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1
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Ben-Oz BM, Machour FE, Nicola M, Argoetti A, Polyak G, Hanna R, Kleifeld O, Mandel-Gutfreund Y, Ayoub N. A dual role of RBM42 in modulating splicing and translation of CDKN1A/p21 during DNA damage response. Nat Commun 2023; 14:7628. [PMID: 37993446 PMCID: PMC10665399 DOI: 10.1038/s41467-023-43495-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 11/10/2023] [Indexed: 11/24/2023] Open
Abstract
p53-mediated cell cycle arrest during DNA damage is dependent on the induction of p21 protein, encoded by the CDKN1A gene. p21 inhibits cyclin-dependent kinases required for cell cycle progression to guarantee accurate repair of DNA lesions. Hence, fine-tuning of p21 levels is crucial to preserve genomic stability. Currently, the multilayered regulation of p21 levels during DNA damage is not fully understood. Herein, we identify the human RNA binding motif protein 42 (RBM42) as a regulator of p21 levels during DNA damage. Genome-wide transcriptome and interactome analysis reveals that RBM42 alters the expression of p53-regulated genes during DNA damage. Specifically, we demonstrate that RBM42 facilitates CDKN1A splicing by counteracting the splicing inhibitory effect of RBM4 protein. Unexpectedly, we also show that RBM42, underpins translation of various splicing targets, including CDKN1A. Concordantly, transcriptome-wide mapping of RBM42-RNA interactions using eCLIP further substantiates the dual function of RBM42 in regulating splicing and translation of its target genes, including CDKN1A. Collectively, our data show that RBM42 couples splicing and translation machineries to fine-tune gene expression during DNA damage response.
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Affiliation(s)
- Bella M Ben-Oz
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Feras E Machour
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Marian Nicola
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Amir Argoetti
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Galia Polyak
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Rawad Hanna
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Oded Kleifeld
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Yael Mandel-Gutfreund
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel
| | - Nabieh Ayoub
- Department of Biology, Technion - Israel Institute of Technology, Haifa, 3200003, Israel.
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2
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Trauernicht M, Rastogi C, Manzo S, Bussemaker H, van Steensel B. Optimisation of TP53 reporters by systematic dissection of synthetic TP53 response elements. Nucleic Acids Res 2023; 51:9690-9702. [PMID: 37650627 PMCID: PMC10570033 DOI: 10.1093/nar/gkad718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2023] [Revised: 07/24/2023] [Accepted: 08/22/2023] [Indexed: 09/01/2023] Open
Abstract
TP53 is a transcription factor that controls multiple cellular processes, including cell cycle arrest, DNA repair and apoptosis. The relation between TP53 binding site architecture and transcriptional output is still not fully understood. Here, we systematically examined in three different cell lines the effects of binding site affinity and copy number on TP53-dependent transcriptional output, and also probed the impact of spacer length and sequence between adjacent binding sites, and of core promoter identity. Paradoxically, we found that high-affinity TP53 binding sites are less potent than medium-affinity sites. TP53 achieves supra-additive transcriptional activation through optimally spaced adjacent binding sites, suggesting a cooperative mechanism. Optimally spaced adjacent binding sites have a ∼10-bp periodicity, suggesting a role for spatial orientation along the DNA double helix. We leveraged these insights to construct a log-linear model that explains activity from sequence features, and to identify new highly active and sensitive TP53 reporters.
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Affiliation(s)
- Max Trauernicht
- Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
- Oncode Institute, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
| | - Chaitanya Rastogi
- Department of Biological Sciences, Columbia University, New York, NY, USA
| | - Stefano G Manzo
- Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
- Oncode Institute, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
- Department of Biosciences, University of Milan “La Statale”, 20133 Milan, Italy
| | - Harmen J Bussemaker
- Department of Biological Sciences, Columbia University, New York, NY, USA
- Department of Systems Biology, Columbia University Medical Center, New York, NY, USA
| | - Bas van Steensel
- Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
- Oncode Institute, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
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3
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Löffler T, Krüger A, Zirak P, Winterhalder MJ, Müller AL, Fischbach A, Mangerich A, Zumbusch A. Influence of chain length and branching on poly(ADP-ribose)-protein interactions. Nucleic Acids Res 2023; 51:536-552. [PMID: 36625274 PMCID: PMC9881148 DOI: 10.1093/nar/gkac1235] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 11/16/2022] [Accepted: 12/10/2022] [Indexed: 01/11/2023] Open
Abstract
Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR-protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53-and potentially also APLF-can form complex multivalent PAR-protein structures. In conclusion, our study gives detailed and quantitative insight into PAR-protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.
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Affiliation(s)
| | | | - Peyman Zirak
- Department of Chemistry, Universität Konstanz, Konstanz D-78457, Germany
| | | | - Anna-Lena Müller
- Department of Chemistry, Universität Konstanz, Konstanz D-78457, Germany
| | - Arthur Fischbach
- Department of Biology, Universität Konstanz, Konstanz D-78457, Germany
| | - Aswin Mangerich
- To whom correspondence should be addressed. Tel: +49 33200 88 5301;
| | - Andreas Zumbusch
- Correspondence may also be addressed to Andreas Zumbusch. Tel: +49 7531 882027;
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4
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Unraveling the Structural Changes in the DNA-Binding Region of Tumor Protein p53 ( TP53) upon Hotspot Mutation p53 Arg248 by Comparative Computational Approach. Int J Mol Sci 2022; 23:ijms232415499. [PMID: 36555140 PMCID: PMC9779389 DOI: 10.3390/ijms232415499] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 11/09/2022] [Accepted: 11/16/2022] [Indexed: 12/13/2022] Open
Abstract
The vital tissue homeostasis regulator p53 forms a tetramer when it binds to DNA and regulates the genes that mediate essential biological processes such as cell-cycle arrest, senescence, DNA repair, and apoptosis. Missense mutations in the core DNA-binding domain (109-292) simultaneously cause the loss of p53 tumor suppressor function and accumulation of the mutant p53 proteins that are carcinogenic. The most common p53 hotspot mutation at codon 248 in the DNA-binding region, where arginine (R) is substituted by tryptophan (W), glycine (G), leucine (L), proline (P), and glutamine (Q), is reported in various cancers. However, it is unclear how the p53 Arg248 mutation with distinct amino acid substitution affects the structure, function, and DNA binding affinity. Here, we characterized the pathogenicity and protein stability of p53 hotspot mutations at codon 248 using computational tools PredictSNP, Align GVGD, HOPE, ConSurf, and iStable. We found R248W, R248G, and R248P mutations highly deleterious and destabilizing. Further, we subjected all five R248 mutant-p53-DNA and wt-p53-DNA complexes to molecular dynamics simulation to investigate the structural stability and DNA binding affinity. From the MD simulation analysis, we observed increased RMSD, RMSF, and Rg values and decreased protein-DNA intermolecular hydrogen bonds in the R248-p53-DNA than the wt-p53-DNA complexes. Likewise, due to high SASA values, we observed the shrinkage of proteins in R248W, R248G, and R248P mutant-p53-DNA complexes. Compared to other mutant p53-DNA complexes, the R248W, R248G, and R248P mutant-p53-DNA complexes showed more structural alteration. MM-PBSA analysis showed decreased binding energies with DNA in all five R248-p53-DNA mutants than the wt-p53-DNA complexes. Henceforth, we conclude that the amino acid substitution of Arginine with the other five amino acids at codon 248 reduces the p53 protein's affinity for DNA and may disrupt cell division, resulting in a gain of p53 function. The proposed study influences the development of rationally designed molecular-targeted treatments that improve p53-based therapeutic outcomes in cancer.
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5
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Alimba CG, Rudrashetti AP, Sivanesan S, Krishnamurthi K. Landfill soil leachates from Nigeria and India induced DNA damage and alterations in genes associated with apoptosis in Jurkat cell. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2022; 29:5256-5268. [PMID: 34417692 DOI: 10.1007/s11356-021-15985-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 08/11/2021] [Indexed: 06/13/2023]
Abstract
Landfill soil leachates, containing myriad of xenobiotics, increase genotoxic and cytotoxic stress-induced cell death. However, the underlying mechanism involved in the elimination of the damaged cells is yet to be fully elucidated. This study investigated the apoptotic processes induced in lymphoma (Jurkat) cells by landfill soil leachates from Olusosun (OSL, Nigeria) and Nagpur (NPL, India). Jurkat was incubated with sub-lethal concentrations of OSL and NPL for 24 h and analyzed for DNA fragmentation and apoptosis using agarose gel electrophoresis and Hoechst 33258-PI staining, respectively. Complementary DNA expression profiling of some pro-apoptotic and anti-apoptotic genes regulating apoptosis was also analyzed using real-time PCR (RT-PCR) method. Agarose gel electrophoresis revealed DNA fragmentations in OSL and NPL-treated cells. Hoecsht-33258 - Propidium Iodide (PI) based apoptotic analysis confirmed apoptotic cell death in exposed Jurkat. RT-PCR analysis revealed different fold changes in the pro- and anti-apoptotic genes in OSL and NPL-treated Jurkat. There was significant increase in fold change of the up-regulated genes; apoptosis inducing factor mitochondrion-associated 2 (AIFM2), Fas-associated death domain (FADD), Caspase-2, Caspase-6, BH3 interacting domain death agonist (BID), tumor suppressor (p53), and BCL2 associated agonist of cell death (BAD) and down-regulation of apoptosis inhibitor 5 (API5). Results suggest that OSL and NPL elicited genotoxic stress-related apoptosis in Jurkat. The dysregulation in the expression of genes involved in apoptotic processes in wildlife and human exposed to landfill emissions may increase aetiology of various pathological diseases including cancer.
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Affiliation(s)
- Chibuisi G Alimba
- Cell Biology and Genetics Unit, Department of Zoology, University of Ibadan, Ibadan, Nigeria.
- Department of Toxicology, Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, 44139, Dortmund, Germany.
| | - Ashwinkumar P Rudrashetti
- Environmental Biotechnology and Genomic Division, CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
| | - Saravanadevi Sivanesan
- Health and Toxicity Cell (HTC), CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
- Academy of Scientific, Innovative Research (AcSIR), Ghaziabad, U.P., India
| | - Kannan Krishnamurthi
- Health and Toxicity Cell (HTC), CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India.
- Academy of Scientific, Innovative Research (AcSIR), Ghaziabad, U.P., India.
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6
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Rizzotto D, Englmaier L, Villunger A. At a Crossroads to Cancer: How p53-Induced Cell Fate Decisions Secure Genome Integrity. Int J Mol Sci 2021; 22:ijms221910883. [PMID: 34639222 PMCID: PMC8509445 DOI: 10.3390/ijms221910883] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 09/29/2021] [Accepted: 10/01/2021] [Indexed: 12/12/2022] Open
Abstract
P53 is known as the most critical tumor suppressor and is often referred to as the guardian of our genome. More than 40 years after its discovery, we are still struggling to understand all molecular details on how this transcription factor prevents oncogenesis or how to leverage current knowledge about its function to improve cancer treatment. Multiple cues, including DNA-damage or mitotic errors, can lead to the stabilization and nuclear translocation of p53, initiating the expression of multiple target genes. These transcriptional programs may be cell-type- and stimulus-specific, as is their outcome that ultimately imposes a barrier to cellular transformation. Cell cycle arrest and cell death are two well-studied consequences of p53 activation, but, while being considered critical, they do not fully explain the consequences of p53 loss-of-function phenotypes in cancer. Here, we discuss how mitotic errors alert the p53 network and give an overview of multiple ways that p53 can trigger cell death. We argue that a comparative analysis of different types of p53 responses, elicited by different triggers in a time-resolved manner in well-defined model systems, is critical to understand the cell-type-specific cell fate induced by p53 upon its activation in order to resolve the remaining mystery of its tumor-suppressive function.
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Affiliation(s)
- Dario Rizzotto
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; (D.R.); (L.E.)
| | - Lukas Englmaier
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; (D.R.); (L.E.)
- Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases (LBI-RUD), 1090 Vienna, Austria
| | - Andreas Villunger
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; (D.R.); (L.E.)
- Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases (LBI-RUD), 1090 Vienna, Austria
- Institute for Developmental Immunology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria
- Correspondence:
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7
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Kara A, Özgür A, Nalbantoğlu S, Karadağ A. DNA repair pathways and their roles in drug resistance for lung adenocarcinoma. Mol Biol Rep 2021; 48:3813-3825. [PMID: 33856604 DOI: 10.1007/s11033-021-06314-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 03/24/2021] [Indexed: 01/24/2023]
Abstract
Lung cancer is the leading cancer type of death rate. The lung adenocarcinoma subtype is responsible for almost half of the total lung cancer deaths. Despite the improvements in cancer treatment in recent years, lung adenocarcinoma patients' overall survival rate remains poor. Immunetherapy and chemotherapy are two of the most widely used options for the treatment of cancer. Although many cancer types initially respond to these treatments, the development of resistance is inevitable. The rapid development of drug resistance mainly characterizes lung adenocarcinoma. Despite being the subject of many studies in recent years, the resistance initiation and progression mechanism is still unclear. In this review, we have examined the role of the primary DNA repair pathways (non-homologous end joining (NHEJ) pathway, homologous-recombinant repair (HR) pathway, base excision repair (BER) pathway, and nucleotide excision repair (NER) pathway and transactivation mechanisms of tumor protein 53 (TP53) in drug resistance development. This review suggests that mentioned pathways have essential roles in developing the resistance against chemotherapy and immunotherapy in lung adenocarcinoma patients.
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Affiliation(s)
- Altan Kara
- Molecular Oncology Laboratory, Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Kocaeli, Turkey.
| | - Aykut Özgür
- Laboratory and Veterinary Health Program, Department of Veterinary Medicine, Artova Vocational School, Tokat Gaziosmanpaşa University, Tokat, Turkey
| | - Sinem Nalbantoğlu
- Molecular Oncology Laboratory, Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Kocaeli, Turkey
| | - Abdullah Karadağ
- Molecular Oncology Laboratory, Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Kocaeli, Turkey
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8
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Hamad SH, Brinkman MC, Tsai YH, Mellouk N, Cross K, Jaspers I, Clark PI, Granville CA. Pilot Study to Detect Genes Involved in DNA Damage and Cancer in Humans: Potential Biomarkers of Exposure to E-Cigarette Aerosols. Genes (Basel) 2021; 12:genes12030448. [PMID: 33809907 PMCID: PMC8004185 DOI: 10.3390/genes12030448] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 03/18/2021] [Accepted: 03/19/2021] [Indexed: 11/16/2022] Open
Abstract
There is a paucity of data on how gene expression enables identification of individuals who are at risk of exposure to carcinogens from e-cigarette (e-cig) vaping; and how human vaping behaviors modify these exposures. This pilot study aimed to identify genes regulated from acute exposure to e-cig using RT-qPCR. Three subjects (2M and 1F) made three visits to the lab (nTOT = 9 visits); buccal and blood samples were collected before and immediately after scripted vaping 20 puffs (nTOT = 18 samples); vaping topography data were collected in each session. Subjects used their own e-cig containing 50:50 propylene glycol (PG):vegetable glycerine (VG) +3-6 mg/mL nicotine. The tumor suppressor TP53 was significantly upregulated in buccal samples. TP53 expression was puff volume and flow rate dependent in both tissues. In blood, the significant downregulation of N-methylpurine DNA glycosylase (MPG), a base excision repair gene, was consistent across all subjects. In addition to DNA repair pathway, cell cycle and cancer pathways were the most enriched pathways in buccal and blood samples, respectively. This pilot study demonstrates that vaping 20 puffs significantly alters expression of TP53 in human tissues; vaping behavior is an important modifier of this response. A larger study is needed to confirm these relationships.
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Affiliation(s)
- Samera H. Hamad
- UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA;
- Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA;
- Correspondence: (S.H.H.); (P.I.C.); (C.A.G.); Tel.: +1-608-217-2829 (S.H.H.); +1-443-791-3553 (P.I.C.); +1-614-607-2766 (C.A.G.)
| | | | - Yi-Hsuan Tsai
- UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA;
| | - Namya Mellouk
- National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA;
| | - Kandice Cross
- Gad Consulting Services, Risk Assessment, Consulting in Raleigh, Raleigh, NC 27609, USA;
| | - Ilona Jaspers
- Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA;
- Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Pamela I. Clark
- School of Public Health, University of Maryland, College Park, MD 20742, USA
- Correspondence: (S.H.H.); (P.I.C.); (C.A.G.); Tel.: +1-608-217-2829 (S.H.H.); +1-443-791-3553 (P.I.C.); +1-614-607-2766 (C.A.G.)
| | - Courtney A. Granville
- Drug Information Association, Washington, DC 20036, USA
- Correspondence: (S.H.H.); (P.I.C.); (C.A.G.); Tel.: +1-608-217-2829 (S.H.H.); +1-443-791-3553 (P.I.C.); +1-614-607-2766 (C.A.G.)
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9
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Farkas M, Hashimoto H, Bi Y, Davuluri RV, Resnick-Silverman L, Manfredi JJ, Debler EW, McMahon SB. Distinct mechanisms control genome recognition by p53 at its target genes linked to different cell fates. Nat Commun 2021; 12:484. [PMID: 33473123 PMCID: PMC7817693 DOI: 10.1038/s41467-020-20783-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Accepted: 12/15/2020] [Indexed: 12/21/2022] Open
Abstract
The tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome.
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Affiliation(s)
- Marina Farkas
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Hideharu Hashimoto
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Yingtao Bi
- Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Ramana V Davuluri
- Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | | | | | - Erik W Debler
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Steven B McMahon
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
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10
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Choi SH, Cho SY, Song J, Hur MW. KLHL4, a novel p53 target gene, inhibits cell proliferation by activating p21 WAF/CDKN1A. Biochem Biophys Res Commun 2020; 530:588-596. [PMID: 32753315 DOI: 10.1016/j.bbrc.2020.07.100] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Accepted: 07/22/2020] [Indexed: 12/12/2022]
Abstract
KLHL4 is a member of the KLHL protein family, many of whom bind the Cul3 E3 ligase, and mediate the ubiquitination of interacting proteins. The KLHL4 gene, localized on the X chromosome, associates with a disorder known as X-linked cleft palate (CPX). However, the biological functions of KLHL4 are largely unknown. In this study, microarray analysis of HEK293A embryonic kidney cells, expressing ectopic p53, showed a 3-fold increase of KLHL4 mRNA. Moreover, both KLHL4 mRNA and protein expression were elevated by p53 or DNA damage, suggesting that KLHL4 might be a p53 target gene. We also found that KLHL4 activates transcription of p21WAF/CDKN1A, a p53 target gene encoding a major negative regulator of the cell-cycle. KLHL4 interacted with p53 to increase its binding to p53 response element of the p21WAF/CDKN1A gene, resulting in transcriptional upregulation. Furthermore, we observed that KLHL4 can interact with the Cul3 ubiquitin ligase, to possibly play a role in ubiquitin-mediated proteasomal degradation, and Klhl4 knocked-out MEF mouse embryonic fibroblasts proliferated faster than WT MEF cells. These results suggest that KLHL4 upregulation by p53 may inhibit cell proliferation, by activating p21WAF/CDKN1A.
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Affiliation(s)
- Seo-Hyun Choi
- Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute, Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, 50-1 Yonsei-Ro, SeoDaeMoon-Ku, Seoul, 03722, Republic of Korea
| | - Su-Yeon Cho
- Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute, Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, 50-1 Yonsei-Ro, SeoDaeMoon-Ku, Seoul, 03722, Republic of Korea
| | - Jiyang Song
- Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute, Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, 50-1 Yonsei-Ro, SeoDaeMoon-Ku, Seoul, 03722, Republic of Korea
| | - Man-Wook Hur
- Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute, Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, 50-1 Yonsei-Ro, SeoDaeMoon-Ku, Seoul, 03722, Republic of Korea.
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11
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Pajares MÁ. PDRG1 at the interface between intermediary metabolism and oncogenesis. World J Biol Chem 2017; 8:175-186. [PMID: 29225734 PMCID: PMC5714802 DOI: 10.4331/wjbc.v8.i4.175] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Revised: 11/14/2017] [Accepted: 11/19/2017] [Indexed: 02/05/2023] Open
Abstract
PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the p53 and DNA damage-regulated gene 1 (PDRG1) gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase II complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored.
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Affiliation(s)
- María Ángeles Pajares
- Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas (CSIC), Madrid 28040, Spain
- Instituto de Investigación Sanitaria La Paz (IdiPAZ), Madrid 28046, Spain
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12
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Pappas K, Xu J, Zairis S, Resnick-Silverman L, Abate F, Steinbach N, Ozturk S, Saal LH, Su T, Cheung P, Schmidt H, Aaronson S, Hibshoosh H, Manfredi J, Rabadan R, Parsons R. p53 Maintains Baseline Expression of Multiple Tumor Suppressor Genes. Mol Cancer Res 2017; 15:1051-1062. [PMID: 28483946 DOI: 10.1158/1541-7786.mcr-17-0089] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2017] [Revised: 04/21/2017] [Accepted: 05/02/2017] [Indexed: 02/07/2023]
Abstract
TP53 is the most commonly mutated tumor suppressor gene and its mutation drives tumorigenesis. Using ChIP-seq for p53 in the absence of acute cell stress, we found that wild-type but not mutant p53 binds and activates numerous tumor suppressor genes, including PTEN, STK11(LKB1), miR-34a, KDM6A(UTX), FOXO1, PHLDA3, and TNFRSF10B through consensus binding sites in enhancers and promoters. Depletion of p53 reduced expression of these target genes, and analysis across 18 tumor types showed that mutation of TP53 associated with reduced expression of many of these genes. Regarding PTEN, p53 activated expression of a luciferase reporter gene containing the p53-consensus site in the PTEN enhancer, and homozygous deletion of this region in cells decreased PTEN expression and increased growth and transformation. These findings show that p53 maintains expression of a team of tumor suppressor genes that may together with the stress-induced targets mediate the ability of p53 to suppress cancer development. p53 mutations selected during tumor initiation and progression, thus, inactivate multiple tumor suppressor genes in parallel, which could account for the high frequency of p53 mutations in cancer.Implications: In this study, we investigate the activities of p53 under normal low-stress conditions and discover that p53 is capable of maintaining the expression of a group of important tumor suppressor genes at baseline, many of which are haploinsufficient, which could contribute to p53-mediated tumor suppression. Mol Cancer Res; 15(8); 1051-62. ©2017 AACR.
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Affiliation(s)
- Kyrie Pappas
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York.,Department of Pharmacology, Columbia University Medical Center, New York, New York
| | - Jia Xu
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Sakellarios Zairis
- Department of Systems Biology, Columbia University Medical Center, New York, New York
| | - Lois Resnick-Silverman
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Francesco Abate
- Department of Systems Biology, Columbia University Medical Center, New York, New York.,Department of Biomedical Informatics, Columbia University Medical Center, New York, New York
| | - Nicole Steinbach
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York.,Graduate School of Arts and Sciences, Columbia University Medical Center, New York, New York
| | - Sait Ozturk
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Lao H Saal
- Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden.,Lund University Cancer Center, Lund, Sweden.,CREATE Health Strategic Center for Translational Cancer Research, Lund University, Lund, Sweden
| | - Tao Su
- Department of Pathology, Columbia University Medical Center, New York, New York
| | - Pamela Cheung
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Hank Schmidt
- The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York.,Dubin Breast Center, The Mount Sinai Hospital, New York, New York.,Department of Surgery, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Stuart Aaronson
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Hanina Hibshoosh
- Department of Pathology, Columbia University Medical Center, New York, New York
| | - James Manfredi
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York.,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Raul Rabadan
- Department of Systems Biology, Columbia University Medical Center, New York, New York.,Department of Biomedical Informatics, Columbia University Medical Center, New York, New York
| | - Ramon Parsons
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York. .,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York
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13
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Fischer M, Quaas M, Nickel A, Engeland K. Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex. Oncotarget 2015; 6:41402-17. [PMID: 26595675 PMCID: PMC4747163 DOI: 10.18632/oncotarget.6356] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Accepted: 11/11/2015] [Indexed: 12/15/2022] Open
Abstract
The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1.
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Affiliation(s)
- Martin Fischer
- Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germany
- Department of Medical Oncology, Dana–Farber Cancer Institute, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
| | - Marianne Quaas
- Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germany
| | - Annina Nickel
- Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germany
| | - Kurt Engeland
- Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germany
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14
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Hao Q, Cho WC. Battle against cancer: an everlasting saga of p53. Int J Mol Sci 2014; 15:22109-22127. [PMID: 25470027 PMCID: PMC4284697 DOI: 10.3390/ijms151222109] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2014] [Revised: 10/23/2014] [Accepted: 11/25/2014] [Indexed: 02/07/2023] Open
Abstract
Cancer is one of the most life-threatening diseases characterized by uncontrolled growth and spread of malignant cells. The tumor suppressor p53 is the master regulator of tumor cell growth and proliferation. In response to various stress signals, p53 can be activated and transcriptionally induces a myriad of target genes, including both protein-encoding and non-coding genes, controlling cell cycle progression, DNA repair, senescence, apoptosis, autophagy and metabolism of tumor cells. However, around 50% of human cancers harbor mutant p53 and, in the majority of the remaining cancers, p53 is inactivated through multiple mechanisms. Herein, we review the recent progress in understanding the molecular basis of p53 signaling, particularly the newly identified ribosomal stress-p53 pathway, and the development of chemotherapeutics via activating wild-type p53 or restoring mutant p53 functions in cancer. A full understanding of p53 regulation will aid the development of effective cancer treatments.
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Affiliation(s)
- Qian Hao
- School of Continuing Studies, Tulane University, New Orleans, LA 70118, USA.
| | - William C Cho
- Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong 999077, China.
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15
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Hsieh JC, Kuta R, Armour CR, Boehmer PE. Identification of two novel functional p53 responsive elements in the herpes simplex virus-1 genome. Virology 2014; 460-461:45-54. [PMID: 25010269 DOI: 10.1016/j.virol.2014.04.019] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Revised: 12/17/2013] [Accepted: 04/14/2014] [Indexed: 12/19/2022]
Abstract
Analysis of the herpes simplex virus-1 (HSV-1) genome reveals two candidate p53 responsive elements (p53RE), located in proximity to the replication origins oriL and oriS, referred to as p53RE-L and p53RE-S, respectively. The sequences of p53RE-L and p53RE-S conform to the p53 consensus site and are present in HSV-1 strains KOS, 17, and F. p53 binds to both elements in vitro and in virus-infected cells. Both p53RE-L and p53RE-S are capable of conferring p53-dependent transcriptional activation onto a heterologous reporter gene. Importantly, expression of the essential immediate early viral transactivator ICP4 and the essential DNA replication protein ICP8, that are adjacent to p53RE-S and p53RE-L, are repressed in a p53-dependent manner. Taken together, this study identifies two novel functional p53RE in the HSV-1 genome and suggests a complex mechanism of viral gene regulation by p53 which may determine progression of the lytic viral replication cycle or the establishment of latency.
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Affiliation(s)
- Jui-Cheng Hsieh
- Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, 425 North 5th Street, Phoenix, AZ 85004-2157, USA.
| | - Ryan Kuta
- Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, 425 North 5th Street, Phoenix, AZ 85004-2157, USA
| | - Courtney R Armour
- Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, 425 North 5th Street, Phoenix, AZ 85004-2157, USA
| | - Paul E Boehmer
- Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, 425 North 5th Street, Phoenix, AZ 85004-2157, USA
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16
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Adikesavan AK, Karmakar S, Pardo P, Wang L, Liu S, Li W, Smith CL. Activation of p53 transcriptional activity by SMRT: a histone deacetylase 3-independent function of a transcriptional corepressor. Mol Cell Biol 2014; 34:1246-61. [PMID: 24449765 PMCID: PMC3993559 DOI: 10.1128/mcb.01216-13] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression.
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Affiliation(s)
| | - Sudipan Karmakar
- Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Patricia Pardo
- Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Liguo Wang
- Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Shuang Liu
- Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Wei Li
- Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Carolyn L. Smith
- Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
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17
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Abstract
This issue marks the 50th anniversary of the release of the U.S. Surgeon General's Report on Smoking and Health. Perhaps no other singular event has done more to highlight the effects of smoking on the development of cancer. Tobacco exposure is the leading cause of cancers involving the oral cavity, conductive airways, and the lung. Owing to the many carcinogens in tobacco smoke, smoking-related malignancies have a high genome-wide burden of mutations, including in the gene encoding for p53. The p53 protein is the most frequently mutated tumor suppressor in cancer, responsible for a range of critical cellular functions that are compromised by the presence of a mutation. Herein, we review the epidemiologic connection between tobacco exposure and cancer, the molecular basis of p53 mutation in lung cancer, and the normal molecular and cellular roles of p53 that are abrogated during lung tumor development and progression as defined by in vitro and in vivo studies. We also consider the therapeutic potential of targeting mutant p53 in a clinical setting based upon the cellular role of mutant p53 and data from genetic murine models.
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Affiliation(s)
- Don L Gibbons
- University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Box 0432, Houston, Texas 77030.
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18
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Fischer M, Steiner L, Engeland K. The transcription factor p53: not a repressor, solely an activator. Cell Cycle 2014; 13:3037-58. [PMID: 25486564 PMCID: PMC4612452 DOI: 10.4161/15384101.2014.949083] [Citation(s) in RCA: 115] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2014] [Accepted: 07/10/2014] [Indexed: 12/12/2022] Open
Abstract
The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway.
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Key Words
- CDE, cell cycle-dependent element
- CDKN1A
- CHR, cell cycle genes homology region
- ChIP, chromatin immunoprecipitation
- DREAM complex
- DREAM, DP, RB-like, E2F4, and MuvB complex
- E2F/RB complex
- HPV, human papilloma virus
- NF-Y, Nuclear factor Y
- cdk, cyclin-dependent kinase
- genome-wide meta-analysis
- p53
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Affiliation(s)
- Martin Fischer
- Molecular Oncology; Medical School; University of Leipzig; Leipzig, Germany
| | - Lydia Steiner
- Center for Complexity & Collective Computation; Wisconsin Institute for Discovery; Madison, WI USA
- Computational EvoDevo Group & Bioinformatics Group; Department of Computer Science and Interdisciplinary Center for Bioinformatics; University of Leipzig; Leipzig, Germany
| | - Kurt Engeland
- Molecular Oncology; Medical School; University of Leipzig; Leipzig, Germany
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19
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Another fork in the road--life or death decisions by the tumour suppressor p53. EMBO Rep 2013; 14:414-21. [PMID: 23588418 DOI: 10.1038/embor.2013.25] [Citation(s) in RCA: 126] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2012] [Accepted: 02/13/2013] [Indexed: 12/18/2022] Open
Abstract
In response to cellular stress signals, the tumour suppressor p53 accumulates and triggers a host of antineoplastic responses. For instance, DNA damage activates two main p53-dependent responses: cell cycle arrest and attendant DNA repair or apoptosis (cell death). It is broadly accepted that, in response to DNA damage, the function of p53 as a sequence-specific transcription factor is crucial for tumour suppression. The molecular determinants, however, that favour the initiation of either a p53-dependent cell cycle arrest (life) or apoptotic (death) transcriptional programme remain elusive. Gaining a clear understanding of the mechanisms controlling cell fate determination by p53 could lead to the identification of molecular targets for therapy, which could selectively sensitize cancer cells to apoptosis. This review summarizes the literature addressing this important question in the field. Special emphasis is given to the role of the p53 response element, post-translational modifications and protein-protein interactions on cell fate decisions made by p53 in response to DNA damage.
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20
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Reaz S, Mossalam M, Okal A, Lim CS. A single mutant, A276S of p53, turns the switch to apoptosis. Mol Pharm 2013; 10:1350-9. [PMID: 23402381 DOI: 10.1021/mp300598k] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The tumor suppressor protein p53 induces apoptosis, cell cycle arrest, and DNA repair along with other functions in a transcription-dependent manner [Vousden, K. H. Cell 2000, 103(5), 691-694]. The selection of these functions depends on sequence-specific recognition of p53 to a target decameric sequence of gene promoters [Kitayner, M.; et al. Mol. Cell 2006, 22(6), 741-753]. Amino acid residues in p53 that directly bind to DNA were analyzed, and the replacement of A276 in p53 with selected amino acids elucidated its importance in promoter transcription. For most apoptotic and cell cycle gene promoters, position 9 of the target decameric sequence is a cytosine, while for DNA repair gene promoters, thymine is found instead. Therefore, selective binding to the cytosine at the ninth position may transcribe apoptotic gene promoters and thus can induce apoptosis and cell cycle arrest. Molecular modeling with PyMOL indicated that substitution of a hydrophilic residue, A276S, would prefer binding to cytosine at the ninth position of the target decameric sequence, whereas substitution of a hydrophobic residue (A276F) would fail to do so. Correspondingly, A276S demonstrated higher transcription of PUMA, PERP, and p21(WAF1/CIP1)gene promoters containing a cytosine at the ninth position and lower transcription of GADD45 gene promoter containing a thymine at the ninth position compared to wild-type p53. Cell cycle analysis showed that A276S maintained similar G1/G0 phase arrest as wild-type p53. Additionally, A276S induced higher apoptosis than wild-type p53 as measured by DNA segmentation and 7-AAD assay. Since the status of endogenous p53 can influence the activity of the exogenous p53, we examined the activity of A276S in HeLa cells (wild-type endogenous p53) in addition to T47D cells (mutated and mislocalized endogenous p53). The same apoptotic trend in both cell lines suggested A276S can induce cell death regardless of endogenous p53 status. Cell proliferation assay depicted that A276S efficiently reduced the viability of T47D cells more than wild-type p53 over time. We conclude that the predicted preferred binding of A276S to cytosine at the ninth position better transactivates a number of apoptotic gene promoters. Higher induction apoptosis than wild-type p53 makes A276S an attractive candidate for therapy to eradicate cancer.
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Affiliation(s)
- Shams Reaz
- Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA
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21
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Park JY, Song JY, Kim HM, Han HS, Seol HS, Jang SJ, Choi J. p53-Independent expression of wild-type p53-induced phosphatase 1 (Wip1) in methylmethane sulfonate-treated cancer cell lines and human tumors. Int J Biochem Cell Biol 2012; 44:896-904. [PMID: 22405851 DOI: 10.1016/j.biocel.2012.02.013] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2011] [Revised: 02/08/2012] [Accepted: 02/15/2012] [Indexed: 01/03/2023]
Abstract
Wild-type p53-induced phosphatase 1 (Wip1, PPM1D) is induced by p53 in response to various stressors and dephosphorylates cellular target proteins involved in DNA repair and cell cycle checkpoint pathways. The Wip1 gene is frequently amplified or overexpressed in human cancers, promoting tumor growth by switching off major checkpoint kinases and p53. To explore wild-type p53-independent Wip1 induction, Wip1 promoter activity and its transcript level were evaluated by luciferase assay and real-time PCR, after methylmethane sulfonate (MMS) treatment in breast cancer cell lines and p53-null cell lines. Wip1 promoter activities in response to UV irradiation and various anti-cancer agents were compared between wild-type and a p53-response element (p53RE) mutated construct. Wip1 expression and its effects were examined in primary non-small cell lung cancer (NSCLC) and colon tumor cells by using Wip1-specific siRNA. MMS induced Wip1 promoter activity in Hs578T, MDA-MB-231, and SK-BR-3 cells expressing DNA binding-deficient p53 mutants. A549-E6 and HCT116 (p53(-/-)) cells retained substantial Wip1 induction. Wip1 promoter activity was reduced, but not eliminated, in cells expressing a promoter containing a mutated p53-response element. Wip1 induction was not blocked by SB202190 or SP600125. MMS increased Wip1 expression in primary non-small cell lung cancer cells expressing a p53 R175H mutant. Our data indicate that Wip1 is induced in the absence of functional p53, like p38 MAPK and JNK, as a stress response terminator.
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Affiliation(s)
- Ji-Young Park
- Department of Pathology, University of Ulsan College of Medicine, Asan Medical center, Seoul, Republic of Korea
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22
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Using targeted transgenic reporter mice to study promoter-specific p53 transcriptional activity. Proc Natl Acad Sci U S A 2012; 109:1685-90. [PMID: 22307631 DOI: 10.1073/pnas.1114173109] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
The p53 transcription factor modulates gene expression programs that induce cell cycle arrest, senescence, or apoptosis, thereby preventing tumorigenesis. However, the mechanisms by which these fates are selected are unclear. Our objective is to understand p53 target gene selection and, thus, enable its optimal manipulation for cancer therapy. We have generated targeted transgenic reporter mice in which EGFP expression is driven by p53 transcriptional activity at a response element from either the p21 or Puma promoter, which induces cell cycle arrest/senescence and apoptosis, respectively. We demonstrate that we could monitor p53 activity in vitro and in vivo and detect variations in p53 activity depending on the response element, tissue type, and stimulus, thereby validating our reporter system and illustrating its utility for preclinical drug studies. Our results also show that the sequence of the p53 response element itself is sufficient to strongly influence p53 target gene selection. Finally, we use our reporter system to provide evidence for p53 transcriptional activity during early embryogenesis, showing that p53 is active as early as embryonic day 3.5 and that p53 activity becomes restricted to embryonic tissue by embryonic day 6.5. The data from this study demonstrate that these reporter mice could serve as powerful tools to answer questions related to basic biology of the p53 pathway, as well as cancer therapy and drug discovery.
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23
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Chen K, Perez-Stable C, D'Ippolito G, Schiller PC, Roos BA, Howard GA. Human bone marrow-derived stem cell proliferation is inhibited by hepatocyte growth factor via increasing the cell cycle inhibitors p53, p21 and p27. Bone 2011; 49:1194-204. [PMID: 21907315 DOI: 10.1016/j.bone.2011.08.023] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/06/2011] [Revised: 08/22/2011] [Accepted: 08/23/2011] [Indexed: 12/22/2022]
Abstract
Human bone marrow-derived stem cells (hMSCs) are a major source of osteoprogenitors. Hepatocyte growth factor (HGF), a glycoprotein constitutively produced by hMSCs, is reported to act on differentiated osteoblasts and also osteoclasts. Moreover, HGF has been shown by us and others to enhance osteoblastic differentiation from hMSCs. Typically, the pro-differentiation effects of HGF have required cooperative action with regulatory factors such as vitamin D or bone matrix material. Here, we have pursued the molecular mechanisms underlying the osteogenic effect of HGF on hMSCs, the principal precursors to bone forming cells. HGF treatment of hMSCs reduced the cell number over time and increased G1/S cell-cycle arrest compared to control (non-treated) cells. RT-qPCR showed treatment with HGF increased gene expression of the cell-cycle inhibitors p53, p21, and p27, possibly explaining the cell growth inhibition and G1 arrest, a step critical to phenotypic differentiation. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect on hMSCs and the HGF-mediated increase in p53, p21, and p27, strongly supporting a role for these cell-cycle inhibitors in HGF's regulation of hMSCs. HGF in combination with a known inducer of osteogenic differentiation, 1,25-dihydroxyvitamin D, significantly increased cell maturation/differentiation as indicated by an increase in several osteoblast markers. Taken together these results demonstrate that HGF significantly enhances hMSC osteoblast differentiation by 1,25-dihydroxyvitamin D.
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Affiliation(s)
- Ketian Chen
- GRECC and Research Service, Veterans Affairs Medical Center, Miami, FL 33125, USA.
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24
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Abstract
The p53 tumor suppressor protein is a key transcription factor that regulates several signaling pathways involved in the cell's response to stress. Through stress-induced activation, p53 accumulates and triggers the expression of target genes that protect the genetic integrity of all cells including hematopoietic stem cells (HSCs). These protective mechanisms include cell-cycle arrest, DNA repair, induction of apoptosis, or initiation of senescence. In addition to its function under stress conditions, p53 has important functions during steady-state hematopoiesis, regulating HSC quiescence and self-renewal. In addition, it appears that p53 levels affect HSC competition for the hematopoietic niche, with the less p53 activated HSCs preferentially surviving. The specific genes and precise mechanisms underlying p53's effects on normal HSCs are slowly being clarified. p53 also plays an important role in leukemia stem cell (LSC) behavior, with p53 loss affecting drug resistance and disease progression. Pharmacologic activation of p53 function could overcome the adverse impact of p53 inactivation in LSCs. Thus, understanding the p53 regulatory mechanisms active in HSCs and LSCs may promote the development of new therapeutic strategies that could eliminate the population of largely quiescent LSCs.
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Affiliation(s)
- Takashi Asai
- Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065
| | - Yan Liu
- Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065
| | - Narae Bae
- Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065
| | - Stephen D Nimer
- Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065
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25
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Rinn JL, Huarte M. To repress or not to repress: this is the guardian's question. Trends Cell Biol 2011; 21:344-53. [PMID: 21601459 DOI: 10.1016/j.tcb.2011.04.002] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2011] [Revised: 04/11/2011] [Accepted: 04/13/2011] [Indexed: 10/18/2022]
Abstract
p53 is possibly the most central tumor suppressor gene of our cells, integrating stress signals to activate a transcriptional program responsible for maintaining cellular homeostasis. Many of the downstream effects of p53 are a consequence of its activity as a transcription factor, resulting in the induction of multiple target genes. In addition to gene activation, however, gene repression is an essential part of the p53 cellular response. Despite extensive research efforts towards the elucidation of p53 functions, the molecular mechanisms and biological consequences of gene repression by p53 have not been studied extensively. We review our current knowledge of the mechanisms and biological consequences of p53 repression, with special attention to recently discovered mechanisms of repression that involve non-coding RNA molecules, an emerging aspect of regulation in the p53 cellular network.
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Affiliation(s)
- John L Rinn
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA
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26
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Abstract
It has been suggested that adenosine monophosphate-activated protein kinase (AMPK) and 12 AMPK-related kinases (ARK), including novel (nua) kinase family 1 (NUAK1), are activated by master kinase LKB1, a major tumor suppressor. Apart from evidence to suggest that NUAK1 participates in induction of tumor survival, invasion and p53-independent cellular senescence, its detailed biological functions remain unclear. Here we showed that in the presence of wild-type LKB1, NUAK1 directly interacts with and phosphorylates p53 in vitro and in vivo. The phosphorylation of p53 induced by LKB1 required the kinase activity of NUAK1 and phosphorylation of NUAK1 at Thr211 by LKB1 was essential for its kinase activity, which leads to the conclusion that LKB1 activates NUAK1 and regulates phosphorylation of p53 through the NUAK1 kinase, at least partially. LKB1/NUAK1 activation leads to cell cycle arrest at the G(1)/S border by inducing expression of p21/WAF1. Under the regulation of LKB1, NUAK1 interacts with p53 in the nucleus and binds to the p53-responsive element of p21/WAF1 promoter. These findings have highlighted a novel role for NUAK1 in LKB1-related signaling pathways; NUAK1 can regulate cell proliferation and exert tumor suppression through direct interaction with p53.
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27
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Stik G, Laurent S, Coupeau D, Coutaud B, Dambrine G, Rasschaert D, Muylkens B. A p53-dependent promoter associated with polymorphic tandem repeats controls the expression of a viral transcript encoding clustered microRNAs. RNA (NEW YORK, N.Y.) 2010; 16:2263-2276. [PMID: 20881002 PMCID: PMC2957064 DOI: 10.1261/rna.2121210] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2010] [Accepted: 08/13/2010] [Indexed: 05/29/2023]
Abstract
The tumor suppressor protein p53 plays a role in cellular responses to cancer-initiating events by regulating progress through the cell cycle. Several recent studies have shown that p53 transactivates expression of the members of the proapoptotic microRNA-34 family, which are underexpressed in several cancers. We demonstrate here that the latency-associated cluster of microRNAs (miRNA) encoded by an oncogenic herpesvirus, gallid herpesvirus 2 (GaHV-2), is a direct target of p53. Robust transcriptional activity was induced in three avian cell lines by a sequence mapping 600 base pairs (bp) upstream of the cluster of miRNAs. We found transcription start sites for the pri-miRNA transcript at the 3' end of this transcription-inducing sequence. The promoter has no consensus core promoter element, but is organized into a variable number of tandem repeats of 60-bp harboring p53-responsive elements (RE). The minimal functional construct consists of two tandem repeats. Mutagenesis to change the sequence of the p53 RE abolished transcriptional activity, whereas p53 induction enhanced mature miRNA expression. The identification of a viral miRNA promoter regulated by p53 is biologically significant, because all avirulent GaHV-2 strains described to date lack the corresponding regulatory sequence, whereas all virulent, very virulent, and hypervirulent strains possess at least two tandem repeats harboring the p53 RE.
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Affiliation(s)
- Grégoire Stik
- Transcription, Lymphome Viro-Induit, University François Rabelais, UFR Sciences et Techniques, Parc de Grandmont, F-37200 Tours, France
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28
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Sarkari F, Sheng Y, Frappier L. USP7/HAUSP promotes the sequence-specific DNA binding activity of p53. PLoS One 2010; 5:e13040. [PMID: 20885946 PMCID: PMC2946354 DOI: 10.1371/journal.pone.0013040] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2010] [Accepted: 09/03/2010] [Indexed: 12/02/2022] Open
Abstract
The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity.
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Affiliation(s)
- Feroz Sarkari
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Yi Sheng
- Department of Biology, York University, Toronto, Ontario, Canada
| | - Lori Frappier
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- * E-mail:
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29
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Chandrachud U, Gal S. Three assays show differences in binding of wild-type and mutant p53 to unique gene sequences. Technol Cancer Res Treat 2010; 8:445-53. [PMID: 19925028 DOI: 10.1177/153303460900800606] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Cancer-associated mutations in the p53 gene often change amino acids in the protein's DNA binding domain. We used three different binding assays specifically gel shift, DNA binding scintillation proximity assay and a streptavidin magnetic bead assay to analyze the DNA binding of the tumor suppressor p53 from 4 human cell lines with different DNA sequences from the mdm2, p21 and cyclin G genes and a mutant form of the cyclin G sequence. Treatment of MCF-7 cells having wild-type p53 with hydrogen peroxide increased the binding of p53 to DNA as detected using all three assays, but to different extents. The p53 proteins from the thyroid cancer cell lines with different p53 mutations (ARO, WRO and NPA) have comparable binding reactions in the three assays, but show different specificities for the sequences. Here we show that multiple different binding assays allow us to generate a more complete picture of the function of DNA transcription factors in diseases such as cancer.
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Affiliation(s)
- Uma Chandrachud
- Department of Biological Sciences, Binghamton University, Binghamton, NY 13902, USA.
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Gomes NP, Espinosa JM. Differential regulation of p53 target genes: it's (core promoter) elementary. Genes Dev 2010; 24:111-4. [PMID: 20080948 DOI: 10.1101/gad.1893610] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
p53 is a pleiotropic transcription factor driving a flexible transcriptional program that mediates disparate cellular responses to stress, including cell cycle arrest and apoptosis. The mechanisms by which p53 differentially regulates its diverse target genes remain poorly understood. In this issue of Genes & Development, Morachis and colleagues (pp. 135-147) demonstrate the critical role of core promoter elements at p53 target loci, in that they dictate differential RNA polymerase II recruitment and activity in a p53-autonomous fashion.
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Affiliation(s)
- Nathan P Gomes
- Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, The University of Colorado at Boulder, Boulder, Colorado 80309, USA
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31
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Müller GA, Engeland K. The central role of CDE/CHR promoter elements in the regulation of cell cycle-dependent gene transcription. FEBS J 2009; 277:877-93. [PMID: 20015071 DOI: 10.1111/j.1742-4658.2009.07508.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The cell cycle-dependent element (CDE) and the cell cycle genes homology region (CHR) control the transcription of genes with maximum expression in G(2) phase and in mitosis. Promoters of these genes are repressed by proteins binding to CDE/CHR elements in G(0) and G(1) phases. Relief from repression begins in S phase and continues into G(2) phase and mitosis. Generally, CDE sites are located four nucleotides upstream of CHR elements in TATA-less promoters of genes such as Cdc25C, Cdc2 and cyclin A. However, expression of some other genes, such as human cyclin B1 and cyclin B2, has been shown to be controlled only by a CHR lacking a functional CDE. To date, it is not fully understood which proteins bind to and control CDE/CHR-containing promoters. Recently, components of the DREAM complex were shown to be involved in CDE/CHR-dependent transcriptional regulation. In addition, the expression of genes regulated by CDE/CHR elements is mostly achieved through CCAAT-boxes, which bind heterotrimeric NF-Y proteins as well as the histone acetyltransferase p300. Importantly, many CDE/CHR promoters are downregulated by the tumor suppressor p53. In this review, we define criteria for CDE/CHR-regulated promoters and propose to distinguish two classes of CDE/CHR-regulated genes. The regulation through transcription factors potentially binding to the CDE/CHR is discussed, and recently discovered links to central pathways regulated by E2F, the pRB family and p53 are highlighted.
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Affiliation(s)
- Gerd A Müller
- Molecular Oncology, Department of Obstetrics and Gynecology, University of Leipzig, Germany
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32
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Downregulation of p53 by phosphatase of regenerating liver 3 is mediated by MDM2 and PIRH2. Life Sci 2009; 86:66-72. [PMID: 19945467 DOI: 10.1016/j.lfs.2009.11.010] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2009] [Revised: 11/05/2009] [Accepted: 11/16/2009] [Indexed: 11/24/2022]
Abstract
AIMS The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. MAIN METHODS To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. KEY FINDINGS We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. SIGNIFICANCE These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.
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Capocefalo A, Franceschi V, Whitelaw CBA, Vasey DB, Lillico SG, Cavirani S, Donofrio G. p21(Waf1/Cip1) as a molecular sensor for BoHV-4 replication. J Virol Methods 2009; 161:308-11. [PMID: 19540267 DOI: 10.1016/j.jviromet.2009.06.012] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2009] [Revised: 05/27/2009] [Accepted: 06/10/2009] [Indexed: 10/20/2022]
Abstract
BoHV-4 replication cycle is dependent on the S-phase of the cell-cycle at the stage of viral DNA synthesis. Because p21 is a rate-limiting regulator of the G1/S-phase transition and up-regulated by DNA-damaging agents, in this study p21 expression in BoHV-4 infected cells was investigated. The p21 promoter was found to be highly activated in a dose- and time-dependent manner following BoHV-4 infection only in cells which are permissive for BoHV-4 replication. Thus p21 expression reports on BoHV-4 replication and could represent a host cell defensive response to infection-associated cellular damage.
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Affiliation(s)
- A Capocefalo
- Facoltà di Medicina Veterinaria, Dipartimento di Salute Animale, Sezione di Malattie Infettive degli Animali, Parma, Italy
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34
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Abstract
Long interspersed nuclear elements-1 (L1s) are highly repetitive DNA elements that are capable of altering the human genome through retrotransposition. To protect against L1 retroposition, the cell downregulates the expression of L1 proteins by various mechanisms, including high-density cytosine methylation of L1 promoters and DICER-dependent destruction of L1 mRNAs. In this report, a large number of p53 responsive elements, or p53 DNA binding sites, were detected in L1 elements within the human genome. At least some of these p53 responsive elements are functional and can act to increase the levels of L1 mRNA expression. The p53 protein can directly bind to a short 15-nucleotide sequence within the L1 promoter. This p53 responsive element within L1 is a recent addition to evolution, appearing approximately 20 million years ago. This suggests an interplay between L1 elements, which have a rich history of causing changes in the genome, and the p53 protein, the function of which is to protect against genomic changes. To understand these observations, a model is proposed in which the increased expression of L1 mRNAs by p53 actually increases, rather than decreases, the genomic stability through amplification of p53-dependent processes for genomic protection.
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35
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Huang V, Lu X, Jiang Y, Wang JYJ. Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73. BMC Biol 2009; 7:35. [PMID: 19558638 PMCID: PMC2711048 DOI: 10.1186/1741-7007-7-35] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2009] [Accepted: 06/26/2009] [Indexed: 12/24/2022] Open
Abstract
Background The p53 tumor suppressor and its related protein, p73, share a homologous DNA binding domain, and mouse genetics studies have suggested that they have overlapping as well as distinct biological functions. Both p53 and p73 are activated by genotoxic stress to regulate an array of cellular responses. Previous studies have suggested that p53 and p73 independently activate the cellular apoptotic program in response to cytotoxic drugs. The goal of this study was to compare the promoter-binding activity of p53 and p73 at steady state and after genotoxic stress induced by hydroxyurea. Results We employed chromatin immunoprecipitation, the NimbleGen promoter arrays and a model-based algorithm for promoter arrays to identify promoter sequences enriched in anti-p53 or anti-p73 immunoprecipitates, either before or after treatment with hydroxyurea, which increased the expression of both p53 and p73 in the human colon cancer cell line HCT116-3(6). We calculated a model-based algorithm for promoter array score for each promoter and found a significant correlation between the promoter occupancy profiles of p53 and p73. We also found that after hydroxyurea treatment, the p53-bound promoters were still bound by p73, but p73 became associated with additional promoters that that did not bind p53. In particular, we showed that hydroxyurea induces the binding of p73 but not p53 to the promoter of MLH3, which encodes a mismatch repair protein, and causes an up-regulation of the MLH3 mRNA. Conclusion These results suggest that hydroxyurea exerts differential effects on the promoter-binding functions of p53 and p73 and illustrate the power of model-based algorithm for promoter array in the analyses of promoter occupancy profiles of highly homologous transcription factors.
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Affiliation(s)
- Vera Huang
- Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0820, USA.
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36
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PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53. Proc Natl Acad Sci U S A 2009; 106:7852-7. [PMID: 19416861 DOI: 10.1073/pnas.0812148106] [Citation(s) in RCA: 98] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2alpha) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.
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37
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Ivanov VN, Zhou H, Partridge MA, Hei TK. Inhibition of ataxia telangiectasia mutated kinase activity enhances TRAIL-mediated apoptosis in human melanoma cells. Cancer Res 2009; 69:3510-9. [PMID: 19351839 DOI: 10.1158/0008-5472.can-08-3883] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The aim of the present study was to elucidate the effects of ataxia telangiectasia mutated (ATM) kinase on the regulation of the extrinsic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We revealed that total ATM protein levels were high in some human melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser(1981) were also detectable in many melanoma lines and could be further up-regulated by gamma-irradiation. Pretreatment of several melanoma lines just before gamma-irradiation with the inhibitor of ATM kinase KU-55933 suppressed p53 and nuclear factor-kappaB (NF-kappaB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, gamma-irradiation in the presence of KU-55933 rendered TRAIL-resistant HHMSX melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in melanoma cells. Besides p53 and NF-kappaB, crucial regulators of DR5 expression, transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser(727) and Tyr(705) phosphorylation of STAT3 by KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3beta also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human melanoma cells to TRAIL-mediated apoptosis.
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Affiliation(s)
- Vladimir N Ivanov
- Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
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38
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Rossi M, Demidov ON, Anderson CW, Appella E, Mazur SJ. Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites. Nucleic Acids Res 2008; 36:7168-80. [PMID: 19015127 PMCID: PMC2602757 DOI: 10.1093/nar/gkn888] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.
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Affiliation(s)
- Matteo Rossi
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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39
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Min SH, Kim DM, Heo YS, Kim YI, Kim HM, Kim J, Han YM, Kim IC, Yoo OJ. New p53 target, phosphatase of regenerating liver 1 (PRL-1) downregulates p53. Oncogene 2008; 28:545-54. [PMID: 18997816 DOI: 10.1038/onc.2008.409] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Most of the p53 target genes, all except MDM2, COP1 and PIRH2, perform functions in apoptosis, differentiation and cell cycle arrest. The aforementioned oncogenes downregulate p53 through a negative feedback mechanism, and thus contribute to tumor development. In this study, we report a new p53 target, PRL-1, which is believed to be a significant regulator in the development and metastasis of a variety of cancer types. Phosphatase of regenerating liver 1 (PRL-1) overexpression reduced the levels of endogenous and exogenous p53 proteins, and inhibited p53-mediated apoptosis. On the other hand, the ablation of PRL-1 by small interfering RNA (siRNA) increased p53 protein levels. The p53 downregulation was mediated by p53 ubiquitination and subsequent proteasomal degradation. Furthermore, p53 ubiquitination by PRL-1 was achieved through two independent pathways, by inducing PIRH2 transcription and by inducing MDM2 phosphorylation through Akt signaling. In addition, we showed that the PRL-1 gene harbors a p53 response element in the first intron, and its transcription is regulated by the p53 protein. These findings imply that the new oncogenic p53 target, PRL-1, may contribute to tumor development by the downregulation of p53 by a negative feedback mechanism.
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Affiliation(s)
- S-H Min
- Department of Biological Sciences, Biomedical Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
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40
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Radiosensitization of melanoma cells through combined inhibition of protein regulators of cell survival. Apoptosis 2008; 13:790-802. [PMID: 18454317 DOI: 10.1007/s10495-008-0212-y] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The incidence of melanoma continues to dramatically increase in most Western countries with predominantly Caucasian populations. However, only limited therapies for the metastatic stage of the disease are currently available. The main purpose of this study is to determine approaches that can substantially increase radiosensitivity of melanoma cells. The PI3K-AKT, NF-kappaB and COX-2 pathways, which are involved in the radioprotective response, are highly active in melanoma cells. Pharmacological suppression of COX-2 and PI3K-AKT, or RNAi-mediated knockdown of COX-2, substantially increased levels of G2/M arrest of the cell cycle and decreased clonogenic survival of gamma-irradiated melanomas, predominantly via a necrotic mechanism. On the other hand, resveratrol, a polyphenolic phytoalexin, selectively targets numerous cell signaling pathways, decreasing clonogenic survival primarily via an apoptotic mechanism. In melanoma cells, resveratrol inhibits STAT3 and NF-kappaB-dependent transcription, culminating in suppression of cFLIP and Bcl-xL expression, while activating the MAPK- and the ATM-Chk2-p53 pathways. Resveratrol also upregulates TRAIL promoter activity and induces TRAIL surface expression in some melanoma cell lines, resulting in a rapid development of apoptosis. Sequential treatment of melanoma cells, first with gamma-irradiation to upregulate TRAIL-R surface expression, and then with resveratrol to suppress antiapoptotic proteins cFLIP and Bcl-xL and induce TRAIL surface expression, had dramatic effects on upregulation of apoptosis in some melanoma lines, including SW1 and WM35. However, for melanoma lines exhibiting suppressed translocation of TRAIL to the cell surface, a necrotic mechanism of cell death was primarily involved in radiation response. Hence, surface expression of TRAIL induced by resveratrol appears to be a decisive event, one which determines an apoptotic versus a necrotic response of melanoma cells to sequential treatment.
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41
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Espinosa JM. Mechanisms of regulatory diversity within the p53 transcriptional network. Oncogene 2008; 27:4013-23. [PMID: 18278067 PMCID: PMC2914794 DOI: 10.1038/onc.2008.37] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2007] [Revised: 01/28/2008] [Accepted: 01/28/2008] [Indexed: 01/18/2023]
Abstract
p53 is arguably the most intensively studied protein to date, yet there is much that we ignore about its function as a transcription factor. The p53-dependent transcriptional program is remarkably flexible, as it varies with the nature of p53-activating stimuli, the cell type and the duration of the activation signal. This flexibility may allow cells to mount alternative responses to p53 activation, such as cell cycle arrest or apoptosis. Here, I organize the available data into two alternative models to explain how this regulatory diversity is achieved.
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Affiliation(s)
- J M Espinosa
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA.
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42
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Jordan JJ, Menendez D, Inga A, Nourredine M, Bell D, Resnick MA. Noncanonical DNA motifs as transactivation targets by wild type and mutant p53. PLoS Genet 2008; 4:e1000104. [PMID: 18714371 PMCID: PMC2518093 DOI: 10.1371/journal.pgen.1000104] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2008] [Accepted: 05/22/2008] [Indexed: 12/31/2022] Open
Abstract
Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network. Within human cells, the tumor suppressor p53 is the central node of regulation required to elicit multiple biological responses that include cell cycle arrest and death in response to stress or DNA damage, where mutations in p53 are a hallmark of cancer. As a master regulatory gene, p53 controls the action of target genes within its network by directly interacting with a widely accepted consensus DNA binding sequence, composed of two decamer ½-sites that can be separated by up to 13 bases. While mismatches from consensus sequence are frequent, the canonical consensus sequence places a limitation upon the organization and number of target genes within the p53 transcriptional network. Using yeast and human cell systems, our goal was to further understand how the DNA sequence, DNA organization, and level of p53 expression might influence the inclusion of genes within the p53 regulatory network. We found that increases in spacer beyond a few bases greatly reduce responsiveness to p53. Importantly, we established that p53 can function from noncanonical sequences comprising only a decamer ½-site or a ¾-site. These findings further define and expand the universe of potential downstream target genes which may be regulated by p53 and bring further diversity into the p53 regulatory network.
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Affiliation(s)
- Jennifer J. Jordan
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Daniel Menendez
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
| | - Alberto Inga
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
- Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research, IST, Genoa, Italy
| | - Maher Nourredine
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
| | - Douglas Bell
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
| | - Michael A. Resnick
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail:
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Ivanov VN, Partridge MA, Johnson GE, Huang SXL, Zhou H, Hei TK. Resveratrol sensitizes melanomas to TRAIL through modulation of antiapoptotic gene expression. Exp Cell Res 2007; 314:1163-76. [PMID: 18222423 DOI: 10.1016/j.yexcr.2007.12.012] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2007] [Revised: 12/11/2007] [Accepted: 12/11/2007] [Indexed: 12/01/2022]
Abstract
Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-kappaB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-kappaB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while gamma-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by gamma-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas.
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Affiliation(s)
- Vladimir N Ivanov
- Center for Radiological Research, Columbia University, New York, NY 10032, USA.
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Menendez D, Inga A, Jordan JJ, Resnick MA. Changing the p53 master regulatory network: ELEMENTary, my dear Mr Watson. Oncogene 2007; 26:2191-201. [PMID: 17401428 DOI: 10.1038/sj.onc.1210277] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The p53 master regulatory network provides for the stress-responsive direct control of a vast number of genes in humans that can be grouped into several biological categories including cell-cycle control, apoptosis and DNA repair. Similar to other sequence-specific master regulators, there is a matrix of key components, which provide for variation within the p53 master regulatory network that include p53 itself, target response element sequences (REs) that provide for p53 regulation of target genes, chromatin, accessory proteins and transcription machinery. Changes in any of these can impact the expression of individual genes, groups of genes and the eventual biological responses. The many REs represent the core of the master regulatory network. Since defects or altered expression of p53 are associated with over 50% of all cancers and greater than 90% of p53 mutations are in the sequence-specific DNA-binding domain, it is important to understand the relationship between wild-type or mutant p53 proteins and the target response elements. In the words of the legendary detective Sherlock Holmes, it is 'Elementary, my dear Mr. Watson'.
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Affiliation(s)
- D Menendez
- Laboratory of Molecular Genetics, Chromosome Stability Section, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA
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45
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Gal S, Cook JR, Howells L. Scintillation proximity assay for DNA binding by human p53. Biotechniques 2006; 41:303-8. [PMID: 16989090 DOI: 10.2144/000112222] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.
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Affiliation(s)
- Susannah Gal
- Department of Biological Sciences, SUNY-Binghamton and BioLife Solutions, Binghamton, NY 13902, USA.
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Kaneshiro K, Tsutsumi S, Tsuji S, Shirahige K, Aburatani H. An integrated map of p53-binding sites and histone modification in the human ENCODE regions. Genomics 2006; 89:178-88. [PMID: 17085012 DOI: 10.1016/j.ygeno.2006.09.001] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2006] [Revised: 08/30/2006] [Accepted: 09/06/2006] [Indexed: 10/23/2022]
Abstract
TP53 (tumor protein p53; p53) regulates its target genes under various cellular stresses. By combining chromatin immunoprecipitation with oligonucleotide microarrays, we have mapped binding sites of p53 (p53-BS) in the genome of HCT116 human colon carcinoma cells, along with those of acetylated H3, acetylated H4, and methylated H3-K4. We analyzed a 30-Mb portion of the human genome selected as a representative model by the ENCODE Consortium. In the region, we found 37 p53-BS, of which the p53-binding motif was present in 32 (86%). Acetylated histone H3 and H4 were detected at 14 (38%) and 33 (89%) of the p53-BS, respectively. A significant portion (58%) of H4 acetylation in the p53-BS was not accompanied by H3 acetylation. Acetyl H3 were preferentially located at the 5' and 3' ends of genes, whereas acetyl H4 were distributed widely across the genome. These results provide novel insights into how p53 binding coordinates with histone modification in human.
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Affiliation(s)
- Kiyofumi Kaneshiro
- Genome Science Division, Research Center for Advanced Science and Technology (RCAST), University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan
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47
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Chao C, Wu Z, Mazur SJ, Borges H, Rossi M, Lin T, Wang JYJ, Anderson CW, Appella E, Xu Y. Acetylation of mouse p53 at lysine 317 negatively regulates p53 apoptotic activities after DNA damage. Mol Cell Biol 2006; 26:6859-69. [PMID: 16943427 PMCID: PMC1592865 DOI: 10.1128/mcb.00062-06] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.
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Affiliation(s)
- Connie Chao
- Section of Molecular Biology, Division of Biological Sciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA
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48
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Kitayner M, Rozenberg H, Kessler N, Rabinovich D, Shaulov L, Haran TE, Shakked Z. Structural basis of DNA recognition by p53 tetramers. Mol Cell 2006; 22:741-753. [PMID: 16793544 DOI: 10.1016/j.molcel.2006.05.015] [Citation(s) in RCA: 323] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2006] [Revised: 04/18/2006] [Accepted: 05/10/2006] [Indexed: 12/28/2022]
Abstract
The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.
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Affiliation(s)
- Malka Kitayner
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100
| | - Haim Rozenberg
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100
| | - Naama Kessler
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100
| | - Dov Rabinovich
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100
| | - Lihi Shaulov
- Department of Biology, Technion, Technion City, Haifa 32000, Israel
| | - Tali E Haran
- Department of Biology, Technion, Technion City, Haifa 32000, Israel.
| | - Zippora Shakked
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100.
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49
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Stambolsky P, Weisz L, Shats I, Klein Y, Goldfinger N, Oren M, Rotter V. Regulation of AIF expression by p53. Cell Death Differ 2006; 13:2140-9. [PMID: 16729031 DOI: 10.1038/sj.cdd.4401965] [Citation(s) in RCA: 151] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
The tumor suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing genomic stability, cell cycle arrest or apoptosis. AIF is a mitochondrial protein, which, upon translocation to the nucleus, can participate in apoptosis, primarily in a caspase-independent contexts. We now report that AIF gene expression is subject to positive transcriptional regulation by p53. Interestingly, unlike most known p53 target genes, the AIF gene is regulated by basal levels of p53, and activation of p53 by genotoxic stress does not result in a substantial further increase in AIF expression. The AIF gene harbors a p53 responsive element, which is bound by p53 within cells. p53 drives efficient induction of large-scale DNA fragmentation, a hallmark of AIF activity. Importantly, caspase-independent death is compromised in cells lacking functional p53, in line with the known role of AIF in this process. Thus, in addition to its documented effects on caspase-dependent apoptosis, p53 may also sensitize cells to caspase-independent death through positive regulation of AIF expression. Moreover, in the absence of overt apoptotic signals, the constitutive induction of AIF by p53 may underpin a cytoprotective maintenance role, based on the role of AIF in ensuring proper mitochondrial function.
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Affiliation(s)
- P Stambolsky
- Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
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50
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Liu W, Konduri SD, Bansal S, Nayak BK, Rajasekaran SA, Karuppayil SM, Rajasekaran AK, Das GM. Estrogen receptor-alpha binds p53 tumor suppressor protein directly and represses its function. J Biol Chem 2006; 281:9837-40. [PMID: 16469747 DOI: 10.1074/jbc.c600001200] [Citation(s) in RCA: 126] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Estrogen receptor-alpha (ERalpha) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERalpha and p53. Ionizing radiation together with ERalpha knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the pro-proliferative role of ERalpha.
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Affiliation(s)
- Wensheng Liu
- Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Sts., Buffalo, NY 14263, USA
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