Interestingly, our results show that TMZ as well as brequinar alone decreased the levels of MGMT in the TMZ-sensitive SF188 cells. (D, E) Relative proliferation of LN229 and GBM9 in the presence of TMZ, brequinar (Breq.) 47S pre-rRNA, 28S and 18S levels were normalized by ACTIN mRNA levels. Tables including values of the experiments represented in S4 Fig. M.C-S is the Virginia Murchison Linthicum Scholar in Medical Research. For cell proliferation, six-well or 12-well plates were seeded with 1.5 x 105 or 50,000 cells, respectively. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm. 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. Copyright: © 2020 Lafita-Navarro et al. DNA double-strand breaks are marked by S139 phosphorylation of H2AX (known as γ-H2AX) . The lysates were passed through a 20G syringe and incubated on ice for 15 min with resuspending every 5 min. Surprisingly, treatment with ML390 or brequinar, which did not increase DNA damage (Fig 2F, 2G and 2K), did cause an increase in p53 protein levels that was reversed by the addition of uridine (Fig 2F, 2G, 2K and 2M–2O, and S2F, S2G and S2J Fig). (B) Relative proliferation of LN229 cells upon DHODH knockdown with or without uridine. Writing – review & editing, * E-mail: Maralice.ConacciSorrell@UTSouthwestern.edu, Affiliations It is possible that VEGFA expression is regulated by other mechanisms in the LN229 xenograft tumors. 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. 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. However, brequinar potentiated the effect of TMZ on the phosphorylation of H2AX, and this increase was reduced by the addition of uridine (Fig 2F and 2G and S2F and S2G Fig). (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. De novo pyrimidine synthesis occurs in the cytosol of cells in all tissues. (E) Correlation of pre-rRNA, mature 28S and 18S rRNA, and ACTIN RNA levels with tumor weight. Consequently, the impairment of the pre-rRNA synthesis upon DHODH inhibition leads to nucleolar stress specifically in glioblastoma cells but not in non-transformed cells. This lecture explains about the mechanism of pyrimidine biosynthesis in cell. p21 expression was not elevated in SF188 and GBM9 cells upon DHODH inhibition, indicating that additional mechanisms are responsible for reduced proliferation in these cells upon brequinar and ML390 treatment. Media with drugs was replaced every 2 days for 6 days. 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. At the end of the experiment the mice were weighed, and the tumors were harvested and weighed. Unlike purine synthesis, pyrimidines are synthesized as bases and latter it is added to ribose sugar, i.e., the ring is completed before being it is linked to ribose-5-phosphate. Numerical values for each of the experiments represented are available in S1 Data. In fact, the mRNA levels of the human vascular endothelial growth factor A (VEGFA), known to increase vascularity in glioblastoma , were lower in the brequinar-treated group after 55 days of treatment (Fig 3F, right panel). PRPP is made by the activation of riboseâ5âphosphate. RNA levels were measured by quantitative PCR with the iTaq Universal SYBR Green Supermix (BIO-RAD). Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America, Roles Active rDNA transcription governs the overall organization of the nucleolus . ), which is mutated in the DNA binding domain (G266E). Last brequinar injection was 3 h before harvesting the tissues. (B) Relative proliferation of LN229, GBM9 and SF188 cells with or without brequinar and uridine. Starting the gradient column by the most concentrated sucrose solution. (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. (K) Cell cycle representation of results in Fig 2L. In fact, the size of the nucleolus positively correlates with rRNA synthesis, cell growth, and malignancy of tumors . https://doi.org/10.1371/journal.pgen.1009117.s015. Cells treated with brequinar had fewer monosomes in comparison with the 40S and 60S subunits fractions (Fig 5G). For brequinar quantification, the Analyst software Sciex MS computer was used to calculate the compound concentrations. https://doi.org/10.1371/journal.pgen.1009117.g005. Furthermore, we compared the effects of TMZ and brequinar on DNA damage in the TMZ-sensitive and TMZ-resistant SF188 cells. Importantly, different cancer types, such as acute and chronic myeloid leukemia (AML and CML) [14, 15], multiple myeloma , melanoma , K-Ras–driven pancreatic cancer , PTEN-mutated breast cancer or glioblastoma , and chemotherapy-resistant triple-negative breast cancer cells , have been found to be vulnerable to the inhibition of DHODH. Then, cells were resuspended in 0.5 mL PBS and fixed by adding 70% cold ethanol in drop-wise manner on a vortex. Moreover, UMP and uridine levels in the serum (UDP and UTP were undetectable) of brequinar-treated mice were also unaffected (Fig 4F). Roles Uridine did not rescue the effects of 5-FU in proliferation. (A) Relative proliferation of p14 ARF-/- human astrocytes, LN229, GBM9 and SF188 cells with increasing amounts of brequinar. After 24 h of treatment, cells were trypsinized and harvested and washed with PBS. Pyrimidine synthesis takes place in cytoplasm. Is the Subject Area "Uridine" applicable to this article? (F, G) Western blot of LN229 and GBM9 for γ-H2AX and p53. Proliferation was assessed by crystal violet staining after 4 days of siRNA transfection. 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). The TMZ-resistant cells showed decreased basal levels of γ-H2AX, which is in agreement with the acquisition of TMZ resistance in glioblastoma that is due to an increase in the levels of DNA repair enzymes such as MGMT (Fig 2K) . Scale bar is 5 μm. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, Affiliation Cells were imaged with a Zeiss LSM780 inverted confocal microscope. recycling of the bases. Lysates were set on ice for 15 min and resuspended every 5 min. Surprisingly, the levels of 47S pre-rRNA positively correlated with tumor size only in the control group and not in the brequinar-treated group (S5E Fig). 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). Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. Tables including numerical values of the experiments represented in Fig 3. https://doi.org/10.1371/journal.pgen.1009117.s011. This effect was reversed by the addition of uridine. Media with drugs were changed every 2 days for 6 days. Bioinformatics Core Facility, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, Roles Pyrimidines are synthesized from carbamoyl phosphate and aspartate. De novo synthesis and salvage pathway are the two pathways for nucleic acid synthesis in vivo (Evans and Guy, 2004). (C) Amounts of brequinar, UMP, UDP, UTP and uridine measured by LC-MS/MS in the brain tissues of mice used for xenografts. While inhibition of DHODH causes a decrease in rRNA production in glioblastoma cells, it does not affect ACTIN abundance at short incubation periods. Media with drugs was replaced the day after seeding, and cell proliferation was measured 4 days later. Data were analyzed using GraphPad for nucleotide quantification. This antibody detects the 47S pre-rRNA as well as the mature rRNA. (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. Nonetheless, HIF1α, a transcription factor known to induce the expression of VEGFA in hypoxia , was not detectable in either the control or brequinar-treated tumors (Fig 3G). As expected, brequinar was only present in the brain, liver, and serum of the treated mice. 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-5â-Decarboxylase /Uridine Monophos- 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). (C) Representation of the effects of temozolomide (TMZ) and the depletion of nucleotides causing DNA double-strand breaks and the phosphorylation of H2AX (γ-H2AX). 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. (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. siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen). (C) Brequinar levels quantified by LC-MS/MS in LN229 cells treated with 0.1 μM brequinar. Tables including values of the experiments represented in S3 Fig. Also see Supplementary S2F and S2G Fig. â¦ https://doi.org/10.1371/journal.pgen.1009117.g003. 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]. It is possible that the residual DHODH levels after siRNA transfection are enough to maintain the production of pyrimidines through the de novo pathway. Protein involved in the biosynthesis of pyrimidine, a nitrogenous heterocyclic base, e.g. No, Is the Subject Area "Glioblastoma multiforme" applicable to this article? Biosynthesis of pyrimidines is simple than that of purines. 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. 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. ADVERTISEMENTS: Let us make an in-depth study of the biosynthesis of purine nucleotides, pyrimidine nucleotides and deoxyribonucleotides. 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. https://doi.org/10.1371/journal.pgen.1009117.g001. https://doi.org/10.1371/journal.pgen.1009117.g002. (F) Immunofluorescence of rRNA in LN229 cells by using the anti-rRNA Y10b antibody with or without 0.1 μM brequinar and with or without 100 μM uridine for 24 h. Brequinar decreased the amounts of rRNA, which was rescued by uridine. For all the experiments, media with drugs and uridine were replaced the day after seeding. Pyrimidines that have already been formed â¦ 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. 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). N = 3. Citation: Lafita-Navarro MC, Venkateswaran N, Kilgore JA, Kanji S, Han J, Barnes S, et al. Also see Supplementary S2H Fig. Therefore, we asked whether the combination of TMZ with brequinar synergized to kill glioblastoma cells. There are two different pathways for both purine and pyrimidine biosynthesis, which are de novo and salvage pathways. (E) Relative proliferation of the LN229 and GBM9 cells in the presence of increasing amounts of temozolomide (TMZ). Similarly, treatment with 2 μM ML390 activated apoptosis in GBM9 but not in ARPE or LN229 (Fig 2N). 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. Methodology, Affiliation The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. SB is the Mays Family Foundation Distinguished Chair in Oncology. UniProtKB (345,440) Reviewed (4,568) Swiss-Prot. Mice were treated with 15 mg/kg brequinar every 3 days by intraperitoneal injections (IP). (C) Tumor weight of LN229 xenografts at day 60 (end of experiment). Glioblastomas are grade IV gliomas and the most frequent malignant tumors of the brain. Pyrimidine Biosynthesis 1. Similar to the tumor volumes, tumor weights (measured at the endpoint) were also lower in the brequinar-treated mice (Fig 3C and 3D and S5B Fig). For the experiments in vitro, expression levels for 47S pre-rRNA, 18S rRNA and 28S rRNA were normalized to β-actin expression. 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. We, therefore, generated a TMZ-resistant SF188 cell line by treating cells for approximately 3 weeks with increasing concentrations of TMZ until the population growth was resistant to the addition of 100 μM TMZ (Fig 2H and 2I). Then, the supernatant was collected as the serum fraction. Asterisks indicate p-values ≦ 0.05. In summary, our data show that the inhibition of the de novo pyrimidine biosynthesis by targeting the enzymatic activity of DHODH leads to efficient inhibition of rRNA production and nucleolar stress and therefore, decreases proliferation specifically in glioblastoma cells. Uridine did not rescue the effects of DHODH knockdown in proliferation. β-actin levels were normalized to RNA load for the cDNA production. Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway Proc Natl Acad Sci U S A. Proliferation was assessed by crystal violet staining after 4 days of treatment. It is possible that uridine is used to synthesize pyrimidines by the salvage pathway (Fig 1C) in this differentiated tissue. Overall, our findings suggest new therapeutic possibilities for glioblastoma patients by blocking the production of ribosomes through the specific inhibition of the de novo pyrimidine biosynthesis pathway. 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). (D) Relative proliferation of ARPE, LN229 and GBM9 cells with or without brequinar and with or without uridine. We then compared the amounts of 47S pre-rRNA, 28S and 18S rRNAs and ACTIN levels with each tumor weight to see whether a larger tumor size correlated with higher levels of rRNAs. (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. Importantly, TMZ-resistant cells were still sensitive to brequinar and responsive to the addition of uridine (Fig 2J and S2H Fig). Type . Lower panels in (A) and (B) show UBF and NPM1 3D representation from Z-stack images. Pyrimidine biosynthesis can occur both inside a living organism and outside, or in vivo and in vitro. Total RNA was extracted with TRI Reagent solution (Sigma) following the manufacturer’s instructions, and cDNA was produced with the iScript RT Supermix for RT-qPCR (BIO-RAD). Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, Affiliation Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States of America, Anti-phosphorylated H2AX antibody shows non-ubiquitinated (~ 15 KDa) and ubiquitinated (~ 25 KDa) γ-H2AX. Yes See also Supplementary S5B and S5C Fig. To generate nucleotide standards curves, supernatants from control tumor, serum and brain samples were diluted 1:5000 in Resuspension Solution and used for the nucleotide standard curves. TMZ is an alkylating agent that methylates adenine and guanine residues in DNA, leading to DNA damage and double-strand breaks (Fig 2C) . Cell proliferation was measured by crystal violet staining and 595 nm absorbance quantification of the solubilized die. 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