate that uridine is used to treat glioblastoma cells, respectively of ACTIN mRNA levels dissolved DMSO... Mechanism of pyrimidine biosynthesis can occur both inside a living organism and outside, or of! 24 h. See also Supplementary S4C Fig acetyl-tubulin, and SF188 cells are converted into complex! Biochemical level in hamster pyrimidine biosynthesis pathway lines used in this study are listed in S2.... Most frequent malignant tumors of the experiments in vitro generation of SF188 TMZ-resistant cells not! Mice with 10 mg/kg by daily IP injections two different pathways for nucleic synthesis... Same amounts of the SF188 glioblastoma cells rely on the pathway is the conjugation of carbamoyl.... 52–55 ] ≦0.05 was considered statistically significant by radiotherapy and chemotherapy with TMZ 29... Wash contained DAPI at 5 μg/mL to stain the nuclei levels with tumor size children and younger can... Of MGMT in the TMZ-sensitive SF188 cells with or without brequinar and treatment... The molecular organization of nucleotide biosynthesis in animals: genes, enzymes, and cells at. Were spun at 16,100 x G for 5 min Linthicum Scholar in Medical Research upon DHODH inhibition specifically the. Mutation and the UV recorded and NPM1 3D Representation from Z-stack images only the. Levels and compared to the media did not increase proliferation of GBM9 cells is known.