• Proteomic response of Schizosaccharomyces pombe to static and oscillating extremely low-frequency electromagnetic fields.

      Sinclair, John; Weeks, Mark E; Butt, Amna; Worthington, Jessica L; Akpan, Akunna; Jones, Nic; Waterfield, Michael D; Allan, Donald; Timms, John F; Ludwig Institute for Cancer Research, Department of Biochemistry and Molecular Biology University College London, UK. (2006-09)
      There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.
    • Proteomics profiling of interactome dynamics by colocalisation analysis (COLA).

      Mardakheh, F; Sailem, H; Kümper, S; Tape, C; McCully, R; Paul, A; Anjomani-Virmouni, S; Jørgensen, Claus; Poulogiannis, G; Marshall, C; et al. (2016-11-08)
      Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein-protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.
    • Proteomics techniques and their application to hematology.

      Cristea, Ileana M; Gaskell, Simon J; Whetton, Anthony D; Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester, United Kingdom. (2004-05-15)
      The recent sequencing of a number of genomes has raised the level of opportunities for studies on proteins. This area of research has been described with the all-embracing term, proteomics. In proteomics, the use of mass spectrometric techniques enables genomic databases to be used to establish the identity of proteins with relatively little data, compared to the era before genome sequencing. The use of related analytical techniques also offers the opportunity to gain information on regulation, via posttranslational modification, and potential new diagnostic and prognostic indicators. Relative quantification of proteins and peptides in cellular and extracellular material remains a challenge for proteomics and mass spectrometry. This review presents an analysis of the present and future impact of these proteomic technologies with emphasis on relative quantification for hematologic research giving an appraisal of their potential benefits.
    • A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

      Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Sanderson, Helen S; Gardiner, Fiona; Kiseleva, Elena; Goldberg, Martin W; Drummond, Sheona P; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk (2007)
      This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.
    • A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).

      Kiseleva, Elena; Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Morozova, Ksenia N; Gardiner, Fiona; Goldberg, Martin W; Drummond, Sheona P; Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ru (2007)
      This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.
    • A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy.

      Murray, Stephen M; Kiseleva, Elena; TEM Service Facility, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2008)
      This article describes a protocol that details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immunogold labelling of proteins, and visualization by Field Emission Scanning Electron Microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high resolution microscopy. The nuclear isolation step is performed by enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of nuclei by centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs), and associated cytoskeletal structures. Samples, once processed for FESEM, can be stored under vacuum for weeks, allowing considerable time for image acquisition.
    • Proton nuclear magnetic resonance imaging as a predictor of the outcome of photodynamic therapy of tumours.

      Moore, James V; Dodd, Nicholas J F; Wood, B; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester. (1989-09)
    • Proton therapy in supradiaphragmatic lymphoma: predicting treatment-related mortality to help optimize patient selection

      Ntentas, G.; Dedeckova, K.; Andrlik, M.; Aznar, Marianne Camille; Shakir, R.; Ramroth, J.; Begum, R.; Kubeš, J.; Darby, S. C.; Mikhaeel, N. G.; et al. (2021)
      Purpose: In some Hodgkin lymphoma (HL) patients, proton beam therapy (PBT) may reduce the risk of radiation-related cardiovascular disease (CVD) and second cancers (SC) compared with photon radiotherapy (photon-RT). Our aim was to identify those who benefit most from PBT in terms of predicted 30-year absolute mortality risks (AMR30) from CVD and SC, taking into account individual background, chemotherapy, radiation and smoking-related risks. Methods and materials: Eighty patients with supradiaphragmatic HL treated with PBT during 2015-2019 were re-planned using optimal photon-RT. To identify patients predicted to derive the greatest benefit from PBT compared to Photon-RT, doses and AMR30 from CVD and SC of the lung, breast and esophagus were compared for all patients and across patient subgroups. Results: For patients with mediastinal disease below the origin of the left main coronary artery (n=66, 82%), PBT reduced mean dose to heart, left ventricle and heart valves by 1.0, 2.7 and 3.6 Gray (Gy) respectively. Based on US mortality rates, PBT reduced CVD AMR30 by 0.2%, from 5.9% to 5.7%. The benefit was larger if the mediastinal disease overlapped longitudinally with the heart by ≥40% (n=23, 29%), where PBT reduced mean dose to heart, left ventricle and heart valves by 3.2, 5.6, and 5.1Gy respectively, and reduced CVD AMR30 by 0.8%, from 7.0% to 6.2%. For patients with axillary disease (n=25, 31%), PBT reduced mean lung dose by 2.8Gy and lung cancer AMR30 by 0.6%, from 2.7% to 2.1%. Breast and esophageal doses were also lower with PBT but effects on AMR30 were negligible. The effect of smoking on CVD and lung cancer AMR30 was much larger than radiation and chemotherapy and the differences between radiation modalities. Conclusions: The predicted benefit of PBT is not universal and is limited to certain categories of lymphoma patients with lower mediastinal or axillary disease. Smoking cessation should be strongly encouraged in smokers requiring thoracic radiotherapy.
    • A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence.

      Komseli, E; Pateras, I; Krejsgaard, T; Stawiski, K; Rizou, S; Polyzos, A; Roumelioti, F; Chiourea, M; Mourkioti, I; Paparouna, E; et al. (2018-01-10)
      Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now.
    • Proximal 6q, a region showing allele loss in primary breast cancer.

      Orphanos, Vassilis; McGown, Gail; Hey, Yvonne; Boyle, John M; Santibanez-Koref, Mauro F; Cancer Research Campaign Department of Cancer Genetics, Christie CRC Research Centre, Manchester, UK. (1995-02)
      To define regions of deletion of chromosome 6q in breast cancer, we scored 18 (CA)n microsatellites for allelic imbalance (AI) in 42 paired blood/tumour samples. Heterozygosity frequencies of the markers in the sample population ranged from 31% to 92% (mean 68%). Two regions of the chromosome arm showed AI values greater than the background range of 10-22% (mean 17%) of informative cases that was observed with five markers spanning 6q21-q25.2. Firstly, seven markers gave AI values that averaged 35% in a region flanked by D6S313 (AI = 10%) at 6q13 and D6S283 (AI = 17%) at 6q16.3-21. The second region showed marginally increased AI at 6q25.2-q27 and included D6S193, previously shown to be close to a tumour-suppressor gene involved in ovarian carcinoma. Since AI of 6q in breast cancer was shown previously to be due predominantly to loss of heterozygosity, our results suggest the presence of at least two tumour-suppressor genes on 6q that are involved in breast cancer. The proximal region has not been recognised in breast cancer before and is involved in a higher frequency of tumours than the distal region.
    • A PSP94-derived peptide PCK3145 inhibits MMP-9 secretion and triggers CD44 cell surface shedding: implication in tumor metastasis.

      Annabi, Borhane; Bouzeghrane, Mounia; Currie, Jean-Christophe; Hawkins, Robert E; Dulude, Hélène; Daigneault, Luc; Ruiz, Marcia; Wisniewski, Jan; Garde, Seema; Rabbani, Shafaat A; et al. (2005)
      PURPOSE: PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. EXPERIMENTAL DESIGN: HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). RESULTS: We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. CONCLUSIONS: Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.
    • PtdIns5P and Pin1 in oxidative stress signaling.

      Keune, Willem-Jan; Jones, David R; Divecha, Nullin; Inositide Laboratory CRUK, The Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom (2013-05)
      Oxidative signaling is important in cellular health, involved in aging and contributes to the development of several diseases such as cancer, neurodegeneration and diabetes. Correct management of reactive oxygen species (ROS) prevents oxidative stress within cells and is imperative for cellular wellbeing. A key pathway that is regulated by oxidative stress is the activation of proline-directed stress kinases (p38, JNK). Phosphorylation induced by these kinases is often translated into cellular outcome through the recruitment of the prolyl-isomerase Pin1. Pin1 binds to phosphorylated substrates using its WW-domain and can induce conformational changes in the target protein through its prolyl-isomerase activity. We show that exposure of cells to UV irradiation or hydrogen peroxide (H2O2), induces the synthesis of the phosphoinositide second messenger PtdIns5P in part by inducing the interaction between phosphatidylinositol-5-phosphate 4-kinase (PIP4K) enzymes that remove PtdIns5P, with Pin1. In response to H2O2 exposure, Murine Embryonic Fibroblasts (MEFs) derived from Pin1(-/-) mice showed increased cell viability and an increased abundance of PtdIns5P compared to wild-type MEFs. Decreasing the levels of PtdIns5P in Pin1(-/-) MEFs decreased both their viability in response to H2O2 exposure and the expression of genes required for cellular ROS management. The decrease in the expression of these genes manifested itself in the increased accumulation of cellular ROS. These data strongly argue that PtdIns5P acts as a stress-induced second messenger that can calibrate how cells manage ROS.
    • PtdIns5P is an oxidative stress-induced second messenger that regulates PKB activation.

      Jones, David R; Foulger, Rebecca; Keune, Willem-Jan; Bultsma, Yvette; Divecha, Nullin; Cancer Research UK Inositide Laboratory, The Paterson Institute for Cancer Research, University of Manchester, Manchester UK. (2012-12-14)
      Oxidative stress initiates signaling pathways, which protect from stress-induced cellular damage, initiate apoptosis, or drive cells into senescence or into tumorigenesis. Oxidative stress regulates the activity of the cell survival factor PKB, through the regulation of PtdIns(3,4,5)P(3) synthesis. Whether oxidative stress regulates other phosphoinositides to control PKB activation is not clear. Here we show that PtdIns5P is a redox-regulated second messenger. In response to hydrogen peroxide (H(2)O(2)), we measured an increase in PtdIns5P in cells derived from human osteosarcoma, U2OS (5-fold); breast tumors, MDA-MB-468 (2-fold); and fibrosarcoma, HT1080 (3-fold); and in p53-null murine embryonic fibroblasts (8-fold). In U2OS cells, the increase in H(2)O(2)-dependent PtdIns5P did not require mTOR, PDK1, PKB, ERK, and p38 signaling or PIKfyve, a lipid kinase that increases PtdIns5P in response to osmotic and oncogenic signaling. A reduction in H(2)O(2)-induced PtdIns5P levels by the overexpression of PIP4K revealed its role in PKB activation. Suppression of H(2)O(2)-induced PtdIns5P generation reduced PKB activation and, surprisingly, reduced cell sensitivity to growth inhibition by H(2)O(2). These data suggest that inhibition of PIP4K signaling might be useful as a novel strategy to increase the susceptibility of tumor cells to therapeutics that function through increased oxidative stress.-Jones, D. R., Foulger, R., Keune, W.-J., Bultsma, Y., Divecha, N. PtdIns5P is an oxidative stress-induced second messenger that regulates PKB activation.
    • PTPsigma promotes retinal neurite outgrowth non-cell-autonomously.

      Sajnani, Gustavo; Aricescu, A Radu; Jones, E Yvonne; Gallagher, John T; Alete, Daniel; Stoker, Andrew; Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK. (2005-10)
      The receptor-like protein tyrosine phosphatase (RPTP) PTPsigma controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPsigma have been thought to be cell-autonomous, the possibility that RPTPs related to PTPsigma also have non-cell-autonomous signaling functions during axon development has also been supported genetically. Here we report that a cell culture substrate made from purified PTPsigma ectodomains supports retinal neurite outgrowth in cell culture. We show that a receptor for PTPsigma must exist on retinal axons and that binding of PTPsigma to this receptor does not require the known, heparin binding properties of PTPsigma. The neurite-promoting potential of PTPsigma ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPsigma. Furthermore, we demonstrate that heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPsigma substrate, by competing for binding to this same domain. This is the first direct evidence of a non-cell-autonomous, neurite-promoting function of PTPsigma and of a potential role for heparin-related oligosaccharides in modulating neurite promotion by an RPTP.
    • Publisher Correction: Breast cancer management pathways during the COVID-19 pandemic: outcomes from the UK 'Alert Level 4' phase of the B-MaP-C study

      Dave, R. V.; Kim, B.; Courtney, A.; O'Connell, R.; Rattay, T.; Taxiarchi, V. P.; Kirkham, J. J.; Camacho, E. M.; Fairbrother, P.; Sharma, N.; et al. (2021)
    • Publisher correction: genomic instability in mutant p53 cancer cells upon entotic engulfment.

      Mackay, H; Moore, D; Hall, Callum; Birkbak, N; Jamal-Hanjani, M; Karim, S; Phatak, V; Piñon, L; Morton, J; Swanton, C; et al. (2018-08-28)
      The original version of this article incorrectly omitted an affiliation of Patricia A. J. Muller: 'Cancer Research UK Manchester Institute, The University of Manchester | Alderley Park, Manchester, SK10 4TG, UK'. This has been corrected in both the PDF and HTML versions of the Article.
    • Publisher Correction: In silico prediction of housekeeping long intergenic non-coding RNAs reveals HKlincR1 as an essential player in lung cancer cell survival

      Memon, Danish; Bi, J; Miller, Crispin J; RNA Biology Group, CRUK Manchester Institute, The University of Manchester, Alderley Park, Manchester, SK10 4TG, (2019)
    • Publisher correction: Novel pleiotropic risk loci for melanoma and nevus density implicate multiple biological pathways.

      Duffy, DL; Zhu, G; Li, X; Sanna, M; Iles, MM; Jacobs, LC; Evans, DM; Yazar, S; Beesley, J; Law, MH; et al. (2019)
      The original version of this Article contained errors in the spelling of the authors Fan Liu and M. Arfan Ikram, which were incorrectly given as Fan Lui and Arfan M. Ikram. In addition, the original version of this Article also contained errors in the author affiliations which are detailed in the associated Publisher Correction.
    • Publisher correction: ultraviolet radiation-induced DNA damage is prognostic for outcome in melanoma

      Trucco, Lucas D; Mundra, Piyushkumar A; Hogan, Kate; Garcia-Martinez, Pablo; Viros, Amaya; Mandal, Amit Kumar; Macagno, N; Gaudy-Marqueste, C; Allan, D; Baenke, Franziska; et al. (2018)
      In the version of this article originally published, Extended Data Fig. 3 was incorrect. A duplicate of Extended Data Fig. 4 was uploaded in place of Extended Data Fig. 3. Extended Data Fig. 3 has now been uploaded. The error has been fixed in the PDF and HTML versions of this article.
    • Pulse irradiation of aqueous solutions containing ferrous and chloride ions: Reaction between Cl2- and HO2.

      Gilbert, C W; Ingalls R B; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1977)