Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses
2019; Springer Nature; Volume: 38; Issue: 20 Linguagem: Inglês
10.15252/embj.2019102096
ISSN1460-2075
AutoresOleg Timofeev, Boris Klimovich, Jean Schneikert, Michael Wanzel, Evangelos Pavlakis, Julia Han Noll, Samet Mutlu, Sabrina Elmshäuser, Andrea Nist, Marco Mernberger, Boris Lamp, Ulrich Wenig, Alexander Brobeil, Stefan Gattenlöhner, Kernt Köhler, Thorsten Stiewe,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle4 September 2019Open Access Source DataTransparent process Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses Oleg Timofeev Oleg Timofeev Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Boris Klimovich Boris Klimovich Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Jean Schneikert Jean Schneikert Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Michael Wanzel Michael Wanzel Institute of Molecular Oncology, Philipps-University, Marburg, Germany German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center, Marburg, Germany Search for more papers by this author Evangelos Pavlakis Evangelos Pavlakis Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Julia Noll Julia Noll Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Samet Mutlu Samet Mutlu Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Sabrina Elmshäuser Sabrina Elmshäuser Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Andrea Nist Andrea Nist Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Marco Mernberger Marco Mernberger Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Boris Lamp Boris Lamp Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Ulrich Wenig Ulrich Wenig Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Alexander Brobeil Alexander Brobeil Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Stefan Gattenlöhner Stefan Gattenlöhner Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Kernt Köhler Kernt Köhler Institute of Veterinary Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Thorsten Stiewe Corresponding Author Thorsten Stiewe [email protected] orcid.org/0000-0003-0134-7826 Institute of Molecular Oncology, Philipps-University, Marburg, Germany German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center, Marburg, Germany Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Oleg Timofeev Oleg Timofeev Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Boris Klimovich Boris Klimovich Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Jean Schneikert Jean Schneikert Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Michael Wanzel Michael Wanzel Institute of Molecular Oncology, Philipps-University, Marburg, Germany German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center, Marburg, Germany Search for more papers by this author Evangelos Pavlakis Evangelos Pavlakis Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Julia Noll Julia Noll Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Samet Mutlu Samet Mutlu Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Sabrina Elmshäuser Sabrina Elmshäuser Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Andrea Nist Andrea Nist Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Marco Mernberger Marco Mernberger Institute of Molecular Oncology, Philipps-University, Marburg, Germany Search for more papers by this author Boris Lamp Boris Lamp Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Ulrich Wenig Ulrich Wenig Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Alexander Brobeil Alexander Brobeil Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Stefan Gattenlöhner Stefan Gattenlöhner Institute of Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Kernt Köhler Kernt Köhler Institute of Veterinary Pathology, Justus Liebig University, Giessen, Germany Search for more papers by this author Thorsten Stiewe Corresponding Author Thorsten Stiewe [email protected] orcid.org/0000-0003-0134-7826 Institute of Molecular Oncology, Philipps-University, Marburg, Germany German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center, Marburg, Germany Genomics Core Facility, Philipps University, Marburg, Germany Search for more papers by this author Author Information Oleg Timofeev1, Boris Klimovich1, Jean Schneikert1, Michael Wanzel1,2, Evangelos Pavlakis1, Julia Noll1, Samet Mutlu1, Sabrina Elmshäuser1, Andrea Nist3, Marco Mernberger1, Boris Lamp3, Ulrich Wenig4, Alexander Brobeil4, Stefan Gattenlöhner4, Kernt Köhler5 and Thorsten Stiewe *,1,2,3 1Institute of Molecular Oncology, Philipps-University, Marburg, Germany 2German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Center, Marburg, Germany 3Genomics Core Facility, Philipps University, Marburg, Germany 4Institute of Pathology, Justus Liebig University, Giessen, Germany 5Institute of Veterinary Pathology, Justus Liebig University, Giessen, Germany *Corresponding author. Tel: +49 6421 28 26280; E-mail: [email protected] The EMBO Journal (2019)38:e102096https://doi.org/10.15252/embj.2019102096 See also: JJ Manfredi (October 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53−/− mice. Surprisingly, stabilization of p53R178E in Mdm2−/− mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53R178E;Mdm2−/− embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53R178E mice develop tumors indistinguishably from Trp53−/− mice and tumors retain and even stabilize the p53R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53R178E tumors exhibit remarkably better chemotherapy responses than Trp53−/− ones, resulting in enhanced eradication of p53-mutated tumor cells. Together, this provides genetic proof-of-principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy. Synopsis In addition to its role as a transcription factor, p53 has non-transcriptional activities. A DNA binding cooperativity mutant p53 mouse genetically separates these functions and reveals a prominent role for non-transcriptional apoptotic p53 activities in development and cancer therapy responses. p53 cooperativity mutant Trp53R178E is deficient for DNA binding and target gene activation. Trp53R178E-mutant mice develop tumors as fast and extensively as Trp53-knockout mice. Constitutive stabilization of Trp53R178E in Mdm2-knockout mice triggers widespread apoptosis and embryonic lethality. Stabilized Trp53R178E and human TP53R181E induce non-transcriptional apoptosis in response to DNA damage and sensitize tumors to chemotherapy. Introduction The TP53 gene, encoding the tumor-suppressive transcription factor p53, is mutated in about half of all human cancers. The presence of TP53 mutations correlates in many cancer types with enhanced metastasis and aggressiveness, reduced responses to chemotherapeutic drugs, and, thus, a poor prognosis (Robles et al, 2016; Sabapathy & Lane, 2018). More than 85% of all amino acid positions were found to be mutated in cancer patients, generating a "rainbow" of > 2,000 distinct missense variants (Sabapathy & Lane, 2018). Mutations cluster in the central DNA binding domain (DBD), suggesting that tumorigenesis selects against p53′s DNA binding function (Muller & Vousden, 2014; Stiewe & Haran, 2018). In support of this, mutant frequency was found to directly correlate with loss of transactivation function (Kato et al, 2003). However, TP53 mutations show a remarkable preference for missense mutations, although DNA binding can be disrupted equally well by nonsense or frameshift mutations. Furthermore, missense mutants are unstable in normal unstressed cells, but become constitutively stabilized in tumors by Hsp90 which protects mutant p53 from degradation by Mdm2 and CHIP (Terzian et al, 2008; Alexandrova et al, 2015). The preferential selection of missense mutants together with their excessive stabilization therefore points at additional mechanisms that promote tumor development beyond a mere loss of DNA binding activity: Missense mutants exhibit dominant-negative effects on remaining wild-type p53 and display neomorphic properties that—like an oncogene—actively drive tumor development to a metastatic and drug-resistant state (Freed-Pastor & Prives, 2012; Muller & Vousden, 2014; Kim & Lozano, 2018; Stiewe & Haran, 2018). Missense mutations therefore enhance tumor development and progression in three ways: the loss of wild-type-like DNA binding activity (loss of function, LOF), dominant-negative effects on wild-type p53, and the gain of new tumor-promoting oncogenic properties (gain of function, GOF) (Stiewe & Haran, 2018). p53 missense mutants are broadly classified as either "structural" or "DNA contact" mutants (Bullock & Fersht, 2001). Structural mutants destabilize the inherently low stability of the DBD resulting in its denaturation at body temperature and therefore likely affect also those non-transcriptional functions which are mediated by the DBD through, for instance, protein–protein interactions. In contrast, DNA contact mutants affect single DNA-interacting residues and retain an intact native fold (Bullock & Fersht, 2001). We and others have previously described a new class of non-structural mutations affecting the DBD surface residues E180 and R181 which form a reciprocal salt bridge between two adjacent p53 subunits in the tetrameric DNA-bound complex (Klein et al, 2001; Kitayner et al, 2010). This salt bridge is essential for p53 to bind DNA in a cooperative manner so that mutations at these sites are referred to as cooperativity mutations (Dehner et al, 2005; Schlereth et al, 2010a). Despite being no mutational hot-spots, cooperativity mutations at residues E180 and R181 are estimated to account for 34,000 cancer cases per year (Leroy et al, 2014). Importantly, distinct cooperativity mutations reduce p53 DNA binding to different extents without changing the overall DBD structure determined by NMR spectroscopy (Dehner et al, 2005). Of all mutations of the double salt bridge, the R181E mutant disrupts formation of the intermolecular salt bridge most effectively (Schlereth et al, 2010a). Although R181E retains a native fold, it is entirely DNA binding deficient as assessed by electrophoretic mobility shift assays and genome-wide chromatin immunoprecipitation analysis (Dehner et al, 2005; Schlereth et al, 2010a, 2013). In R181E, the double salt bridge residues 180 and 181 are both glutamic acid (E), so that we refer to this mutant in short as "EE". Mutations engineered into the mouse Trp53 gene locus are valuable tools to delineate in vivo tumor suppressor functions in tumorigenesis and cancer therapy (Bieging et al, 2014; Mello & Attardi, 2018). Besides mutations derived from cancer patients, especially non-naturally occurring mutations of post-translational modification sites (Sluss et al, 2004; Slee et al, 2010; Li et al, 2012) or functional domains (Toledo et al, 2006; Brady et al, 2011; Hamard et al, 2013; Simeonova et al, 2013) have yielded substantial mechanistic insight into the pathways required for tumor suppression. To explore the relevance of DNA binding cooperativity for p53′s anti-tumor activities, we therefore generated the "EE" mouse carrying the human R181E-equivalent R178E mutation at the endogenous Trp53 gene locus. Cistrome and transcriptome analysis confirms the EE mutant as DNA binding deficient in vivo. Phenotype analysis demonstrates a knock-out-like appearance characterized by undetectable p53 target gene regulation and widespread, early-onset tumorigenesis, indicating that DNA binding cooperativity is essential for DNA binding and tumor suppression in vivo. Surprisingly, the EE mutation—different from the p53-knock-out—does not rescue the embryonic lethality of the Mdm2 knock-out and triggers massive apoptotic cell death providing support for residual cytotoxic activities upon constitutive stabilization. An essential role of caspases, localization of EE to the mitochondria, and sensitization to mitochondrial outer membrane permeabilization point toward the intrinsic apoptosis pathway as the cause of cell death. Importantly, apoptosis was also triggered in vitro and in vivo by DNA-damaging chemotherapy of tumor cells expressing constitutively or pharmacologically stabilized EE. This translated into improved survival under chemotherapy. Similar results were obtained with the human R181L cooperativity mutant, which has been recurrently identified in cancer patients. Together, these findings highlight that mutant p53, in principle, can retain residual apoptotic activities that are insufficient to prevent tumorigenesis and not efficiently counter-selected during tumor evolution. Stabilization of such a p53 mutant in combination with chemotherapy is capable to trigger mutant p53-mediated cytotoxicity resulting in improved anti-cancer responses and increased survival. Results p53EE is deficient for DNA binding and target gene activation We previously showed that the DNA binding cooperativity mutant p53R181E (EE) fails to bind p53 response elements in vitro and when exogenously expressed in p53-null cells (Schlereth et al, 2010a, 2013). To address how ablation of DNA binding cooperativity affects p53 functions in vivo, we generated a conditional knock-in mouse, carrying the R178E (EE) mutation in exon 5 of the endogenous mouse Trp53 gene locus (Fig EV1A–D). DNA binding deficiency of the EE mutation in the context of the mouse p53 protein was confirmed by electrophoretic mobility shift assays using nuclear extracts of homozygous p53EE/EE mouse embryonic fibroblasts (MEFs) and a high-affinity, consensus-like p53 response element (Fig EV1E). Next, DNA binding was assessed genome-wide by sequencing chromatin immunoprecipitated with a p53 antibody from MEFs under untreated conditions and following p53 stabilization with the Mdm2 inhibitor Nutlin-3a (Nutlin) (ChIP-seq, Fig 1A). We identified a total of 468 p53-specific peaks in Nutlin-treated p53+/+ MEFs (Figs 1A and EV1F). Validating the quality of the ChIP-seq, these peaks were strongly enriched for the p53 consensus motif at the peak center and significantly annotated with multiple Molecular Signatures Database (MSigDB) gene sets related to p53 function (Fig 1B and G). Only 3 peaks were identified in Nutlin-treated p53EE/EE MEFs that were, however, also called in p53−/− MEFs and therefore considered non-specific (Fig EV1F). Thus, the p53 binding pattern observed in p53EE/EE MEFs was indistinguishable from p53−/− MEFs, irrespective of Nutlin treatment, and therefore validated the p53EE mutant expressed from the endogenous Trp53 gene locus to be DNA binding deficient in cells. Click here to expand this figure. Figure EV1. Generation and characterization of the Trp53R178E knock-in mouse Trp53 targeting strategy. Asterisk indicates the R178E (EE) point mutation in exon 5; LSL, lox-stop-lox cassette. Southern blot, showing integration of the construct in a correctly targeted 129/SvEv embryonic stem cell clone. Genomic DNA was digested with SspI and hybridized with the 3′ probe shown in (A). LSL-EE denotes the targeted allele carrying a lox-stop-lox (LSL) cassette and R178E (EE) mutation. The 10.3 kb SspI fragment corresponds to the wild-type and the 8.4 kb fragment to the targeted allele. Sanger sequencing of a Trp53 exon 5-6 PCR amplicon confirms the presence of the Arg->Glu mutation in a tiptail biopsy from a heterozygous founder mouse. PCR used for genotyping of mouse biopsies and cells. Asterisk indicates unspecific PCR product. Electrophoretic mobility shift assay (EMSA) performed with a radiolabeled oligonucleotide containing a p53 consensus binding site incubated with in vitro translated full-length p53 protein (IVT p53WT, left) or nuclear extracts from primary MEFs with indicated p53 genotypes treated with 10 μM Nutlin o/n (right). For supershift analysis, anti-p53 antibody (FL-393, Santa Cruz) was added; asterisks denote disrupted and shifted bands, respectively. Arrowhead, specific p53-DNA complex. ret lys—reticulocyte lysate; specific comp—non-radiolabeled consensus binding site oligonucleotide as competitor; scrambled comp—non-radiolabeled sequence-scrambled competitor oligonucleotide; ns—non-specific. Venn diagram illustrating number and overlap of peaks called in the p53 ChIP-seq datasets from Nutlin-treated MEFs of indicated genotypes. Only peaks present in p53+/+—but not in p53−/− MEFs—were considered p53-specific. Hypergeometric enrichment showing that genes in the vicinity of p53 ChIP-seq peaks from Nutlin-treated p53+/+ MEFs are significantly enriched for p53-related gene sets from the Molecular Signatures Database (MSigDB). Shown is the -log10 of the P value adjusted for multiple comparisons using Benjamini–Hochberg correction. Nutlin-regulated gene expression in primary MEFs of indicated p53 genotypes. Scatter plot shows the log2-fold change of the 1,000 top-regulated genes. Box and whiskers indicate the interquartile range and 5–95 percentiles, respectively. Significance was tested by ordinary ANOVA with Sidak's multiple comparisons test. p53EE fails to regulate non-canonical tumor-suppressive target genes identified by transcriptional profiling of transactivation domain mutant mice (Brady et al, 2011). Shown are the z-transformed RNA expression values (FPKM). Download figure Download PowerPoint Figure 1. p53EE is deficient for DNA binding and target gene activation A. p53 ChIP-seq in MEFs of the indicated genotype treated with or without 10 μM Nutlin-3a (Nutlin) for 16 h. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53−/− or p53EE/EE MEFs. For p53EE/EE MEFs, three independent replicates are shown. B. De novo motif search using MEME-Chip was performed on the 468 p53 binding peaks (as in A). The best hit motif is reported with corresponding E-value and logo (upper part). Graphs depict a CentriMo enrichment analysis for the best MEME motif (middle) and for known transcription factor binding sites (bottom). The top two hits are shown with corresponding E-values. C. RNA-seq was performed with MEFs of the indicated genotype untreated or treated with 10 μM Nutlin for 16 h. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM). D, E. RNA-seq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes. (E) Summary of GSEA results for p53-related gene sets. NES, normalized enrichment score; nom P, nominal P value. F. Reverse transcription–quantitative PCR (RT–qPCR) analysis of p53 target genes in MEFs treated for 24 h with 1 μg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n = 6). P values were calculated by 2-way ANOVA with Sidak's multiple comparisons test. G, H. Western blots of protein lysates prepared from MEFs treated for 24 h with (G) 10 μM Nutlin or (H) 0.4 μg/ml Doxo. Download figure Download PowerPoint When global gene expression was profiled by RNA-seq, Nutlin exerted a significantly stronger effect on global gene expression in p53+/+ versus either p53EE/EE or p53−/− MEFs, while Nutlin effects on p53EE/EE and p53−/− cells showed no significant difference (Fig EV1H). Furthermore, we observed in p53+/+ but not in p53EE/EE or p53-null MEFs a strong Nutlin-inducible expression of a p53 signature including both bona fide p53 pathway genes (MSigDB Hallmarks_P53_Pathway) and non-canonical targets previously identified to be critical mediators of tumor suppression (Figs 1C and EV1I) (Brady et al, 2011). Gene set enrichment analysis (GSEA) showed a highly significant enrichment of a p53 target gene signature (MSigDB P53_Downstream_Pathway) in p53+/+ cells compared to either p53EE/EE or p53−/− MEFs, but no enrichment in p53EE/EE versus p53−/− MEFs (Fig 1D). The same was observed for multiple other p53-related gene sets (Fig 1D). The lack of p53 target gene activation in p53EE/EE MEFs was confirmed also under conditions of DNA damage induced with doxorubicin (Fig 1F). Western blots revealed increased p53 expression in p53EE/EE versus p53+/+ MEFs, which was further augmented by Nutlin or doxorubicin—yet in the absence of detectable expression of the p53 targets p21 and Mdm2 (Fig 1G and H). We conclude from these data that the murine p53EE mutant lacks detectable sequence-specific DNA binding and p53 target gene activation. p53EE fails to induce apoptosis, cell cycle arrest, and senescence In response to various types of stress, wild-type p53 elicits cell cycle arrest and senescence mediated by transcriptional activation of target genes, such as Cdkn1a/p21. Consistent with the inability of p53EE to induce target genes, p53EE/EE and p53-null MEFs comparably failed to undergo cell cycle arrest in response to doxorubicin-triggered DNA damage (Fig 2A) or to enter senescence upon enforced expression of oncogenic Ras (Fig EV2A) or in vitro passaging (Figs 2B and EV2B). Figure 2. p53EE fails to induce apoptosis, cell cycle arrest, and senescence A. Proliferation of primary MEFs. Cells were treated o/n with 0.2 μg/ml doxorubicin (Doxo) and pulse-labeled with 32 μM 5-bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n = 4. B. Long-term proliferation assay with primary MEFs of indicated genotypes. +/+ and −/−: n = 3; EE/EE: n = 6. C. MEFs were immortalized with the adenoviral oncogene E1A.12S (E1A MEF) and treated with 0.4 μg/ml Doxo for 17 h. Apoptosis (annexin V) was quantified by flow cytometry. +/+ and −/−: n = 3; EE/EE: n = 6. Western blots show expression of E1A and β-actin as loading control. D. Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 μM dexamethasone as a control for p53-independent apoptosis. Cell survival relative to untreated samples was analyzed using CellTiter-Glo assay (Promega). n = 11 for each time point and genotype. E. mRNA expression analysis (RT–qPCR) of p53 target genes in thymocytes 6 h after 6 Gy irradiation. Shown are expression values normalized to β-actin. F–I. Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU 2 h before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL) and (G) proliferation (BrdU); scale bars 50 μm. Red arrowheads highlight TUNEL-positive apoptotic cells. (H,I) Quantification for n = 3 mice/genotype (150 crypts/mouse). Data information: All data are shown as mean ± SD. P values were calculated by 2-way ANOVA with Sidak's multiple comparisons test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cells and tissues from p53EE mice show deficiency in cell cycle arrest, senescence, and apoptosis Oncogenic HRasG12V was overexpressed in primary MEFs, and cells were stained for senescence-associated β-galactosidase (SA β-gal) to detect oncogene-induced senescence (positive cells are marked with arrowheads). Western blots show expression of H-Ras and β-actin as loading control. Senescence caused by cell culture stress was assessed in primary MEFs at passage 8 by SA β-gal staining (arrowheads). Left, 3D structure of the double salt bridge formed between the H1 helices of two adjacent p53WT monomers. Residues glutamate 177 (177E) and arginine 178 (178R) are labeled. Right, schematic illustration of interactions between the two H1 helices of p53 molecules with wild-type and mutated Glu177 and Arg178. Primary thymocytes were isolated from mice with indicated genotypes and irradiated ex vivo with 6 Gy X-ray. Cellular survival at indicated time points was analyzed using CellTiter-Glo assay (Promega). Note, homozygous EE and homozygous RR mutant thymocytes are apoptosis-deficient, while compound EE/RR mutant thymocytes are sensitive to irradiation. Data are shown as mean ± SD. Apoptosis (detected by IHC staining of cleaved caspase-3) in thymus of control or irradiated mice (6 Gy X-ray) after 6 h. Note massive apoptosis in both p53+/+ and p53EE/RR mice. The absence of p53EE expression in small intestine of unstressed mice and accumulation at indicated time points after irradiation. p53+/+ are shown for comparison. Dynamics of apoptosis (TUNEL staining) in small intestine at indicated time points after whole-body irradiation (6 Gy X-ray). Arrowheads mark TUNEL-positive apoptotic cells. Cell proliferation in samples from (G) as detected by immunohistochemical staining for BrdU incorporation. Data information: All scale bars denote 50 μm. Download figure Download PowerPoint Besides cell cycle arrest, p53 is capable of inducing apoptotic cell death upon severe DNA damage. While immortalization with adenoviral E1A.12S strongly sensitized p53+/+ MEFs to apoptosis, E1A-expressing p53EE/EE and p53-null MEFs remained refractory to apoptosis induction by genotoxic damage or Nutlin (Fig 2C). Likewise, p53EE/EE thymocytes were as resistant as p53-null cells to apoptosis triggered by ionizing radiation, despite retaining the ability to undergo p53-independent apoptosis thereby excluding a general failure of the apoptosis machinery (Fig 2D). The apoptosis defect corresponded with a deficiency in transactivating not only Cdkn1a/p21 but also the key pro-apoptotic target genes Pmaip1/Noxa and Bbc3/Puma (Fig 2E). Of note, we have previously reported a similar but more selective apoptosis defect in p53RR mice carrying the E177R (RR) cooperativity mutation (Fig EV2C) (Timofeev et al, 2013). p53RR forms a p53WT-like salt bridge with p53EE which enables formation of stably DNA-bound and transcriptionally active p53EE/p53RR heterotetramers (Fig EV2C) (Dehner et al, 2005; Schlereth et al, 2010a, 2013). We therefore crossed p53EE/EE mice to p53RR/RR mice and obtained compound p53EE/RR animals that launched an apoptotic DNA damage response like p53+/+ animals in thymocytes ex vivo (Fig EV2D) and upon whole-body irradiation in vivo (Fig EV2E). Rescue of the apoptosis deficiency of p53EE/EE mice by the equally apoptosis-defective p53RR mutant proves that the p53EE loss-of-function phenotype is directly linked to the inability to form the salt bridge responsible for cooperative DNA binding and in turn further excludes global DBD misfolding or secondary local structural alterations at the DNA binding surface as an underlying cause. Like p53WT and the hot-spot mutant p53R172H (Terzian et al, 2008), p53EE was undetectable in vivo by immunostaining in all tissues analyzed, but rapidly stabilized in response to whole-body ionizing radiation (Fig EV2F). This suggests that the elevated p53EE protein level observed in MEF cultures (Fig 1G and H) reflects a stabilization in response to unphysiological culture stress. p53 stabilization upon whole-body irradiation triggered waves of cell cycle inhibition and apoptosis in intestinal crypts and other radiosensitive organs of p53+/+ animals (Figs 2F–I and EV2G and H). None of these effects were recorded in p53EE/EE or p53-null mice (Figs 2F–I and EV2G and H), indicating a complete defect of p53EE regarding classical p53 effector functions in vivo. Constitutive p53EE stabilization triggers ROS-dependent senescence When passaging p53EE/EE MEFs for longer time periods, we noted that—unlike p53−/− MEFs—the proliferation rate of p53EE/EE MEFs eventually declined and the cells started to express the senescence marker SA-β-galactosidase (Fig EV3A and B). This was accompanied by a progressive increase in p53EE protein levels, but without the increased expression in p53 target genes that was detectable in p53+/+ MEFs (Fig EV3C and D). Spontaneous (Fig EV3C) or CRISPR-enforced (Fig EV3E) deletion of p53EE caused
Referência(s)