• Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase.

      Dunstan, M; Barkauskaite, Eva; Lafite, P; Knezevic, C; Brassington, A; Ahel, Marijan; Hergenrother, P; Leys, D; Ahel, I; Manchester Interdisciplinary Biocentre, Princess Street 131, M1 7DN, Manchester, UK. (2012)
      Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1''-2') ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.
    • Structure and regulation of the erythroid system at the level of progenitor cells.

      Testa, Nydia G; Department of Experimental Hematology, Paterson Institute for Cancer Research, Manchester, England. (1989)
      Considerable ground has been covered since the first clonal assays for hemopoietic cells were described. The possibility of studying populations of progenitor cells and the regulatory factors that influence them has already thrown considerable light on our understanding of the structure and physiology of the normal erythroid system and its alterations in disease. The relative importance of humoral and short-range factors and of possible cell-to-cell interactions in the regulation of proliferation and differentiation in the erythroid cell lineage is now being studied actively in several laboratories. The possibility of analyzing possible regulatory networks involving such highly reactive cells as lymphocytes and monocytes-macrophages in defined in vitro conditions now exists. As these studies are being extended to the diseased state, concepts related to alterations in regulatory mechanisms in syndromes with abnormal cell proliferation can be tested. Clinical applications in the treatment of patients with hematological disease are being contemplated. The usefulness of Epo for the treatment of the anemia of renal disease has been demonstrated already.
    • Structure of an aprataxin-DNA complex with insights into AOA1 neurodegenerative disease.

      Tumbale, P; Appel, C; Kraehenbuehl, Rolf; Robertson, P; Williams, J; Krahn, J; Ahel, Ivan; Williams, R; Laboratory of Structural Biology, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, North Carolina, USA. (2011-11)
      DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5'-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5'-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn(2+) complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove-binding C(2)HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5'-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5'-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).
    • Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism.

      Herbert, Andrew P; Deakin, Jon A; Schmidt, Christoph Q; Blaum, Bärbel S; Egan, Claire; Ferreira, Viviana P; Pangburn, Michael K; Lyon, Malcolm; Uhrín, Dusan; Barlow, Paul N; et al. (2007-06-29)
      A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.
    • Structure, function and assembly of the nuclear pore complex.

      Drummond, Sheona P; Allen, Terence D; Departmemt of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester. (2004)
    • Structure-activity relationship of heparan sulphate.

      Gallagher, John T; Department of Medical Oncology, Paterson Institute for Cancer Research, Withington, Manchester, U.K. (1997-11)
      HS influences fundamental cellular properties and biochemical processes at the cell surface. In addition to the issues already discussed, it has a profound effect on cell adhesion and migration through its interaction with many extracellular matrix proteins, most notably fibronectin and thrombospondin; it is closely linked to lipid metabolism through its capacity to bind low-density lipoprotein and lipoprotein lipase; and aberrations in HS structure and degradation are linked to human malignancy and Alzheimer's disease [26,27]. The subtle variations in HS structure enable it to distinguish between families of related proteins such as the FGFs, the chemokines [28] and the TGF beta s [29]. The multifunctional nature of HS is the result of its structural diversity and strategic positioning in the pericellular domain. The biosynthesis of HS, in common with other complex carbohydrates, is not directed by any known template yet the system is clearly subject to quite precise control so that in general, the HS family has a common domain organization that is finely tuned at the cellular level to produce HS species of variable length, fine structure and biological properties. A major challenge for future research will be to unravel the regulatory mechanisms that determine the molecular structure of HS. It remains unclear whether these mechanisms are entirely intrinsic in nature or subject to substantial modulation by the cellular microenvironment.
    • Structure-activity studies on 2-aryl-4H-3,1-benzoxazin-4-ones.

      Hadfield, John A; Pavlidis, V H; McGown, Alan T; Whitworth, C; Perry, P J; Fox, Brian W; CRC Department of Experimental Chemotherapy, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1994-10)
      Eight benzoxazin-4-ones related in structure to NSC 341964 (1) have been tested for cytotoxicity in two different cell systems. Two of the benzoxazin-4-ones (3 and 10) showed good cytotoxicity (ID50 = 9.9 and 8.9 microM) in P388 cells. The nitrobenzoxazin-4-one (10) caused a significant alteration in cell cycle distribution when administered to P388 cells and was an inhibitor of porcine pancreatic elastase. Structure-activity relationships are discussed.
    • Structure-based development of anticancer drugs: complexes of NAD(P)H:quinone oxidoreductase 1 with chemotherapeutic quinones.

      Faig, Margarita; Bianchet, Mario A; Winski, Shannon; Hargreaves, Robert H J; Moody, Christopher J; Hudnott, Anna R; Ross, David; Amzel, L Mario; Department of Biophysics and Biophysical Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, USA. (2001-08)
      BACKGROUND: NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1's ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. RESULTS: Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. CONCLUSIONS: The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.
    • Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations.

      Zhang, F; Tsunoda, M; Suzuki, K; Kikuchi, Y; Wilkinson, O; Millington, C; Margison, Geoffrey P; Williams, D; Czarina M; Takénaka, A; et al. (2013-04-10)
      N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O(6)-CMG]ATTCGCG) and d(CGC[O(6)-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson-Crick-type pair of O(6)-CMG with thymine, but might also accept the reversed wobble pair of O(6)-CMG with cytosine. Thus, O(6)-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O(6)-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.
    • Studies of the effect of 1,25-dihydroxycholecalciferol on the proliferation and differentiation of the human promyelocytic leukaemia cell line HL-60.

      Djulbegović, B; Christmas, Stephen E; Evans, Gareth S; Moore, Michael (1986)
      Treatment of the human promyelocytic leukaemia cell line HL-60 with 1,25(OH)2D3, the active metabolite of vitamin D3, led to a dose- and time-dependent inhibition of growth and 3H-TdR incorporation at the population level. A similar effect was noted at the single cell level in clonogenic assays and autoradiographic experiments. Flow cytometry indicated that there was an arrest of cells in the G0/G1 phase of the cell cycle. Parallel to the loss of proliferative capacity 1,25(OH)2D3 induced differentiation of HL-60 into monocyte/macrophages as measured by the enzyme NSE and the macrophage membrane antigen recognised by the monoclonal antibody EB11 as well as by morphological changes. These findings reinforce the concept of concordant induction of differentiation and loss of proliferative capacity and demonstrate that the latter occurs not only at the population level but also at the single cell level in this system. In limiting dilution assays in liquid culture there was evidence for positive interactions between HL-60 cells as untreated cells gave less colonies at low dilutions than would have been expected by Poisson statistical analysis. In the presence of 10(-8) M 1,25(OH)2D3 more complex growth parameters were noted indicating the involvement of both positive and negative cellular interactions.
    • Studies of the enhancement of natural cytotoxicity by the streptococcal immunopotentiator OK432.

      Christmas, Stephen E; Meager, A; Moore, Michael; Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK. (1986)
      The immunopotentiator OK432, a product of Streptococcus pyogenes A3, enhanced the natural killer (NK) activity of unseparated human peripheral blood mononuclear cells (PBM) and separated, nonadherent lymphocytes (PBL) containing less than 0.5% contamination with monocytes. Following treatment with OK432, both unseparated and non-adherent cell preparations produced interferon (IFN) alpha and gamma and low levels of interleukin-2 (IL-2). However, in the presence of neutralising amounts of anti-IFN alpha and gamma antisera, the NK enhancing effect of OK432 was not fully inhibited. Filtered supernatants derived from OK432-treated PBM or PBL also enhanced the NK activity of fresh PBM. In this instance, in most experiments, the NK enhancing effect of supernatants (from non-adherent cells) was fully inhibited by anti-IFN antisera. However, in some experiments, enhancement of NK activity by anti-IFN antisera-treated supernatants was still found. It is suggested that the presence of IL-2 or, possibly, other as yet uncharacterized factors secreted following OK432 treatment, account for this residual enhancement of NK activity.
    • Studies on ehrlich ascites tumour cells: DNA synthesis, and template activity of chromatin for DNA polymerase.

      Baghdjian, R B; Itzhaki, S; Ockey, Charles H; Itzhaki, Ruth F; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK (1982-12-10)
      Chromatin obtained from Ehrlich ascites cells on different days after cell inoculation has been assayed for its template activity with added DNA polymerase I. We have found that the template activity is 2 times higher in 7-8 day cell chromatin than in 4-day chromatin. Studies with added polylysine indicate that this increase reflects an increase in initiation sites rather than in accessibility to the enzyme. We have measured the growth fraction, mitotic index and rate of DNA chain growth in the intact cells. The results show that there is a large decrease in growth fraction with age of tumour, the number of cells dropping out of cycle approximately doubling over the period studied. The overall rate of chain growth decreases in the later stages of growth but in a small proportion of cells there is an increase in rate with fewer replicons involved in DNA synthesis. We suggest that in the ascites cells there is a decrease in level of repair and replicative enzymes with age of tumour; this would account both for the increase in initiation sites in the chromatin DNA, for the decrease in number of cells in cycle and for the overall decreased rate of chain growth.
    • Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

      Galbraith, A; Itzhaki, Ruth F (1979-12)
      A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.
    • Studies on liver chromatin from rats treated with dimethylnitrosamine.

      Cooper, Helen K; Itzhaki, Ruth F; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1975)
    • Studies on liver chromatin RNA from rats treated with alkylating agents.

      Itzhaki, Ruth F; Barker, Malcolm; Billington, Robert W (1976-12-01)
      We have examined firstly some properties of rat liver chromatin RNA and nuclear sap RNA and secondly the incorporation of [3H]orotic acid into the RNA in vivo in control rats and in rats treated with the alkylating agents, N,N-dimethylnitrosamine or methyl methane sulphonate. Half or more of the nuclear RNA is associated with the chromatin and consists mainly of two species: one is labelled and probably comprises "nascent" RNA, and the other is unlabelled and of lower molecular weight. Neither species is attributable to cytoplasmic contamination. Studies with added polylysine with RNAase A and with DNAase I suggest that both species are ironically bound to protein and that the labelled species is not associated with the part of the chromatin DNA most readily degraded by DNAase I. After dimethylnitrosamine treatment, the amount of unlabelled RNA remains constant but the amount of labelled RNA increases after a low dose, and decreases after a high dose. After methyl methane sulphonate treatment, no change occurs in either species. These results can be explained by changes in extent of association of the DNA and protein within the chromatin complex.
    • Studies on nucleic acids in lymphocytes of chronic lymphocytic leukaemia.

      Billington, R; Itzhaki, Ruth F; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1975)
      The resting lymphocytes of patients with chronic lymphocytic leukaemia (CLL) contain the same amount of RNA as do those of normal individuals, and in terms of different molecular species, as analyzed by polyacrylamide gels, there is very little to distinguish the leukaemic cells. However, a relatively large amount of low molecular weight RNA, similar to the SnRNA found in several other tissues, is present in the CLL cells. The products of transcription of the leukaemic cell nucleus have been studied by the incorporation of labelled uridine and methionine. The leukaemic lymphocytes show a build-up and apparent delay in processing of ribosomal RNA precursor, when compared to normals, but studies of methylation reveal that the production of mature ribosomal RNA occurs at a normal rate. The nature of the stable high molecular weight material produced is now being studied. The production of proteins on the ribosome of CLL cells seems likely to be faulty, as evidenced by the deficiency of active ribosomes in the leukaemic cells. Studies with selective inhibitors will show whether this is due to some fault in transcription of messenger RNA.
    • Studies on the in vitro culture of murine fetal thymus: effect of factors promoting epithelial growth.

      Garland, J M; Dexter, T Michael (1981)
      Fourteen-day fetal thymus was grown in organ culture under conditions reportedly optimizing epidermal/epithelial cell growth. While the fibroblastoid component is stimulated by epidermal growth factor and fibroblastoid growth factor, embryonic thymic epithelial cells do not grow significantly under any conditions, although they persist in organ explants. The major cell types cultured are fibroblastoid and macrophage. In organ explant cultures, epithelial cells appear to be oriented by the presence of a cell coat demonstrated by electron microscopy. This coat is absent from certain ciliated cells consistently found in deeper regions of the explant. Using amniotic fluid as a growth factor, almost pure cultures of thymic macrophages can be obtained.
    • Studies on the in vivo production of a lymphokine activity, interleukin 3 (IL-3) elaborated by lymphocytes and a myeloid leukaemic line in vitro and the fate of IL-3 dependent cell lines.

      Garland, John M; Aldridge, A; Wagstaffe, J; Dexter, T Michael; Paterson Laboratories and Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX (1983-08)
      Interleukin 3 (IL-3) is produced constitutively by WEHI-3b leukaemic cells and stimulated lymphoid cell populations in vitro. We have investigated the in vivo production of IL-3 in mice rendered leukaemic with WEHI-3b cells and mice stimulated by acute graft versus host disease (GVHD). In leukaemic mice, IL-3 was not found in serum or sonicates of 18-day spleens or bone marrow, although cells from the leukaemic organs were fully competent to elaborate IL-3 in vitro. Further, elaboration of IL-3 by WEHI cells in vitro was not affected by co-culture with normal haemopoietic cells. However, intracellular IL-3 was detected in leukaemic nodules isolated from the liver. Inhibitors specific for IL-3 were not found, although liver-cell conditioned medium and leukaemic nodule sonicates contained potent non-specific inhibitors of cell growth. At 21 days, intracellular IL-3 was also present in spleens and correlated with the presence of non-specific inhibitors. In GVHD, no evidence for IL-3 elaboration in vivo was found, nor did lymphoid populations affected by GVHD spontaneously elaborate it in vitro; however, their competence to produce it was unaffected, as IL-3 was elaborated on subsequent mitogen stimulation in vitro. We also investigated the recovery and circulation of in vitro 111Indium-labelled IL-3 dependent cells after injection in vivo and the half-life of semi-purified IL-3. Dependent cells were not recovered after injection into irradiated recipients, although the cells recirculated for at least 24 hours. Inability to recover dependent cells was explicable on general cytotoxicity which masked potential recovery. The serum half-life of injected partially purified material with IL-3 activity was short (less than 30 min). We conclude that the elaboration of IL-3 by leukaemic WEHI-3b is not an in vitro artifact and these results are discussed in relationship to other growth factors and the leukaemic state, and the origin of IL-3 dependent lines.
    • Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug.

      Cummings, J; Graham, M A; Hoey, Brigid M; Butler, John; Fry, A M; Hickson, I D; Leonard, G; French, R; Smyth, J F; Imperial Cancer Research Fund, Western General Hospital, Edinburgh, U.K. (1992-08-04)
      GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.