• Novel syntheses of cis and trans isomers of combretastatin A-4.

      Gaukroger, Keira; Hadfield, John A; Hepworth, Lucy A; Lawrence, Nicholas J; McGown, Alan T; CRC Drug Development Group and CRC Radiochemical Targeting and Imaging Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, U.K. (2001-11-30)
      A high-yielding, two-step stereoselective synthesis of the anticancer drug (Z)-combretastatin A-4 (1) has been devised. The method uses the Perkin condensation of 3,4,5-trimethoxyphenylacetic acid and 3-hydroxy-4-methoxybenzaldehyde followed by decarboxylation of the cinnamic acid intermediate using copper and quinoline. The iodine-catalyzed isomerization of the Z isomer 1 results in complete conversion to the E isomer. The Suzuki cross-coupling of an aryl boronic acid and vinyl bromide has also been successfully employed to produce both Z and E isomers of combretastatin A-4 stereoselectively. Both methods are far superior to the current five-step Wittig synthesis in which both isomers are produced nonstereoselectively.
    • Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT).

      Shibata, Takayuki; Glynn, Nicola; McMurry, T Brian H; McElhinney, R Stanley; Margison, Geoffrey P; Williams, David M; Department of Chemistry, Centre for Chemical Biology, Richard Roberts Building, University of Sheffield, Sheffield, S3 7HF, UK. (2006)
      The human DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O6-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O6-alkylguanines. One such ODN containing O6-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC50 of 0.1 nM.
    • Novel therapeutic strategies by regulatory T cells in allergy.

      Elkord, Eyad; Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. eelkord@picr.man.ac.uk (2008)
      Natural CD4+CD25+FoxP3+ regulatory T cells (Treg) actively suppress physiological and pathological responses, therefore playing a critical role in controlling peripheral tolerance to self antigens and maintaining immune homeostasis. In normal individuals, natural Treg and interleukin- 10-secreting Treg are able to suppress Th2 responses to allergens, whereas lower levels of Treg or defect in their functionality have been described as potential mechanisms for inducing allergic diseases. In animal models, adoptive transfer of CD4+CD25+Treg has been shown as a promising strategy for preventing or treating allergic disorders. Recent studies show that induction of Treg activity is associated with suppression of allergic responses in allergic patients treated with specific immunotherapy. Herein, I review the potential of Treg as exciting targets for developing new immunotherapeutic strategies for treating allergic diseases.
    • Novel therapeutic targets in lung cancer: Inhibitor of apoptosis proteins from laboratory to clinic.

      Dean, Emma J; Ranson, Malcolm R; Blackhall, Fiona H; Holt, Sarah V; Dive, Caroline; Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom. emma.dean@christie-tr.nwest.nhs.uk <emma.dean@christie-tr.nwest.nhs.uk> (2007-04)
      Lung cancer is the leading cause of cancer death worldwide. Despite the introduction of new agents and schedules, chemotherapy still obtains unsatisfactory overall response rates, rare complete remissions and responses of relatively short duration. The inhibitor of apoptosis proteins (IAPS) are a family of caspase inhibitors that selectively bind and inhibit caspases-3, -7, and -9. As caspase activation is central to apoptosis, novel therapeutic drugs that target IAPs enabling apoptosis to occur have potential as a treatment of malignancy. Several agents that target core components of the apoptotic signalling pathway are currently at an early stage of development. This review reports the progress being made in characterising the IAP family, with a focus on the available data relevant to the treatment of lung cancer.
    • A novel TP53 splicing mutation in a Li-Fraumeni syndrome family: a patient with Wilms' tumour is not a mutation carrier.

      Varley, Jennifer; McGown, Gail; Thorncroft, Mary R; White, Gavin R M; Tricker, K J; Kelsey, Anna M; Birch, Jillian M; Evans, D Gareth R; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, UK. (1998-10)
      We report a Li-Fraumeni syndrome family in which we have detected a splice acceptor mutation in intron 3 of TP53. The mutation affects one of the invariant residues at the splice acceptor site, as a result of which two aberrant transcripts are produced. A child with Wilms' tumour aged 3 years in this family was shown not to be a mutation carrier.
    • Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies.

      Alduaij, Waleed; Ivanov, Andrei; Honeychurch, Jamie; Cheadle, Eleanor J; Potluri, Sandeep; Lim, Sean H; Shimada, Kazuyuki; Chan, Claude H T; Tutt, Alison L; Beers, Stephen A; et al. (2011-04-28)
      The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.
    • Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter.

      Perez-Campo, Flor-Maria; Spencer, Helen L; Elder, Rhoderick H; Stern, Peter L; Ward, Christopher M; Stem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK. (2007-10-01)
      The use of gene mutation/knock-out strategies in mouse embryonic stem (ES) cells has revolutionized the study of gene function in ES cells and embryonic development. However, the construction of vectors for homologous recombination strategies requires considerable expertise and time. We describe two novel vectors that can generate site specific knock-out or EGFP knock-in ES cells within 6 weeks from construct design to identification of positive ES cell clones. As proof-of-principle, we have utilized the knock-out targeting vector to modify the NEIL2 locus in ES cells. In addition, using the knock-in vector, we have inserted EGFP downstream of the 5T4 oncofetal antigen promoter in ES cells (5T4-GFP ES cells). Undifferentiated 5T4-GFP ES cells lack EGFP and maintain expression of the pluripotent markers OCT-4 and NANOG. Upon differentiation, EGFP expression is increased in 5T4-GFP ES cells and this correlates with 5T4 transcript expression of the unmodified allele, loss of Nanog and Oct-4 transcripts and upregulation of differentiation-associated transcripts. Furthermore, we demonstrate that fluorescent activated cell sorting of 5T4-GFP ES cells allows isolation of pluripotent or differentiated cells from a heterogeneous population. These vectors provide researchers with a rapid method of modifying specific ES cell genes to study cellular differentiation and embryonic development.
    • Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex.

      Scharfenberger, Maren; Ortiz, Jennifer; Grau, Nicole; Janke, Carsten; Schiebel, Elmar; Lechner, Johannes; Biochemie-Zentrum Heidelberg Ruprecht-Karls Universität, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. (2003-12-15)
      We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore. When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids. In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity. The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex. However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does. Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity. Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity.
    • Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy.

      Gant, T M; Goldberg, Martin W; Allen, Terence D; Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. tmgant@welchlink.welch.jhu.edu (1998-06)
      At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.
    • Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract.

      Zhang, C; Jenkins, H; Goldberg, Martin W; Allen, Terence D; Hutchison, C J; Department of Biological Sciences, University of Dundee, Scotland, UK. (1996-09)
      Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from "lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.
    • Nuclear localization and regulation of Id protein through an E protein-mediated chaperone mechanism.

      Deed, Richard W; Armitage, Suzanne; Norton, John D; Cancer Research Campaign Department of Gene Regulation, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Wilmslow Road, Manchester M20 9BX, United Kingdom. (1996-09-27)
      Members of the Id family of helix-loop-helix proteins function as negative regulators of DNA binding, E protein, helix-loop-helix transcription factors in the control of cell growth, differentiation, and development. By using transient transfection analysis of COS cells, we show that in the absence of its E protein target, the Id3 protein is localized exclusively to the cytoplasm/perinuclear region. Co-transfection with E protein (E47) results in nuclear translocation of the Id3 protein, a process requiring both a functional Id helix-loop-helix dimerization domain and an E protein nuclear localization signal. Id3 that is associated with E protein displays an extended half-life, while the E protein itself is more rapidly turned over. These observations demonstrate that E protein, by nuclear chaperoning Id, can regulate the available cellular pool of its own inhibitory partner.
    • Nuclear localization of the pre-mRNA associating protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP).

      El Bounkari, Omar; Guria, Anuja; Klebba-Faerber, Sabine; Claussen, Maike; Pieler, Tomas; Griffiths, John R; Whetton, Anthony D; Koch, Alexandra; Tamura, Teruko; Institut fuer Biochemie, OE4310 Medizinische Hochschule Hannover, Hannover, Germany. (2009-01-05)
      THOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50-137, amino acid numbers) binds to the N-terminal portion (1-199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.
    • Nuclear membrane disassembly and rupture.

      Cotter, Laura A; Allen, Terence D; Kiseleva, Elena; Goldberg, Martin W; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. (2007-06-08)
      The nuclear envelope consists of two membranes traversed by nuclear pore complexes. The outer membrane is continuous with the endoplasmic reticulum. At mitosis nuclear pore complexes are dismantled and membranes disperse. The mechanism of dispersal is controversial: one view is that membranes feed into the endoplasmic reticulum, another is that they vesiculate. Using Xenopus egg extracts, nuclei have been assembled and then induced to breakdown by addition of metaphase extract. Field emission scanning electron microscopy was used to study disassembly. Strikingly, endoplasmic reticulum-like membrane tubules form from the nuclear surface after the addition of metaphase extracts, but vesicles were also observed. Microtubule inhibitors slowed but did not prevent membrane removal, whereas Brefeldin A, which inhibits vesicle formation, stops membrane disassembly, suggesting that vesiculation is necessary. Structures that looked like coated buds were observed and buds were labelled for beta-COP. We show that nuclear pore complexes are dismantled and the pore closed prior to membrane rupturing, suggesting that rupturing is an active process rather than a result of enlargement of nuclear pores.
    • Nuclear phosphoinositides and their impact on nuclear functions.

      Shah, Zahid H; Jones, David R; Sommer, Lilly; Foulger, Rebecca; Bultsma, Yvette; D'Santos, C; Divecha, Nullin; Cancer Research UK Inositide Laboratory, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK. (2013-09-24)
      Polyphosphoinositides (PPIn) are important lipid molecules whose levels are deregulated in human diseases such as cancer, neurodegenerative and in metabolic syndromes. PPIn are synthesised and degraded by an array of kinases, phosphatases and lipases which are localised to various subcellular compartments and are subject to regulation in response to both extra- and intracellular cues. Changes in the activities of enzymes that metabolise PPIn lead to changes in the profiles of PPIn in different sub-cellular compartments. Understanding how subcellular PPIn are regulated and how they impact on downstream signaling is critical to understand their roles in human diseases. PPIn are present in the nucleus, and their levels are changed in response to various stimuli, suggesting that they may serve to regulate specific nuclear functions. However, the lack of nuclear downstream targets has hindered the definition of which pathways nuclear PPIn impact on. Over recent years targeted and global proteomic studies have identified a plethora of potential PPIn interacting proteins involved in many aspects of transcription, chromatin remodeling and mRNA maturation suggesting that PPIn signaling within the nucleus represents a largely unexplored novel layer of complexity in the regulation of nuclear functions. This article is protected by copyright. All rights reserved.
    • Nuclear pore clustering is a consistent feature of apoptosis in vitro.

      Reipert, Siegfried; Reipert, Brigit M; Hickman, John A; Allen, Terence D; CRC, Department of Structural Cell Biology, Paterson Institute for Cancer Research and Christie Hospital, Manchester, UK. (1996-01)
      Two cell lines which show different patterns of DNA fragmentation have been examined for variations of their nuclear morphology during apoptosis. FDCP-Mix, a pluripotent murine haemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis either by drugs or withdrawal of growth factor (IL-3) was compared with the human lymphoid leukemia cell line MOLT-4, a cell line which undergoes apoptosis without production of a typical DNA 'ladder'. The nuclear morphology of FDCP-Mix cells was consistent after apoptotic induction by drug or by growth factor withdrawal. Apoptotic nuclear morphology for MOLT-4 and FDCP-Mix showed variations in the distribution, density and texture of the electron dense nuclear marginations. Despite these differences, clustering of nuclear pore complexes (NPCs) after treatment with the topoisomerase II inhibitor etoposide was a common phenomenon for both cell lines. Moreover, pore clustering for FDCP-Mix nuclei occurred independently from the way in which apoptosis was induced, either by growth factor withdrawal or etoposide treatment. In a novel approach, we visualised the clustering of NPCs three-dimensionally by field emission in-lens scanning electron microscopy (FEISEM).
    • The nuclear pore complex and lamina: three-dimensional structures and interactions determined by field emission in-lens scanning electron microscopy.

      Goldberg, Martin W; Allen, Terence D; CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, UK. (1996-04-12)
      Three dimensional surface imaging was used to examine structural details of the nuclear pore complex. Subsurface structures were uncovered by detergent extraction, proteolysis, mechanical fracturing and combinations of these. Features observed in this way were mostly consistent with previous three-dimensional structures with some novel observations. In addition to cytoplasmic and basket filaments attached to each coaxial ring, we have observed radiating filaments within the central channel. New details of basket organization are presented, showing that basket filaments branch and are woven together to form the basket ring. The "central transporter" is shown to be a regular, consistent structure revealed after removal of overlying internal filaments. The lumenal ring is visualised and we present evidence that the lamina may be attached to the spoke ring complex. Finally we show that there is evidence for a novel structure, the "star ring", sandwiched between the cytoplasmic ring and the membrane.
    • Nuclear pore complex disassembly and nuclear envelope breakdown during mitosis may occur by both nuclear envelope vesicularisation and dispersion throughout the endoplasmic reticulum.

      Cotter, Laura A; Goldberg, Martin W; Allen, Terence D; Paterson Institute for Cancer Research, Christie Hospital, Manchester, England. (1998-04)
    • Nuclear pore complex structure in birds.

      Goldberg, Martin W; Solovei, I; Allen, Terence D; Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (1997-08)
      The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.
    • The nuclear pore complex: mediator of translocation between nucleus and cytoplasm.

      Allen, Terence D; Cronshaw, J M; Bagley, Steven; Kiseleva, Elena; Goldberg, Martin W; CRC Structural Cell Biology Group, Paterson Institute, Christie Hospital, Manchester, M20 4BX, UK. (2000-05)
      The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.
    • The nuclear pore complex: structure, function, and dynamics.

      Kiseleva, Elena; Goldberg, Martin W; Cronshaw, J M; Allen, Terence D; CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, UK. (2000)
      A full understanding of nucleocytoplasmic transport depends on knowledge of nuclear pore complex (NPC) structure, the functional roles of NPC components, their interactions during transport and dynamics during the cell cycle. NPC structure is conserved, flexible, and is not simply a tunnel between the nucleus and cytoplasm but appears to be actively involved in the transport process by a series of structural modifications. Transport through the NPC begins in either of its asymmetrical peripheral compartments that are both structurally reorganized during transport in different ways. The central compartment is composed of two symmetrical halves, and functions as a system of transiently open, discrete gates that is not believed to play a role in determining direction. Each NPC subunit has a specific morphology that corresponds to the functional role it plays. A complicated system of vertical and horizontal connections may allow one part of the NPC to transmit a signal to other parts, leading to an ordered series of conformational changes that drive translocation. High-resolution scanning electron microscopy has identified sequential stages of NPC assembly in vitro and revealed how the individual NPC components are assembled into a mature NPC. This review focuses on structural events during transport and on possible mechanisms of NPC assembly.