P53 Laboratory P53Lab

Laboratory, Singapore

P53 Laboratory P53Lab

Laboratory, Singapore
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Terrier O.,University of Dundee | Terrier O.,University of Lyon | Marcel V.,University of Dundee | Cartet G.,University of Lyon | And 4 more authors.
Journal of Virology | Year: 2012

Previous studies have described the role of p53 isoforms, including p53 Isoforms p53ß and Δ133p53α, in the modulation of the activity of fulllength p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53 Isoforms p53ß and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53 Isoforms p53ß and Δ133p53α acting as regulators of viral production in a p53-dependent manner. © 2012, American Society for Microbiology.


Yurlova L.,ChromoTek | Derks M.,Janssen Pharmaceutical | Buchfellner A.,Reutlingen University | Hickson I.,Janssen Pharmaceutical | And 9 more authors.
Journal of Biomolecular Screening | Year: 2014

Protein-protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells.We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53-Mdm2 inhibitors. However, none exhibited intracellular activity on p53-Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53-Mdm2 interaction as well as p53-Mdm4 interaction.Here, we show that the F2H assays enable side-by-side analysis of substances' dual Mdm2-Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells. © 2014 Society for Laboratory Automation and Screening.


Bourdon J.-C.,University of Dundee | Bourdon J.-C.,French Institute of Health and Medical Research | Khoury M.P.,University of Dundee | Khoury M.P.,French Institute of Health and Medical Research | And 14 more authors.
Breast Cancer Research | Year: 2011

Introduction: Normal function of the p53 network is lost in most cancers, often through p53 mutation. The clinical impact of p53 mutations in breast cancer remains uncertain, especially where p53 isoforms may modify the effects of these p53 mutations.Methods: Expression of p53β and p53γ isoforms, the isoforms identified in normal breast tissue, was detected by reverse transcription polymerase chain reaction from a cohort of 127 primary breast tumours. Expression of p53β and p53γ isoforms was analysed in relation to clinical markers and clinical outcomes (5 years) by binary logistic regression, Cox proportional hazards regression and Kaplan-Meier survival analyses.Results: p53β and p53γ were not randomly expressed in breast cancer. p53β was associated with tumour oestrogen receptor (ER) expression, and p53γ was associated with mutation of the p53 gene. The patient group with the mutant p53 breast tumour-expressing p53γ isoform had low cancer recurrence and an overall survival as good as that of patients with wild-type p53 breast cancer. Conversely, patients expressing only mutant p53, without p53γ isoform expression, had a particularly poor prognosis.Conclusions: The determination of p53γ expression may allow the identification, independently of the ER status, of two subpopulations of mutant p53 breast cancer patients, one expressing p53γ with a prognosis as good as the wild-type p53 breast cancer patients and a second one not expressing p53γ with a particularly poor prognosis. The p53γ isoform may provide an explanation of the hitherto inconsistent relationship between p53 mutation, treatment response and outcome in breast cancer. © 2011 Bourdon et al.; licensee BioMed Central Ltd.


Marcel V.,French Institute of Health and Medical Research | Petit I.,University of Nice Sophia Antipolis | Murray-Zmijewski F.,French Institute of Health and Medical Research | Goullet De Rugy T.,French Institute of Health and Medical Research | And 7 more authors.
Cell Death and Differentiation | Year: 2012

In response to stress, p53 binds and transactivates the internal TP53 promoter, thus regulating the expression of its own isoform, Δ133p53α. Here, we report that, in addition to p53, at least four p63/p73 isoforms regulate Δ133p53 expression at transcriptional level: p63β, ΔNp63α, ΔNp63β and ΔNp73γ. This regulation occurs through direct DNA-binding to the internal TP53 promoter as demonstrated by chromatin immunoprecipitation and the use of DNA-binding mutant p63. The promoter regions involved in the p63/p73-mediated transactivation were identified using deleted, mutant and polymorphic luciferase reporter constructs. In addition, we observed that transient expression of p53 family members modulates endogenous Δ133p53α expression at both mRNA and protein levels. We also report concomitant variation of p63 and Δ133p53 expression during keratinocyte differentiation of HaCat cells and induced pluripotent stem cells derived from mutated p63 ectodermal dysplasia patients. Finally, proliferation assays indicated that Δ133p53α isoform regulates the anti-proliferative activities of p63β, ΔNp63α, ΔNp63β and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome. © 2012 Macmillan Publishers Limited. All rights reserved.


Marcel V.,Ninewells Hospital | Fernandes K.,Ninewells Hospital | Terrier O.,Ninewells Hospital | Lane D.P.,P53 Laboratory p53Lab | Bourdon J.-C.,Ninewells Hospital
Cell Death and Differentiation | Year: 2014

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. © 2014 Macmillan Publishers Limited All rights reserved.


Brown C.J.,p53 Laboratory p53Lab | Lim J.J.,p53 Laboratory p53Lab | Leonard T.,Matrix | Lim H.C.A.,Experimental Therapeutics Center | And 3 more authors.
Journal of Molecular Biology | Year: 2011

Eukaryotic initiation factor (eIF)4E is overexpressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins such as c-Myc, cyclin D1, and vascular endothelial growth factor. Inhibition of eIF4E is therefore a potential therapeutic target for human cancers. A novel peptide based on the eIF4E-binding peptide eIF4G1, where the α-helix was stabilized by the inclusion of α-helix inducers as shown by CD measurements, was synthesized. The helically stabilized peptide binds with an apparent Kd of 9.43 ± 2.57 nM, which is ∼ 15.7-fold more potent than the template peptide from which it is designed. The helically stabilized peptide showed significant biological activity at a concentration of 400 μM, unlike the naturally occurring eIFG1 peptide when measured in cell-based cap-dependent translational reporter and WST-1 (4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assays. Fusion of the template peptide and the stabilized peptide to the cell-penetrating peptide TAT produced more active but equally potent inhibitors of cap-dependent translation in cell lines. They also equally disrupted cell metabolism as measured in a WST-1 assay. Propidium iodide staining revealed that the TAT-fused, helically stabilized peptide caused more cell death than the TAT-fused eIF4G1 template peptide with substantial decreases in the G1 and G2 cell populations. Annexin-staining experiments also indicated that the TAT-fused eIF4G1 derivative peptides caused cell death by apoptosis. The results presented should offer further insight into peptidomimetics development for eIF4E. © 2010 Elsevier Ltd. All rights reserved.


PubMed | University of Dundee and p53 Laboratory p53Lab
Type: Journal Article | Journal: Cell death and differentiation | Year: 2014

In addition to the tumor suppressor p53 protein, also termed p53, the TP53 gene produces p53 and p53 through alternative splicing of exons 9 and 9 located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53 and p53 at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9/9. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and variant, supporting our experimental data. Using siRNA specifically targeting exons 9/9, we demonstrate that cell growth can be driven by modulating p53 and p53 expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53 and p53 promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53 enhanced p53 transcriptional activity on the p21 and Bax promoters, while p53 increased p53 transcriptional activity on the Bax promoter only. Moreover, p53 and p53 co-immunoprecipitate with p53 only in the presence of p53-responsive promoter. Interestingly, although p53 and p53 promote apoptosis in MCF7 cells, p53 and p53 maintain cell growth in response to TG003 in a p53-dependent manner. The dual activities of p53 and p53 isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53 and p53 regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.


PubMed | p53 Laboratory p53Lab
Type: Journal Article | Journal: Journal of molecular biology | Year: 2011

Eukaryotic initiation factor (eIF)4E is overexpressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins such as c-Myc, cyclin D1, and vascular endothelial growth factor. Inhibition of eIF4E is therefore a potential therapeutic target for human cancers. A novel peptide based on the eIF4E-binding peptide eIF4G1, where the -helix was stabilized by the inclusion of -helix inducers as shown by CD measurements, was synthesized. The helically stabilized peptide binds with an apparent K(d) of 9.432.57nM, which is 15.7-fold more potent than the template peptide from which it is designed. The helically stabilized peptide showed significant biological activity at a concentration of 400M, unlike the naturally occurring eIFG1 peptide when measured in cell-based cap-dependent translational reporter and WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assays. Fusion of the template peptide and the stabilized peptide to the cell-penetrating peptide TAT produced more active but equally potent inhibitors of cap-dependent translation in cell lines. They also equally disrupted cell metabolism as measured in a WST-1 assay. Propidium iodide staining revealed that the TAT-fused, helically stabilized peptide caused more cell death than the TAT-fused eIF4G1 template peptide with substantial decreases in the G1 and G2 cell populations. Annexin-staining experiments also indicated that the TAT-fused eIF4G1 derivative peptides caused cell death by apoptosis. The results presented should offer further insight into peptidomimetics development for eIF4E.

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