• Variations in chromosome numbers and their possible relationship to the development of 8-azaguanine resistance in V79 Chinese hamster cells.

      Fox, Margaret; Radacic, M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, United Kingdom (1982-01)
    • Variations in supportive care needs of patients after diagnosis of localised cutaneous melanoma: a 2-year follow-up study.

      Beesley, V; Smithers, B; O'Rourke, P; Janda, M; Khosrotehrani, K; Green, Adèle C; Population Health Department, QIMR Berghofer Medical Research Institute, Locked Bag 2000 Royal Brisbane Hospital, Brisbane, QLD, 4029, Australia. (2016-08-25)
      We aimed to describe variations in unmet supportive care needs of patients diagnosed with localised melanoma at high risk of recurrence and factors associated with initial and persisting moderate-to-high needs.
    • The variety of leukemic stem cells in myeloid malignancy.

      Wiseman, Daniel H; Greystoke, Brigit F; Somervaille, Tim C P; Cancer Research UK Leukaemia Biology Laboratory, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK. (2013)
      Human acute myeloid leukemias (AMLs) are sustained by leukemic stem cells (LSCs) that generate through aberrant differentiation the blast cells that make up the bulk of the malignant clone. LSCs were first identified as rare cells with an immunophenotype shared with normal hematopoietic stem cells (HSCs). However, refinements of xenotransplantation assays, alternative methods of quantitation and syngeneic murine models have all led to an appreciation that LSCs display marked variability in frequency, immunophenotype and differentiation potential, both between and even within leukemias. Insights from next-generation sequencing efforts have dramatically extended understanding of the mutational landscape and clonal organization of AML and have added an additional layer of complexity to the biology of LSCs: a requirement to consider the effect of the various recurrently occurring genetic lesions in AML on the initiation and maintenance of leukemic subclones. Despite these advances, cure rates in AML remain substantially unchanged in recent years. A renewed focus on the biological properties of chemotherapy-resistant LSCs, a cellular entity of prime clinical importance, will be required to develop additional therapeutic strategies to enhance patient outcomes.
    • Vascular endothelial growth factor (VEGF) expression is a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix.

      Loncaster, Juliette A; Cooper, Rachel A; Logue, John P; Davidson, Susan E; Hunter, Robin D; West, Catharine M L; CRC Experimental Radiation Oncology Group, Paterson Institute for Cancer Research, Manchester, UK. (2000-09)
      The aim of the study was to evaluate VEGF expression in tumour biopsies as a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix. A retrospective study was carried out on 100 patients. Pre-treatment tumour VEGF expression was examined immunohistochemically in formalin-fixed, paraffin-embedded biopsies using a widely available commercial antibody. A semi-quantitative analysis was made using a scoring system of 0, 1, 2, and 3, for increasing intensity of staining. High VEGF expression was associated with a poor prognosis. A univariate log rank analysis found a significant relationship with overall survival (P = 0.0008) and metastasis-free survival (P = 0.0062), but not local control (P = 0.23). There was no correlation between VEGF expression and disease stage, tumour differentiation, patient age, or tumour radiosensitivity (SF2). In a Cox multivariate analysis of survival VEGF expression was the most significant independent prognostic factor (P = 0.001). After allowing for VEGF only SF2 was a significant prognostic factor (P = 0.003). In conclusion, immunohistochemical analysis of VEGF expression is a highly significant and independent prognostic indicator of overall and metastasis-free survival for patients treated with radiotherapy for advanced carcinoma of the cervix. It is also a rapid and easy method that could be used in the clinical setting, to identify patients at high risk of failure with conventional radiotherapy who may benefit from novel approaches or chemoradiotherapy.
    • Vascular endothelial growth factors and receptors in colorectal cancer: implications for anti-angiogenic therapy.

      Duff, Sarah E; Jeziorska, M; Rosa, Daniela D; Kumar, Shant; Haboubi, Najib; Sherlock, David J; O'Dwyer, Sarah T; Jayson, Gordon C; Department of Surgery, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom. (2006-01)
      There are conflicting associations between growth factor expression and clinicopathological variables in colorectal cancer. This study aimed to define the expression of members of the VEGF family and the receptor, VEGFR2, in primary and metastatic sites of colorectal cancer and their relationship to metastatic potential. Thirty colorectal cancers, 12 lymph node metastases and 9 liver metastases were immunostained for VEGF-A, VEGF-C, VEGF-D and VEGFR2. VEGFR2 was expressed by endothelial cells and by the malignant epithelium. VEGF-C and VEGFR2 were co-expressed in the same territory and correlated throughout the primary tumour and in metastatic lymph nodes, but not in liver metastases. Their expression at the invasive tumour edge correlated with expression in metastatic nodes. The benefit of anti-VEGF antibodies might be increased by directing additional therapies against VEGF-C or against the kinase receptors to target redundancy in the system. A component of the therapeutic benefit might be due to a direct anti-tumour effect as well as an anti-angiogenic effect.
    • Vascular function and the probability of skin necrosis after photodynamic therapy: an experimental study.

      Benstead, K; Moore, James V; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1988-05)
      The clearance of an intradermally-injected solution of 133Xenon in 0.9% saline has been used to study the impairment and recovery of blood flow in mouse tail for 5 days following photodynamic therapy (PDT) with 2mg TPPS i.v. per mouse and a range of doses of white light. Impairment of blood flow was observed within 10 min of light exposure. Blood flow increased between day 1 and day 5 at light doses less than 151J cm-2 and had returned to control levels by day 5 at light doses less than 129J cm-2. In mice treated with a light dose that caused a 50% incidence of necrosis, there was no significant difference in the initial xenon clearance half-time (measured at 10 min and 1 day after PDT) between those mice which developed tail necrosis and those which healed. However, the latter showed significantly greater improvement in vascular function on days 2, 3 and 4. This suggests that the timing and extent of recovery of blood flow determined the risk of necrosis in individual mice.
    • Vascular function and tissue injury in murine skin following hyperthermia and photodynamic therapy, alone and in combination.

      Moore, James V; West, Catharine M L; Haylett, Ann K; Paterson Institute for Cancer Research (Cancer Research Campaign), Christie Hospital (NHS) Trust, Manchester, UK. (1992-12)
      The murine tail has been used as a model for injury to skin when hyperthermia (HT) and photodynamic therapy (PDT) using haematoporphyrin derivative, are used in combination. Skin injury by either agent alone was quantitated by the probability of tail necrosis as a function of dose of agent. 'Tolerance' doses of each modality were given and changes in skin vascular function were measured by the rate of clearance of 133Xenon. This was promptly inhibited but restored to normal by 7 days. The absolute numbers of hypodermal vessels of different sizes were measured in tail cross-sections and capillary numbers were found to be greatly reduced between 1 and 7 days, and restored to normal by 21-28 days. When a tolerance dose of PDT was followed at 1, 7, 21 and 28 days by test doses of HT, or vice versa, marked enhancements in probability of necrosis were observed when the interval was 1 or 7 days (Enhancement ratio (ER)PDT-HT = 1.5 and ERHT-PDT = 1.8). Prolonging the interval between modalities to 21-28 days spared the tissue (ERHT-PDT/21 DAYS = 1.1; ERPDT-HT/28 DAYS = 1.0). Close temporal apposition of PDT and HT, such as has been advocated to improve tumour control, may also increase injury to normal tissue through vascular effects common to both.
    • Vectors with multiple insertion sites for expression of cloned genes by T7 RNA polymerase: expression of a peptide from EBV thymidine kinase (TK).

      Mackett, Mike; Littler, Edward; Michael, R; Gartland, M; Arrand, John R; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Withington, Manchester, UK. (1990-02-25)
    • VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase.

      Robinson, Christopher J; Mulloy, Barbara; Gallagher, John T; Stringer, Sally E; Cancer Research UK and University of Manchester Department of Medical Oncology, Christie Hospital National Health Service Trust, Wilmslow Road, Manchester M20 4BX, United Kindgom. Christopher.Robinson@manchester.ac.uk (2006-01-20)
      The vascular endothelial growth factor (VEGF) family of proteins controls the formation and growth of blood vessels. The most potent and widely expressed isoform, VEGF165, is secreted as a disulfide-linked homodimer with two identical heparin-binding sites. Interactions with heparan sulfate (HS) regulate the diffusion, half-life, and affinity of VEGF165 for its signaling receptors. We have determined a number of key HS structural features that mediate the specific binding of the VEGF165 dimer. Carboxylate groups and 2-O-, 6-O-, and N-sulfation of HS contributed to the strength of the VEGF165 interaction; however, 6-O-sulfates appeared to be particularly important. Cleavage of HS by heparinase, heparitinase, or heparanase severely reduced VEGF165 binding. In contrast, K5 lyase-cleaved HS retained significant VEGF165 affinity, suggesting that binding sites for the growth factor are present within extended stretches of sulfation. Binding studies and molecular modeling demonstrated that an oligosaccharide 6 or 7 residues long was sufficient to fully occupy the heparin-binding site of a VEGF165 monomer. The data presented are consistent with a model whereby the two heparin-binding sites of the VEGF165 dimer interact simultaneously with highly sulfated S-domain regions of the HS chain that can be linked through a stretch of transition sequence.
    • 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.