Department of Neurosurgery, University of Texas Health Science Center, San Antonio, Texas, United States of America, Pyrimidine biosynthesis Stable Identifier. 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. N = 3–4. Here, we demonstrate that glioblastoma cells rely on the de novo biosynthesis of pyrimidines to support rDNA transcription and thus cell growth. Then, cells were resuspended in 0.5 mL PBS and fixed by adding 70% cold ethanol in drop-wise manner on a vortex. LC-MS/MS was used to quantify brequinar, UMP, UDP, UTP, and uridine in all harvested tissues. (B) Table showing the characteristics of the cell lines used in the present manuscript. 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. Moreover, UMP and uridine levels in the serum (UDP and UTP were undetectable) of brequinar-treated mice were also unaffected (Fig 4F). (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. Methodology, The day after seeding, the media were replaced with fresh media containing the appropriate inhibitors and metabolites. 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. Intensity quantification were performed using Image J using integrated density quantification for individual cells. While inhibition of DHODH causes a decrease in rRNA production in glioblastoma cells, it does not affect ACTIN abundance at short incubation periods. 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. In the salvage pathway, UMP can be generated by the conversion of uridine (already present in cells or taken up from the environment) into UMP by the activity of uridine/cytidine kinase 1/2 (UCK1/2) [6]. https://doi.org/10.1371/journal.pgen.1009117.s005. Software, Keyword - Pyrimidine biosynthesis (KW-0665) Map to. No, Is the Subject Area "Biosynthesis" applicable to this article? Numerical values for each of the experiments represented are available in S5 Data. 47S pre-rRNA, 28S and 18S levels were normalized by ACTIN mRNA levels. This effect was more dramatic when both agents were combined (Fig 2K and S2J Fig). All animal experiments were performed following UTSW IACUC guidelines (APN# 2017–101798). 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. Tables including numerical values of the experiments represented in S2 Fig. Numerical values for each of the experiments represented are available in S2 Data. 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). Given that glioblastoma cells exhibited increased levels of the enzymes involved in de novo pyrimidine biosynthesis, we hypothesized that rRNA production in glioblastoma cells would be altered by inhibitors of the de novo pyrimidine biosynthesis enzymes (Fig 1C and 1D). N = 2 for p53, N = 3 for MGMT. Previously, it was shown that SF188 cells were less sensitive to TMZ than other glioblastoma cells [43]. Our results on rRNA and nucleolar stress are in agreement with previous studies showing that de novo purine biosynthesis is necessary for rRNA synthesis and proliferation in glioblastoma [36] and that DHODH inhibition with leflunomide leads to decreased rRNA synthesis and p53 stabilization in mammary and colon cells [63]. Similarly, in the liver tissues of brequinar-treated mice only uridine but not UMP, UDP or UTP, was significantly decreased (Fig 4E). (D) Representative xenograft tumors from control and brequinar-treated mice. https://doi.org/10.1371/journal.pgen.1009117.s016. UMP, UDP, UTP, and uridine levels in the brain tissues were not affected by the treatment with brequinar (Fig 4C and S1 Table). 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