p53 pathway is involved in cell competition during mouse embryogenesis

The function of tumor suppressor p53 has been under intense investigation. Acute stresses such as DNA damage are able to trigger a high level of p53 activity, leading to cell cycle arrest or apoptosis. In contrast, the cellular response of mild p53 activity induced by low-level stress in vivo remains largely unexplored. Murine double minute (MDM)2 and MDM4 are two major negative regulators of p53. Here, we used the strategy of haploinsufficiency of Mdm2 and Mdm4 to induce mild p53 activation in vivo and found that Mdm2+/−Mdm4+/− double-heterozygous mice exhibited normal embryogenesis. However, closer examination demonstrated that the Mdm2+/−Mdm4+/− cells exhibited a growth disadvantage and were outcompeted during development in genetic mosaic embryos that contained wild-type cells. Further study indicated the out-competition phenotype was dependent on the levels of p53. These observations revealed that cells with mild p53 activation were less fit and exhibited altered fates in a heterotypic environment, resembling the cell competition phenomenon first uncovered in Drosophila. By marking unfit cells for elimination, p53 may exert its physiological role to ensure organ and animal fitness.

Download Full-text

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence

Cell Death and Differentiation ◽

10.1038/s41418-021-00917-6 ◽

2021 ◽

Author(s):

Irene Fernández-Duran ◽

Andrea Quintanilla ◽

Núria Tarrats ◽

Jodie Birch ◽

Priya Hari ◽

...

Keyword(s):

Cellular Senescence ◽

Cellular Response ◽

Critical Role ◽

Ras Signaling ◽

Tumor Suppressor P53 ◽

Oncogenic Ras ◽

Cycle Arrest ◽

Immune Sensing ◽

Innate Immune Sensing

AbstractCytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.

Download Full-text

Nucleophosmin Sets a Threshold for p53 Response to UV Radiation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3703-3711.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3703-3711 ◽

Cited By ~ 78

Author(s):

Dony A. Maiguel ◽

Leslie Jones ◽

Devulapalli Chakravarty ◽

Chonglin Yang ◽

France Carrier

Keyword(s):

Uv Radiation ◽

Tumor Suppressor P53 ◽

Cycle Arrest ◽

Early Responder ◽

P53 Response ◽

A Cell ◽

P53 Activation ◽

Uv Dose ◽

P53 Inactivation

ABSTRACT Because activation of p53 can trigger cell cycle arrest and apoptosis, it is necessary for a cell to suppress this activation until it is absolutely required for survival. The mechanisms underlying this important regulatory event are poorly understood. Here we show that nucleophosmin (NPM) acts as a natural repressor of p53 by setting a threshold for p53 activation in response to UV radiation. NPM binds to the p53 N terminus and inhibits p53 transcriptional activity by more than 70%. Our data indicate that the levels of NPM in a cell determine the UV dose at which the tumor suppressor p53 can be phosphorylated on Ser15. Moreover, we show that NPM is a substrate for the UV-activated protein kinase ATR and inhibits the UV-induced p53 phosphorylation at Ser15. In addition, NPM forms a complex with p53 and ATR in vivo. These data suggest that NPM is an early responder to DNA damage that prevents premature activation of p53. In normal cells, NPM could contribute to suppressing p53 activation until its functions are absolutely required while in cancer cells overexpression of NPM could contribute to p53 inactivation and tumor progression.

Download Full-text

Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

Download Full-text

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001520117 ◽

2020 ◽

Vol 117 (30) ◽

pp. 17808-17819 ◽

Cited By ~ 3

Author(s):

Andrea Lees ◽

Alexander J. McIntyre ◽

Nyree T. Crawford ◽

Fiammetta Falcone ◽

Christopher McCann ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Fate ◽

Target Genes ◽

Caspase 8 ◽

Cycle Arrest ◽

P53 Activation ◽

Induced Apoptosis

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Download Full-text

Immunomodulation of ALT-2 and TLR may collude in antigen specific T cell hyporesponsiveness: Proposed mechanism for elevated IL-10 levels in Balb/C mice

Acta Parasitologica ◽

10.2478/s11686-014-0204-8 ◽

2014 ◽

Vol 59 (1) ◽

Cited By ~ 1

Author(s):

Ramanathan Aparnaa ◽

Perumal Kaliraj

Keyword(s):

Cellular Response ◽

High Titer ◽

Cell Mediated Immunity ◽

Control Groups ◽

Igg Antibody ◽

Th1 Cytokine ◽

Protection Efficacy ◽

Ifn Γ ◽

High Level

AbstractALT-2, a novel antigen belonging to the chromadorea ALT-2 family of the filarial nematode is proved to clear filarial parasites in Jirds. In order to increase the protection efficacy by stimulating the cell mediated immunity, MPLA a detoxified derivative of LPS known to induce the cellular response, was used in this study as an adjuvant on mice models. ALT-2+MPLA formulation elicited a high titer of total IgG antibody, with profoundly increased levels of IgG2b. Reduced splenocyte proliferation was observed in immunized group when compared to control groups which could be attributed to many in vivo factors. The levels of IFN-γ were high in unstimulated MPLA group compared to ALT-2 stimulated MPLA group, suggesting that the ALT-2 antigen suppressed the IFN-γ levels. A high level of IL-10 was induced by the ALT-2+MPLA formulation, which inhibited the production of Th2 cytokines (IL-4, IL-5) and also reduced the Th1 cytokine (IFN-γ, IL-2) levels which are not in vogue with the classical MPLA adjuvant formulation. We propose a mechanism for this immunomodulation which involves a diminished expression of TLR-4, by which the filarial parasites have evolved to evade host immune mechanism.

Download Full-text

Design of SARS-CoV-2 RBD mRNA Vaccine Using Novel Ionizable Lipids

10.1101/2020.10.15.341537 ◽

2020 ◽

Author(s):

Uri Elia ◽

Srinivas Ramishetti ◽

Niels Dammes ◽

Erez Bar-Haim ◽

Gonna Somu Naidu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Cellular Response ◽

Humoral Response ◽

The Novel ◽

Receptor Binding Domain ◽

A Cell ◽

Luciferase Mrna ◽

Novel Coronavirus ◽

High Level

AbstractThe novel coronavirus SARS-CoV-2 has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple and rapid platform for immunization, and therefore have been employed in recent studies towards the development of a SARS-CoV-2 vaccine. In this study, we present the design of a lipid nanoparticles (LNP)-encapsulated receptor binding domain (RBD) mRNA vaccine. Several ionizable lipids have been evaluated in vivo in a luciferase mRNA reporter assay, and two leading LNPs formulation have been chosen for the subsequent RBD mRNA vaccine experiment. Intramuscular administration of LNP RBD mRNA elicited robust humoral response, high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. These novel lipids open new avenues for mRNA vaccines in general and for a COVID19 vaccine in particular.

Download Full-text

p53 drives a transcriptional program that elicits a non-cell-autonomous response and alters cell state in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008474117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23663-23673 ◽

Cited By ~ 1

Author(s):

Sydney M. Moyer ◽

Amanda R. Wasylishen ◽

Yuan Qi ◽

Natalie Fowlkes ◽

Xiaoping Su ◽

...

Keyword(s):

Transcriptional Activation ◽

Cellular Response ◽

Target Genes ◽

Transcriptional Program ◽

Sequencing Data ◽

Tissue Specific ◽

Chip Sequencing ◽

Cell State ◽

P53 Activation

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion ofMdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which includedMdm2but notp21. Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

Download Full-text

The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo

Communications Biology ◽

10.1038/s42003-021-01663-8 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Briana Rackley ◽

Chang-Soo Seong ◽

Evan Kiely ◽

Rebecca E. Parker ◽

Manali Rupji ◽

...

Keyword(s):

Malignant Transformation ◽

Malignant Tumors ◽

Light Sheet ◽

High Level Expression ◽

Oncogenic Ras ◽

Cycle Arrest ◽

Metabolic Heterogeneity ◽

High Level ◽

Sustained Activation

AbstractThe genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.

Download Full-text