• The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis.

      Jensen, M; Ernestus, K; Kemshead, John T; Klehr, M; Von Bergwelt-Baildon, M S; Schinköthe, T; Schultze, J L; Berthold, F; Department of Pediatric Oncology and Hematology, University of Cologne, Germany. jensen@uni-koeln.de (2003-11)
      To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 x NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7- effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.
    • Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.

      Huot, Thomas J G; Rowe, Janice; Harland, Mark; Drayton, Sarah; Brookes, Sharon; Gooptu, Chandra; Purkis, Patricia; Fried, Mike; Bataille, Veronique; Hara, Eiji; et al. (2002-12)
      The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.
    • Bibliographies on radiation chemistry: II. Studies of the chemistry of semiquinones.

      Swallow, A John; Rose, A; Helman, P; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England. (1981)
    • Biguanides drugs: Past success stories and promising future for drug discovery

      Grytsai, Oleksandr; Myrgorodska, I.; Rocchi, S.; Ronco, C; Benhida, R.; Université Côte d'Azur, CNRS, Institut de Chimie de Nice UMR 7272, 28 Avenue Valrose, 06108, Nice, France; Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Alderley Park, Macclesfield, SK10 4TG, (2021)
      Biguanides have attracted much attention a century ago and showed resurgent interest in recent years after a long period of dormancy. They constitute an important class of therapeutic agents suitable for the treatment of a wide spectrum of diseases. Therapeutic indications of biguanides include antidiabetic, antimalarial, antiviral, antiplaque, and bactericidal applications. This review presents an extensive overview of the biological activity of biguanides and different mechanisms of action of currently marketed biguanide-containing drugs, as well as their pharmacological properties when applicable. We highlight the recent developments in research on biguanide compounds, with a primary focus on studies on metformin in the field of oncology. We aim to provide a critical overview of all main bioactive biguanide compounds and discuss future perspectives for the design of new drugs based on the biguanide fragment.
    • BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

      Korfi, Koorosh; Smith, Matthew; Swan, Jacqueline; Somervaille, Tim C P; Dhomen, Nathalie; Marais, Richard; Molecular Oncology Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester (2016)
      B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential.
    • Bimolecular disappearance of pyridinyl radicals in water

      Kosower, E; Teuerstein, A; Swallow, A John; Department of Chemistry, Tel-Aviv University, Ramat-aviv, Tel-Aviv, Israel (1973)
    • Bimolecular reactions of pyridinyl radicals in water and the mechanism of NAD+-NADH dehydrogenase reactions.

      Kosower, Edward M; Teuerstein, Avraham; Burrows, Hugh D; Swallow, A John; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1978)
    • Binding mode and selectivity of steroids towards glucose-6-phosphate dehydrogenase from the pathogen Trypanosoma cruzi.

      Ortiz, C; Moraca, F; Medeiros, A; Botta, M; Hamilton, Niall M; Comini, M; Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay (2016)
      Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.
    • Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates.

      Blackhall, Fiona H; Merry, Catherine L R; Lyon, Malcolm; Jayson, Gordon C; Folkman, Judah; Javaherian, Kashi; Gallagher, John T; Department of Medical Oncology, University of Manchester, Cancer Research UK, Christie Hospital NHS Trust, Manchester M20 4BX, UK. (2003-10-01)
      Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st-/- mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential.
    • The binding properties of minimal oligosaccharides reveal a common heparan sulfate/dermatan sulfate-binding site in hepatocyte growth factor/scatter factor that can accommodate a wide variety of sulfation patterns.

      Deakin, Jon A; Blaum, B; Gallagher, John T; Uhrín, D; Lyon, Malcolm; Cancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Rd., Manchester M20 4BX, United Kingdom. (2009-03-06)
      Heparan sulfate (HS)/heparin and dermatan sulfate (DS) both bind with high affinity to hepatocyte growth factor/scatter factor (HGF/SF) and function as necessary co-factors in vitro. How both these two structurally distinct glycosaminoglycans (GAGs) are recognized has remained unclear. We have now reconciled this issue using a panel of minimal tri- and tetrasaccharide sequences of variable but well defined sulfation patterns in combination with further development of the gel mobility shift assay to allow simultaneous comparisons of relative protein affinities/selectivities for different oligosaccharides. From this approach it would seem that a minimum binding sequence is a disulfated trisaccharide comprised of an internal iduronate flanked by monosulfated hexosamine residues and that additional sulfation further enhances affinity. However, the similarity in recognition of HS/heparin and DS seems to arise primarily from a lack of any apparent positional requirement for sulfation. Thus, isomers of HS/heparin tetrasaccharides containing only two sulfates irrespective of whether they are purely N-, 2-O-, or 6-O-sulfates bind with equivalent apparent affinity as a disulfated DS tetrasaccharide. In addition, the NMR chemical shifts induced in NK1 (the truncated variant of HGF/SF comprised of the N-terminal and first Kringle domains) by titration with either heparin or DS oligosaccharides strongly indicate that both bind to essentially the same site. Together, these observations reveal an unexpected degree of flexibility in the GAG-HGF/SF interface, allowing a single binding site in the protein to accommodate iduronate-containing sequences of variable sulfation pattern and/or density from different GAGs.
    • Bio-specific sequences and domains in heparan sulphate and the regulation of cell growth and adhesion.

      Lyon, Malcolm; Gallagher, John T; CRC Department of Medical Oncology, Christie CRC Research Center, Christie Hospital NHS Trust, Manchester, UK. (1998-11)
    • Bioactivity and molecular modelling of diphenylsulfides and diphenylselenides.

      Woods, Julie A; Hadfield, John A; McGown, Alan T; Fox, Brian W; CRC Department of Experimental Chemotherapy, Pateson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1993-11)
      Bis(2-bromo-4,5-dimethoxyphenyl)sulfide (5) and bis(2-bromo-4,5-dimethoxyphenyl) selenide (7) have been shown to block cells in the G2/M phase of the cell cycle, whereas the debromo (4,6) equivalents do not. The biobromoselenide (7) is cytotoxic to tumour cells in vitro and has been shown to increase the mitotic index of treated cells. These biological effects are consistent with disruption of the mitotic apparatus. This agent does not inhibit microtubule assembly in vitro, but does bind to tubulin. Molecular modelling of these structures indicates that their spatial and electronic structures may make an important contribution to the biological activity.
    • Bioavailability and catalytic properties of copper and iron for Fenton chemistry in human cerebrospinal fluid.

      Spasojević, Ivan; Mojović, Milos; Stević, Zorica; Spasić, Snezana D; Jones, David R; Morina, Arian; Spasić, Mihajlo B; Institute for Multidisciplinary Research, University of Belgrade, 11000 Belgrade, Serbia. ivan@cms.bg.ac.rs (2010)
      A breakdown in homeostasis of redox-active metals represents an important factor for neurodegeneration. We have used EPR spectroscopy and BMPO spin-trap to investigate the catalytic properties and ligand modulation of redox activity of copper and iron in human cerebrospinal fluid (CSF). In contrast to iron, copper supplementation provoked a statistically significant increase in hydroxyl free radical generation in CSF treated with H(2)O(2). However, in a binary copper/iron containing Fenton system, iron catalytically activated copper. The chelator EDTA, which represents a model of physiological metal ligands, completely prevented copper's redox activity in CSF, while iron chelation led to a significant increase in hydroxyl radical generation, indicating that copper and iron do not only have diverse catalytic properties in the CSF but also that their redox activities are differently modulated by ligands. The application of DDC reduced hydroxyl radical generation in the CSF containing catalytically active metals (free Cu(2+) or Fe(3+)-EDTA complex). We conclude that chelators, such as DDC, are capable of preventing the prooxidative activity of both metals and may be suitable for reducing hydroxyl radical formation in certain pathophysiological settings.
    • Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells.

      McKenzie, Edward; Young, Kathryn; Hircock, Margaret; Bennett, James; Bhaman, Maina; Felix, Robert; Turner, Paul; Stamps, Alasdair; McMillan, David; Saville, Giles; et al. (2003-07-15)
      The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.
    • Biochemical controls of the G1 phase of a mammalian cell cycle. I. Analysis of chromatin proteins in temperature-sensitive variants.

      Naha, P M; Sorrentino, R; Paterson Laboratories Christie Hospital and Holt Radium Institute, Manchester M20 9BX, U.K. (1980-04)
      Chromatin proteins of Balb/c-3T3 cell line and its temperature sensitive variants A83 and A8, arrested in G1 phase of the cell cycle at the restricted temperature, were analysed on SDS-polyacrylamide gel by electrophoresis to detect major alterations that could be related to the G1 arrest. Appearance of two additional proteins (mol. wt. 80,000 and 20,000) and disappearance of another (mol.wt. 14,000) were thought to be significant. The protein of mol.wt. 20,000 was absent in the G1- derivatives, suggesting a possible relationship to the appearance of the G1 phase.
    • Biochemical controls of the G1 phase of a mammalian cell cycle. II. Synthesis of H1 histones in temperature sensitive mouse cells, arrested in G1 phase.

      Gooderham, K; Naha, P M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK (1981-12)
    • Biochemical manipulation of the nuclear-envelope (ne) characterized by field-emission scanning electron-microscopy (feisem), immunogold labeling and western blotting.

      Goldberg, Martin W; Robson, A; Rutherford, S; Allen, Terence D; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, UK. (1994)
    • The biochemistry and biology of the myeloid haemopoietic cell growth factors.

      Heyworth, Clare M; Vallance, S J; Whetton, Anthony D; Dexter, T Michael; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1990)
      In the adult, blood cell production or haemopoiesis takes place mainly in the bone marrow. The blood cell types produced are a reflection of the needs of the organism at any moment, for example bacterial infection leads to a large increase in neutrophil production. The rate and scale of blood cell production in vivo are regulated, at least in part, by the synthesis and release of specific cytokines both within the bone marrow and also from other tissues. Here we detail the range of cytokines which act directly on haemopoietic stem cells and myeloid progenitor cells. Also cellular systems which will permit the elucidation of the specific interactions between these various cytokines which regulate stem cell self-renewal, differentiation and proliferation are described.
    • Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates.

      Meikle, I; Cummings, Jeffrey; Macpherson, J S; Hadfield, John A; Smyth, J F; Imperial Cancer Research Fund Medical Oncology Unit, Western General Hospital, Edinburgh, U.K. (1995-06-16)
      Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.
    • BioID-based proteomic analysis of the Bid interactome identifies novel proteins involved in cell-cycle-dependent apoptotic priming

      Pedley, R.; King, L. E.; Mallikarjun, V.; Wang, Pengbo; Swift, J.; Brennan, K.; Gilmore, A. P.; Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. (2020)
      Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how it is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to antimitotic drugs. Here, we report an unbiased proximity biotinylation (BioID) screen to identify regulators of apoptotic priming in mitosis, using Bid as bait. The screen primarily identified proteins outside of the canonical Bid interactome. Specifically, we found that voltage-dependent anion-selective channel protein 2 (VDAC2) was required for Bid phosphorylation-dependent changes in apoptotic priming during mitosis. These results highlight the importance of the wider Bcl-2 family interactome in regulating the temporal control of apoptotic priming.