77 results on '"Graff JR"'
Search Results
2. Physical and optical properties of phytoplankton-rich layers in a coastal fjord: a step toward prediction and strategic sampling of plankton patchiness
- Author
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Graff, JR, primary and Menden-Deuer, S, additional
- Published
- 2016
- Full Text
- View/download PDF
3. Photoacclimation of natural phytoplankton communities
- Author
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Graff, JR, primary, Westberry, TK, additional, Milligan, AJ, additional, Brown, MB, additional, Dall’Olmo, G, additional, Reifel, KM, additional, and Behrenfeld, MJ, additional
- Published
- 2016
- Full Text
- View/download PDF
4. Analytical phytoplankton carbon measurements spanning diverse ecosystems
- Author
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Graff, JR, Westberry, TK, Milligan, AJ, Brown, MB, Dall'Olmo, G, van Dongen-Vogels, V, Reifel, KM, Behrenfeld, MJ, Graff, JR, Westberry, TK, Milligan, AJ, Brown, MB, Dall'Olmo, G, van Dongen-Vogels, V, Reifel, KM, and Behrenfeld, MJ
- Abstract
© 2015 The Authors. The measurement of phytoplankton carbon (Cphyto) in the field has been a long-sought but elusive goal in oceanography. Proxy measurements of Cphyto have been employed in the past, but are subject to many confounding influences that undermine their accuracy. Here we report the first directly measured Cphyto values from the open ocean. The Cphyto samples were collected from a diversity of environments, ranging from Pacific and Atlantic oligotrophic gyres to equatorial upwelling systems to temperate spring conditions. When compared to earlier proxies, direct measurements of Cphyto exhibit the strongest relationship with particulate backscattering coefficients (bbp) (R2=0.69). Chlorophyll concentration and total particulate organic carbon (POC) concentration accounted for ~20% less variability in Cphyto than bbp. Ratios of Cphyto to Chl a span an order of magnitude moving across and within distinct ecosystems. Similarly, Cphyto:POC ratios were variable with the lowest values coming from productive temperate waters and the highest from oligotrophic gyres. A strong relationship between Cphyto and bbp is particularly significant because bbp is a property retrievable from satellite ocean color measurements. Our results, therefore, are highly encouraging for the global monitoring of phytoplankton biomass from space. The continued application of our Cphyto measurement approach will enable validation of satellite retrievals and contribute to an improved understanding of environmental controls on phytoplankton biomass and physiology.
- Published
- 2015
5. Dry matter and nitrogen allocation in western redcedar, western hemlock, and Douglas fir seedlings grown in low- and high-N soils 1
- Author
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E. Graff Jr, Joseph, K. Hermann, Richard, B. Zaerr, Joe, and Revues Inra, Import
- Subjects
[SDV.SA.SF]Life Sciences [q-bio]/Agricultural sciences/Silviculture, forestry ,[SDV.SA.SF] Life Sciences [q-bio]/Agricultural sciences/Silviculture, forestry ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 1999
6. Bacterial attachment to phytoplankton in the pelagic marine environment
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Graff, JR, primary, Rines, JEB, additional, and Donaghay, PL, additional
- Published
- 2011
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7. Alternative concepts for dry storage of spent fuel at Morris Operation
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Graff, Jr, W A, primary, Breisch, R C, additional, Judson, B F, additional, and Weech, M E, additional
- Published
- 1980
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8. Ionic balance and organic acids in western redcedar, western hemlock, and Douglas-fir seedlings grown in low- and high-N soils
- Author
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Graff, Jr., J E, primary, Hermann, R K, additional, and Zaerr, J B, additional
- Published
- 1999
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9. Dry matter and nitrogen allocation in western redcedar, western hemlock, and Douglas fir seedlings grown in low- and high-N soils
- Author
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Graff Jr, Joseph E., primary, Hermann, Richard K., additional, and Zaerr, Joe B., additional
- Published
- 1999
- Full Text
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10. Security Technology in U.S. Public Schools.
- Author
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Graff, Jr., David R.
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PUBLIC schools ,NONFICTION - Abstract
The article reviews the book "Security Technology in U.S. Public Schools," by J. K. Coon.
- Published
- 2009
- Full Text
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11. Synechococcus nitrogen gene loss in iron-limited ocean regions.
- Author
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Sharpe G, Zhao L, Meyer MG, Gong W, Burns SM, Tagliabue A, Buck KN, Santoro AE, Graff JR, Marchetti A, and Gifford S
- Abstract
Synechococcus are the most abundant cyanobacteria in high latitude regions and are responsible for an estimated 17% of annual marine net primary productivity. Despite their biogeochemical importance, Synechococcus populations have been unevenly sampled across the ocean, with most studies focused on low-latitude strains. In particular, the near absence of Synechococcus genomes from high-latitude, High Nutrient Low Chlorophyll (HNLC) regions leaves a gap in our knowledge of picocyanobacterial adaptations to iron limitation and their influence on carbon, nitrogen, and iron cycles. We examined Synechococcus populations from the subarctic North Pacific, a well-characterized HNLC region, with quantitative metagenomics. Assembly with short and long reads produced two near complete Synechococcus metagenome-assembled genomes (MAGs). Quantitative metagenome-derived abundances of these populations matched well with flow cytometry counts, and the Synechococcus MAGs were estimated to comprise >99% of the Synechococcus at Station P. Whereas the Station P Synechococcus MAGs contained multiple genes for adaptation to iron limitation, both genomes lacked genes for uptake and assimilation of nitrate and nitrite, suggesting a dependence on ammonium, urea, and other forms of recycled nitrogen leading to reduced iron requirements. A global analysis of Synechococcus nitrate reductase abundance in the TARA Oceans dataset found nitrate assimilation genes are also lower in other HNLC regions. We propose that nitrate and nitrite assimilation gene loss in Synechococcus may represent an adaptation to severe iron limitation in high-latitude regions where ammonium availability is higher. Our findings have implications for models that quantify the contribution of cyanobacteria to primary production and subsequent carbon export., (© 2023. ISME Publications B.V.)
- Published
- 2023
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12. Altered growth and death in dilution-based viral predation assays.
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Knowles B, Bonachela JA, Cieslik N, Della Penna A, Diaz B, Baetge N, Behrenfeld MJ, Naumovitz K, Boss E, Graff JR, Halsey KH, Haramaty L, Karp-Boss L, and Bidle KD
- Subjects
- Animals, Phytoplankton, Atlantic Ocean, Ponds, Predatory Behavior, Viruses
- Abstract
Viral lysis of phytoplankton is one of the most common forms of death on Earth. Building on an assay used extensively to assess rates of phytoplankton loss to predation by grazers, lysis rates are increasingly quantified through dilution-based techniques. In this approach, dilution of viruses and hosts are expected to reduce infection rates and thus increase host net growth rates (i.e., accumulation rates). The difference between diluted and undiluted host growth rates is interpreted as a measurable proxy for the rate of viral lytic death. These assays are usually conducted in volumes ≥ 1 L. To increase throughput, we implemented a miniaturized, high-throughput, high-replication, flow cytometric microplate dilution assay to measure viral lysis in environmental samples sourced from a suburban pond and the North Atlantic Ocean. The most notable outcome we observed was a decline in phytoplankton densities that was exacerbated by dilution, instead of the increased growth rates expected from lowered virus-phytoplankton encounters. We sought to explain this counterintuitive outcome using theoretical, environmental, and experimental analyses. Our study shows that, while die-offs could be partly explained by a 'plate effect' due to small incubation volumes and cells adhering to walls, the declines in phytoplankton densities are not volume-dependent. Rather, they are driven by many density- and physiology-dependent effects of dilution on predation pressure, nutrient limitation, and growth, all of which violate the original assumptions of dilution assays. As these effects are volume-independent, these processes likely occur in all dilution assays that our analyses show to be remarkably sensitive to dilution-altered phytoplankton growth and insensitive to actual predation pressure. Incorporating altered growth as well as predation, we present a logical framework that categorizes locations by the relative dominance of these mechanisms, with general applicability to dilution-based assays., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Knowles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
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13. Imprime PGG Enhances Anti-Tumor Effects of Tumor-Targeting, Anti-Angiogenic, and Immune Checkpoint Inhibitor Antibodies.
- Author
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Chan ASH, Kangas TO, Qiu X, Uhlik MT, Fulton RB, Ottoson NR, Gorden KB, Yokoyama Y, Danielson ME, Jevne TM, Michel KS, Graff JR, and Bose N
- Abstract
Imprime PGG (Imprime) is in late-stage clinical development as a combinatorial agent with several therapeutic modalities. Here we present pre-clinical mechanistic data supportive of Imprime, a soluble yeast β-1,3/1,6-glucan pathogen-associated molecular pattern able to prime innate immune cells in a Dectin-1dependent manner. In tumor-free mice, Imprime evoked broad innate immune responses (type I interferon signature, mobilization of myeloid cells, dendritic cell and monocyte/macrophage expression of co-stimulatory ligands like CD86, and activation of natural killer cells). Imprime-mediated activation of myeloid cells also resulted in functional priming of antigen-specific CD8 T cell response. In tumor-bearing mice, Imprime monotherapy further resulted in activation of systemic and tumor infiltrating macrophages and enhanced cytotoxic CD8 T cell trafficking. Imprime enhanced the anti-tumor activity of several combinatorial agents in mouse cancer models; anti-tyrosinase-related protein 1 antibody in B16F10 melanoma experimental lung metastasis model, anti-vascular endothelial growth factor receptor 2 antibody in H1299 and H441 lung cancer, and anti-programmed cell death protein 1 antibody in MC38 colon cancer models. Mechanistically, combining Imprime with these combinatorial therapeutic agents elicited enhanced innate immune activation, supporting immunological synergy. Finally, Imprime treatment induced similar in vitro phenotypic and functional activation of human innate immune cells. Collectively, these data demonstrate Imprime's potential to orchestrate a broad, yet coordinated, anti-cancer immune response and complement existing cancer immunotherapies., Competing Interests: All authors were employed by Biothera Pharmaceuticals, Inc.. Authors AC, TK, XQ, MD, TJ, KM, NB are currently employed by HiberCell, Inc., (Copyright © 2022 Chan, Kangas, Qiu, Uhlik, Fulton, Ottoson, Gorden, Yokoyama, Danielson, Jevne, Michel, Graff and Bose.)
- Published
- 2022
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14. SAR11 Cells Rely on Enzyme Multifunctionality To Metabolize a Range of Polyamine Compounds.
- Author
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Noell SE, Barrell GE, Suffridge C, Morré J, Gable KP, Graff JR, VerWey BJ, Hellweger FL, and Giovannoni SJ
- Subjects
- Alphaproteobacteria genetics, Alphaproteobacteria metabolism, Bacteria classification, Carbon metabolism, Dissolved Organic Matter, Nitrogen metabolism, Polyamines classification, Seawater chemistry, Bacteria genetics, Bacteria metabolism, Multifunctional Enzymes metabolism, Polyamines metabolism, Seawater microbiology
- Abstract
In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations at seawater pH produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells uptake and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, " Candidatus Pelagibacter" strain HTCC7211 and " Candidatus Pelagibacter ubique" strain HTCC1062. Both strains took up all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but polyamines did not fully substitute for their requirements of glycine (or related compounds) or pyruvate (or related compounds). Our data suggest that potABCD transports all five polyamines and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they metabolize. IMPORTANCE Genome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically, they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality, referring to enzymes that are adapted to have broader substrate and catalytic range than canonically defined, is hypothesized to be an adaptation that increases the range of organic compounds metabolized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells take up and metabolize multiple polyamine compounds and propose that a small set of multifunctional enzymes catalyze this metabolism. We report that polyamine uptake rates can exceed metabolic rates, resulting in both high intracellular concentrations of these nitrogen-rich compounds (in comparison to native polyamine levels) and an increase in cell size.
- Published
- 2021
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15. Evaluation of diagnostic pigments to estimate phytoplankton size classes.
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Chase AP, Kramer SJ, Haëntjens N, Boss ES, Karp-Boss L, Edmondson M, and Graff JR
- Abstract
Phytoplankton accessory pigments are commonly used to estimate phytoplankton size classes, particularly during development and validation of biogeochemical models and satellite ocean color-based algorithms. The diagnostic pigment analysis (DPA) is based on bulk measurements of pigment concentrations and relies on assumptions regarding the presence of specific pigments in different phytoplankton taxonomic groups. Three size classes are defined by the DPA: picoplankton, nanoplankton, and microplankton. Until now, the DPA has not been evaluated against an independent approach that provides phytoplankton size calculated on a per-cell basis. Automated quantitative cell imagery of microplankton and some nanoplankton, used in combination with conventional flow cytometry for enumeration of picoplankton and nanoplankton, provide a novel opportunity to perform an independent evaluation of the DPA. Here, we use a data set from the North Atlantic Ocean that encompasses all seasons and a wide range of chlorophyll concentrations (0.18-5.14 mg m
-3 ). Results show that the DPA overestimates microplankton and picoplankton when compared to cytometry data, and subsequently underestimates the contribution of nanoplankton to total biomass. In contrast to the assumption made by the DPA that the microplankton size class is largely made up of diatoms and dinoflagellates, imaging-in-flow cytometry shows significant presence of diatoms and dinoflagellates in the nanoplankton size class. Additionally, chlorophyll b is commonly attributed solely to picoplankton by the DPA, but Chl b -containing phytoplankton are observed with imaging in both nanoplankton and microplankton size classes. We suggest revisions to the DPA equations and application of uncertainties when calculating size classes from diagnostic pigments., Competing Interests: None declared., (© 2020 The Authors. Limnology and Oceanography: Methods published by Wiley Periodicals LLC. on behalf of Association for the Sciences of Limnology and Oceanography.)- Published
- 2020
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16. Temperate infection in a virus-host system previously known for virulent dynamics.
- Author
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Knowles B, Bonachela JA, Behrenfeld MJ, Bondoc KG, Cael BB, Carlson CA, Cieslik N, Diaz B, Fuchs HL, Graff JR, Grasis JA, Halsey KH, Haramaty L, Johns CT, Natale F, Nissimov JI, Schieler B, Thamatrakoln K, Frede Thingstad T, Våge S, Watkins C, Westberry TK, and Bidle KD
- Subjects
- Haptophyta physiology, Host-Pathogen Interactions, Models, Biological, Virulence, Haptophyta virology, Plant Viruses pathogenicity, Plant Viruses physiology
- Abstract
The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus-host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host-virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host-virus densities.
- Published
- 2020
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17. Small phytoplankton dominate western North Atlantic biomass.
- Author
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Bolaños LM, Karp-Boss L, Choi CJ, Worden AZ, Graff JR, Haëntjens N, Chase AP, Della Penna A, Gaube P, Morison F, Menden-Deuer S, Westberry TK, O'Malley RT, Boss E, Behrenfeld MJ, and Giovannoni SJ
- Subjects
- Biomass, Phytoplankton, Seasons, Cyanobacteria genetics, Diatoms genetics
- Abstract
The North Atlantic phytoplankton spring bloom is the pinnacle in an annual cycle that is driven by physical, chemical, and biological seasonality. Despite its important contributions to the global carbon cycle, transitions in plankton community composition between the winter and spring have been scarcely examined in the North Atlantic. Phytoplankton composition in early winter was compared with latitudinal transects that captured the subsequent spring bloom climax. Amplicon sequence variants (ASVs), imaging flow cytometry, and flow-cytometry provided a synoptic view of phytoplankton diversity. Phytoplankton communities were not uniform across the sites studied, but rather mapped with apparent fidelity onto subpolar- and subtropical-influenced water masses of the North Atlantic. At most stations, cells < 20-µm diameter were the main contributors to phytoplankton biomass. Winter phytoplankton communities were dominated by cyanobacteria and pico-phytoeukaryotes. These transitioned to more diverse and dynamic spring communities in which pico- and nano-phytoeukaryotes, including many prasinophyte algae, dominated. Diatoms, which are often assumed to be the dominant phytoplankton in blooms, were contributors but not the major component of biomass. We show that diverse, small phytoplankton taxa are unexpectedly common in the western North Atlantic and that regional influences play a large role in modulating community transitions during the seasonal progression of blooms.
- Published
- 2020
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18. Immune Pharmacodynamic Responses of the Novel Cancer Immunotherapeutic Imprime PGG in Healthy Volunteers.
- Author
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Bose N, Ottoson NR, Qiu X, Harrison B, Lowe JR, Uhlik MT, Rathmann BT, Kangas TO, Jordan LR, Ertelt KE, Jonas AB, Walsh RM, Chan ASH, Fulton RB, Leonardo SM, Fraser KA, Gorden KB, Matson MA, Graff JR, and Huhn RD
- Subjects
- Antibodies, Fungal blood, Antibodies, Fungal immunology, Chemokines blood, Chemokines immunology, Female, Humans, Male, Adjuvants, Immunologic administration & dosage, Adjuvants, Immunologic chemistry, Adjuvants, Immunologic pharmacokinetics, Fungal Polysaccharides administration & dosage, Fungal Polysaccharides chemistry, Fungal Polysaccharides pharmacokinetics, Immunotherapy, Neoplasms blood, Neoplasms immunology, Neoplasms therapy, Saccharomyces cerevisiae chemistry, beta-Glucans administration & dosage, beta-Glucans chemistry, beta-Glucans pharmacokinetics
- Abstract
Imprime PGG (Imprime) is an i.v. administered, yeast β-1,3/1,6 glucan in clinical development with checkpoint inhibitors. Imprime-mediated innate immune activation requires immune complex formation with naturally occurring IgG anti-β glucan Abs (ABA). We administered Imprime to healthy human volunteers to assess the necessity of ABA for Imprime-mediated immunopharmacodynamic (IPD) changes. Imprime (4 mg/kg) was administered i.v. in single and multiple infusions. Subsets of subjects were premedicated with antihistamine and corticosteroid. Peripheral blood was measured before, during and after Imprime administration for IPD changes (e.g., ABA, circulating immune complexes, complement activation, complete blood counts, cytokine/chemokine, and gene expression changes). IPD changes were analyzed based on pretreatment serum ABA levels: low-ABA (<20 μg/ml), mid-ABA (≥20-50 μg/ml), and high-ABA (≥50 μg/ml). At the end of infusion, free serum ABA levels decreased, circulating immune complex levels increased, and complement activation was observed. At ∼1-4 h after end of infusion, increased expression of cytokines/chemokines, a 1.5-4-fold increase in neutrophil and monocyte counts and a broad activation of innate immune genes were observed. Low-ABA subjects typically showed minimal IPD changes except when ABA levels rose above 20 μg/ml after repeated Imprime dosing. Mild-to-moderate infusion-related reactions occurred in subjects with ABA ≥20 μg/ml. Premedications alleviated some of the infusion-related reactions, but also inhibited cytokine responses. In conclusion, ABA levels, being critical for Imprime-mediated immune activation may provide a plausible, mechanism-based biomarker to identify patients most likely to respond to Imprime-based anticancer immunotherapy., (Copyright © 2019 by The American Association of Immunologists, Inc.)
- Published
- 2019
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19. Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation.
- Author
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Chan AS, Jonas AB, Qiu X, Ottoson NR, Walsh RM, Gorden KB, Harrison B, Maimonis PJ, Leonardo SM, Ertelt KE, Danielson ME, Michel KS, Nelson M, Graff JR, Patchen ML, and Bose N
- Subjects
- Antigen-Antibody Complex immunology, Antineoplastic Agents chemistry, HEK293 Cells, Humans, Immunity, Innate drug effects, Macrophage-1 Antigen metabolism, Receptors, IgG metabolism, beta-Glucans chemistry, beta-Glucans immunology, Antigen-Antibody Complex metabolism, Antineoplastic Agents pharmacology, beta-Glucans pharmacology
- Abstract
Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy., Competing Interests: Anissa S.H. Chan, Adria Bykowski Jonas, Xiaohong Qiu, Nadine R. Ottoson, Richard M. Walsh, Keith B. Gorden, Ben Harrison, Peter J. Maimonis, Steven Leonardo, Kathleen E. Ertelt, Michael E. Danielson, Kyle S. Michel, Mariana Nelson, Jeremy R. Graff, Myra L. Patchen and Nandita Bose are employed by Biothera Pharmaceuticals Inc. All of the authors, with the exception of P.M. and M.N., own stock in Biothera Pharmaceuticals Inc. Imprime is a product in clinical development and has all the associated patents [WO2007146416, WO2015084732, WO2015510271, WO2015510272]. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
- Published
- 2016
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- View/download PDF
20. Cotargeting MNK and MEK kinases induces the regression of NF1-mutant cancers.
- Author
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Lock R, Ingraham R, Maertens O, Miller AL, Weledji N, Legius E, Konicek BM, Yan SC, Graff JR, and Cichowski K
- Subjects
- Anilides administration & dosage, Animals, Cell Line, Tumor, Genes, Neurofibromatosis 1, Humans, Mechanistic Target of Rapamycin Complex 1, Mice, Multiprotein Complexes metabolism, Nerve Sheath Neoplasms metabolism, Nucleocytoplasmic Transport Proteins antagonists & inhibitors, Phosphorylation, Protein Kinase Inhibitors administration & dosage, Pyridines administration & dosage, Signal Transduction, TOR Serine-Threonine Kinases metabolism, MAP Kinase Kinase Kinases antagonists & inhibitors, Mutation, Nerve Sheath Neoplasms drug therapy, Nerve Sheath Neoplasms genetics, Neurofibromin 1 genetics, Protein Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Neurofibromin 1-mutant (NF1-mutant) cancers are driven by excessive Ras signaling; however, there are currently no effective therapies for these or other Ras-dependent tumors. While combined MEK and mTORC1 suppression causes regression of NF1-deficient malignancies in animal models, the potential toxicity of cotargeting these 2 major signaling pathways in humans may necessitate the identification of more refined, cancer-specific signaling nodes. Here, we have provided evidence that MAPK-interacting kinases (MNKs), which converge on the mTORC1 effector eIF4E, are therapeutic targets in NF1-deficient malignancies. Specifically, we evaluated primary human NF1-deficient peripheral nervous system tumors and found that MNKs are activated in the majority of tumors tested. Genetic and chemical suppression of MNKs in NF1-deficient murine tumor models and human cell lines potently cooperated with MEK inhibitors to kill these cancers through effects on eIF4E. We also demonstrated that MNK kinases are important and direct targets of cabozantinib. Accordingly, coadministration of cabozantinib and MEK inhibitors triggered dramatic regression in an aggressive genetically engineered tumor model. The cytotoxicity of this combination required the suppression of MNK-induced eIF4E phosphorylation and was not recapitulated by suppressing other cabozantinib targets. Collectively, these studies demonstrate that combined MNK and MEK suppression represents a promising therapeutic strategy for these incurable Ras-driven tumors and highlight the utility of developing selective MNK inhibitors for these and possibly other malignancies.
- Published
- 2016
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21. Phosphorylation of eIF4E serine 209 is associated with tumour progression and reduced survival in malignant melanoma.
- Author
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Carter JH, Deddens JA, Spaulding NR IV, Lucas D, Colligan BM, Lewis TG, Hawkins E, Jones J, Pemberton JO, Douglass LE, and Graff JR
- Subjects
- Adult, Disease Progression, Female, Humans, Immunohistochemistry, Kaplan-Meier Estimate, Male, Melanoma mortality, Melanoma pathology, Phosphorylation, Retrospective Studies, Biomarkers, Tumor metabolism, Eukaryotic Initiation Factor-4E metabolism, Melanoma metabolism, Serine metabolism
- Abstract
Background: Melanoma is a disease that primarily arises in the skin but is a derivative of the neural crest. Eukaryotic translation initiation factor 4E (eIF4E) regulates translation of multiple malignancy-associated mRNAs and is overexpressed in many epithelial tumours. However, expression in human tumours derived from the neural crest is unknown. Here, we determined the association of eIF4E and phospho-eIF4E expression in melanocytic lesions with malignant conversion, metastatic potential and patient survival., Methods: Archived formalin-fixed, paraffin-embedded surgical specimens from 114 patients with melanocytic lesions were stained immunohistochemically for eIF4E and phospho-eIF4E and evaluated semiquantitatively. The relationship between cytoplasmic and nuclear eIF4E and phospho-eIF4E protein expression, melanocytic lesion subtype and tumour progression was determined. Kaplan-Meier survival analyses and Cox proportional hazard regression were performed., Results: Increased eIF4E and phospho-eIF4E expression was highly associated with malignancy (P<0.0001). High nuclear phospho-eIF4E was associated with synchronous or future metastasis (P=0.0059). Kaplan-Meier analyses demonstrated highly significant associations between high histoscores for cytoplasmic and nuclear phospho-eIF4E and reduced survival in all patients (P=0.0003 and 0.0009, respectively)., Conclusions: Increased melanoma expression of eIF4E and phospho-eIF4E is associated with metastatic potential, reduced survival and increased risk of death.
- Published
- 2016
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22. Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.
- Author
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Thumma SC, Jacobson BA, Patel MR, Konicek BW, Franklin MJ, Jay-Dixon J, Sadiq A, De A, Graff JR, and Kratzke RA
- Subjects
- Carcinoma, Non-Small-Cell Lung drug therapy, Cell Line, Tumor, Cell Proliferation drug effects, Cell Proliferation genetics, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology, Dose-Response Relationship, Drug, Eukaryotic Initiation Factor-4E metabolism, Humans, Lung Neoplasms drug therapy, Oligonucleotides, Antisense genetics, Osteopontin metabolism, Vascular Endothelial Growth Factor A metabolism, Gemcitabine, Carcinoma, Non-Small-Cell Lung genetics, Eukaryotic Initiation Factor-4E genetics, Lung Neoplasms genetics, Molecular Targeted Therapy methods, Oligonucleotides, Antisense pharmacology
- Abstract
Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.
- Published
- 2015
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23. Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.
- Author
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Gkogkas CG, Khoutorsky A, Cao R, Jafarnejad SM, Prager-Khoutorsky M, Giannakas N, Kaminari A, Fragkouli A, Nader K, Price TJ, Konicek BW, Graff JR, Tzinia AK, Lacaille JC, and Sonenberg N
- Subjects
- Adenosine Triphosphatases antagonists & inhibitors, Animals, Autistic Disorder enzymology, Benzofurans therapeutic use, Brain enzymology, Cation Transport Proteins antagonists & inhibitors, Cells, Cultured, Copper-Transporting ATPases, Dendritic Spines pathology, Enzyme Induction drug effects, Female, Fragile X Syndrome enzymology, Fragile X Syndrome genetics, Humans, Male, Matrix Metalloproteinase 9 metabolism, Mice, Inbred C57BL, Mice, Transgenic, Phenotype, Phosphorylation, Protein Processing, Post-Translational, Receptors, Metabotropic Glutamate genetics, Receptors, Metabotropic Glutamate metabolism, Benzofurans pharmacology, Eukaryotic Initiation Factor-4E physiology, Fragile X Syndrome drug therapy, Matrix Metalloproteinase 9 genetics, Protein Biosynthesis drug effects
- Abstract
Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
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24. Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity.
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Campbell RM, Anderson BD, Brooks NA, Brooks HB, Chan EM, De Dios A, Gilmour R, Graff JR, Jambrina E, Mader M, McCann D, Na S, Parsons SH, Pratt SE, Shih C, Stancato LF, Starling JJ, Tate C, Velasco JA, Wang Y, and Ye XS
- Subjects
- Adenosine Triphosphate metabolism, Animals, Anisomycin pharmacology, Binding Sites, Blotting, Western, Cell Line, Cell Line, Tumor, Cells, Cultured, Cytokines metabolism, Dose-Response Relationship, Drug, HeLa Cells, Humans, Imidazoles chemistry, Macrophages drug effects, Macrophages metabolism, Melanoma, Experimental drug therapy, Melanoma, Experimental pathology, Mice, Molecular Structure, Neoplasms genetics, Neoplasms metabolism, Phosphorylation drug effects, Pyridines chemistry, RNA Interference, Treatment Outcome, p38 Mitogen-Activated Protein Kinases genetics, p38 Mitogen-Activated Protein Kinases metabolism, Imidazoles pharmacology, Neoplasms drug therapy, Pyridines pharmacology, Xenograft Model Antitumor Assays, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)(70) = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.
- Published
- 2014
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25. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.
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Jacobson BA, Thumma SC, Jay-Dixon J, Patel MR, Dubear Kroening K, Kratzke MG, Etchison RG, Konicek BW, Graff JR, and Kratzke RA
- Subjects
- Actins genetics, Actins metabolism, Antineoplastic Agents pharmacology, Apoptosis, Cell Count, Cell Line, Tumor, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology, Eukaryotic Initiation Factor-4E genetics, Eukaryotic Initiation Factor-4E metabolism, Eukaryotic Initiation Factor-4F genetics, Eukaryotic Initiation Factor-4F metabolism, Gene Expression, Glutamates pharmacology, Guanine analogs & derivatives, Guanine pharmacology, Humans, Mesothelioma metabolism, Mesothelioma pathology, Molecular Targeted Therapy, Oligonucleotides, Antisense metabolism, Ornithine Decarboxylase genetics, Ornithine Decarboxylase metabolism, Pemetrexed, Poly(ADP-ribose) Polymerases genetics, Poly(ADP-ribose) Polymerases metabolism, Protein Binding, Protein Biosynthesis drug effects, Proto-Oncogene Proteins c-bcl-2 genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Transfection, Gemcitabine, Eukaryotic Initiation Factor-4E antagonists & inhibitors, Eukaryotic Initiation Factor-4F antagonists & inhibitors, Mesothelioma genetics, Oligonucleotides, Antisense genetics, RNA, Messenger antagonists & inhibitors
- Abstract
Background: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma., Methods: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment., Results: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number., Conclusion: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.
- Published
- 2013
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26. LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models.
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Yan SB, Peek VL, Ajamie R, Buchanan SG, Graff JR, Heidler SA, Hui YH, Huss KL, Konicek BW, Manro JR, Shih C, Stewart JA, Stewart TR, Stout SL, Uhlik MT, Um SL, Wang Y, Wu W, Yan L, Yang WJ, Zhong B, and Walgren RA
- Subjects
- Administration, Oral, Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Biological Availability, Blood Vessels drug effects, Blood Vessels pathology, Cell Line, Tumor, Cell Proliferation drug effects, Humans, Indazoles administration & dosage, Indazoles chemistry, Mice, Mutation genetics, Niacinamide administration & dosage, Niacinamide chemistry, Niacinamide pharmacology, Protein Kinase Inhibitors administration & dosage, Protein Kinase Inhibitors chemistry, Proto-Oncogene Proteins c-met metabolism, Receptor Protein-Tyrosine Kinases metabolism, Tetrazoles administration & dosage, Tetrazoles chemistry, Xenograft Model Antitumor Assays, Indazoles pharmacology, Niacinamide analogs & derivatives, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-met antagonists & inhibitors, Receptor Protein-Tyrosine Kinases antagonists & inhibitors, Tetrazoles pharmacology
- Abstract
The HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min(-1) and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).
- Published
- 2013
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27. A common partitioning strategy for photosynthetic products in evolutionarily distinct phytoplankton species.
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Halsey KH, O'Malley RT, Graff JR, Milligan AJ, and Behrenfeld MJ
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- Absorption, Autotrophic Processes drug effects, Carbon Cycle drug effects, Cell Cycle drug effects, Nitrates pharmacology, Phytoplankton drug effects, Phytoplankton growth & development, Biological Evolution, Photosynthesis drug effects, Phytoplankton physiology
- Abstract
· We compare the nutrient-dependent photosynthetic efficiencies of the chlorophyte, Dunaliella tertiolecta, with those of the marine diatom, Thalassiosira weissflogii. Despite considerable evolutionary and physiological differences, these two species appear to use nearly identical growth strategies under a wide range of nutrient limitation. · Using a variety of physiological measurements, we find that, for both species and across all growth rates, 75% of the gross photosynthetic electron flow is invested in carbon fixation and only 30% is retained as net carbon accumulation. A majority of gross photosynthesis (70%) is ultimately used as reductant for biosynthetic pathways and for the generation of ATP. · In both species, newly formed carbon products exhibit much shorter half-lives at slow growth rates than at fast growth rates. We show that this growth rate dependence is a result of increased polysaccharide storage during the S phase of the cell cycle. · We present a model of carbon utilization that incorporates this growth rate-dependent carbon allocation and accurately captures (r(2) = 0.94) the observed time-resolved carbon retention. Together, our findings suggest a common photosynthetic optimization strategy in evolutionarily distinct phytoplankton species and contribute towards a systems-level understanding of carbon flow in photoautotrophs., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)
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- 2013
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28. Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors.
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Altman JK, Szilard A, Konicek BW, Iversen PW, Kroczynska B, Glaser H, Sassano A, Vakana E, Graff JR, and Platanias LC
- Subjects
- Animals, Cell Line, Tumor, Cell Proliferation drug effects, Copper-Transporting ATPases, Down-Regulation drug effects, Humans, K562 Cells, Mice, Neoplastic Stem Cells drug effects, U937 Cells, Xenograft Model Antitumor Assays, Adenosine Triphosphatases antagonists & inhibitors, Antineoplastic Agents therapeutic use, Benzofurans therapeutic use, Cation Transport Proteins antagonists & inhibitors, Leukemia, Myeloid, Acute drug therapy, Protein Kinase Inhibitors therapeutic use
- Abstract
Mnk kinases regulate the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), a protein that plays key roles in the initiation of messenger RNA translation and whose activity is critical for various cellular functions. eIF4E is deregulated in acute myeloid leukemia (AML), and its aberrant activity contributes to leukemogenesis. We determined whether cercosporamide, an antifungal agent that was recently shown to act as a unique Mnk inhibitor, exhibits antileukemic properties. Treatment of AML cells with cercosporamide resulted in a dose-dependent suppression of eIF4E phosphorylation. Such suppression of Mnk kinase activity and eIF4E phosphorylation by cercosporamide resulted in dose-dependent suppressive effects on primitive leukemic progenitors (CFU-L) from AML patients and enhanced the antileukemic properties of cytarabine (Ara-C) or mammalian target of rapamycin (mTOR) complex 1 inhibition. Similarly, the combination of cercosporamide with cytarabine resulted in enhanced antileukemic responses in a xenograft mouse model in vivo. Altogether, this work demonstrates that the unique Mnk inhibitor cercosporamide suppresses phosphorylation of eIF4E and exhibits antileukemic effects, in support of future clinical-translational efforts involving combinations of Mnk inhibitors with cytarabine and/or mTOR inhibitors for the treatment of AML.
- Published
- 2013
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29. Vibrio cholerae Exploits Sub-Lethal Concentrations of a Competitor-Produced Antibiotic to Avoid Toxic Interactions.
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Graff JR, Forschner-Dancause SR, Menden-Deuer S, Long RA, and Rowley DC
- Abstract
Vibrio cholerae is a human pathogenic marine bacterium inhabiting coastal regions and is vectored into human food and water supplies via attachment to particles including detritus, phytoplankton, and zooplankton. Particle colonization by the pathogen is inhibited by an antagonistic interaction with the particle-associated Vibrionales bacterium SWAT3, a producer of the antibiotic andrimid. By analyzing the individual movement behaviors of V. cholerae exposed to a gradient of andrimid in a microfluidics device, we show that the pathogen has a concentration dependent avoidance response to sub-lethal concentrations of the pure antibiotic and to the metabolites produced by a growing colony of SWAT3-wild-type. This avoidance behavior includes a 25% increase in swimming speeds, 30% increase in run lengths, and a shift in the direction of the bacteria away from the andrimid source. Consequently, these behavioral shifts at low concentrations of andrimid would lead to higher diffusivity and result in the dispersion of bacteria away from the competitor and source of the antibiotic. Such alterations in motility were not elicited in response to a non-andrimid-producing SWAT3 mutant, suggesting andrimid may be a negative effector of chemotaxis for V. cholerae. The behavioral response of colonizing bacteria to sub-inhibitory concentrations of competitor-produced antibiotics is one mechanism that can influence microbial diversity and interspecific competition on particles, potentially affecting human health in coastal communities and element cycling in the ocean.
- Published
- 2013
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30. Therapeutic inhibition of MAP kinase interacting kinase blocks eukaryotic initiation factor 4E phosphorylation and suppresses outgrowth of experimental lung metastases.
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Konicek BW, Stephens JR, McNulty AM, Robichaud N, Peery RB, Dumstorf CA, Dowless MS, Iversen PW, Parsons S, Ellis KE, McCann DJ, Pelletier J, Furic L, Yingling JM, Stancato LF, Sonenberg N, and Graff JR
- Subjects
- Animals, Apoptosis drug effects, Base Sequence, Blotting, Western, Cell Proliferation drug effects, Female, Humans, Inhibitory Concentration 50, Intracellular Signaling Peptides and Proteins genetics, Lung Neoplasms drug therapy, Lung Neoplasms pathology, Mice, Mice, Nude, Molecular Sequence Data, Neoplasm Metastasis drug therapy, Phosphorylation, Polymerase Chain Reaction, Protein Serine-Threonine Kinases genetics, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Benzofurans pharmacology, Eukaryotic Initiation Factor-4E metabolism, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Lung Neoplasms metabolism, Protein Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Activation of the translation initiation factor 4E (eIF4E) promotes malignant transformation and metastasis. Signaling through the AKT-mTOR pathway activates eIF4E by phosphorylating the inhibitory 4E binding proteins (4E-BP). This liberates eIF4E and allows binding to eIF4G. eIF4E can then be phosphorylated at serine 209 by the MAPK-interacting kinases (Mnk), which also interact with eIF4G. Although dispensable for normal development, Mnk function and eIF4E phosphorylation promote cellular proliferation and survival and are critical for malignant transformation. Accordingly, Mnk inhibition may serve as an attractive cancer therapy. We now report the identification of a potent, selective and orally bioavailable Mnk inhibitor that effectively blocks 4E phosphorylation both in vitro and in vivo. In cultured cancer cell lines, Mnk inhibitor treatment induces apoptosis and suppresses proliferation and soft agar colonization. Importantly, a single, orally administered dose of this Mnk inhibitor substantially suppresses eIF4E phosphorylation for at least 4 hours in human xenograft tumor tissue and mouse liver tissue. Moreover, oral dosing with the Mnk inhibitor significantly suppresses outgrowth of experimental B16 melanoma pulmonary metastases as well as growth of subcutaneous HCT116 colon carcinoma xenograft tumors, without affecting body weight. These findings offer the first description of a novel, orally bioavailable MNK inhibitor and the first preclinical proof-of-concept that MNK inhibition may provide a tractable cancer therapeutic approach., (©2011 AACR.)
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- 2011
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31. Modulation of 4E-BP1 function as a critical determinant of enzastaurin-induced apoptosis.
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Dumstorf CA, Konicek BW, McNulty AM, Parsons SH, Furic L, Sonenberg N, and Graff JR
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- Adaptor Proteins, Signal Transducing, Animals, Cell Cycle Proteins, Cell Line, Tumor, Drug Screening Assays, Antitumor, Eukaryotic Initiation Factor-4F metabolism, Eukaryotic Initiation Factors, Gene Knockout Techniques, Humans, Mice, Mice, Knockout, Phosphorylation drug effects, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction drug effects, TOR Serine-Threonine Kinases metabolism, Apoptosis drug effects, Carrier Proteins metabolism, Indoles pharmacology, Phosphoproteins metabolism
- Abstract
Enzastaurin (LY317615.HCl) is currently in a phase III registration trial for diffuse large B-Cell lymphoma and numerous phase II clinical trials. Enzastaurin suppresses angiogenesis and induces apoptosis in multiple human tumor cell lines by inhibiting protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K)/AKT pathway signaling. PI3K/AKT pathway signaling liberates eukaryotic translation initiation factor 4E (eIF4E) through the hierarchical phosphorylation of eIF4E binding proteins (4E-BP). When hypophosphorylated, 4E-BPs associate with eIF4E, preventing eIF4E from binding eIF4G, blocking the formation of the eIF4F translation initiation complex. Herein, we show that enzastaurin treatment impacts signaling throughout the AKT/mTOR pathway leading to hypophosphorylation of 4E-BP1 in cancer cells of diverse lineages (glioblastoma, colon carcinoma, and B-cell lymphoma). Accordingly, enzastaurin treatment increases the amount of eIF4E bound to 4E-BP1 and decreases association of eIF4E with eIF4G, thereby reducing eIF4F translation initiation complex levels. We therefore chose to evaluate whether this effect on 4E-BP1 was involved in enzastaurin-induced apoptosis. Remarkably, enzastaurin-induced apoptosis was blocked in cancer cells depleted of 4E-BP1 by siRNAs, or in 4EBP1/2 knockout murine embryonic fibroblasts cells. Furthermore, eIF4E expression was increased and 4E-BP1 expression was decreased in cancer cells selected for reduced sensitivity to enzastaurin-induced apoptosis. These data highlight the importance of modulating 4E-BP1 function, and eIF4F complex levels, in the direct antitumor effect of enzastaurin and suggest that 4E-BP1 function may serve as a promising determinant of enzastaurin activity., (©2010 AACR.)
- Published
- 2010
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32. eIF4E activation is commonly elevated in advanced human prostate cancers and significantly related to reduced patient survival.
- Author
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Graff JR, Konicek BW, Lynch RL, Dumstorf CA, Dowless MS, McNulty AM, Parsons SH, Brail LH, Colligan BM, Koop JW, Hurst BM, Deddens JA, Neubauer BL, Stancato LF, Carter HW, Douglass LE, and Carter JH
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Apoptosis physiology, Cell Cycle physiology, Cell Cycle Proteins, Cell Line, Tumor, Disease Progression, Eukaryotic Initiation Factor-4E genetics, Humans, Immunohistochemistry, Male, Oligonucleotides, Antisense genetics, Phosphoproteins metabolism, Phosphorylation, Prostatic Neoplasms pathology, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Proto-Oncogene Proteins c-bcl-2 metabolism, Proto-Oncogene Proteins c-myc metabolism, Eukaryotic Initiation Factor-4E biosynthesis, Prostatic Neoplasms metabolism
- Abstract
Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.
- Published
- 2009
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33. Molecular pathways involved in the synergistic interaction of the PKC beta inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells.
- Author
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Tekle C, Giovannetti E, Sigmond J, Graff JR, Smid K, and Peters GJ
- Subjects
- Apoptosis drug effects, Blotting, Western, Carcinoma, Non-Small-Cell Lung enzymology, Carcinoma, Non-Small-Cell Lung genetics, Cell Cycle drug effects, Cell Cycle Proteins metabolism, Cell Division drug effects, Cell Line, Tumor, Cyclooxygenase 2 metabolism, Drug Synergism, Guanine pharmacology, Humans, Lung Neoplasms enzymology, Lung Neoplasms genetics, Pemetrexed, Phosphorylation, Polymerase Chain Reaction, Protein Kinase C metabolism, Protein Kinase C beta, Vascular Endothelial Growth Factor A metabolism, Carcinoma, Non-Small-Cell Lung pathology, Folic Acid Antagonists pharmacology, Glutamates pharmacology, Guanine analogs & derivatives, Indoles pharmacology, Lung Neoplasms pathology, Protein Kinase C antagonists & inhibitors, Protein Kinase Inhibitors pharmacology
- Abstract
Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKC beta inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription-PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3 beta and Akt phosphorylation, which was additionally reduced by drug combination (-58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients.
- Published
- 2008
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34. Targeting the eIF4F translation initiation complex for cancer therapy.
- Author
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Konicek BW, Dumstorf CA, and Graff JR
- Subjects
- Animals, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Eukaryotic Initiation Factor-4F genetics, Eukaryotic Initiation Factor-4F metabolism, Humans, Mice, Neoplasms blood supply, Neoplasms metabolism, Oligonucleotides, Antisense genetics, Oligonucleotides, Antisense therapeutic use, Oncogene Proteins genetics, Oncogene Proteins metabolism, RNA, Messenger biosynthesis, Eukaryotic Initiation Factor-4F antagonists & inhibitors, Neoplasms therapy, Neovascularization, Pathologic therapy, Oncogene Proteins antagonists & inhibitors, RNA, Messenger antagonists & inhibitors
- Abstract
In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.
- Published
- 2008
- Full Text
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35. Targeting the eukaryotic translation initiation factor 4E for cancer therapy.
- Author
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Graff JR, Konicek BW, Carter JH, and Marcusson EG
- Subjects
- Animals, Eukaryotic Initiation Factor-4E biosynthesis, Eukaryotic Initiation Factor-4E genetics, Humans, Neoplasms genetics, Neoplasms metabolism, RNA, Messenger biosynthesis, RNA, Messenger genetics, Eukaryotic Initiation Factor-4E antagonists & inhibitors, Neoplasms therapy
- Abstract
The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer.
- Published
- 2008
- Full Text
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36. Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.
- Author
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Graff JR, Konicek BW, Vincent TM, Lynch RL, Monteith D, Weir SN, Schwier P, Capen A, Goode RL, Dowless MS, Chen Y, Zhang H, Sissons S, Cox K, McNulty AM, Parsons SH, Wang T, Sams L, Geeganage S, Douglass LE, Neubauer BL, Dean NM, Blanchard K, Shou J, Stancato LF, Carter JH, and Marcusson EG
- Subjects
- Animals, Apoptosis, Base Sequence, Cells, Cultured, Endothelial Cells metabolism, Eukaryotic Initiation Factor-4E genetics, Humans, Mice, Neoplasms blood supply, Neoplasms pathology, Xenograft Model Antitumor Assays, Eukaryotic Initiation Factor-4E metabolism, Gene Expression Regulation, Neoplastic, Neoplasms metabolism, Neoplasms therapy, Protein Biosynthesis genetics
- Abstract
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
- Published
- 2007
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37. Int7G24A variant of transforming growth factor-beta receptor type I is associated with invasive breast cancer.
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Chen T, Jackson CR, Link A, Markey MP, Colligan BM, Douglass LE, Pemberton JO, Deddens JA, Graff JR, and Carter JH
- Subjects
- Adult, Aged, Aged, 80 and over, Breast Neoplasms metabolism, Breast Neoplasms pathology, Carcinoma, Ductal, Breast genetics, Carcinoma, Ductal, Breast metabolism, Carcinoma, Ductal, Breast secondary, Carcinoma, Lobular genetics, Carcinoma, Lobular metabolism, Carcinoma, Lobular secondary, Case-Control Studies, Disease Progression, Female, Germ-Line Mutation genetics, Humans, Male, Middle Aged, Neoplasm Invasiveness pathology, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Polymorphism, Single-Stranded Conformational, Protein Serine-Threonine Kinases, Receptor, Transforming Growth Factor-beta Type I, Risk Factors, Sequence Deletion genetics, Activin Receptors, Type I genetics, Breast Neoplasms genetics, Genetic Variation, Introns genetics, Neoplasm Invasiveness genetics, Receptors, Transforming Growth Factor beta genetics
- Abstract
Purpose: The transforming growth factor-beta (TGF-beta) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-beta receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study. We hypothesize that this germline variant may be involved in development and progression of breast cancer., Experimental Design: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population. We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls. The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in the TGFBR1 were also analyzed by PCR and single-strand conformational polymorphism., Results: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71 breast cancers., Conclusion: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with a larger sample size is warranted to validate the association of the Int7G24A with human breast cancer.
- Published
- 2006
- Full Text
- View/download PDF
38. The protein kinase Cbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts.
- Author
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Graff JR, McNulty AM, Hanna KR, Konicek BW, Lynch RL, Bailey SN, Banks C, Capen A, Goode R, Lewis JE, Sams L, Huss KL, Campbell RM, Iversen PW, Neubauer BL, Brown TJ, Musib L, Geeganage S, and Thornton D
- Subjects
- Animals, Cell Growth Processes drug effects, Colonic Neoplasms enzymology, Colonic Neoplasms pathology, Female, Glioblastoma enzymology, Glioblastoma pathology, Glycogen Synthase Kinase 3 antagonists & inhibitors, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, HCT116 Cells, Humans, Male, Mice, Mice, Nude, Phosphorylation drug effects, Prostatic Neoplasms drug therapy, Prostatic Neoplasms enzymology, Prostatic Neoplasms pathology, Protein Kinase C beta, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, Ribosomal Protein S6 antagonists & inhibitors, Ribosomal Protein S6 metabolism, Signal Transduction drug effects, Xenograft Model Antitumor Assays, Apoptosis drug effects, Colonic Neoplasms drug therapy, Glioblastoma drug therapy, Indoles pharmacology, Protein Kinase C antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Proto-Oncogene Proteins antagonists & inhibitors
- Abstract
Activation of protein kinase Cbeta (PKCbeta) has been repeatedly implicated in tumor-induced angiogenesis. The PKCbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCbeta has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3betaser9, ribosomal protein S6(S240/244), and AKT(Thr308). Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3beta in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3beta phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3beta phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3beta phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.
- Published
- 2005
- Full Text
- View/download PDF
39. The progression of LNCaP human prostate cancer cells to androgen independence involves decreased FOXO3a expression and reduced p27KIP1 promoter transactivation.
- Author
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Lynch RL, Konicek BW, McNulty AM, Hanna KR, Lewis JE, Neubauer BL, and Graff JR
- Subjects
- Blotting, Western, Cell Cycle Proteins metabolism, Cell Line, Tumor, Cyclin-Dependent Kinase Inhibitor p27, Disease Progression, Forkhead Box Protein O1, Forkhead Transcription Factors, Humans, Male, Phosphorylation, Plasmids metabolism, Promoter Regions, Genetic, Prostatic Neoplasms genetics, Response Elements, Signal Transduction, Transfection, Tumor Suppressor Proteins metabolism, Androgens metabolism, Cell Cycle Proteins genetics, DNA-Binding Proteins metabolism, Prostatic Neoplasms metabolism, Prostatic Neoplasms pathology, Transcription Factors metabolism, Transcriptional Activation, Tumor Suppressor Proteins genetics
- Abstract
The progression of human prostate cancer from the initial androgen-dependent phase to androgen independence involves diminished apoptosis and a release from the cell cycle block triggered by androgen ablation therapy. FOXO transcription factors play a central role in promoting expression of proapoptotic and cell cycle regulatory genes (e.g., FasL and p27KIP1). Reduced FOXO function might, therefore, play a role in androgen-independent progression of human prostate cancer. Herein, we show that FOXO function is compromised in androgen-independent prostate cancer cells (LNAI) versus androgen-dependent LNCaP cells. The FOXO3a protein, the most highly expressed FOXO family member in prostate cancer cells, is hyperphosphorylated in LNAI cells. FOXO3a expression is also markedly reduced in these androgen-independent LNAI cells when compared with parental LNCaP cells. Together, reduced FOXO3a expression coupled to FOXO3a hyperphosphorylation would suppress FOXO transcriptional activity. Accordingly, activity of the FOXO-responsive p27KIP1 promoter is reduced 60% in these LNAI cells when compared with LNCaP cells. Moreover, mutation of a conserved FOXO response element suppresses p27KIP1 promoter activity, substantiating a regulatory role for this FOXO response element in p27KIP1 promoter transactivation. Finally, we show that the activity of a distinct FOXO-responsive promoter, the 3X-IRS promoter, is also reduced in LNAI cells. Collectively, these data show that reduced FOXO3a expression coupled to increased FOXO3a phosphorylation coincide with reduced FOXO-responsive promoter activity in androgen-independent LNAI cells when compared with androgen-dependent LNCaP cells. To the extent that this model reflects human disease, these data suggest that FOXO function may be compromised with androgen-independent progression of human prostate cancer.
- Published
- 2005
- Full Text
- View/download PDF
40. An intronic variant of the TGFBR1 gene is associated with carcinomas of the kidney and bladder.
- Author
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Chen T, Jackson C, Costello B, Singer N, Colligan B, Douglass L, Pemberton J, Deddens J, Graff JR, and Carter JH
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Case-Control Studies, Female, Homozygote, Humans, Kidney, Male, Middle Aged, Mutation genetics, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Polymorphism, Single-Stranded Conformational, Protein Serine-Threonine Kinases, Receptor, Transforming Growth Factor-beta Type I, Urinary Bladder, Activin Receptors, Type I genetics, Carcinoma, Renal Cell genetics, Carcinoma, Transitional Cell genetics, Genetic Variation, Introns genetics, Kidney Neoplasms genetics, Receptors, Transforming Growth Factor beta genetics, Urinary Bladder Neoplasms genetics
- Abstract
TGF-beta signaling is frequently perturbed in many human cancers, including renal cell carcinomas (RCCs) and transitional cell carcinomas (TCCs) of the bladder. Genetic alterations of the TGF-beta type 1 receptor (TGFBR1) may contribute to these perturbations. We therefore examined variations in the TGFBR1 gene by PCR, SSCP and RFLP in carcinomas of the urinary system and in tissues from noncancer, age-matched controls. A G-->A variant 24 bp downstream of the exon/intron 7 boundary of the TGFBR1 gene (Int7G24A) was evident in patients with RCC (46.5%, n = 86) and bladder and upper urinary tract TCC (49.2%, n = 65) significantly more frequently than in age-matched controls (28.3%, n = 113, p < 0.002 by chi2 test). Moreover, 8 homozygous variant carriers were found in the cancer groups, whereas not a single homozygous variant carrier was found in the control group. The Int7G24A allele (both heterozygous G/A and homozygous A/A carriers) was associated with increased RCC incidence (OR = 2.20, 95% CI 1.22-3.96) and TCC incidence (OR = 2.45, 95% CI 1.89-3.16). One somatic mutation of serine to phenylalanine at codon 57 of the TGFBR1 gene was confirmed in an upper urinary tract TCC. In conclusion, the Int7G24A variant in the TGFBR1 gene is significantly more frequent in patients with RCC and TCC than normal age-matched controls, suggesting that it may represent a risk factor for the development of kidney and bladder carcinomas.
- Published
- 2004
- Full Text
- View/download PDF
41. Expression levels of protein kinase C-alpha in non-small-cell lung cancer.
- Author
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Lahn M, Su C, Li S, Chedid M, Hanna KR, Graff JR, Sandusky GE, Ma D, Niyikiza C, Sundell KL, John WJ, Giordano TJ, Beer DG, Paterson BM, Su EW, and Bumol TF
- Subjects
- Carcinoma, Non-Small-Cell Lung pathology, Humans, Lung Neoplasms pathology, Oligonucleotide Array Sequence Analysis, Protein Kinase C-alpha, RNA, Messenger metabolism, Carcinoma, Non-Small-Cell Lung genetics, Gene Expression Profiling, Lung Neoplasms genetics, Protein Kinase C genetics
- Abstract
Current treatments of non-small-cell lung cancer (NSCLC) are inadequate and new therapies are being developed that target specific cellular signaling proteins associated with tumor growth. One potential target is protein kinase C (PKC)-alpha, a signaling molecule with an important role in cell regulation and proliferation. The present study examines the expression levels of PKC-alpha in NSCLC to better understand the distribution of PKC-alpha in NSCLC. We analyzed tumor specimens from an independent tumor tissue bank to determine PKC-alpha protein and messenger RNA gene expression in NSCLC. In addition, we used publicly available gene expression array data to further understand PKC-a-associated gene expression profiles in NSCLC. We found that PKC-alpha is highly expressed in < or = 20% of patients with NSCLC. We also found that PKC-alpha was preferentially expressed in adenocarcinoma compared with squamous cell carcinoma of the lung.
- Published
- 2004
- Full Text
- View/download PDF
42. Pak-1 expression increases with progression of colorectal carcinomas to metastasis.
- Author
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Carter JH, Douglass LE, Deddens JA, Colligan BM, Bhatt TR, Pemberton JO, Konicek S, Hom J, Marshall M, and Graff JR
- Subjects
- Adenoma metabolism, Carcinoma metabolism, Colon pathology, Colonic Neoplasms pathology, Colorectal Neoplasms metabolism, Cytoplasm metabolism, Disease Progression, GTP Phosphohydrolases metabolism, Humans, Immunohistochemistry, Lymphatic Metastasis, Neoplasm Metastasis, Phosphatidylinositol 3-Kinases metabolism, Protein Serine-Threonine Kinases metabolism, Treatment Outcome, cdc42 GTP-Binding Protein metabolism, p21-Activated Kinases, ras Proteins metabolism, Colorectal Neoplasms pathology, Protein Serine-Threonine Kinases biosynthesis
- Abstract
Purpose: The p21-activated kinase-1 (Pak-1) promotes cell motility and invasiveness. Pak-1 is activated by the Rac, Rho, and Cdc42 small GTPases in response to a variety of stimuli including ras and phosphatidylinositol 3'-kinase/AKT pathway activation. Because Pak-1 plays a central role in regulating cell motility and invasiveness, we sought to determine whether Pak-1 may be involved in the malignant progression of colorectal carcinoma., Experimental Design: Pak-1 expression was examined by immunohistochemistry in archived tissues from normal human colons, tubular and tubulovillous adenomas, invasive adenocarcinomas (stages I-III/IV), and lymph node metastases (184 total specimens from 38 patients). Specific cytoplasmic immunostaining was evaluated for overall intensity and uniformity to derive a combined histoscore (stain intensity x percentage of epithelium stained)., Results: Pak-1 expression was increased significantly with colorectal cancer progression from normal tissue to lymph node metastases (P < 0.0001). Furthermore, Pak-1 expression was increased significantly in adenomas, invasive carcinomas, and lymph node metastases compared with normal colon (P < 0.0001). Strikingly, Pak-1 expression was significantly higher in lymph node metastases than in invasive cancers, adenomas, or normal colon (P < 0.0001). Moreover, in patients with multiple lesions representing different stages of disease, Pak-1 expression was increased specifically in the most advanced lesions., Conclusions: This study demonstrates that Pak-1 expression is increased significantly with malignant progression of human colorectal carcinoma. These data, along with numerous functional studies demonstrating a central role for Pak-1 activity in tumor invasiveness and motility, implicate Pak-1 as an exciting target for therapy of colorectal carcinoma.
- Published
- 2004
- Full Text
- View/download PDF
43. eIF-4E expression and its role in malignancies and metastases.
- Author
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De Benedetti A and Graff JR
- Subjects
- Animals, Cell Transformation, Neoplastic, Eukaryotic Initiation Factor-4E antagonists & inhibitors, Eukaryotic Initiation Factor-4E genetics, Fibroblast Growth Factor 2 genetics, Humans, Neoplasms metabolism, Neoplasms therapy, Ornithine Decarboxylase genetics, Protein Biosynthesis, RNA, Messenger genetics, Vascular Endothelial Growth Factor A genetics, Eukaryotic Initiation Factor-4E physiology, Neoplasms etiology
- Abstract
The contribution of the mRNA cap-binding protein, eIF-4E, to malignant transformation and progression has been illuminated over the past decade. eIF-4E overexpression has been demonstrated in human tumors of the breast, head and neck, colon, prostate, bladder, cervix and lung, and has been related to disease progression. Overexpression of eIF-4E in experimental models dramatically alters cellular morphology, enhances proliferation and induces cellular transformation, tumorigenesis and metastasis. Conversely, blocking eIF-4E function by expression of antisense RNA, or overexpression of the inhibitory eIF-4E binding proteins (4E-BPs), suppresses cellular transformation, tumor growth, tumor invasiveness and metastasis. Although eIF-4E regulates the recruitment of mRNA to ribosomes, and thereby globally regulates cap-dependent protein synthesis, eIF-4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs--those that generally encode key proteins involved in cellular growth, angiogenesis, survival and malignancy (e.g. cyclin D1, c-myc, vascular endothelial growth factor, matrix metalloprotease 9). A deeper understanding of the role of eIF-4E in regulating the translation of the diverse gene products involved in all aspects of malignancy will improve the capacity to exploit eIF-4E as a therapeutic target and as a marker for human cancer progression.
- Published
- 2004
- Full Text
- View/download PDF
44. The selective estrogen receptor modulator trioxifene (LY133314) inhibits metastasis and extends survival in the PAIII rat prostatic carcinoma model.
- Author
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Neubauer BL, McNulty AM, Chedid M, Chen K, Goode RL, Johnson MA, Jones CD, Krishnan V, Lynch R, Osborne HE, and Graff JR
- Subjects
- Adenocarcinoma metabolism, Adenocarcinoma pathology, Animals, Body Weight drug effects, Cell Division drug effects, Disease Models, Animal, Estrogen Receptor alpha, Estrogen Receptor beta, Genitalia, Male drug effects, Luciferases antagonists & inhibitors, Luciferases metabolism, Lung Neoplasms prevention & control, Lung Neoplasms secondary, Lymphatic Metastasis, Male, Organ Size drug effects, Prostatic Neoplasms metabolism, Prostatic Neoplasms pathology, Pyrrolidines metabolism, Rats, Receptors, Androgen biosynthesis, Receptors, Estrogen biosynthesis, Receptors, Estrogen metabolism, Selective Estrogen Receptor Modulators metabolism, Testis anatomy & histology, Testis drug effects, Adenocarcinoma drug therapy, Prostatic Neoplasms drug therapy, Pyrrolidines pharmacology, Selective Estrogen Receptor Modulators pharmacology
- Abstract
Trioxifene (LY133314) is a selective estrogen receptor modulator (SERM) with competitive binding activity against estradiol for estrogen receptor alpha (ERalpha) and antagonistic activity against ERalpha-mediated gene expression. The PAIII rat prostatic adenocarcinoma (PCa) is an androgen receptor-negative, ERalpha- and ERbeta-positive, spontaneously metastatic rodent tumor cell line. After s.c. implantation of 10(6) PAIII cells in the tail, s.c. administration of trioxifene (2.0, 4.0, 20.0, or 40.0 mg/kg-day) for 30 days produced significant (P < 0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximum nodal weight decreases, 86% and 88% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by trioxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P < 0.05) reduced by trioxifene administration in a dose-related manner (maximal reduction, 98% from control values). Continual administration of the compound significantly (P < 0.05) extended survival of PAIII-bearing rats. Trioxifene inhibited the proliferation of PAIII cells at micromolar levels in vitro but did not slow growth of the primary tumor growth in the tail. Trioxifene administration also produced regression of male accessory sex organs. In PAIII-tumor-bearing animals, trioxifene administration produced a maximal regression of 76% for ventral prostate and 64% for seminal vesicle (P < 0.05 for both). SERMs may be preferable to estrogens given their efficacy in experimental PCa models and relative lack of side effects observed in clinical trials. Our data support the contention that trioxifene represents a SERM with potential antimetastatic efficacy for the treatment of androgen-independent, metastatic PCa.
- Published
- 2003
45. Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs.
- Author
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Graff JR and Zimmer SG
- Subjects
- Animals, Disease Progression, Humans, Up-Regulation, Eukaryotic Initiation Factor-4E physiology, Gene Expression Regulation, Neoplastic, Neoplasm Metastasis genetics, Protein Biosynthesis, RNA, Messenger metabolism
- Abstract
To form metastases, tumors must break from the primary tumor site, invade surrounding tissues, enter and survive within the circulation and ultimately colonize a distal tissue. Each of these steps requires the cooperative function of numerous proteins--proteins that facilitate angiogenesis (e.g., VEGF), cell survival (e.g., Bcl-2), invasion (e.g., MMPs), and autocrine growth stimulation (e.g., c-myc, cyclin D1). Although expression of these proteins is regulated at many levels by disparate stimuli, translation of these key malignancy-related proteins is regulated primarily by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex. By binding the cap structure at the 5' terminus of cellular mRNAs, eIF-4E recruits mRNAs to the eIF-4F complex, which then scans from the 5' cap through the untranslated region (5'UTR), unwinding secondary structure to reveal the translation initiation codon and to enable ribosome loading. Messenger RNAs with short unstructured 5' UTRs are more easily translated than mRNAs harboring lengthy, highly structured 5' UTRs, as these prohibit efficient scanning and start codon recognition. As such, the translation of these mRNAs, which typically encode proteins involved in angiogenesis (e.g., VEGF), tumor growth (cyclin D1) and survival (Bcl-2), is suppressed except when eIF-4E is engaged with the eIF-4F complex--a common event in many human and experimental cancers. This review focuses on the hypothesis that enhanced eIF-4E function contributes to metastatic progression by selectively upregulating the translation of key malignancy-related proteins that together conspire to drive the metastatic process.
- Published
- 2003
- Full Text
- View/download PDF
46. Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma.
- Author
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Jiang J, Neubauer BL, Graff JR, Chedid M, Thomas JE, Roehm NW, Zhang S, Eckert GJ, Koch MO, Eble JN, and Cheng L
- Subjects
- Adenocarcinoma pathology, Aged, Group II Phospholipases A2, Humans, Male, Middle Aged, Neoplasm Staging, Phospholipases A2, Prostatic Intraepithelial Neoplasia chemistry, Prostatic Intraepithelial Neoplasia pathology, Prostatic Neoplasms pathology, Adenocarcinoma enzymology, Phospholipases A analysis, Prostate enzymology, Prostatic Intraepithelial Neoplasia enzymology, Prostatic Neoplasms enzymology
- Abstract
Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.
- Published
- 2002
- Full Text
- View/download PDF
47. Emerging targets in the AKT pathway for treatment of androgen-independent prostatic adenocarcinoma.
- Author
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Graff JR
- Subjects
- Animals, Apoptosis drug effects, Humans, Male, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-akt, Adenocarcinoma drug therapy, Adenocarcinoma pathology, Antineoplastic Agents therapeutic use, Prostatic Neoplasms drug therapy, Prostatic Neoplasms pathology, Protein Serine-Threonine Kinases, Proto-Oncogene Proteins physiology, Signal Transduction drug effects
- Abstract
Prostatic adenocarcinoma (CaP) is the most common, non-cutaneous malignancy and the second-leading cause of cancer death in men. The disease has two distinct phases: the androgen-dependent phase, which can be treated effectively with androgen ablation therapies, and the androgen-independent phase, for which there is no effective life-prolonging therapy. An estimated 32,000 men will die this year from androgen-independent, metastatic CaP. Efforts to understand the metastatic progression of CaP and the emergence of androgen-independent disease have begun to illuminate the molecular events involved. Recent work suggests that CaP progression to androgen-independent, metastatic disease involves a dampened apoptotic response, a release from the cell cycle block that initially follows androgen withdrawal and a shift from dependence on paracrine-derived growth and survival factors to autonomous production of these key proteins. Functional loss of the tumour suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) and subsequent activation of the AKT pathway, have been prominently implicated in the progression of CaP to androgen-independence. Activation of the AKT pathway can suppress the apoptotic response, undermine cell cycle control and selectively enhance the production of key growth and survival factors. Though many proteins and intracellular signalling pathways can influence these biological processes, activation of the AKT pathway may be a particularly potent signal involved in CaP progression to androgen-independence and therefore presents a series of potential targets for therapy of advanced androgen-independent CaP.
- Published
- 2002
- Full Text
- View/download PDF
48. Expression of group IIa secretory phospholipase A2 increases with prostate tumor grade.
- Author
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Graff JR, Konicek BW, Deddens JA, Chedid M, Hurst BM, Colligan B, Neubauer BL, Carter HW, and Carter JH
- Subjects
- Androgens pharmacology, Arachidonic Acid metabolism, Cell Division, Disease Progression, Humans, Immunohistochemistry, Isoenzymes metabolism, Male, Phospholipases A2, Prostatic Hyperplasia enzymology, Prostatic Hyperplasia pathology, Prostatic Neoplasms pathology, Transplantation, Heterologous, Tumor Cells, Cultured, Phospholipases A metabolism, Prostatic Neoplasms enzymology
- Abstract
Purpose: Arachidonate release contributes to prostate tumor progression as arachidonate is metabolized into prostaglandins and leukotrienes, potent mediators of immune suppression, cellular proliferation, tumor motility, and invasion. The group IIa sPLA2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids. We therefore sought to determine whether sPLA2-IIa expression might be related to the development or progression of prostatic adenocarcinoma (CaP)., Experimental Design: sPLA2-IIa expression was examined by Western blot analyses of CaP cells and xenografts and by immunohistochemistry of benign prostatic hyperplasias and primary human CaPs (n = 101) using a sPLA2-IIa-specific polyclonal antibody., Results: sPLA2-IIa expression was increased dramatically in the androgen-independent CWR-22R and LNAI CaP cells versus the androgen-dependent CWR-22 and LNCaP cells. Immunohistochemical analyses revealed that sPLA2-IIa expression was also significantly increased with CaP development and advancing disease (trend analysis; Pearson correlation coefficient, P = 0.016). High-grade CaPs showed intense, uniform staining for sPLA2-IIa that was significantly different from that in adjacent benign prostatic hyperplasias (Fisher's exact test, P = 0.021) or low-grade CaP (P = 0.013), both of which showed only focal or weak sPLA2-IIa staining. Further, uniform sPLA2-IIa expression was directly related to the increased proliferative index that typifies advancing disease (P = 0.001). Most significantly, enhanced sPLA2-IIa expression was inversely related to 5-year patient survival (P = 0.015)., Conclusions: These data show that sPLA2-IIa expression increases with progression to androgen-independence and is highest in the most poorly-differentiated, highest-grade primary human CaP samples.
- Published
- 2001
49. AKT-1, -2, and -3 are expressed in both normal and tumor tissues of the lung, breast, prostate, and colon.
- Author
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Zinda MJ, Johnson MA, Paul JD, Horn C, Konicek BW, Lu ZH, Sandusky G, Thomas JE, Neubauer BL, Lai MT, and Graff JR
- Subjects
- Breast metabolism, Breast pathology, Breast Neoplasms genetics, Breast Neoplasms pathology, Colon metabolism, Colon pathology, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Female, Gene Expression, Gene Expression Regulation, Neoplastic, Humans, Lung metabolism, Lung pathology, Lung Neoplasms genetics, Lung Neoplasms pathology, Male, Neoplasms pathology, Oncogene Proteins genetics, Prostate metabolism, Prostate pathology, Prostatic Neoplasms genetics, Prostatic Neoplasms pathology, Protein Serine-Threonine Kinases genetics, Proto-Oncogene Proteins c-akt, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Neoplasm genetics, Reverse Transcriptase Polymerase Chain Reaction, Tissue Distribution, Tumor Cells, Cultured, Neoplasms genetics, Proto-Oncogene Proteins genetics, RNA, Neoplasm metabolism
- Abstract
Purpose: The AKT/PKB kinase controls many of the intracellular processes that are dysregulated in human cancer, including the suppression of apoptosis and anoikis and the induction of cell cycle progression. Three isoforms of AKT have been identified: AKT-1, -2, and -3. Selective up-regulation of AKT-3 RNA expression has been reported in hormone-independent breast and prostate cancer cell lines suggesting that AKT-3 expression may be increased with breast or prostate tumor progression. To determine whether AKT-3 RNA expression is selectively up-regulated in human cancers and whether the patterns of AKT RNA expression may change with tumor development, we examined AKT isoform expression by RT-PCR in human cancer cell lines, primary human cancers, and normal human tissues., Experimental Design: AKT-1, -2, and -3 RNA expression was examined by RT-PCR. Because up-regulated AKT-3 expression has been implicated in human breast and prostate cancer progression, we also examined AKT-3 expression levels by semiquantitative RT-PCR using matched normal/tumor first-strand cDNA pairs from colon, breast, prostate, and lung cancers., Results: Our data reveal that the overwhelming majority of both normal and tumor tissues express all three of the AKT isoforms. Moreover, semiquantitative RT-PCR of matched normal/tumor pairs confirmed similar AKT-3 RNA expression levels in both normal and tumor tissue., Conclusions: Our data show that both normal and tumor tissues express all three of the AKT isoforms and indicate that tumorigenesis does not involve a dramatic shift in the RNA expression patterns of the three AKT isoforms.
- Published
- 2001
50. Integrin-linked kinase expression increases with prostate tumor grade.
- Author
-
Graff JR, Deddens JA, Konicek BW, Colligan BM, Hurst BM, Carter HW, and Carter JH
- Subjects
- Adenocarcinoma enzymology, Apoptosis, Cell Division, Disease Progression, Humans, Immunohistochemistry, In Situ Nick-End Labeling, Ki-67 Antigen analysis, Male, Mitotic Index, Prostatic Neoplasms enzymology, Adenocarcinoma pathology, Prostatic Neoplasms pathology, Protein Serine-Threonine Kinases biosynthesis
- Abstract
Purpose: Integrin-linked kinase (ILK) overexpression can suppress anoikis, promote anchorage-independent cell cycle progression, and induce tumorigenesis and invasion. Inhibition of ILK in prostatic adenocarcinoma (CaP) cells elicits cell cycle arrest and induces apoptosis. Furthermore, ILK expression increases with androgen-independent progression of human CaP cell lines, suggesting that increased ILK expression may be associated with CaP progression., Experimental Design: To assess whether ILK expression may be related to CaP development and/or progression, we have evaluated ILK expression by immunohistochemistry in 100 human prostate tissues., Results: We show that ILK expression increases significantly with CaP progression. ILK immunostaining is specifically increased in high-grade, primary human CaP relative to adjacent benign prostatic hyperplasia (P < 0.001), benign prostatic hyperplasia from patients without cancer (P < 0.002), and low-grade CaP (P = 0.003). ILK overexpression is specifically associated with the increased proliferative index (P = 0.001) that typifies CaP progression. Strikingly, intense uniform ILK immunostaining was inversely related to 5-year patient survival (P = 0.004)., Conclusions: ILK expression increases dramatically with CaP progression. ILK expression is also specifically related to the disproportionately increased proliferative index that contributes to the net gain of CaP cells during progression. Finally, enhanced ILK expression is inversely related to 5-year patient survival. These data therefore implicate increased ILK expression in prostate tumor progression.
- Published
- 2001
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