Here, we demonstrate that glioblastoma cells rely on the de novo biosynthesis of pyrimidines to support rDNA transcription and thus cell growth. (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. Pyrimidine rings are assembled from bicarbonate, aspartate, and Ammonia. Pyrimidine synthesis takes place in cytoplasm. Then, the supernatant was collected as the serum fraction. https://doi.org/10.1371/journal.pgen.1009117.s006. * indicates p-values ≦ 0.05. 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. 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). siRNAs used in this study are listed in S2 Table. To generate a brequinar standard curve, varying concentrations of brequinar standards were spiked into control tumor homogenate, blank commercial serum, or blank brain homogenate and processed like samples. It is possible that uridine is used to synthesize pyrimidines by the salvage pathway (Fig 1C) in this differentiated tissue. (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. 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. (D) qPCR of 47S pre-rRNA and 28S and 18S rRNAs in the brain tissues of the mice used for LN229 xenografts. 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. Briefly, 150,000 cells were seeded and transfected with 3 μL siRNA (20 μM) and 3 μL Lipofectamine previously mixed on OPTI-MEM for 15 min. Media with drugs and metabolites were replaced every 2 days for 6 days for the experiments shown in Fig 2A, and 2B and S2A, S2B and S2C Fig. 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. All reactions are thus annotated here only in the forward direction. 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). Also see Supplementary S2F and S2G Fig. (F) Western blot quantification by Image J of Fig 2F and additional experimental replicates. N = 2–6. Furthermore, brequinar and TMZ treatment leads to decreased expression of the DNA damage repair enzyme MGMT. 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. The oncogene MYC upregulates the expression of CAD and DHODH [7, 8]. https://doi.org/10.1371/journal.pgen.1009117.s019. 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