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• W2118833232 abstract "The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p5325,26, is severely compromised for transactivation of most p53 target genes, and, moreover, p5325,26 cannot induce G1-arrest or apoptosis in response to acute DNA damage. Surprisingly, p5325,26 retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p5325,26,53,54 mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression—a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression. The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p5325,26, is severely compromised for transactivation of most p53 target genes, and, moreover, p5325,26 cannot induce G1-arrest or apoptosis in response to acute DNA damage. Surprisingly, p5325,26 retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p5325,26,53,54 mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression—a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression. Full p53 transactivation potential is required for acute DNA-damage responses Robust transactivation of most known p53 targets is dispensable for tumor suppression p53 transactivation function is critical for tumor suppression This tumor suppression is associated with activation of a set of new target genes The facts that over half of all human cancers sustain mutations in the p53 tumor suppressor gene and that p53 null mice display a dramatic early-onset, completely penetrant cancer predisposition together underscore the fundamental importance of p53 for tumor suppression (Kenzelmann Broz and Attardi, 2010Kenzelmann Broz D. Attardi L.D. In vivo analysis of p53 tumor suppressor function using genetically engineered mouse models.Carcinogenesis. 2010; 31: 1311-1318Crossref PubMed Scopus (61) Google Scholar, Vogelstein et al., 2000Vogelstein B. Lane D. Levine A.J. Surfing the p53 network.Nature. 2000; 408: 307-310Crossref PubMed Scopus (5798) Google Scholar). p53 serves as a cellular stress sentinel, responding to myriad stresses by restricting cellular expansion under unfavorable conditions (Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2315) Google Scholar). The best characterized p53 functions are in inducing cell-cycle arrest or apoptosis in response to acute DNA-damage signals. The ability of p53 to eliminate cells that have encountered acute genotoxic stress is thought to be an ancestral function, as this response is conserved through lower eukaryotes, including Drosophila melanogaster and Caenorhabditis elegans, where it is critical for culling damaged cells to preserve germline and tissue integrity (Lu and Abrams, 2006Lu W.J. Abrams J.M. Lessons from p53 in non-mammalian models.Cell Death Differ. 2006; 13: 909-912Crossref PubMed Scopus (44) Google Scholar). In higher eukaryotes, oncogene expression can also activate p53, leading to cellular senescence or apoptosis as safeguards against neoplasia (Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2315) Google Scholar). The extent to which the ability of p53 to respond to DNA damage is involved in mediating tumor suppression has been controversial. Analysis of early human neoplastic lesions has revealed molecular marks of activated DNA-damage components, leading to a model whereby oncogene-induced hyperproliferation results in replication fork collapse, DNA double-strand break formation, checkpoint kinase activation, and p53 induction to ultimately impose a barrier to tumor development (Halazonetis et al., 2008Halazonetis T.D. Gorgoulis V.G. Bartek J. An oncogene-induced DNA damage model for cancer development.Science. 2008; 319: 1352-1355Crossref PubMed Scopus (1397) Google Scholar). However, studies in mouse models of DNA-damage-induced lymphomas and fibrosarcomas have suggested that to serve as a tumor suppressor, p53 responds not to acute DNA damage but rather to oncogene-induced expression of the p19ARF tumor suppressor, which directly activates p53 through sequestration and inhibition of its negative regulator Mdm2 (Christophorou et al., 2006Christophorou M.A. Ringshausen I. Finch A.J. Swigart L.B. Evan G.I. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression.Nature. 2006; 443: 214-217Crossref PubMed Scopus (337) Google Scholar, Efeyan et al., 2006Efeyan A. Garcia-Cao I. Herranz D. Velasco-Miguel S. Serrano M. Tumour biology: policing of oncogene activity by p53.Nature. 2006; 443: 159Crossref PubMed Scopus (99) Google Scholar). A clearer understanding of the role of DNA-damage-triggered, p53-induced cell-cycle arrest or apoptosis in tumor suppression would come from elucidating the underlying molecular mechanisms for p53 action in the contexts of acute DNA damage versus tumor suppression. Moreover, illuminating any distinct downstream aspects to these pathways has critical therapeutic implications, as many of the deleterious side effects of genotoxic chemotherapies result from p53-driven apoptosis in radiosensitive tissues, and therefore identifying strategies to mitigate these side effects without compromising tumor suppressor function throughout the organism would be broadly valuable for cancer therapy (Gudkov and Komarova, 2003Gudkov A.V. Komarova E.A. The role of p53 in determining sensitivity to radiotherapy.Nat. Rev. Cancer. 2003; 3: 117-129Crossref PubMed Scopus (461) Google Scholar). The molecular underpinnings for p53 action in tumor suppression have remained elusive. p53 serves as a transcriptional activator of numerous target genes, but several other biochemical activities have also been ascribed to p53 (Green and Kroemer, 2009Green D.R. Kroemer G. Cytoplasmic functions of the tumour suppressor p53.Nature. 2009; 458: 1127-1130Crossref PubMed Scopus (841) Google Scholar, Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2315) Google Scholar). Moreover, no p53 target gene knockout mouse strain recapitulates the dramatic cancer predisposition of p53 null mice, illustrating our incomplete understanding of p53 networks involved in tumor suppression and suggesting that other pathways could be involved (Lozano and Zambetti, 2005Lozano G. Zambetti G.P. What have animal models taught us about the p53 pathway?.J. Pathol. 2005; 205: 206-220Crossref PubMed Scopus (59) Google Scholar). Defining the role of transactivation in tumor suppression by p53 is complicated by the fact that p53 contains two distinct transcriptional activation domains (comprising residues 1–40 and 40–83, respectively), whose discrete functions and relative contributions to p53 function are not understood (Candau et al., 1997Candau R. Scolnick D.M. Darpino P. Ying C.Y. Halazonetis T.D. Berger S.L. Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity.Oncogene. 1997; 15: 807-816Crossref PubMed Scopus (124) Google Scholar, Venot et al., 1999Venot C. Maratrat M. Sierra V. Conseiller E. Debussche L. Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.Oncogene. 1999; 18: 2405-2410Crossref PubMed Scopus (60) Google Scholar, Zhu et al., 1998Zhu J. Zhou W. Jiang J. Chen X. Identification of a novel p53 functional domain that is necessary for mediating apoptosis.J. Biol. Chem. 1998; 273: 13030-13036Crossref PubMed Scopus (156) Google Scholar). Parsing out the specific roles of these two domains for p53 function in vivo could reveal distinct transcriptional requirements for acute DNA-damage responses and tumor suppression and lead to the discovery of p53 target genes principally important for tumor suppression. Here, we investigate the mechanism of p53-mediated tumor suppression and its relationship to acute DNA-damage responses by deciphering the p53 transactivation requirements for function in these contexts. We generate a series of transactivation domain (TAD) mutant knockin mouse strains, with alterations in the first, second, and both TADs. Knockin mice, in which the mutant genes are expressed from the native p53 promoter, uniquely enable the study of both primary cells ex vivo and tumor development in the physiological context of the organism. Intriguingly, our studies reveal that different p53 transcriptional activation requirements, associated with different target gene expression programs, are important in the settings of acute genotoxic stress and oncogenic stimuli. Our findings thereby provide genetic evidence that the mechanisms through which p53 engages responses to these signals are different and lend fundamental new insight into the networks involved in p53-mediated tumor suppression. To decipher the discrete roles of the two p53 TADs in DNA-damage responses and tumor suppression in vivo, we generated a panel of p53 mutant knockin mouse strains with alterations in the first (p5325,26), second (p5353,54), or both TADs (p5325,26,53,54). L25Q;W26S knockin mice were generated previously, and analysis of a small set of p53 target genes in mouse embryo fibroblasts (MEFs) derived from these mice demonstrated compromised transactivation of these genes, except Bax (Johnson et al., 2005Johnson T.M. Hammond E.M. Giaccia A. Attardi L.D. The p53QS transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality.Nat. Genet. 2005; 37: 145-152Crossref PubMed Scopus (112) Google Scholar). Here, we have generated mouse strains bearing either the F53Q;F54S mutations found to incapacitate the second p53 TAD in vitro, or mutations in both TADs (L25Q;W26S;F53Q;F54S; Figures 1A–1D ) (Candau et al., 1997Candau R. Scolnick D.M. Darpino P. Ying C.Y. Halazonetis T.D. Berger S.L. Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity.Oncogene. 1997; 15: 807-816Crossref PubMed Scopus (124) Google Scholar, Venot et al., 1999Venot C. Maratrat M. Sierra V. Conseiller E. Debussche L. Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.Oncogene. 1999; 18: 2405-2410Crossref PubMed Scopus (60) Google Scholar, Zhu et al., 1998Zhu J. Zhou W. Jiang J. Chen X. Identification of a novel p53 functional domain that is necessary for mediating apoptosis.J. Biol. Chem. 1998; 273: 13030-13036Crossref PubMed Scopus (156) Google Scholar). All alleles carried a transcriptional stop element flanked by LoxP sites (Lox-Stop-Lox or LSL) in the first p53 intron to allow regulatable p53 expression (Figures 1A–1D). To initially characterize this set of p53 TAD mutant proteins, we cultured homozygous p53LSL-mut MEFs, infected them with adenoviruses expressing Cre recombinase (Ad-Cre), and assayed p53 protein levels and localization (Figures 1E–1G). Using this approach we typically observed over 90% p53 positivity, and in all experiments, we verified widespread p53 expression in the population being examined. Furthermore, MEFs expressed p53 only after Cre introduction, indicating effective silencing of the locus by the LSL cassette (Figure 1G), and allowing us to use MEFs infected with empty adenoviruses (Ad-empty) as convenient p53 null controls. Although basal p5325,26 and p5325,26,53,54 protein levels were elevated relative to wild-type (WT) p53 levels because mutation of residues 25/26 inhibits binding of the Mdm2 ubiquitin ligase (Lin et al., 1994Lin J. Chen J. Elenbaas B. Levine A.J. Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein.Genes Dev. 1994; 8: 1235-1246Crossref PubMed Scopus (580) Google Scholar), protein levels were in a physiological range, accumulating to levels only slightly higher than those of wild-type p53 after DNA damage (Figures 1F and 1G). Additionally, p5353,54 basal levels were slightly increased relative to wild-type p53, consistent with the reported contribution of residues 53/54 to the p53-Mdm2 interaction (Chi et al., 2005Chi S.W. Lee S.H. Kim D.H. Ahn M.J. Kim J.S. Woo J.Y. Torizawa T. Kainosho M. Han K.H. Structural details on mdm2-p53 interaction.J. Biol. Chem. 2005; 280: 38795-38802Crossref PubMed Scopus (125) Google Scholar). All mutant proteins displayed clear nuclear localization (Figure 1G). To delineate the relative contributions of the respective TADs to overall p53 transactivation function, we examined the activity of the p53 mutants both on a genome-wide scale and quantitatively at select target genes. Initially, we performed gene expression profiling experiments using a model for oncogenic Hras (HrasV12)-driven, p53-dependent senescence in MEFs (Serrano et al., 1997Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3942) Google Scholar). By comparing HrasV12;p53 wild-type and HrasV12;p53 null MEFs using Significance Analysis of Microarrays (Tusher et al., 2001Tusher V.G. Tibshirani R. Chu G. Significance analysis of microarrays applied to the ionizing radiation response.Proc. Natl. Acad. Sci. USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9751) Google Scholar), we defined a group of p53-dependent genes, including numerous established p53 targets such as p21 and Mdm2 (Figure 2A ). Expression of these genes in the mutant MEFs was compared by heat map analysis and Northern blotting to reveal notable differences in the activities of the three mutants. First, expression profiles of p5353,54/53,54 cells resembled those of cells expressing wild-type p53, suggesting that p5353,54 retains p53 transactivation function (Figures 2A and 2B). In contrast, analysis of p5325,26/25,26 MEFs revealed gene expression profiles intermediate between those observed in wild-type and p53 null cells (Figure 2A). To elaborate on this pattern, we focused on individual established p53-inducible genes, to ensure analysis of direct p53 targets. This analysis demonstrated that p5325,26 merely drives extremely low-level expression of p21, Noxa, and Puma but induces efficient expression of Bax comparable to wild-type p53, suggesting that p5325,26 is severely impaired for transactivation of most but not all p53 target genes (Figures 2B and 2D). Finally, the expression profile of p5325,26,53,54/25,26,53,54 MEFs closely resembled that of p53 null MEFs, suggesting that mutation of both TADs abolishes transactivation activity (Figures 2A, 2B, and 2D). Chromatin immunoprecipitation (ChIP) demonstrated that p53 TAD mutants bind p53 target gene promoters in cells, indicating that these mutations selectively disrupt transactivation function but not chromatin association (Figure 2C; see Figure S1A available online). qRT-PCR on DNA-damage-treated MEFs echoed the microarray results, but emphasized how minimally p5325,26 activated targets relative to p53 null MEFs and further revealed that p5325,26,53,54 retained a slight capacity to activate Bax (Figure 2D; Figure S1B). Together, our analyses identify an allelic series of p53 transactivation mutants that can be used to elucidate the contribution of different extents of transactivation to p53 biological function downstream of acute genotoxic and oncogenic stresses.Figure S1Analysis of DNA Binding and Transactivation Potential of p53 TAD Mutants, Related to Figure 2Show full caption(A) Chromatin Immunoprecipitation (ChIP) analysis of homozygous MEFs expressing different p53 mutants, using p53 or control antibodies. p53 null MEFs were used as an additional control to indicate the specificity of the p53 antibody. Enrichment for wild-type p53 and each p53 transactivation mutant was observed at the p53 response element in the p21 promoter. Results represent the average values relative to input from triplicate PCR reactions from 1–2 ChIP experiments per sample ± SD.(B) Quantitative RT-PCR analysis of Perp in MEFs either untreated (white bars) or treated with 0.2 μg/ml dox for 8 hr (black bars). Graph indicates averages ± SEM of quantities normalized first to β-actin and then to wild-type untreated sample values from three independent MEF lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Chromatin Immunoprecipitation (ChIP) analysis of homozygous MEFs expressing different p53 mutants, using p53 or control antibodies. p53 null MEFs were used as an additional control to indicate the specificity of the p53 antibody. Enrichment for wild-type p53 and each p53 transactivation mutant was observed at the p53 response element in the p21 promoter. Results represent the average values relative to input from triplicate PCR reactions from 1–2 ChIP experiments per sample ± SD. (B) Quantitative RT-PCR analysis of Perp in MEFs either untreated (white bars) or treated with 0.2 μg/ml dox for 8 hr (black bars). Graph indicates averages ± SEM of quantities normalized first to β-actin and then to wild-type untreated sample values from three independent MEF lines. In the face of acute genotoxic stress, p53 triggers either cell-cycle arrest or apoptosis to limit the propagation of damaged cells. We first defined transactivation requirements for p53-dependent G1 cell-cycle arrest using a classical MEF model in which cell-cycle profiles are examined 18 hr after 5 Gy γ-irradiation (Kastan et al., 1992Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2927) Google Scholar). Whereas wild-type and p5353,54/53,54 cells arrested efficiently, both p5325,26/25,26 and p5325,26,53,54/25,26,53,54 cells failed to arrest, indicating that activity of the first, but not the second, p53 TAD is essential for DNA-damage-induced G1 arrest (Figures 3A and 3B ). To assess p53 transactivation function in DNA-damage-triggered apoptosis, we examined p53-dependent apoptosis in radiosensitive tissues in vivo upon exposure to ionizing radiation (Lowe et al., 1993Lowe S.W. Schmitt E.M. Smith S.W. Osborne B.A. Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes.Nature. 1993; 362: 847-849Crossref PubMed Scopus (2764) Google Scholar, Merritt et al., 1994Merritt A.J. Potten C.S. Kemp C.J. Hickman J.A. Balmain A. Lane D.P. Hall P.A. The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.Cancer Res. 1994; 54: 614-617PubMed Google Scholar). Using Rosa26CreERT2 mice allowed widespread p53 expression in the small intestine and less efficient expression in the thymus upon tamoxifen administration (Figure 3C) (Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Restoration of p53 function leads to tumour regression in vivo.Nature. 2007; 445: 661-665Crossref PubMed Scopus (1392) Google Scholar). Mice homozygous for each p53 allele were exposed to 5 Gy of whole-body ionizing radiation, and apoptosis in the small intestine and thymus was examined 6 hr later. We found that p5325,26 failed to promote apoptosis in vivo, consistent with our previous analyses examining doxorubicin-induced apoptosis in E1A-MEFs and studies in cultured lymphocytes (Chao et al., 2000Chao C. Saito S. Kang J. Anderson C.W. Appella E. Xu Y. p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage.EMBO J. 2000; 19: 4967-4975Crossref PubMed Scopus (227) Google Scholar, Johnson et al., 2005Johnson T.M. Hammond E.M. Giaccia A. Attardi L.D. The p53QS transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality.Nat. Genet. 2005; 37: 145-152Crossref PubMed Scopus (112) Google Scholar, Gaidarenko and Xu, 2009Gaidarenko O. Xu Y. Transcription activity is required for p53-dependent tumor suppression.Oncogene. 2009; 28: 4397-4401Crossref PubMed Scopus (14) Google Scholar) (Figures 3D–3F). Moreover, compound mutation of both TADs did not alter this phenotype, as p5325,26,53,54 also failed to induce apoptosis. In contrast, mutation of the second TAD alone did not greatly affect p53-dependent apoptosis. Together, these experiments underscore the vital roles of the first TAD and robust transactivation for p53 to promote responses to acute DNA damage and further reveal that the second TAD plays little to no role in these responses. To elucidate p53 transactivation requirements in the context of oncogenic signals, we examined the allelic series of p53 mutants in HrasV12-induced cellular senescence (Serrano et al., 1997Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3942) Google Scholar). HrasV12 was expressed in MEFs, and cell-cycle arrest was assessed by BrdU labeling. As expected, few cells with wild-type p53 incorporated BrdU, whereas those lacking p53 proliferated efficiently upon HrasV12 expression (Figure 4A ). Interestingly, both HrasV12;p5325,26/25,26 and HrasV12;p5353,54/53,54 MEFs underwent a proliferative arrest with time and displayed hallmarks of cellular senescence, including flattened, enlarged morphologies and both transcriptional induction of Pml and widespread Pml nuclear body accumulation (de Stanchina et al., 2004de Stanchina E. Querido E. Narita M. Davuluri R.V. Pandolfi P.P. Ferbeyre G. Lowe S.W. PML is a direct p53 target that modulates p53 effector functions.Mol. Cell. 2004; 13: 523-535Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, Ferbeyre et al., 2000Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. PML is induced by oncogenic ras and promotes premature senescence.Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar) (Figures 4A–4C and 4E, Figures S2A and S2B ). Additionally, all arresting cells displayed strong nuclear staining for the histone variant macroH2A, another indicator of senescence (Kennedy et al., 2010Kennedy A.L. McBryan T. Enders G.H. Johnson F.B. Zhang R. Adams P.D. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci.Cell Div. 2010; 5: 16Crossref PubMed Scopus (45) Google Scholar) (Figure 4D). Unlike their wild-type and p5353,54/53,54 counterparts, however, senescent p5325,26/25,26 cells were negative for SA-β-galactosidase, another common senescence marker (Dimri et al., 1995Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc. Natl. Acad. Sci. USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5707) Google Scholar), suggesting that full p53 transactivation potential is essential for this activity (Figure 4E). Thus, despite being highly impaired for transactivation of most p53 target genes, including the senescence-relevant targets p21 and Pai-1 (Brown et al., 1997Brown J.P. Wei W. Sedivy J.M. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts.Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar, Kortlever et al., 2006Kortlever R.M. Higgins P.J. Bernards R. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.Nat. Cell Biol. 2006; 8: 877-884Crossref PubMed Scopus (425) Google Scholar) (Figure 4B), p5325,26 can induce senescence in the context of oncogenic signals. This surprising finding indicates that full p53 transactivation potential is dispensable for p53 to promote senescence. While either single TAD mutant alone was able to induce senescence, mutation of both domains completely reversed this phenotype, as indicated by the high level of proliferation throughout the time course, the paucity of Pml bodies, and the absence of macroH2A staining in HrasV12;p5325,26,53,54/25,26,53,54 cells, mimicking HrasV12;p53−/− MEFs (Figures 4A, 4C, and 4D). The observation that p5325,26,53,54 cannot engage the senescence program indicates that, although the full p53 transactivation program is dispensable, some limited level of p53 transactivation is required for promoting senescence. Together, our findings suggest fundamentally distinct requirements for transcriptional activation between the acute DNA damage and senescence responses: full p53 transactivation is paramount for proper p53 action downstream of genotoxic stress, in both G1 checkpoint function and apoptosis, whereas more selective p53 transactivation function suffices for senescence.Figure S2Detailed Analysis of Senescence Markers in HrasV12 MEFs, Related to Figure 4Show full caption(A) Low magnification images of Pml staining in HrasV12 MEFs to highlight population-wide Pml nuclear body accumulation in genotypes of cells undergoing cell-cycle arrest. Inset is magnification from larger image. DAPI staining marks nuclei.(B) Phase contrast photomicrographs showing additional examples of senescent cell morphology in HrasV12 MEFs expressing wild-type p53, p5325,26, and p5353,54, but not in HrasV12 MEFs expressing p5325,26,53,54 or no p53.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Low magnification images of Pml staining in HrasV12 MEFs to highlight population-wide Pml nuclear body accumulation in genotypes of cells undergoing cell-cycle arrest. Inset is magnification from larger image. DAPI staining marks nuclei. (B) Phase contrast photomicrographs showing additional examples of senescent cell morphology in HrasV12 MEFs expressing wild-type p53, p5325,26, and p5353,54, but not in HrasV12 MEFs expressing p5325,26,53,54 or no p53. To identify the mechanisms of p53 action downstream of oncogenic signals in vivo, we interrogated the contribution of p53 transactivation to tumor suppression. We employed a model for human non-small cell lung cancer (NSCLC) driven by expression of oncogenic KrasG12D from its endogenous promoter after Cre-mediated excision of an upstream Lox-Stop-Lox element (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1427) Google Scholar). p53 loss promotes the progression of these Kras-driven lung tumors to more advanced lesions, making this an optimal system to query the mechanism of p53 action (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1427) Google Scholar, Johnson et al., 2001Johnson L. Mercer K. Greenbaum D. Bronson R.T. Crowley D. Tuveson D.A. Jacks T. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice.Nature. 2001; 410: 1111-1116Crossref PubMed Scopus (949) Google Scholar). Cohorts of KrasLSL-G12D/+ mice homozygous for the different p53 alleles were subjected to intranasal Ad-Cre instillation, and tumor burden was assessed 12 weeks later. KrasG12D/+;p53−/− mice exhibited visible tumors studding the lung surfaces, while lungs from KrasG12D/+;p53+/+ mice appeared grossly normal (Figures 5A and 5B ). Additionally, the average tumor burden was dramatically increased in KrasG12D/+;p53−/− mice compared to KrasG12D/+;p53+/+ mice (Figures 5C and 5D). Strikingly, KrasG12D/+;p5325,26/25,26 and KrasG12D/+;p5353,54/53,54 lungs were macroscopically normal with mini" @default.
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• W2118833232 date "2011-05-01" @default.
• W2118833232 modified "2023-10-16" @default.
• W2118833232 title "Distinct p53 Transcriptional Programs Dictate Acute DNA-Damage Responses and Tumor Suppression" @default.
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