Acquisition of aneuploidy drives mutant p53-associated gain-of-function phenotypes

Abstractp53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.

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Mutant p53 oncogenicity: dominant-negative or gain-of-function?

Carcinogenesis ◽

10.1093/carcin/bgaa117 ◽

2020 ◽

Author(s):

Yan Stein ◽

Ronit Aloni-Grinstein ◽

Varda Rotter

Keyword(s):

P53 Protein ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Mutant P53 ◽

Gain Of Function ◽

In Vivo Models ◽

Oncogenic Activity ◽

The Impact

Abstract The p53 protein is mutated in about 50% of human cancers. Aside from losing its tumor-suppressive activities, mutant p53 may acquire pro-oncogenic activity, which is facilitated by two underlying mechanisms. The first mechanism is the inhibition of co-expressed wild-type p53 (WTp53) activity, dubbed the dominant-negative effect (DNE). The second mechanism is a neomorphic pro-oncogenic activity that does not involve the inhibition of WTp53, termed gain-of-function (GOF). Throughout the years, both mechanisms were demonstrated in a plethora of in vitro and in vivo models. However, whether both account for protumorigenic activities of mutant p53 and in which contexts is still a matter of ongoing debate. Here, we discuss evidence for both DNE and GOF in a variety of models. These models suggest that both GOF and DNE can be relevant, but are highly dependent on the specific mutation type, genetic and cellular context and even the phenotype that is being assessed. In addition, we discuss how mutant and WTp53 might not exist as two separate entities, but rather as a continuum that may involve a balance between the two forms in the same cells, which could be tilted by various factors and drugs. Further elucidation of the factors that dictate the balance between the WT and mutant p53 states, as well as the factors that govern the impact of DNE and GOF in different cancer types, may lead to the development of more effective treatment regimens for cancer patients.

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Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

Infection and Immunity ◽

10.1128/iai.00292-20 ◽

2020 ◽

Vol 88 (10) ◽

Author(s):

Xuyao Jiao ◽

Sarah Smith ◽

Gabrielle Stack ◽

Qi Liang ◽

Allan Bradley ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Typhoid Fever ◽

Severe Disease ◽

Genetically Engineered ◽

Human Monoclonal Antibodies ◽

Genetically Engineered Mice ◽

Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

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CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

Journal of Biological Chemistry ◽

10.1074/jbc.m401854200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45887-45896 ◽

Cited By ~ 45

Author(s):

Mark J. Demma ◽

Serena Wong ◽

Eugene Maxwell ◽

Bimalendu Dasmahapatra

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

P53 Protein ◽

Binding Activity ◽

Mutant P53 ◽

Dependent Manner ◽

Wild Type ◽

Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3740-3747.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3740-3747

Author(s):

H W Stürzbecher ◽

C Addison ◽

J R Jenkins

Keyword(s):

Complex Formation ◽

Protein Complexes ◽

P53 Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Mutant P53 ◽

Cos Cells

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

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Nucleophosmin Is Required for DNA Integrity and p19Arf Protein Stability

Molecular and Cellular Biology ◽

10.1128/mcb.25.20.8874-8886.2005 ◽

2005 ◽

Vol 25 (20) ◽

pp. 8874-8886 ◽

Cited By ~ 143

Author(s):

Emanuela Colombo ◽

Paola Bonetti ◽

Eros Lazzerini Denchi ◽

Paola Martinelli ◽

Raffaella Zamponi ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Genetic Alterations ◽

Dna Integrity ◽

Senescent Phenotype ◽

Tumor Suppressive Function ◽

Proliferation In Vitro

ABSTRACT Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19Arf and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.

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Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3740 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3740-3747 ◽

Cited By ~ 40

Author(s):

H W Stürzbecher ◽

C Addison ◽

J R Jenkins

Keyword(s):

Complex Formation ◽

Protein Complexes ◽

P53 Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Mutant P53 ◽

Cos Cells

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In vivo and in vitro characterization of immediate release dosage forms containing n-acetylcysteine using the dynamic open flow-through test apparatus

10.26226/morressier.57d6b2b7d462b8028d88dd29 ◽

2016 ◽

Author(s):

Maximilian Sager

Keyword(s):

Immediate Release ◽

Dosage Forms ◽

Test Apparatus ◽

Open Flow ◽

In Vitro Characterization ◽

Flow Through

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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