Extended Data Fig. 4: p53 modulates urea cycle metabolism in vitro and in vivo.
From: p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis
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. f–h, 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. i–l, 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 (b–e, i–k), 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 (b–d, i–l) or two-way ANOVA followed by Tukey’s multiple comparisons test (e, m). See Supplementary Fig. 1 for gel source data.
