• A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

      Janke, Carsten; Magiera, Maria M; Rathfelder, Nicole; Taxis, Christof; Reber, Simone; Maekawa, Hiromi; Moreno-Borchart, Alexandra; Doenges, Georg; Schwob, Etienne; Schiebel, Elmar; et al. (2004-08)
      Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.
    • Viability of haemopoietic progenitors from whole blood, bone marrow and leukapheresis product: effects of storage media, temperature and time.

      Pettengell, Ruth; Woll, Penella J; O'Connor, D A; Dexter, T Michael; Testa, Nydia G; CRC Department of Experimental Haematology, Christie Hospital, Manchester, UK. (1994-11)
      High-dose cytotoxic chemotherapy can be supported with autologous haemopoietic cells. Cryopreserved bone marrow has conventionally been used for this but blood stem cells are now in common use. We have examined different storage conditions for haemopoietic cells from bone marrow, leukapheresis product and whole blood primed with chemotherapy and filgrastim. The mean number of GM-CFC surviving cryopreservation was 80% in leukapheresis product (95% CI 66-96). At 4 degrees C, GM-CFC viability in all three sources of haemopoietic progenitors declined at the same rate, with mean recovery at 24 h of 90% (95% CI 82-98), at 48 h of 68% (95% CI 61-75) and at 72 h of 47% (95% CI 40-53). Progenitors remained viable for longer in autologous serum and citrate phosphate dextrose or Iscove's medium than in phosphate buffered saline. There was no significant difference in GM-CFC recovery from whole blood or whole blood buffy layer at 4 degrees C. The capacity to generate and sustain haemopoiesis in long-term culture is a feature of the more primitive progenitor cells. This capacity was similar in cryopreserved bone marrow and leukapheresis product, cryopreserved or stored for up to 5 days at 4 degrees C, suggesting that long-term culture-initiating cells are more resilient than colony-forming cells when cryopreserved or stored at 4 degrees C. These data indicate that primed whole blood, in addition to leukapheresis product and bone marrow, could be stored at 4 degrees C and used to support multicyclic high-dose chemotherapy.
    • Vinculins interaction with talin is essential for mammary epithelial differentiation

      Wang, Pengbo; Wu, J; Wood, A; Jones, M; Pedley, R; Li, W; Ross, RS; Ballestrem, C; Gilmore, AP; Streuli, CH; et al. (2019)
      Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.
    • Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication.

      Laman, Heike; Coverley, Dawn; Krude, Torsten; Laskey, Ronald; Jones, Nic; Gene Regulation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom. (2001-01)
      The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.
    • Viral-encoded cyclins.

      Laman, Heike; Mann, David J; Jones, Nic; Molecular Oncology Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK. (2000-02)
      D-type cyclin homologs have been found in the genomes of herpesviruses associated with neoplasias. They appear to exploit features of G(1) cyclins but extend their properties to allow for deregulation of the cell cycle. Advances in the study of the molecular basis for these novel features as well as the potential role of viral cyclins in tumorigenesis are addressed.
    • Visualization and Image Analysis of Yeast Cells.

      Bagley, Steven; Imaging and Cytometry, Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester (2016)
      When converting real-life data via visualization to numbers and then onto statistics the whole system needs to be considered so that conversion from the analogue to the digital is accurate and repeatable. Here we describe the points to consider when approaching yeast cell analysis visualization, processing, and analysis of a population by screening techniques.
    • Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities.

      Barkauskaite, Eva; Brassington, A; Tan, E; Warwicker, J; Dunstan, M; Banos, Benito; Lafite, P; Ahel, M; Mitchison, T; Ahel, Ivan; et al. (2013-08-06)
      Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.
    • Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells.

      Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; 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)
      Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3-5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging.
    • Vitamin C and Doxycycline: a synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs).

      De Francesco, Ernestina M; Bonuccelli, G; Maggiolini, M; Sotgia, F; Lisanti, M; Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy (2017-06-09)
      Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.This type of metabolic Achilles' heel will allow us and others to more effectively "starve" the CSC population.
    • Vitamin D Pathway Gene Polymorphisms and Keratinocyte Cancers: A Nested Case-Control Study and Meta-Analysis.

      Von Schuckmann, L; Law, M; Montgomery, G; Green, Adèle C; Van der Pols, J; The University of Queensland, School of Public Health, Herston, Australia (2016-05)
      The vitamin D endocrine system is implicated in skin carcinogenesis and polymorphisms in genes associated with the vitamin D receptor (VDR) gene may alter the risk of keratinocyte cancers (basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)).
    • Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization.

      Kagermeier-Schenk, B; Wehner, D; Ozhan-Kizil, G; Yamamoto, H; Li, J; Kirchner, K; Hoffmann, C; Stern, Peter L; Kikuchi, A; Schambony, A; et al. (2011-12-13)
      Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.
    • WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment.

      Wang, Pengbo; Dreger, M; Madrazo, E; Williams, C; Samaniego, R; Hodson, N; Monroy, F; Baena, Esther; Sánchez-Mateos, P; Hurlstone, A; et al. (2018-07-09)
      Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.
    • Weekly platinum chemotherapy for recurrent ovarian cancer.

      Clamp, Andrew R; Jayson, Gordon C (2002-01-07)
    • Welcome to Tumour Virus Research

      Stern, Peter L; Banks, L.; Manchester Cancer Research Centre, University of Manchester, Manchester, M20 4G (2021)
    • What are mast cells for?

      Dexter, T Michael; Stoddart, R; Quazzaz, S; Department of Experimental Haematology, Christie Hospital and Holt Radium Institute, Manchester. (1981-05-14)
    • What Do the Guidelines Say for Metastatic Prostate Cancer Starting Androgen Deprivation Therapy? National Comprehensive Cancer Network, European Society for Medical Oncology, and European Association of Urology recommendations

      Yu, EY; Gillessen, Silke; Mottet, N; Department of Medicine, Division of Oncology, University of Washington, Seattle, WA, USA; (2018)
      Clinical trial data forms the foundation of how we treat men with metastatic prostate cancer who are initiating therapy. However, clinical trial data does not answer everything; hence, good clinical practice, pragmatism, and occasionally extrapolation drives how we manage these patients. Fortunately, multiple international guideline committees meet regularly and offer clinical guidance. In this mini-review, we focus on the United States National Comprehensive Cancer Network, European Society for Medical Oncology, and European Association of Urology (EAU) recommendations for the initial treatment of metastatic prostate cancer.
    • What is an apoptotic index measuring? A commentary.

      Potten, Christopher S; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1996-12)
    • What is apoptosis, and why is it important?

      Renehan, Andrew G; Booth, Catherine; Potten, Christopher S; Cancer Research Campaign Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. arenehan@picr.man.ac.uk (2001-06-23)
    • What's new in the management of cutaneous T-cell lymphoma?

      McFarlane, V; Friedmann, P S; Illidge, Timothy M; Southampton Oncology Centre, Southampton University NHS Trust, Southampton S016 6YD, UK. (2005-05)
      The aetiology and clinical management of primary cutaneous T-cell lymphoma (CTCL) and specifically of mycosis fungoides and Sezary syndrome are poorly defined. Interesting new insights into CTCL disease biology as well as a number of emerging of novel therapeutic interventions make this an increasingly interesting area for dermatologists and oncologists involved in the treatment of CTCL. This review article covers much of this new information including new drugs, such as denileukin diftitox (Ontak) a targeted cytotoxic biological agent, Bexarotene an RXR selective retinoid, anti-CD4 monoclonal antibodies (mAb), new cytotoxics agents and vaccines.