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Tan B.X.,P53 Laboratory | Khoo K.H.,P53 Laboratory | Lim T.M.,National University of Singapore | Lane D.P.,P53 Laboratory
Oncotarget | Year: 2014

Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment. Key Points Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.


Goh A.M.,p53 Laboratory | Coffill C.R.,Institute of Molecular and Cell Biology | Lane D.P.,p53 Laboratory
Journal of Pathology | Year: 2011

Mutations in the TP53 (p53) gene are present in a large fraction of human tumours, which frequently express mutant p53 proteins at high but heterogeneous levels. The clinical significance of this protein accumulation remains clouded. Mouse models bearing knock-in mutations of p53 have established that the mutant p53 proteins can drive tumour formation, invasion and metastasis through dominant negative inhibition of wild-type p53 as well as through gain of function or 'neomorphic' activities that can inhibit or activate the function of other proteins. These models have also shown that mutation alone does not confer stability, so the variable staining of mutant proteins seen in human cancers reflects tumour-specific activation of p53-stabilizing pathways. Blocking the accumulation and activity of mutant p53 proteins may thus provide novel cancer therapeutic and diagnostic targets, but their induction by chemotherapy may paradoxically limit the effectiveness of these treatments. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Hu K.,CAS Hefei Key Laboratory of Materials for Energy Conversion | Schmidt N.W.,University of California at Los Angeles | Zhu R.,CAS Hefei Key Laboratory of Materials for Energy Conversion | Jiang Y.,CAS Hefei Key Laboratory of Materials for Energy Conversion | And 8 more authors.
Macromolecules | Year: 2013

Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a "minimal" mimic, a well-characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small-angle X-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs. © 2013 American Chemical Society.


Wong J.S.,P53 Laboratory | Wong J.S.,A-Life Medical | Warbrick E.,A-Life Medical | Vojtesk B.,Masaryk Memorial Cancer Institute | And 2 more authors.
Oncotarget | Year: 2013

c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4°C when compared to 37°C. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met a-chain.


Lane D.P.,p53 Laboratory | Cheok C.F.,p53 Laboratory | Brown C.J.,p53 Laboratory | Madhumalar A.,Agency for Science, Technology and Research Singapore | And 2 more authors.
Cell Cycle | Year: 2010

The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the T. adhaerens to man. In common with D. melanogaster and C. elegans, there is a single copy of the p53 gene in T. adhaerens, while in the vertebrates three p53-like genes can be found: p53, p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C. elegans and D. melanogaster. However, it is present in Placazoanand the presence of multiple distinct p53 genes in the Sea anemone N. vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53-like genes in the Arachnid loxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer Tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53. © 2010 Landes Bioscience.


Tay Y.,p53 Laboratory | Ho C.,p53 Laboratory | Droge P.,p53 Laboratory | Ghadessy F.J.,p53 Laboratory
Nucleic acids research | Year: 2010

In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage lambda integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the lambda integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications.


Guo L.,p53 Laboratory | Chua J.,p53 Laboratory | Vijayakumar D.,p53 Laboratory | Lee K.C.,p53 Laboratory | And 5 more authors.
Cell Cycle | Year: 2010

Isoforms of p53 have been described in both zebrafish and human systems based on sensitive analysis of RNA using pCR-based methods. Despite consistent evidence of the existence of these isoforms at the RNA level it has been difficult to detect the endogenous proteins in a physiological setting. In the zebrafish we have previously shown that the mRNA encoding the Δ113p53 is abundantly induced in whole embryos following induction of the p53 response by radiation, CDK inhibitors and chemotherapeutic drugs. Using a set of monoclonal antibodies raised against different domains of ZFp53 we now show for the first time clear evidence for the controlled expression of a truncated form of the ZFp53 protein, Δ113p53. the protein is present at very low levels but is induced by transcriptionally active full-length ZFp53 following the exposure of zebrafish embryos to the CDK inhibitor roscovitine. Induction of the protein is completely ZFp53 dependent and morpholinos that specifically block the expression of endogenous Δ113p53 protein selectively enhance the expression of some but not all ZFp53 responsive genes. We map the p53 response elements in the Δ113p53 promoter using functional assays and identify a region at 1593-1612 in intron 4 of ZFp53, as being crucial in the full-length promoter. thus the endogenous Δ113p53 protein, which oligomerises with the full-length ZFp53 protein, can act as a selective dominant negative inhibitor of the ZFp53 response, creating a distinct feedback response that varies the nature of the p53 response over time after exposure to an inducing signal. © 2010 Landes Bioscience.


Goh W.,p53 Laboratory | Lane D.,p53 Laboratory | Ghadessy F.,p53 Laboratory
Cell Cycle | Year: 2010

The p53 tumor suppressor plays a critical role in cancer biology, functioning as a transcription factor capable of directing cell fate. It interacts with specific DNA response elements (REs) to regulate the activity of target genes. We describe here a novel, non-radioactive assay to measure p53-DNA binding which involves the sequential use of in vitro transcription/ translation (IVT), immunoprecipitation and real-time PCR. The method reliably enables the detection of sequencespecific DNA binding of full-length p53 at low concentrations of physiologically relevant REs (<5 nM). Furthermore, we demonstrate multiplexing of 4 different REs in a single binding reaction. The use of IVT precludes the requirement for purified protein, enabling rapid characterization of the binding properties of p53 variants. Uniquely, it also offers the opportunity to add compounds during translation that might modulate and activate p53. When compared to prevailing protein-DNA binding assays, this method exhibits comparable or higher sensitivity, in addition to an expansive dynamic range afforded by the use of real-time PCR. A further extrapolation of its utility is demonstrated when the addition of a peptide known to activate p53 increased its binding to a consensus RE, consistent with published data. © 2010 Landes Bioscience.


Nirantar S.R.,P53 Laboratory | Ghadessy F.J.,P53 Laboratory
Proteomics | Year: 2011

Emulsion technology has been successfully applied to the fields of next-generation high-throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two-hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two-hybrid iCLIP library for interactants by "PCR-pulldown," using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high-throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two-hybrid system. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


PubMed | p53 Laboratory and Nanyang Technological University
Type: Journal Article | Journal: Protein engineering, design & selection : PEDS | Year: 2015

Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using -lactamase and the -lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin. Using this method, we generated integrase variants displaying improved in vitro recombination of a non-cognate substrate present in the human genome. One generalist integrase variant displaying enhanced catalytic activity was further used in a facile, single-step transformation method to introduce transgenes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.

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