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Masaryk Memorial Cancer Institute and University of Dundee | Date: 2013-07-26

The present invention provides methods and compositions for modulating cell senescence and cell proliferation using isoforms of the p53 tumor suppressor protein. The methods and compositions of the invention find use in inhibiting cancer cell growth or in generating populations of cells for tissue regeneration through the modulation of cell senescence and proliferation.

Palecek E.,Academy of Sciences of the Czech Republic | Tkac J.,Slovak Academy of Sciences | Bartosik M.,Masaryk Memorial Cancer Institute | Bertok T.,Slovak Academy of Sciences | And 2 more authors.
Chemical Reviews | Year: 2015

Significant progress has been done in the electrochemical (EC) analysis of practically all proteins, based on the electroactivity of amino acid (aa) residues in proteins. From a transducer point of view, it can be anticipated that many different ways of how nanomaterials can be integrated into the EC detection platform of detection will be developed. This can be done by direct modification of electroactive surfaces by nanomaterials or by advanced patterning protocol and by using nanomaterials as amplification tags, helping to produce lectin biosensors/biochips working in an ultrasensitive and selective way. Further, it can be anticipated that EC-based biosensors will compete in a future with instrumental techniques or lectin microarrays only if such devices are integrated into a biochip format offering multiplexed glycan measurements. Moreover, it has been shown that some glucosamine-containing poly- and oligosaccharides are electroactive under conditions close to physiological and that most polysaccharides and glycans can be transformed into electrochemically active substances by a simple chemical modification. Usually 4-10 biomarkers have to be detected to obtain good specificity and selectivity of detection. EC detection appears particularly advantageous for the preparation of low-density chips with this number of biomarkers.

Pohanka M.,University of Hradec Kralove | Dobes P.,Masaryk Memorial Cancer Institute
International Journal of Molecular Sciences | Year: 2013

Caffeine is an alkaloid with a stimulant effect in the body. It can interfere in transmissions based on acetylcholine, epinephrine, norepinephrine, serotonin, dopamine and glutamate. Clinical studies indicate that it can be involved in the slowing of Alzheimer disease pathology and some other effects. The effects are not well understood. In the present work, we focused on the question whether caffeine can inhibit acetylcholinesterase (AChE) and/or, butyrylcholinesterase (BChE), the two enzymes participating in cholinergic neurotransmission. A standard Ellman test with human AChE and BChE was done for altering concentrations of caffeine. The test was supported by an in silico examination as well. Donepezil and tacrine were used as standards. In compliance with Dixon's plot, caffeine was proved to be a non-competitive inhibitor of AChE and BChE. However, inhibition of BChE was quite weak, as the inhibition constant, Ki, was 13.9 ± 7.4 mol/L. Inhibition of AChE was more relevant, as Ki was found to be 175 ± 9 μmol/L. The predicted free energy of binding was -6.7 kcal/mol. The proposed binding orientation of caffeine can interact with Trp86, and it can be stabilize by Tyr337 in comparison to the smaller Ala328 in the case of human BChE; thus, it can explain the lower binding affinity of caffeine for BChE with reference to AChE. The biological relevance of the findings is discussed. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Muller P.,Masaryk Memorial Cancer Institute | Ruckova E.,Masaryk Memorial Cancer Institute | Halada P.,Academy of Sciences of the Czech Republic | Coates P.J.,University of Dundee | And 3 more authors.
Oncogene | Year: 2013

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-β in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment. © 2013 Macmillan Publishers Limited. All rights reserved.

Matoulkova E.,Masaryk Memorial Cancer Institute | Michalova E.,Masaryk Memorial Cancer Institute | Vojtesek B.,Masaryk Memorial Cancer Institute | Hrstka R.,Masaryk Memorial Cancer Institute
RNA Biology | Year: 2012

The untranslated regions (UTRs) at the 3′ end of mRNA transcripts contain important sequences that influence the fate of mRNA and thus proteosynthesis. In this review, we summarize the information known to date about 3′ end processing, sequence characteristics including related binding proteins and the role of 3′ UTRs in several selected signaling pathways to delineate their importance in the regulatory processes in mammalian cells. In addition to reviewing recent advances in the more well-known aspects, such as cleavage and polyadenylation processes that influence mRNA stability and location, we concentrate on some newly emerging concepts of the role of the 3′ UTR, including alternative polyadenylation sites in relation to proliferation and differentiation and the recognition of the multi-functional properties of non-coding RNAs, including miRNAs that commonly target the 3′ UTR. The emerging picture is of a highly complex set of regulatory systems that include autoregulation, cooperativity and competition to fine tune proteosynthesis in context-dependent manners. © 2012 Landes Bioscience.

Pastorek J.,Slovak Academy of Sciences | Pastorekova S.,Slovak Academy of Sciences | Pastorekova S.,Masaryk Memorial Cancer Institute
Seminars in Cancer Biology | Year: 2015

The tumor microenvironment includes a complicated network of physiological gradients contributing to plasticity of tumor cells and heterogeneity of tumor tissue. Hypoxia is a key component generating intratumoral oxygen gradients, which affect the cellular expression program and lead to therapy resistance and increased metastatic propensity of weakly oxygenated cell subpopulations. One of the adaptive responses of tumor cells to hypoxia involves the increased expression and functional activation of carbonic anhydrase IX (CA IX), a cancer-related cell surface enzyme catalyzing the reversible conversion of carbon dioxide to bicarbonate ion and proton. Via its catalytic activity, CA IX participates in regulation of intracellular and extracellular pH perturbations that result from hypoxia-induced changes in cellular metabolism producing excess of acid. Through the ability to regulate pH, CA IX also facilitates cell migration and invasion. In addition, CA IX has non-catalytic function in cell adhesion and spreading. Thus, CA IX endows tumor cells with survival advantages in hypoxia/acidosis and confers an increased ability to migrate, invade and metastasize. Accordingly, CA IX is expressed in a broad range of tumors, where it is associated with prognosis and therapy outcome. Its expression pattern and functional implications in tumor biology make CA IX a promising therapeutic target, which can be hit either by immunotherapy with monoclonal antibodies or with compounds inhibiting its enzyme activity. The first strategy has already reached the clinical trials, whereas the second one is still in preclinical testing. Both strategies indicate that CA IX can become a clinically useful anticancer target, but urge further efforts toward better selection of patients for immunotherapy and deeper understanding of tumor types, clinical situations and synthetic lethality interactions with other treatment approaches. © 2014.

Fraser J.A.,University of Edinburgh | Vojtesek B.,Masaryk Memorial Cancer Institute | Hupp T.R.,University of Edinburgh
Journal of Biological Chemistry | Year: 2010

p53 is a thermodynamically unstable protein containing a conformationally flexible multiprotein docking site within the DNA-binding domain. A combinatorial peptide chip used to identify the novel kinase consensus site RXSΦ(K/D) led to the discovery of a homologous phosphorylation site in the S10 β-strand of p53 at Ser269. Overlapping peptide libraries confirmed that Ser269 was a phosphoacceptor site in vitro, and immunochemical approaches evaluated whether p53 is phosphorylated in vivo at Ser269. Mutation or phosphorylation of p53 at Ser269 attenuates binding of the p53-specific monoclonal antibody DO-12, identifying an assay for measuring Ser269 phosphorylation of p53 in vivo. The mAb DO-12 epitope of p53 is masked via phosphorylation in a range of human tumor cells with WT p53 status, as defined by increased mAb DO-12 binding to endogenous p53 after phosphatase treatment. Phospho-Ser269-specific monoclonal antibodies were generated and used to demonstrate that p53 phosphorylation is induced at Ser269 after irradiation with kinetics similar to those of p53 protein induction. Phosphomimetic mutation at Ser 269 inactivated the transcription activation function and clonogenic suppressor activity of p53. These data suggest that the dynamic equilibrium between native and unfolded states of WT p53 can be modulated by phosphorylation of the conformationally flexible multiprotein binding site in the p53 DNA-binding domain. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Bartosik M.,Masaryk Memorial Cancer Institute
Chemicke Listy | Year: 2014

Electrochemistry could be a valuable tool in nucleic acids research by offering relatively inexpensive instrumentation, short assay times and high sensitivity. Interesting applications can be found in the literature, including detection of oncogenes or tumor suppressor genes, analysis of point mutations in DNA, or determination of viruses and bacteria. Moreover, new strategies are being developed for detection of microRNAs, or for analysis of DNA methylation, both important in carcinogenesis. These novel approaches usually involve the use of various electroactive labels, nanomaterials, enzymes or magnetic particles, rendering them more sensitive, selective and efficient. In addition, electrochemistry was applied also in studies of DNA damage and interactions with other molecules. Numerous chemicals damage DNA in such a way that they alters electrochemical properties of DNA itself, such as oxygen radicals, aromatic hydrocarbons, alkylating agents and pesticides. DNA-modified electrodes could thus serve as simple biosensors for detection of environmental pollutants as well as potential antitumor drugs.

Temporal arteries are typically below detectable levels of PET scanners, which repeatedly showed to be limiting in finding increased F-FDG accumulation even in histologically proven cases of giant cell arteritis. In 2010, Gaemperli and coworkers showed metabolic active inflammation in temporal arteries in an experimental study using PET with [C]-PK11195 combined with CT angiography. Herein, we present the case where an increased accumulation of routinely used tracer F-FDG can be identified directly in temporal and occipital arteries and even in smaller branches using a common hybrid PET/CT scanner if a brain acquisition protocol is applied.

Mlynarczyk C.,University Paris Diderot | Fahraeus R.,University Paris Diderot | Fahraeus R.,Masaryk Memorial Cancer Institute
Nature Communications | Year: 2014

Endoplasmic reticulum (ER) stress occurs in poorly perfused tissues and activates the p53 isoform p53/47 to promote G2 arrest via 14-3-3σ. This contrasts with the p21 CDKN1A -dependent G1 arrest caused by p53 following DNA damage. It is not known how cells respond to conditions when both pathways are activated. Here we show that p53/47 prevents p53-induced p21 transcription during ER stress and that both isoforms repress p21 mRNA translation. This prevents p21 from promoting COP1-mediated 14-3-3σ degradation and leads to G2 arrest. DNA damage does not result in p53-dependent induction of p21 during ER stress and instead results in an increase in p53-induced apoptosis. This illustrates how p53 isoforms target an intrinsic balance between the G1 and G2 checkpoints for cell cycle coordination and demonstrates an ER stress-dependent p53 pathway that suppresses p21 and lowers the apoptotic threshold to genotoxic drugs. © 2014 Macmillan Publishers Limited. All rights reserved.

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