Letter | Published:

p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis

Abstract

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.

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Change history

  • 14 May 2019

    In Fig. 1c of this Letter, the labels p53+/+ and p53−/− were inadvertently swapped. The original figure has been corrected online.

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Acknowledgements

We thank H. Wu, Y. Liu, W. He, Y. Wang, J. Na, S. Fu and W. Wu for materials. We thank G. Alderton for discussion. We thank J. Zhang, J. Cheng, L. Li, X. Xiao, F. Sun, N. Li, H. Li and all of the Jiang laboratory members for technical assistance and/or discussion. We thank Y. Ding, W. Wang, Y. Tian and Z. Hu for help with the LC–MS/MS experiments. This research was supported by the 1000 Talents Program for Young Scholars, the Tsinghua University Initiative Scientific Research Program, Tsinghua-Peking Center for Life Sciences, and National Natural Science Foundation of China (31571470; 81722035) to P.J.

Author information

Le Li performed all experiments except those mentioned below. Le Li and Lijia Li conducted the gene expression experiments with the help of M.Z. and J.W. Le Li performed polysome profiling assays with assistance from Y.M. and L.Z. L.Y. provided help for the autophagy study. W.D. supplied reagents. P.J. conceived, designed and supervised the research. P.J. wrote the manuscript. All authors commented on the manuscript.

Correspondence to Peng Jiang.

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Competing interests

The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Schematic depicting the urea cycle and polyamine metabolic pathways.

The urea cycle consists of five reactions catalysed by carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase 1 (ARG1), respectively. Through these reactions, one carbon atom from HCO3 and two amino groups (one from NH4+ generated by the transamination of glutamine or glutamate, and one from aspartate) are converted to urea at the cost of three ATPs. Black and red arrows indicate urea cycle reactions and polyamine metabolic reactions, respectively.

Extended Data Fig. 2 p53 transcriptionally regulates urea cycle gene expression.

a, Gene expression in U2OS cells expressing p53 shRNA or control shRNA was analysed by qRT–PCR and normalized to ACTB. b, HEK293 cells were transfected with vector control plasmid (0 μg) or increasing amount of Flag-tagged p53 plasmids for 24 h. The expression of CPS1, OTC, ARG1, p53, p21 and ACTB was determined by qRT–PCR. c, The abundance of CPS1, OTC, ARG1, p53 and p21 transcripts in p53−/− HCT116 cells that were transfected with the p53(A138V) mutant and cultured at 32 °C or 37 °C was determined by qRT–PCR analysis (bottom). The expression of p53 and p21 was further analysed by western blot (top). d, Western blot analysis of CPS1, OTC or ARG1 expression in p53-depleted and control HCT116 or U2OS cells as indicated. e, Protein expression of CPS1, OTC, ARG1, p53 and p21 in human hepatoblastoma HuH-6 cells control CRISPR and p53 CRISPR cells was determined by western blot analysis. f, Western blot analysis of CPS1, OTC, ARG1 and AKT expression in different tissues derived from either p53+/+ or p53−/− mice. g, Enzyme activities of CPS1, OTC and ARG1 in U2OS cells expressing p53 shRNA or control shRNA were determined (see Supplementary Methods). Data are mean ± s.d. of one representative experiment, with n = 5 or n = 4 technical replicates. Experiments were repeated three times with similar results. h, The genomic structure of human CPS1, OTC and ARG1. Shown are the exon and intron organization, and the consensus p53 response element. i, j, Luciferase reporter assay with indicated response element constructs in human HEK293T cells co-transfected with or without the p53 expression vector. Data are mean ± s.d., n = 3. Consistent with previous findings, p53 is shown to suppress the luciferase expression driven by the genomic regions of ME1 and PDK2, and promote luciferase expression driven by the EPCAM genomic region. Renilla vector pRL-CMV was used as a transfection internal control. Relative luciferase activity (fold change) is shown. k, qRT–PCR analysis of mRNA expression of CPS1, OTC and ARG1 in HCT116 cells left untreated (0 μM) or treated with increasing amounts of the p53 inhibitor pifithrin-α (PFT-α) for 24 h (top). The expression of p53 and p21 was analysed by western blot (bottom). l, qRT–PCR analysis of mRNA expression of CPS1, OTC, ARG1, p53 and p21 in HepG2 cells left untreated (0 μM) or treated with increasing amounts of Nutlin-3 for 24 h (top). The expression of p53 and p21 was analysed by western blot (bottom). Data are mean ± s.d. of three biologically independent samples per treatment (a), or n = 3 technical replicates of one out of three independent experiments (b, c, il). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test (ac, g, i, j) or two-way ANOVA followed by Tukey’s multiple comparisons test (k, l). See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 3 Mutant p53 minimally affects expression of urea cycle genes.

a, Transcript levels of p53 and urea cycle genes (CPS1, OTC, ASS1, ASL, ARG1 and ORNT1) were measured by qRT–PCR in DU145 cells transfected with control siRNA or p53 siRNA targeting mutant p53 (that is, P223L and V274F substitutions). Data are mean ± s.d. of n = 3 biologically independent samples per treatment. **P < 0.01, two-tailed Student’s t-test. b, mRNA and protein from p53−/− HCT116 cells stably expressing mutant p53(R175H) or vector control were analysed for the expression of urea cycle genes, p53 and p21 by qRT–PCR and/or western blot as indicated. Data are mean ± s.d. of one representative experiment (n = 3 technical replicates). ***P < 0.001, two-tailed Student’s t-test. Experiments were performed three times with similar results. c, p53−/− HCT116 cells engineered to conditionally express mutant p53(R175H) (Tet-On) were treated with 0.5 μg ml−1 doxycycline for 24 h, and the expression of urea cycle enzymes and p53 was determined by western blot analysis. See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 4 p53 modulates urea cycle metabolism in vitro and in vivo.

a, Atom-transition map showing that the isotope nitrogen-15 (15N) transfers from (amide-15N)-glutamine and 15N-aspartate through the urea cycle pathway. Open circles represent carbon, red and green circles indicate 15N from (amide-15N)-glutamine and 15N-aspartate, respectively. b, p53+/+ and p53−/− HCT116 cells were cultured in medium containing 3 mM 15N-aspartate for 12 h. The abundance of 15N-citrulline and 15N-arginine was determined and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). c, d, p53−/− HCT116 cells engineered to conditionally express wild-type p53 (Tet-On p53) were treated with 0.5 μg ml−1 doxycycline for 12 h and cultured in medium containing 3 mM 15N-glutamine (c) or 15N-aspartate (d) for another 24 h in the presence of 0.5 μg ml−1 doxycycline. The abundance of 15N-citrulline and 15N-arginine was determined and quantified by LC–MS/MS. e, Urea release from p53+/+ and p53−/− HCT116 cells (left), U2OS cells expressing p53 shRNA or control shRNA (middle), or HepG2 control CRISPR and p53 CRISPR cells (right) in the presence of increasing amounts of NH4Cl (an exogenous source of ammonia) was determined and quantified using a colorimetric method as per milligrams of protein per day. fh, Western blot analysis of p53, phosphorylated p53 (p-p53; S15) and p21 in p53+/+ and p53−/− HCT116 cells (f), U2OS cells expressing p53 shRNA or control shRNA (g), or HepG2 control CRISPR and p53 CRISPR cells (h) untreated (0 mM) or treated with increasing amounts of NH4Cl (5 mM, 10 mM) for 24 h. il, The rates of ureagenesis (i), glutamine consumption (j), glutamate production (k), and the cellular levels of ammonia (l) in several cell lines were measured as indicated (see Supplementary Methods). m, Urea levels in liver, serum and urine derived from p53+/+ and p53−/− mice loaded with or without 0.25 M NH4Cl for 6 days. Data are mean ± s.d., n = 5 mice in each treatment. n, Western blot analysis of p53, phosphorylated p53, p21 and urea cycle enzymes in liver, lung and intestinal tissues derived from p53+/+ and p53−/− mice loaded with or without 0.25 M NH4Cl for 6 days. As controls, lysates from Ampk+/+ (Ampk is also known as Prkaa2) and Ampk−/− mouse embryonic fibroblasts (MEFs) were used. In addition, to determine whether these tissue samples are well homogenized, less protein was loaded and analysed for actin expression (bottom panel, see Supplementary Fig. 1). Data are mean ± s.d. of n = 3 biologically independent samples per treatment (be, ik), or n = 3 technical replicates of one out of three independent experiments (l). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test (bd, il) or two-way ANOVA followed by Tukey’s multiple comparisons test (e, m). See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 5 Effects of CPS1, OTC and ARG1 on ureagenesis, cellular ammonia levels and p53 activity, and a role for MDM2 in urea cycle-mediated p53 inactivation.

ac, Ureagenesis (a), ammonia levels (b) and protein expression (c) in HCT116 cells treated with indicated siRNAs were analysed. df, HCT116 cells stably expressing siRNA-resistant CPS1, OTC or ARG1 were transfected with control siRNA, CPS1 siRNA, OTC siRNA or ARG1 siRNA as indicated. The levels of cellular ammonia (d), urea release (e), and expression of p53, phosphorylated p53, p21, CPS1, OTC and ARG1 (f) were determined. g, h, HCT116 cells transfected with CPS1, OTC and ARG1 siRNAs (triple knockdown) or control siRNA for 48 h, and analysed by western blot (g) and qRT–PCR (h) for MDM2 and p53 as indicated. i, HCT116 cells were transfected with control siRNA or triple knockdown siRNAs. After 48 h, cells were treated with increasing amounts of indicated molecules (ornithine, citrulline and/or arginine) for another 24 h. The protein levels of p53, p21, CPS1, OTC and ARG1 were analysed by western blot. In a, data are mean ± s.d. of n = 3 biological replicates. In b, d, e and h, data are mean ± s.d. (n = 3 technical replicates) of one representative experiment. Experiments were repeated three times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test (h), one-way ANOVA followed by Dunnett’s multiple comparisons test (a, b) or two-way ANOVA followed by Tukey’s multiple comparisons test (d, e). See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 6 CPS1, OTC and ARG1 promote tumour growth, are highly expressed in many human cancers, and inversely correlate with patients survival and p53 expression.

a, Related to Fig. 2g. Western blot analyses of CPS1, OTC, ARG1 and p53 in xenograft tumours derived from p53+/+ and p53−/− HCT116 cells triply transfected with CPS1, OTC and ARG1 siRNAs, or control siRNA are shown. b, Colony formation assay of p53+/+ and p53−/− HCT116 cells triply transfected with CPS1, OTC and ARG1 siRNAs, or control siRNA. Numbers of colonies with a diameter greater than 20 μm were quantified (left). Data are mean ± s.d., n = 6 cultures. **P < 0.01, ***P < 0.001, one-way ANOVA followed by followed by Dunnett’s multiple comparisons test. Representative images of colonies stained with crystal violet at day 14 are shown (right). c, Average weights of xenograft tumours (2 weeks, mean ± s.d., n = 5 tumours in each treatment) generated by p53+/+ HCT116 cells stably expressing CPS1, OTC and ARG1 individually. *P < 0.05, one-tailed t-test. df, Box plot comparing transcript levels of CPS1 (d), OTC (e) and ARG1 (f) in liver cancer, leukaemia, prostate cancer, oesophageal adenocarcinoma and their normal counterparts. Data were generated from Oncomine data. In d, liver (maximum = 1.219, median = −0.018, minimum = −2.018, percentile: 90th = 0.709, 75th = 0.348, 25th = −0.423, 10th = −0.88); hepatocarcinoma (maximum = 2.745, median = 0.933, minimum = −0.678, percentile: 90th = 2.269, 75th = 1.638, 25th = 0.449, 10th = −0.005); bone marrow (maximum = −0.997, median = −1.255, minimum = −1.53, percentile: 90th = −0.997, 75th = −0.997, 25th = −1.42, 10th = −1.53); leukaemia (maximum = 2.932, median = 0.298, minimum = −1.391, percentile: 90th = 1.319, 75th = 0.797, 25th = −0.114, 10th = −0.49); oesophagus (maximum = 3.235, median = 1.57, minimum = 0.321, percentile: 90th = 2.446, 75th = 1.878, 25th = 1.266, 10th = 1.076); Barrett’s oesophagus (maximum = 7.71, median = 5.52, minimum = 3.153, percentile: 90th = 7.026, 75th = 6.537, 25th = 4.385, 10th = 3.32). In e, oesophagus (maximum = −0.188, median = -0.314, minimum = −0.581, percentile: 90th = −0.212, 75th = −0.253, 25th = −0.394, 10th = −0.533); Barrett’s oesophagus (maximum = 5.926, median = 4.337, minimum = −0.235, percentile: 90th = 5.42, 75th = 5.217, 25th = 1.976, 10th = 1.542); liver (maximum = 4.132, median = 1.67, minimum = −0.928, percentile: 90th = 3.603, 75th = 3.308, 25th = 0.366, 10th = 0.135); hepatocellular carcinoma (maximum = 4.919, median = 3.918, minimum = −1.121, percentile: 90th = 4.628, 75th = 4.427, 25th = 3.537, 10th = 2.469); CD4-positive T lymphocyte (maximum = −3.268, median = −3.726, minimum = −4.225, percentile: 90th = −3.268, 75th = −3.268, 25th = −3.868, 10th = −4.225); acute adult T cell leukaemia/lymphoma (maximum = −0.441, median = −3.143, minimum = −4.466, percentile: 90th = −1.807, 75th = −2.239, 25th = −3.621, 10th = −3.935). In f, B lymphocyte T lymphocyte (maximum = −2.17, median = −3.004, minimum = −4.31, percentile: 90th = −2.17, 75th = −2.17, 25th = −3.981, 10th = −4.31); centroblast (maximum = −1.586, median = −4.123, minimum = −4.826, percentile: 90th = −1.586, 75th = −1.586, 25th = −4.219, 10th = −4.826); memory B lymphocyte (maximum = −2.355, median = −4.453, minimum = −6.27, percentile: 90th = −2.355, 75th = −2.355, 25th = −5.531, 10th = −6.27); naive pregerminal centre B lymphocyte (maximum = −3.153, median = −4.42, minimum = −5.084, percentile: 90th = −3.153, 75th = −3.153, 25th = −4.439, 10th = −5.084); small cleaved follicle centre cell (maximum = −3.161, median = −3.55, minimum = −5.71, percentile: 90th = −3.161, 75th = −3.161, 25th = −4.39, 10th = −5.71); hairy cell leukaemia (maximum = −3.268, median = −0.612, minimum = −5.659, percentile: 90th = 1.593, 75th = 0.94, 25th = −1.585, 10th = −2.735); prostate (maximum = 0.684, median = −0.708, minimum = −1.331, percentile: 90th = 0.684, 75th = 0.684, 25th = −0.855, 10th = −1.331); benign prostatic hyperplasia (maximum = 1.251, median = 0.92, minimum = 0.25, percentile: 90th = 1.251, 75th = 1.251, 25th = 0.465, 10th = 0.25). n indicates number of samples. g, Kaplan–Meier survival curves of patients with colon cancer based on CPS1 (left), OTC (middle) or ARG1 (right) expression. Data were generated from Gene Expression Omnibus (GEO) ‘the human colon cancer Marisa dataset ’(series GSE39582) via ‘R2: Genomics Analysis and Visualization Platform’. h, Correlation plots of wild-type p53 expression with CPS1, OTC or ARG1 expression with significant Pearson’s correlation in the colon cancer dataset (n = 371 patients) are shown. r, Pearson’s correlation coefficient; x and y axes denote the respective genes being analysed. Data were generated from The Cancer Genome Atlas (TCGA) ‘Liver Hepatocellular Carcinoma’ dataset (ID: LIHC) via ‘R2: Genomics Analysis and Visualization Platform’. See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 7 p53 induces ammonia accumulation to suppress tumour cell proliferation and polyamine biosynthesis.

a, Ammonia levels in p53+/+ and p53−/− HCT116 cells, U2OS cells expressing p53 shRNA or control shRNA, or control CRISPR and p53 CRISPR HepG2 cells was quantified. b, p53+/+ and p53−/− HCT116 cells, or U2OS p53 shRNA and U2OS control shRNA cells were treated with 0, 5, or 10 mM NH4Cl as indicated for 24 h. The levels of cellular ammonia were assayed. c, Ammonia levels in liver derived from p53+/+ and p53−/− mice loaded with or without 0.25 M NH4Cl for 6 days (n = 5 mice per treatment, total n = 20). d, p53+/+ and p53−/− HCT116 cells, or U2OS cells transfected with p53 shRNA or control shRNA were treated with 0, 5 or 10 mM NH4Cl as indicated for 24 h. Proliferation inhibition was assayed. e, p53+/+ and p53−/− HCT116 cells treated with control siRNA, or with CPS1, OTC and ARG1 siRNAs as indicated were incubated with increasing amounts of NH4Cl (0, 5 or 10 mM) for 24 h. Proliferation inhibition was determined by cell counting. f, Colony formation assay of p53+/+ and p53−/− HCT116 cells treated with increasing amounts of NH4Cl (0, 5 or 10 mM) as indicated. Data are mean ± s.d. of one representative experiment (n = 6 cultures per treatment). Colonies were stained with crystal violet at day 14. Representative images of the colonies are given. g, Related to Fig. 3c. p53+/+ and p53−/− HCT116 cells were treated with increasing amounts of NH4Cl for 24 h. The levels of putrescine were determined and quantified by high-performance liquid chromatography (HPLC) and peak images for each of the polyamines are given. 1,8-Diaminooctane was used as an internal control. h, k, l, U2OS cells stably expressing control shRNA or p53 shRNA were treated with increasing amounts of NH4Cl as indicated for 24 h. The levels of putrecine (h), spermidine (k) and spermine (l) were determined and quantified by HPLC. i, j, Related to Fig. 3c. The levels of spermidine (i) and spermine (j) in p53+/+ and p53−/− HCT116 cells treated with increasing amounts of NH4Cl for 24 h were determined and quantified by HPLC. Data are mean ± s.d. of n = 3 (a, b, d, hl) or n = 2 (e) technical replicates of one out of three independent experiments, or n = 3 biologically replicates (hl). *P < 0.05, **P < 0.01, two-tailed Student’s t-test (a), two-way ANOVA (bd, hl) or one-way (f) ANOVA followed by multiple t-test using Tukey’s or Dunnett’s method. Soft agar experiments (f) were performed twice. Liver ammonia assay (c) was performed once. All other experiments were performed three times. Source data

Extended Data Fig. 8 Polyamine biosynthesis is critical for tumour cell proliferation.

a, HCT116 cells treated with control siRNA, triple knockdown siRNA (CPS1, OTC and ARG1 siRNA; siTriple) or 200 μM DFMO were cultured in medium containing increasing amounts of putrescine (0, 100 and 500 μM) for 6 days. Cell proliferation was determined by cell counting. b, Colony formation assay of p53+/+ HCT116 cells treated with control siRNAs, triple knockdown siRNA or DFMO were cultured in medium containing increasing amounts of putrescine as indicated. Numbers of colonies were quantified (left). Data are mean ± s.d. Colonies were stained with crystal violet at day 12. Representative images of colonies are shown (right). c, p53+/+ and p53−/− HCT116 cells were cultured in medium containing DMSO or 0.25 mM DMFO for indicated times. Cell proliferation was determined by cell counting. d, Colony formation assay of p53+/+ and p53−/− HCT116 cells treated with DMSO (0 mM) or increasing amounts of DMFO (0.25 mM, 2 mM) as indicated. Representative images of colonies stained with crystal violet at day 14 are shown. e, Colony formation assay of p53+/+ and p53−/− HCT116 cells transfected with two different sets of ODC1 siRNAs (siODC #1 and #2) or control siRNA as indicated. Numbers of colonies with a diameter greater than 15 μm were quantified (left, bottom). ODC knockdown efficiency was determine by western blot analysis (left, top). Representative images of colonies stained with crystal violet at day 12 are shown (right). f, g, Colony formation assay of A549 cells (f) and H1299 cells (g) transfected with two different sets of ODC1 siRNAs or control siRNA as indicated. Numbers of colonies were quantified (left, top) and ODC knockdown efficiency was determined by western blot analysis (left, bottom). Representative images of colonies stained with crystal violet at day 14 are shown (right panel). h, Related to Fig. 3f. Expression of ODC and p53 in xenograft tumours derived from p53+/+ and p53−/− HCT116 transfected with control siRNA or ODC1 siRNA. Data are mean ± s.d. of one representative experiment with n = 3 (a, b), n = 6 (d, e) or n = 4 (f, g) cultures out of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons test (a, b), or one-way ANOVA followed by Dunnett’s multiple comparisons test (dg). See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 9 Ammonia induces a reduction in ODC protein, and this is transcription-independent.

a, Lysates from p53+/+ and p53−/− HCT116 cells treated with 0, 5 or 10 mM NH4Cl for 24 h were subject to western blot analysis. b, ODC, p53 and p21 transcripts in p53+/+ and p53−/− HCT116 cells untreated (0 mM) or treated with increasing amounts of NH4Cl (5 mM, 10 mM) for 24 h were analysed by qRT–PCR and normalized to ACTB. c, p53-depleted and control HCT116, U2OS or HepG2 cells were assayed for ODC expression. d, Gene expression in p53+/+ and p53−/− HCT116 cells was analysed by qRT–PCR and normalized to ACTB. Data are mean ± s.d. of one representative experiment with n = 3 cultures out of at least two independent experiments. **P < 0.01, ***P < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons test (b), or two-tailed Student’s t-test (d). All experiments were performed two or three times. See Supplementary Fig. 1 for gel source data. Source data

Extended Data Fig. 10 Ammonia induces a reduction in ODC protein, and this is independent of proteasome- and autophagy-mediated degradation.

a, HCT116 cells were untreated (0 mM) or treated with 5 mM NH4Cl for 24 h and then cultured in medium containing 100 μg ml−1 cycloheximide (CHX) and/or 15 μM MG132 as indicated. Cells were collected at indicated time points and analysed by western blot. b, p53+/+ HCT116 and U2OS cells were treated with increasing amounts of NH4Cl for 18 h in the presence or absence of 15 μM MG132 for another 6 h. Cell lysates were prepared and subjected to western blot analysis. Protein expression is shown. c, HCT116 cells were transfected with AZ1 (also known as CEP131) siRNA, NQO1 siRNA or control siRNA as indicated. After 24 h, cells were treated with increasing amounts of NH4Cl for another 24 h. Cell lysates were prepared and subjected to western blot analysis. d, HEK293 cells were transfected with haemagglutinin (HA)-tagged ubiquitin (Ub) and/or Flag-tagged ODC in the absence (0 mM) or presence of increasing amounts of NH4Cl (5 mM, 10 mM). Cell lysates were incubated with anti-Flag M2 beads (Sigma). Input and immunoprecipitates were analysed by western blot. The levels of ODC ubiquitination were analysed by immunoblotting with antibodies against ubiquitin, HA or Flag as indicated. e, Lysates from HEK293 cells transfected with HA–ODC and/or Flag–ODC in the presence or absence of NH4Cl were subjected to immunoprecipitation using anti-Flag antibody. The interaction between HA–ODC and Flag–ODC was determined by immunoblotting with anti-HA and anti-Flag antibodies. f, Extracts of p53+/+ and p53−/− HCT116 cells cultured in medium containing no NH4Cl (0 mM) or 5 mM NH4Cl for 24 h were treated with or without 5 mM disuccinimidyl suberate (DSS) and analysed by western blot. The positions of various forms (that is, monomers (×1), dimers (×2) and tetramers (×4)) of ODC and p53 are indicated. g, HCT116 cells were transfected with control siRNA or two different sets of ATG5 siRNA (siATG5 #1 and #2) individually as indicated. After 48 h, cells were treated with increasing amounts of NH4Cl for another 24 h. The expression of ODC, LC3, ATG5 and actin was determined by western blot. h, Lysates from HCT116 cells treated with increasing amounts of NH4Cl in the presence or absence of 20 mM methyl pyruvate (MP) were subjected to western blot analysis. i, A proposed model for the p53–urea cycle–ODC pathway in ammonia metabolism, ureagenesis, polyamine biosynthesis and tumour growth. j, qPCR amplification of urea cycle genes in various tissues derived from mice. The amplified DNA was measured by measuring fluorescence at threshold cycles. Data are mean ± s.d., and defined in the Source Data. See Supplementary Fig. 1 for gel source data. Source data

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Supplementary Information

This file contains Supplementary Methods, Supplementary References, Supplementary Figure 1 and Supplementary Tables 1-2.

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Fig. 1: Identification of urea cycle genes that are targets for p53.
Fig. 2: p53 regulates urea cycle metabolism, and a role for the urea cycle in tumour growth.
Fig. 3: p53 induction of ammonia accumulation via urea cycle inhibition results in a block to polyamine biosynthesis and tumour growth.
Fig. 4: Ammonia represses ODC1 mRNA translation.
Extended Data Fig. 1: Schematic depicting the urea cycle and polyamine metabolic pathways.
Extended Data Fig. 2: p53 transcriptionally regulates urea cycle gene expression.
Extended Data Fig. 3: Mutant p53 minimally affects expression of urea cycle genes.
Extended Data Fig. 4: p53 modulates urea cycle metabolism in vitro and in vivo.
Extended Data Fig. 5: Effects of CPS1, OTC and ARG1 on ureagenesis, cellular ammonia levels and p53 activity, and a role for MDM2 in urea cycle-mediated p53 inactivation.
Extended Data Fig. 6: CPS1, OTC and ARG1 promote tumour growth, are highly expressed in many human cancers, and inversely correlate with patients survival and p53 expression.
Extended Data Fig. 7: p53 induces ammonia accumulation to suppress tumour cell proliferation and polyamine biosynthesis.
Extended Data Fig. 8: Polyamine biosynthesis is critical for tumour cell proliferation.
Extended Data Fig. 9: Ammonia induces a reduction in ODC protein, and this is transcription-independent.
Extended Data Fig. 10: Ammonia induces a reduction in ODC protein, and this is independent of proteasome- and autophagy-mediated degradation.

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