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Cancer Stem Cells
DNER, an Epigenetically Modulated Gene, Regulates Glioblastoma-Derived Neurosphere Cell Differentiation and Tumor Propagation†‡
Article first published online: 9 APR 2009
DOI: 10.1002/stem.89
Copyright © 2009 AlphaMed Press
Volume 27, Issue 7, pages 1473–1486, July 2009
Additional Information
How to Cite
Sun, P., Xia, S., Lal, B., Eberhart, C. G., Quinones-Hinojosa, A., Maciaczyk, J., Matsui, W., DiMeco, F., Piccirillo, S. M., Vescovi, A. L. and Laterra, J. (2009), DNER, an Epigenetically Modulated Gene, Regulates Glioblastoma-Derived Neurosphere Cell Differentiation and Tumor Propagation. STEM CELLS, 27: 1473–1486. doi: 10.1002/stem.89
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Author contributions: P.S.: conception and design, collection/assembly of data, data analysis/interpretation, manuscript writing, final approval; S.X.: collection/assembly of data, data analysis/interpretation, final approval; B.L.: collection/assembly of data, data analysis/interpretation, final approval; C.E.: conception and design, final approval; A.Q.: provision of study material, final approval; J.M.: provision of study material, final approval; W.M.: collection/assembly of data, final approval; F.D.: provision of study material, final approval; S.P.: provision of study material, final approval; A.V.: provision of study material, final approval; J.L.: conception and design, collection/assembly of data, data analysis/interpretation, manuscript writing, final approval.
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First published online in STEM CELLS EXPRESS April 9, 2009.
Publication History
- Issue published online: 6 JUL 2009
- Article first published online: 9 APR 2009
- Accepted manuscript online: 9 APR 2009 12:00AM EST
- Manuscript Accepted: 1 APR 2009
- Manuscript Received: 14 OCT 2008
Funded by
- NIH. Grant Numbers: NS43987, NS055089
- The Maryland Stem Cell Research Fund
- The Brain Tumor Funders Collaborative
- Histone acetylation;
- Glioma;
- Stem cell;
- Oncosphere
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies. STEM CELLS 2009;27:1473–1486
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
During the past decade, there has been a re-emergence of the concept that cancers harbor small populations of malignant stem-like cells that sustain tumor growth and recurrence. These "neoplastic stem cells" [1], which share the defining phenotypic characteristics of self-renewal, multipotential differentiation at least equivalent to that in the primary tumor, and long-term tumor propagation [2, 3], have been identified in leukemia and multiple myeloma [4–6], and more recently in solid tumors, including mammary carcinoma [7] and primary brain tumors [3, 8–10]. The potential role for multipotent stem/progenitor cells in brain cancer initiation is supported by the fact that directing oncogenic growth signals to nestin-positive or glial fibrillary acidic protein (GFAP)+ neural progenitor cells in rodents causes high-grade glioma [11, 12]. Independent of the potential contribution of stem cells to tumor initiation, a current view is that neoplastic cells with stem-like properties underpin the maintenance and therapeutic resistance of malignant glioma. This concept is based on the identification of tumor cells that express stem cell markers (CD133, aldehyde dehydrogenase [ALDH], side-population), grow in vitro as neurospheres under serum-free conditions that support the growth of normal neural stem cells, resist ionizing radiation, and propagate tumor xenografts with high efficiency [3, 8–10, 13–15]. Understanding the molecular mechanisms that regulate the stem-like tumorigenic phenotype of these cells will improve our understanding of malignant brain tumors and lead to novel stem cell-targeting therapeutics [16–18].
Epigenetic mechanisms of gene expression regulation play an important role in the oncogenic phenotype and in the maintenance/differentiation of normal neural stem cells [19–21]. One of the most prominent of these epigenetic mechanisms involves chromosome modification by histone acetylation, which is determined by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs) [22, 23]. Acetylation of lysine residues in the histone tail promotes gene transcription by relaxing the chromosome, allowing the transcriptional machinery to access DNA. Conversely, histone deacetylation often followed by methylation compacts chromatin and represses gene expression [24]. Neural stem cell proliferation can be regulated by histone acetylation/deacetylation via numerous molecular mechanisms, including cell cycle regulation, apoptosis control, and the activation of differentiation [20, 25–27]. Oncogenesis is associated with a relative decrease in histone acetylation, leading to the transcriptional repression of tumor suppressor genes, a common event contributing to tumor formation [28].
In light of the accelerating interface between brain cancer and neural stem cells and the sensitivity of both to histone modification, we used HDAC inhibitors (HDACIs) to explore the biological and molecular basis of the glioblastoma (GBM) stem-like cell phenotype. HDACIs were found to inhibit the growth of GBM-derived neurospheres, induce neurosphere cell apoptosis, and deplete neurosphere cultures of cells expressing stem cell markers. HDACIs induced GBM-derived neurosphere cell differentiation and inhibited the ability of neurosphere cells to propagate tumor xenografts. Biological responses to HDAC inhibition were mediated, in part, by upregulating the noncanonical Notch ligand Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), which was found to inhibit GBM-derived neurosphere formation, induce neurosphere cell differentiation, and inhibit the growth of neurosphere-derived tumor xenografts. This is the first evidence that the DNER signaling pathway has differentiating and tumor-suppressing actions on glioma-derived stem-like cells.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
All reagents were purchased from Sigma Chemical Co. (St. Louis, http://www.sigmaaldrich.com) unless stated otherwise. Trichostatin A (TSA) and MS-275 (N-(2-aminophenyl)-4-[N-(3-pyridineyl-methoxycarbonyl)aminomethyl]-benzamide) were prepared as stock solutions in dimethylsulfoxide (DMSO). In all the experiments, the final DMSO concentration was <0.1%.
Cell Culture
The human GBM-derived neurosphere lines HSR-GBM1A (20913), HSR-GBM 1B (10627), and 050,509 were derived by Vescovi and colleagues and maintained in serum-free medium supplemented with EGF and fibroblast growth factor (neurosphere medium), as previously described [10, 29, 30]. The DM140207 neurosphere line was derived from a GBM at the University of Freiburg and primary JHH225 neurospheres were derived from a malignant glioma at Johns Hopkins University (Baltimore, Maryland) using the same methods and culture conditions. JHH225 neurospheres were used at passage ≤9. All human materials were obtained and used in compliance with the Johns Hopkins institutional review board.
Cell Growth and Clonogenic Assays
Cell growth was measured by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Madison, WI, http://www.promega.com). Briefly, 50 μl of a stock solution of MTS was added to each cell culture well to achieve a final concentration of 333 μg/ml. The reduction in MTS, reflecting the number of viable cells per well, was measured after 3 hours by quantifying the absorbance at 490 nm.
For soft agar clonogenic assays, agarose (1%; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in Dulbecco's modified Eagle's medium was cast on the bottom of plastic six-well plates. Dissociated neurosphere cells were suspended in neurosphere culture medium containing 0.5% agarose equilibrated at 37°C and plated at 5 × 103 cells per well above a bottom layer of 1% agarose. Cells were incubated in a humidified incubator containing 5% CO2 and 95% air at 37°C for 2 hours and then the medium was replaced with that containing 200 nM TSA. Cultures were incubated in 5% CO2 and 95% air at 37°C for 14 days and then colony formation was scored by measuring the number of spheres ≥100 μm in diameter in nine random microscopic fields per well.
Transient Transfection
Neurosphere cells were transfected with hemagglutinin-tagged full-length DNER expression plasmid, a kind gift of Mineko Kengaku (RIKEN Brain Science Institute, Saitama, Japan) [31], using Amaxa nucleofection technology (Amaxa, Koeln, Germany, http://www.amaxa.com). Cells were suspended in Amaxa Primary Neurons Kit solution, according to Amaxa guidelines. Briefly, a 100-μl suspension of 2-5 × 106 cells was mixed with 3 μg plasmid DNA and subjected to nucleofection using an Amaxa Nucleofector apparatus. Cells were then immediately transferred into six- or 12-well tissue culture plates containing neurosphere culture medium prewarmed to 37°C. Cells were cultured for an additional ∼72 hours before experimental analyses. Transfection efficiencies were routinely ∼60% as measured by parallel transfection with a green fluorescent protein reporter under identical conditions.
Immunoblot Analysis
SDS-PAGE was performed on 30 μg of cellular protein per lane using 4%–20% gradient Tris-glycine gels according to the method of Towbin et al. with some modifications [32, 33]. Proteins were electrophoretically transferred to nitrocellulose membranes (GE Healthcare, San Diego, http://www.gehealthcare.com). Membranes, except for those to be probed with anti-DNER, were incubated for 1 hour in Odyssey Licor Blocking Buffer (LI-COR Biosciences, Lincoln, NE, http://www.licor.com) at room temperature and then overnight with anti-glyceraldehyde-3-phosphate dehydrogenase (1:7,500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) or anti-β-actin (1:6,000; Sigma), anti-Deltex-1 (1:300; Santa Cruz Biotechnology), anti-GFAP (1:500; Dako, Carpinteria, CA, http://www.dakousa.com), anti-TuJ1 (1:1,000; Millipore Corporation, Billerica, MA, http://www.millipore.com), anti-histone-4 (1:1,500; Upstate, Charlottesville, VA, http://www.upstate.com), or anti-scetyl-histone-4 (1:1,000; Upstate) at 4°C in Odyssey Blocking Buffer (LI-COR Biosciences). After rinsing, membranes were incubated with IRDye secondary antibodies (1:15,000; LI-COR Biosciences) and protein expression changes were quantified by dual wavelength immunofluorescence Odyssey Infrared Imaging System (LI-COR Biosciences) scanning of the membranes. Immunoblots probed with anti-DNER (1:500; Abnova Corporation, Taipei City, Taiwan, http://www.abnova.com) utilized peroxidase-conjugated secondary antibodies, essentially as previous described [34].
Quantitative Real-Time Polymerase Chain Reaction
Total cellular RNA was extracted using the RNeasy Mini kit (Qiagen, Inc., Chatsworth, CA, http://www1.qiagen.com) and purified using RNeasy columns according to the manufacturer's instructions. The purity and amount of total RNA were spectrophotometrically estimated by measuring absorbance at 260 nm and 280 nm. The integrity of rRNA was checked using agarose gel electrophoresis. Total RNA (1 μg) was reverse-transcribed (RT) using the oligo(dT)12-18 primer and Superscript II (Invitrogen) according to the manufacturer's instructions. DNER primer sequences were: 5′-CTCCATTTCTGCATGGGTCT-3′ and 5′-GAGGAAACCTTGCCAAAACA-3′. MAG primer sequences were: 5′-AGCCCCTACCCCAAGAACTA-3′ and 5′GACGATATCCAGGACGCTGT-3′. HES1 primer sequences were 5′-AGCGGGCGCAGATGAC-3′ and 5′-CGTTCATGCACTCGCTGAA-3. Quantitative real-time polymerase chain reaction (PCR) was performed with an Applied Biosystems Prism 7900 HT Sequence Detection System using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The thermal cycling conditions were as follows: hold for 10 minutes at 95°C, followed by three-step PCR for 40 cycles of 95°C for 15 seconds, 55°C for 25 seconds, and 72°C for 30 seconds. Samples were amplified in triplicate and data were analyzed using the Applied Biosystems Prism Sequencer Detection Software Version 2.3 (Applied Biosystems). Human 18S rRNA was amplified as an endogenous control. Standard curves were prepared for each mRNA and 18S rRNA for each amplification to normalize relative gene expression to the 18S rRNA control.
Gene Expression Knockdown
Predesigned small interfering (si)RNA for human DNER was purchased from Ambion (ID 125934; Ambion, Austin, TX, http://www.ambion.com). The resulting double-stranded siRNA (50 nM) was transfected into cultured cells using siPort Lipid transfection agent (Ambion). Neurosphere cells were plated in duplicate in a six-well plate (105 cells per well) format for RNA analysis and in 48-well plates (1.5 × 104 cells per well) for growth assays. For each transfection, 32 μl siPort transfection reagent was mixed with 1.1 ml Optimum Medium (Gibco, Grand Island, NY, http://www.invitrogen.com) and incubated at room temperature for 20 minutes. Three microliters of 20 μM negative control siRNA (Silencer Negative Control #1 siRNA; Ambion) or DNER siRNA solution was added to the mixture, which was incubated at room temperature for another 20 minutes. The mixture was then added to cell medium and incubated for 48 hours prior to MTS assay or protein extraction. The silencing effect of the siRNA construct on DNER expression was confirmed by immunoblot analysis. The SiDNER sequence one (SiDNER.1) sense siRNA strand was 5′-GUGUGACCCCCCUUCAGGCtt-3′ and the antisense siRNA strand was 3′-ttCACACUGGGGGGAAGUCCG-5′; the SiDNER.2 sense siRNA strand was 5′-GCAGUACGUGGGUACUUUCtt-3′ and antisense siRNA strand was 3′-ctCGUCAUGCACCCAUGAAAG-5′. The SiDeltex-1.1 sense siRNA strand was 5′-GGAUGUGGUUCGAAGAUACtt-3′ and the antisense siRNA strand was 3′-ctCCUACACCAAGCUUCUAUG-5′; the SiDeltex-1.2 sense siRNA strand was 5′-GCACCUUAAAAAGAGUAAGtt-3′ and the antisense siRNA strand was 3′-ttCGUGGAAUUUUUCUCAUUC-5′.
Tumor Xenografts
Female 4- to 6-week-old athymic nude mice were injected s.c. in the flank with 5 × 106 viable cells in 0.1 ml of phosphate-buffered saline (PBS). When tumors reached ∼50 mm3, the mice were randomly divided into groups and treated with TSA (500 μg/kg in 0.2 ml PBS) or with solvent only as a control, i.p. daily as previously described [35]. Tumor sizes were determined weekly by measuring two dimensions—length (a) and width (b)—and volume (V) was estimated using the formula V = ab2/2 [36]. At the end of each experiment, tumors were excised and weighed.
For intracranial xenografts, SCID/NCR immunodeficient mice received 5,000 viable neurosphere cells in 5 μl of culture medium by stereotactic injection to the right caudate/putamen. In certain experiments, cells were treated with TSA (200 nM) for 3 days or transfected with DNER expression plasmid 48 hours prior to implantation. Cell viability was determined by trypan blue dye exclusion. Groups of mice (n = 10) were sacrificed at the indicated times and the brains were removed for histologic studies. Tumor sizes were quantified by measuring tumor cross-sectional areas on hematoxylin and eosin-stained cryostat sections from perfusion-fixed brains using computer-assisted image analysis as previously described [36]. Volumes were estimated based on the formula: volume = (square root of maximum cross-sectional area)3 as previously described [36]. All animal protocols used in this study were approved by the Johns Hopkins School of Medicine Animal Care and Use Committee.
Immunofluorescence and Immunohistochemistry
The expression and cellular localization of CD133 (Santa Cruz Biotechnology), GFAP, and TuJ1 were determined by immunofluorescence. Neurosphere cells were plated on coverslips or collected by cytospin onto glass slides. The cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with PBS containing 7.5% glycine and 0.5% Triton X-100 for 30 minutes. The cells were then incubated with primary antibody in staining solution (1% bovine serum albumin [BSA] with 0.1% NP-40) for 2 hours and then incubated with appropriate corresponding secondary antibodies (fluorescein isothiocyanate [FITC]-conjugated goat anti-mouse and Texas Red-conjugated goat anti-rabbit) for 30 minutes. Coverslips were placed with Vectashield Antifade solution containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Immunofluorescence was detected by fluorescent microscopy using Axiovision software (Carl Zeiss, Jena, Germany, http://www.zeiss.com).
Flow Cytometry
Flow cytometry was performed to determine the percentage of cells expressing ALDH using the Aldefluor reagent according to the manufacturer's specifications (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). Aldefluor substrate (0.625 g/ml) was added to neurosphere cells suspended in Aldefluor assay buffer (106 cells/ml). Cells were then incubated for 20-30 minutes at 37°C to allow the conversion of Aldefluor substrate to its intracellular fluorescent product [37]. Cell fluorescence was measured using an LSR flow cytometer equipped with 424/44 nm band pass and 670 nm long pass optical filters (Omega Optical, Brattleboro, VT, http://www.omegafilters.com). ALDH values are presented as mean fluorescence intensity.
Flow cytometry analysis of CD133 expression was performed with phycoerythrin-conjugated anti-CD133 antibody (clone 293C3; Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) following the manufacturer's protocol. Briefly, up to 5 × 106 dissociated neurosphere cells were suspended in 100 μl assay buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA) and 10 μl of the CD133 antibody were added. The cells were gently rotated for 10 minutes in the dark at 4°C, washed by adding 1 ml of assay buffer, and then pelleted at 300g for 10 minutes. The cell pellet was resuspended in PBS and analyzed by a FACSCalibur fluorescence-activated cell sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Data were quantified using CellQuest software (BD Biosciences).
Cell death was quantified using the annexin V-FITC/propidium iodide (PI) apoptosis kit (BD Bioscieneces, San Diego, http://www.bdbiosciences.com) according to the manufacturer's instructions. Briefly, dissociated cells were resuspended in annexin V binding buffer (150 mM NaCl, 18 mM CaCl2, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2). FITC-conjugated annexin V (1 μg/ml) and PI (50 μg/ml) were added to cells, which were incubated for 30 minutes at room temperature in the dark and subjected to FACScan analysis to quantify the annexin V-positive/PI−negative cells.
Statistical Analysis
Data were analyzed using parametric statistics with one-way analysis of variance. Post hoc tests included Student's t-test and the Tukey multiple comparison tests as appropriate using Prizm (GraphPad Software Inc., San Diego, CA, http://www.graphpad.com). All experiments reported here represent at least three independent replications. All data are represented as the mean value ± standard error of mean. Significance was set at p < .05.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
HDAC Inhibition Inhibits GBM-Derived Neurosphere Formation
Histone modification influences the phenotype of normal stem and progenitor cells and may play substantial roles in the initiation, growth, and malignant progression of cancer. We examined the effect of histone deacetylase inhibition on the growth of GBM-derived neurosphere cells under neurosphere growth conditions. TSA and MS-275 each inhibited neurosphere cell growth in a concentration- and time-dependent manner. When neurosphere cells were treated for 72 hours, the 50% inhibitory concentration (IC50) values for TSA and MS-275 were ∼200 nM and ∼3.5 μM, respectively, as determined by MTS assay (supporting information Fig. 1A). Exposing neurosphere cultures to either drug at its approximate IC50 for 144 hours inhibited neurosphere cell growth by 82%–86% (p < .001) (Fig. 1A).
TSA and MS-275 potently inhibited GBM neurosphere cell growth in immobilized colony formation assays (Fig. 1B). Treating cultures with TSA (200 nM) continuously for 14 days inhibited the formation of colonies (≥100 μm diameter) by 95%–98%. Pretreating cells with TSA for 3 days immediately prior to initiating the assay inhibited colony formation by 88%–90%. Both TSA and MS-275 increased GBM neurosphere cell histone acetylation three- to fivefold under these experimental conditions, consistent with their HDAC inhibitory actions (supporting information Fig. 1B).
HDAC Inhibition Induces GBM-Derived Neurosphere Cell Differentiation and Depletes Neurospheres of Stem-Like Cells
Possible mechanisms by which HDACIs reduce GBM-derived neurosphere cell growth and neurosphere formation include the inhibition of neurosphere proliferation, the induction of neurosphere cell death, the induction of neurosphere cell differentiation, and the depletion of the stem-like cell pool in heterogeneous neurosphere cultures. We examined the effects of HDAC inhibition on GBM neurosphere cell expression of markers associated with the neoplastic stem-like cell phenotype [9, 38]. Under control neurosphere growth conditions, ALDH was expressed by ∼2%–3% of neurosphere cells, as determined by quantitative Aldefluor flow cytometry assay. The percentage of ALDH-expressing cells diminished approximately tenfold to ∼0.2%–0.4% when cultured for 72 hours with either TSA or MS-275 (Fig. 2A, 2B). CD133 has been linked to stem-like, tumor-initiating GBM cells, and nestin is an intermediate filament protein expressed by both neurosphere stem and progenitor cells. We therefore also examined the effects of HDAC inhibition on CD133 and nestin. Flow cytometry analysis showed that CD133 was expressed by ∼45% of neurosphere cells under control neurosphere growth conditions, and the percentage of CD133+ cells dropped approximately threefold to ∼15% when cultured for 72 hours with TSA (Fig. 2C). The depletion of CD133+ cells was confirmed by immunofluorescence (Fig. 2D). In contrast, the number of nestin-expressing cells was relatively preserved following treatment with TSA (Fig. 2D).
We also examined the effects of TSA and MS-275 on neurosphere cell expression of the astrocyte marker GFAP and the neuronal marker β-tubulin III (TuJ1). Expression levels of both differentiation markers were substantially increased 72 hours after adding either HDACI to standard neurosphere culture medium, as detected by immunofluorescence (Fig. 3A, 3B). Immunoblot analyses confirmed that GFAP and TuJ1 protein expression increased in response to HDAC inhibition (Fig. 3C).
HDAC Inhibition Abrogates the Tumor-Forming Capacity of GBM-Derived Neurospheres
A defining phenotype of neoplastic stem-like cells is their ability to propagate and maintain malignant tumors in vivo. We and others have shown that GBM-derived neurospheres, including the neurospheres used in this current study, form tumor xenografts that recapitulate many features of human GBM (e.g., high degree of invasiveness, necrosis) [17, 29, 39]. We examined the effect of TSA on the tumorigenicity of GBM-derived neurosphere cells in immune-deficient mice. s.c. xenografts were used in these initial experiments to allow us to both assess tumor formation/growth in real time and culture primary neurospheres from resected xenografts. Animals received equal numbers of viable (trypan blue-negative) control- or TSA-treated neurosphere cells (4 × 106 cells per implantation site). The effect of in vivo TSA treatment was also examined. Results are shown in Figure 4A. Control neurospheres formed measurable tumors in 70% of animals. Pretreating neurospheres with TSA (200 nM) for 4 days prior to cell implantation reduced tumorigenicity to 30%. Implanting control neurosphere cells and then treating animals with TSA (0.5 mg/kg per day i.p.) beginning on postimplantation day 3 reduced tumorigenicity to 20%. Combining in vitro and in vivo TSA therapy generated measurable tumors in only 10% of animals. Furthermore, when mice bearing pre-established neurosphere-derived xenografts (9 weeks postimplantation) were treated with TSA (0.5 mg/kg per day i.p.), tumors regressed within 10 days of treatment initiation (Fig. 4B). We asked if TSA similarly affects the growth of neurosphere-derived orthotopic tumor xenografts. Treating GBM-derived neurosphere cultures with TSA (200 nM) for 4 days prior to cell implantation reduced tumorigenicity from 80% to 50%. On postimplantation day 60, tumors that formed from TSA-treated cells were ∼50% smaller than control tumors (Fig. 4C).
The reduction in tumor initiation and tumor growth rates suggested that xenografts derived from TSA-treated cells might be relatively depleted of their tumor-propagating stem-like cells. TSA treatment significantly depleted tumor xenografts of cells with the capacity to generate large neurospheres (>100 μm diameter) (Fig. 4D), and the magnitude of depletion mirrored the effect of TSA on tumor initiation rates (compare Fig. 4A with Fig. 4D). The effects of TSA on tumor xenograft cell expression of CD133 and the differentiation markers GFAP and TuJ1 were assessed by immunoblot analysis of xenograft protein extracts. Both GFAP and TuJ1 expression increased four- to sevenfold and CD133 expression decreased approximately two- to threefold in response to TSA treatment (Fig. 4E). These in vivo effects parallel the effects of HDAC inhibition on neurosphere cells observed in vitro.
DNER Signaling Is Induced by HDAC Inhibition and Regulates GBM-Derived Neurosphere Cell Growth and Differentiation
The effects of HDAC inhibition on GBM-derived neurosphere formation, differentiation, and tumor propagation led us to use HDACIs to identify gene expression patterns that regulate the tumor-initiating phenotype of GBM-derived neurospheres. We examined the effect of TSA on the gene expression profiles of two GBM-derived neurosphere lines using Affymetrix gene expression arrays. The expression levels of 36 genes were found to be changed threefold of greater by TSA in both neurosphere lines. One of these genes, DNER, was induced five- to eightfold, as determined by quantitative RT-PCR (supporting information Fig. 2A). Noncanonical Notch pathway activation by DNER is mediated, in part, by the transcription factor Deltex. Immunoblot and immunofluorescence analyses showed that both DNER and Deltex-1 were induced by TSA treatment in established GBM-derived neurosphere lines and in low passage neurospheres derived from a human malignant glioma (Fig. 5A–5C). Deltex-1 and DNER were similarly induced by MS-275. We found that both TSA and DNER directly activated noncanonical Notch signaling in GBM-derived neurosphere cell lines, as evidenced by Deltex-dependent induction of the myelin-related protein MAG [40, 41]. MAG expression increased ∼15-fold in response to TSA and ∼13-fold following transfection with DNER (Fig. 5D, 5E). Neither TSA nor DNER transfection induced expression of the canonical Mastermind-dependent, Deltex-independent Notch pathway target Hes1 (supporting information Fig. 3A, 3B) [42]. siRNA-mediated Deltex expression knockdown (supporting information Fig. 2D) inhibited MAG induction by both TSA and DNER (Fig. 5D, 5E, using SiDeltex-1.1; supporting information Fig. 3C, 3D, using another siRNA, SiDeltex-1.2). Taken together, these findings indicate that HDAC inhibition activates a noncanonical DNER/Deltex signaling pathway in GBM-derived neurospheres.
We used a gene expression knockdown approach to determine if DNER mediates the GBM neurosphere response to HDAC inhibition. Transient transfection of GBM neurospheres with anti-DNER siRNA (SiDNER) potently inhibited the low level of basal DNER expression and also inhibited DNER induction in response to TSA (supporting information Fig. 2B, 2C). We asked if SiDNER altered neurosphere growth inhibition following HDAC inhibition. GBM neurosphere cells were treated with TSA (200 nM, 72 hours) with or without SiDNER (50 nM, 48 hours). The cells were then transferred to standard neurosphere growth medium for 7 days. Results are shown in Figure 6A. Neurosphere formation was inhibited by TSA and not affected by SiDNER alone. However, cells treated with both TSA and SiDNER formed threefold more neurospheres than cells treated with TSA and control siRNA. Thus, preventing DNER induction with SiDNER partially blocked the growth inhibitory effects of TSA.
DNER was found to induce the differentiation of GBM neurosphere cells as assessed by GFAP and TuJ1 expression (Fig. 6B, 6D). GFAP and TuJ1 expression levels increased approximately threefold and approximately fourfold, respectively, as determined by immunoblot analysis. Forced expression of DNER in neurosphere cells reduced their proportion of CD133+ cells by ∼50% (Fig. 6C). Mirroring the ability of SiDNER to reverse the inhibitory effect of TSA on neurosphere cell colony formation, SiDNER was also found to reverse TSA-induced neurosphere cell differentiation, as determined by GFAP and TuJ1 immunofluorescence (Fig. 6D, using SiDNER.1, and supporting information Fig. 4, using another siRNA, SiDNER.2). Conversely, transfecting neurosphere cells with DNER cDNA induced GFAP and TuJ1 expression, mimicking the response to TSA treatment (Fig. 6D). In contrast to its effects on neurosphere cell differentiation, DNER had no detectable effect on neurosphere cell viability or apoptosis. SiDNER did not alter the viability of control- or TSA-treated neurosphere cells (Fig. 6E). Forced DNER expression did not alter the viability or annexin V staining of neurosphere cells (Fig. 6F). Together, these loss- and gain-of function results strongly suggest that DNER signaling pathways modulate GBM stem-like cell growth, differentiation, and possibly tumor propagation and maintenance.
Finally, we examined the effect of DNER upregulation on the growth of orthotopic tumor xenografts derived from GBM neurospheres. Mice received equal numbers of viable GBM neurosphere cells by stereotaxic injection to the caudate/putamen 72 hours after the cells were transfected with control or full-length DNER plasmid expression vectors. Tumor volumes assessed on postimplantation day 68 showed that tumor xenografts derived from DNER-transfected cells were ∼60% smaller than controls (Fig. 7).
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Glioblastoma multiforme is the most common and aggressive of the primary brain cancers. Despite dramatic reductions in tumor burden with surgery, radiotherapy, and chemotherapy, tumor recurrence is almost universal [43]. The high frequency of GBM recurrence reflects the inherent chemo-/radiation resistance of at least a subset of the invasive cancer cells that inevitably remain after aggressive surgery. According to the cancer stem-cell hypothesis, only a small number or, at its extreme interpretation, a single remaining tumor cell with stem-like properties can ultimately result in the recurrence of a heterogeneous GBM. There has been considerable progress in identifying the biological and molecular features of GBM cell subpopulations with stem-like properties since they were isolated using neurosphere growth conditions and shown to efficiently propagate tumor xenografts contemporaneously in 2004 by Singh et al. [3] and Galli et al. [10]. Perhaps the most clinically relevant features of GBM-derived stem-like cells identified to date are their ability to efficiently form tumor xenografts with the complex histologic features of human GBM and their inherent resistance to chemotherapy and radiation therapy. The resistance of neoplastic stem-like cells to DNA-damaging agents has been attributed in part to Chk1/2-dependent DNA repair mechanisms in GBM-derived cells [14] and to Wnt/β-catenin signaling in breast cancer-derived cells [44]. Neurodevelopmental pathways regulate GBM-derived stem-like cells. Among these are the Hh and canonical Notch/Mastermind pathways, found to maintain the cancer stem-cell phenotype, and bone morphogenetic proteins, which induce their differentiation and inhibit their tumor-initiating potential [17, 29, 39, 45]. Effort is under way to translate these discoveries to reduce the tumor-initiating potential of the stem-like cell subpopulation through either differentiation and/or chemo-/radiation sensitization in conjunction with conventional treatments.
We now show that histone deacetylase inhibition induces GBM-derived stem-like cells to differentiate, undergo apoptosis, and loose their capacity to both grow as neurospheres and initiate tumor xenografts. These cell responses mimic, to some degree, the responses of normal neural stem/progenitor cells to HDAC inhibition [20, 26, 46], supporting the current concepts regarding shared regulatory mechanisms and possibly overlapping origins between normal neural and GBM-derived neoplastic stem cells. Our results are interesting in the context of the recent findings of Lee et al. [17], showing that epigenetic gene regulation via promoter methylation can alter the differentiation and tumor-initiating capacity of GBM stem-like cells. The HDACIs used in our experiments, MS-275 (now available as Sndx-275; Syndax Pharmaceuticals, Waltham, MA http://www.syndax.com) and TSA, also have antitumor activity against tumor xenograft models derived from cancer cells maintained under traditional serum-containing culture conditions [47, 48]. HDAC inhibition can broadly induce tumor suppressors and inhibit tumor promoters regardless of cell stemness. It is also possible that the antitumor effects of HDACIs in traditional xenograft models reflect, at least in part, the targeting of tumor-initiating cell subpopulations with stem-like characteristics within traditional cancer cell lines [49, 50]. HDACIs, including Sndx-275 and suberoylanilide hydroxamic acid, are currently in clinical trials for solid malignancies. Our findings expand the influences of HDACIs to GBM stem-like cells and the nonclassical Notch ligand DNER. The ability of HDACIs to cross the blood–brain barrier makes them applicable to clinical testing in brain tumor patients [51].
Our conclusions that HDAC inhibition and DNER, an HDACI-induced gene product, differentiate GBM stem-like cells and inhibit their tumor-initiating phenotype are based on complementary biological and molecular endpoints. In contrast to hematopoietic neoplastic stem cells, cell surface markers that reliably identify tumor-initiating stem cells in solid cancers are lacking. Therefore, our approach emphasized experimental endpoints that are well accepted to consistently associate with the neoplastic stem-like phenotype, namely, the formation of large neurospheres in vitro and tumor xenograft initiation by neurosphere-derived cells. CD133 and ALDH, markers that correlate with neoplastic neurosphere growth and tumor initiation, were also examined and found to be consistently lost, whereas the neuronal and glial differentiation markers TuJ1 and GFAP were consistently induced in response to HDAC inhibition and DNER expression. That similar results were obtained using (a) multiple human GBM-derived neurosphere lines and low passage neurosphere cultures established at different institutions, (b) in vitro and in vivo model systems, (c) two chemically distinct HDACIs, and (d) both DNER gain and loss of function renders confidence in their broad applicability to cellular models in vitro and more biologically complex tumors in vivo.
The effects of HDAC inhibition can be broad and not limited to proliferation inhibition and differentiation. Consistent with this is our finding that HDAC inhibition also induced GBM neurosphere cell death via a predominantly apoptotic mechanism. In contrast to its effects on cell differentiation, the HDAC-induced gene product DNER had no apparent effects of neurosphere cell viability. We are currently exploring in more detail the transcriptional events downstream of HDAC inhibition specifically responsible for GBM neurosphere cell death. Understanding the molecular determinants that control whether GBM neurosphere cells die or differentiate will likely translate to more effective strategies for targeting neoplastic stem-like cells.
We used the effects of HDAC inhibition on gene expression to identify specific gene products that potentially regulate the GBM stem-like cell phenotype. We show, for the first time, that DNER functions as a tumor suppressor and differentiating factor in GBM-derived neurosphere cells. DNER is a single-pass transmembrane Notch ligand containing 10 extracellular EGF-like repeats and lacking the Delta, Serrate, Lag-2 (DSL) Notch-binding motif that characterizes classical Notch ligands [52]. DNER is strongly expressed by cerebellar Purkinje cells and regulates cerebellar development and neurodevelopmental interactions between Purkinje cells and Bergmann glia that express Notch. Notch ligands induce Notch receptor cleavage by metalloprotease and γ-secretase releasing the Notch intracellular domain (NICD) that transduces to canonical (CSL/NICD/Mastermind transcriptional complex) and noncanonical (NF-κB/NICD and CSL/NICD/Deltex transcriptional complex) pathways. Signaling through the classical Notch pathway has multiple contextual functions, such as stem cell self-renewal, progenitor cell fate determination, and terminal differentiation of proliferating cells [53–55]. DNER promotes Bergmann glial process formation in vitro via a Deltex-dependent mechanism. Our findings that Deltex-1 is induced along with DNER by HDAC inhibition and that DNER induces expression of the Deltex target gene MAG support a role for a noncanonical signaling mechanism.
A complex role for Notch signaling in central nervous system malignancies is emerging. High-grade gliomas express the DSL-containing Notch ligand Jagged, Notch receptors, and downstream target genes induced by CSL/NICD/Mastermind in patterns that have been found to correlate with malignant grade and poor prognosis [56, 57]. Fan et al. [45] found that inhibiting canonical Notch signaling with γ-secretase inhibitors can deplete embryonal brain tumor cell lines of their neoplastic stem-like cells and tumor-initiating capacity. These findings support an oncogenic role for classical Notch signaling in certain brain tumor subtypes and contrast the tumor suppressive effects of DNER reported here in GBM-derived neurospheres. Developing a detailed framework for the oncogenic and tumor-suppressing effects of canonical and noncanonical Notch signaling in brain cancer stem-like cells will require a more complete understanding of the cell type- and context-dependent actions and interactions of the numerous Notch ligands, Notch receptors, and their downstream effectors.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
This work was funded by NIH grants NS43987 (J.L.) and NS055089 (C.G.E.), The Maryland Stem Cell Research Fund (J.L.), and The Brain Tumor Funders Collaborative.
- Top of page
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- Acknowledgements
- Supporting Information
The authors indicate no potential conflicts of interest.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
- 1Cancer research. Mutant stem cells may seed cancer. Science 2003; 301: 1308–1310..
- 2A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648., , et al.
- 3Cancer stem cells in nervous system tumors. Oncogene 2004; 23: 7267–7273., , et al.
- 4Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105–111., , et al.
- 5Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737., .
- 6Mouse myeloma tumor stem cells: A primary cell culture assay. J Natl Cancer Inst 1971; 46: 411–422., , .
- 7Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 3983–3988., , et al.
- 8Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002; 39: 193–206., , et al.
- 9Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci USA 2003; 100: 15178–15183., , et al.
- 10Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 2004; 64: 7011–7021., , et al.
- 11A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev 1998; 12: 3675–3685., , et al.
- 12PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 2001; 15: 1913–1925., , et al.
- 13Making a tumour's bed: Glioblastoma stem cells and the vascular niche. Nat Rev Cancer 2007; 7: 733–736., .
- 14Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006; 444: 756–760., , et al.
- 15Cellular and genetic characterization of human adult bone marrow-derived neural stem-like cells: A potential antiglioma cellular vector. Cancer Res 2003; 63: 8877–8889., , et al.
- 16Cancer stem cells in solid tumors. Curr Opin Biotechnol 2007; 18: 460–466., .
- 17Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cell 2008; 13: 69–80., , et al.
- 18Brain tumour stem cells: Possibilities of new therapeutic strategies. Expert Opin Biol Ther 2007; 7: 1129–1135., .
- 19Histone deacetylase activity is necessary for oligodendrocyte lineage progression. J Neurosci 2002; 22: 10333–10345., , et al.
- 20Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci USA 2004; 101: 16659–16664., , et al.
- 21Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells. Genes Dev 2004; 18: 2963–2972., .
- 22Histone deacetylases (HDACs): Characterization of the classical HDAC family. Biochem J 2003; 370: 737–749., , et al.
- 23Histone deacetylases: Unique players in shaping the epigenetic histone code. Ann N Y Acad Sci 2003; 983: 84–100., , et al.
- 24Histones and histone modifications. Curr Biol 2004; 14: R546–R551., .
- 25Use of a novel histone deacetylase inhibitor to induce apoptosis in cell lines of acute lymphoblastic leukemia. Haematologica 2004; 89: 419–426., , et al.
- 26Effects of histone deacetylation inhibition on neuronal differentiation of embryonic mouse neural stem cells. Neuroscience 2006; 143: 939–951., , et al.
- 27Sirt1 contributes critically to the redox-dependent fate of neural progenitors. Nat Cell Biol 2008; 10: 385–394., , et al.
- 28Histone-deacetylase inhibitors: Novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002; 1: 287–299..
- 29Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 2007; 25: 2524–2533., , et al.
- 30Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 1999; 156: 71–83., , et al.
- 31DNER acts as a neuron-specific Notch ligand during Bergmann glial development. Nat Neurosci 2005; 8: 873–880., , et al.
- 32Transcription-dependent epidermal growth factor receptor activation by hepatocyte growth factor. Mol Cancer Res 2008; 6: 139–150., , et al.
- 33Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350–4354., , .
- 34Sensitization of glioma cells to Fas-dependent apoptosis by chemotherapy-induced oxidative stress. Cancer Res 2005; 65: 5248–5255., , .
- 35Histone deacetylase inhibitors upregulate plakoglobin expression in bladder carcinoma cells and display antineoplastic activity in vitro and in vivo. Int J Cancer 2005; 113: 841–848., , et al.
- 36Targeting the c-Met pathway potentiates glioblastoma responses to gamma-radiation. Clin Cancer Res 2005; 11: 4479–4486., , et al.
- 37Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res 2008; 68: 190–197., , et al.
- 38Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells. Blood 1990; 75: 1947–1950., , et al.
- 39Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 2006; 444: 761–765., , et al.
- 40NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes. J Biol Chem 2004; 279: 25858–25865., , et al.
- 41Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor. J Biol Chem 2001; 276: 45031–45040., , et al.
- 42Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol 2002; 22: 7688–7700., , et al.
- 43Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352: 987–996., , et al.
- 44Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Rev 2007; 3: 157–168., , et al.
- 45Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. Cancer Res 2006; 66: 7445–7452., , et al.
- 46Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells. Proc Natl Acad Sci USA 2007; 104: 14982–14987., , et al.
- 47Evaluation of the in vitro and in vivo antitumor activity of histone deacetylase inhibitors for the therapy of retinoblastoma. Clin Cancer Res 2008; 14: 3113–3123., , .
- 48A synthetic inhibitor of histone deacetylase, MS-27–275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci USA 1999; 96: 4592–4597., , et al.
- 49Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation. Proc Natl Acad Sci USA 2008; 105: 9751–9756., , et al.
- 50Enhanced radiation-induced cell killing and prolongation of gammaH2AX foci expression by the histone deacetylase inhibitor MS-275. Cancer Res 2004; 64: 316–321., , et al.
- 51The benzamide MS-275 is a potent, long-lasting brain region-selective inhibitor of histone deacetylases. Proc Natl Acad Sci USA 2006; 103: 1587–1592., , et al.
- 52Delta/Notch-like epidermal growth factor (EGF)-related receptor, a novel EGF-like repeat-containing protein targeted to dendrites of developing and adult central nervous system neurons. J Biol Chem 2002; 277: 25400–25407., , et al.
- 53The role of Notch in tumorigenesis: Oncogene or tumour suppressor? Nat Rev Cancer 2003; 3: 756–767., .
- 54Notch signaling: Cell fate control and signal integration in development. Science 1999; 284: 770–776., , .
- 55Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 2006; 442: 823–826., , et al.
- 56Contribution of Notch signaling activation to human glioblastoma multiforme. J Neurosurg 2007; 106: 417–427., , et al.
- 57A role for the transcription factor HEY1 in glioblastoma. J Cell Mol Med 2009; 13: 136–146., , et al.
Supporting Information
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- Supporting Information
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Reba Mcentire - Rumor Has It Lyrics
Reba Mcentire – Rumor Has It Lyrics
I overheard a conversation your name was mentioned in
But they didn't talk about us, only her again
I didn't want to listen but I hung on every word
They said some things I wish I hadn't heard
Rumor has it she has you
Rumor has it you love her too
Talk is cheap but the price is high when it's true
Rumor has it she has you
This town's so small a whisper can be heard a mile away
And people here will gossip when there's nothing else to say
I wouldn't have believed my ears but I see it in your eyes
The stories goin' 'round this town aren't lies
Rumor has it she has you
Rumor has it you love her too
Talk is cheap but the price is high when it's true
Rumor has it she has you
Whoa….rumor has it she loves you…
[Reba Mcentire - Rumor Has It Lyrics]
Writer(s): Larry Shell, Bruce Burch, Vern Dant Copyright: Millhouse Music, Ensign Music, Sheddhouse Music Lyrics powered by www.musiXmatch.com
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My experience is long-range. I taught my five youngest children at home from 1990 until 2006. Once they entered public schools, I went back to school to earn my Master's Degree in Applied Criminology with a policy emphasis. I am also a part-time prison minister. I have an Associates of Applied Science degree in Business Information Technology, a certificate in Medical Office, classes in Medical Coding and Transcription, business, and a certificate in ministry, as well as a Bachelors of Applied Arts and Sciences in Business Management. Right now I am a professor at Texas State Technical College teaching Computer Applications 1 and Reading/Writing 1. I can teach all elementary subjects, as well as computer proficiency.
I have a great degree of patience and have also worked with the elderly for many years doing Bible study. I've taught or co-taught many children's programs, AWANAS, Good News Bears, Young Pioneers, etc as well as teaching children's church and Sunday School. I have also been involved in homeschool associations conducting field trips, teaching crochet and hand/machine sewing.
I have a certificate in ministry from the Foursquare church. I have my own prison ministry. I taught Bible study for five years with the elderly. I have taught different children's programs, AWANA, Good News Bears, Young Pioneers, vacation Bible school, as well as taught children's church and Sunday School. I also write Christian Children's stories, and wrote nursery Bible curriculum for small children.
I have a master's of applied science in applied criminology with a policy emphasis. I have also been a prison minister for 17 years. I also create parole files, advocate for inmates with the Ombudsman, and the Health Liaison Agency, and help to find transitional housing when paroled.
My bachelor's is in business information technology of which desktop publishing was a class I needed to test out in and develop a portfolio. I also had my own business, an upholstery business where I created my own website, brochures, as well as business cards. I also have a ministry where I have done the same. I also teach computer applications 1 at TSTC, part of which is creating spreadsheets, visio charts, and floor plans as well as PowerPoint and Microsoft Office applications.
I teach Computer Applications 1 at Texas State Technical College, which includes all of Microsoft Office 2010 applications. I also maintain a client data base for my other job as an administrative assistant using ACCESS.
My AAS is in Business Information Technology which utilized Microsoft Publisher throughout the class. I have also used Microsoft Publisher throughout my career while owning my own upholstery shop, R&J Upholstery of Reagor Springs. I created banners, brochures, greeting cards, business cards, invitations and signs. I've also utilized this application when designing my own resumes, as well as for clients who paid me for administrative work.
I teach Computer Applications 1 at Texas State Technical College, which includes Windows 7. I have used all of Windows operating systems since the 1990's. I also have an associate's degree in business information technology.
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Supreme Court rejects Sahara's review plea
NEW DELHI (Reuters) - The Supreme Court of India has rejected an appeal by the Sahara conglomerate seeking a review of the court's order last year to repay billions of dollars it raised from small investors by selling outlawed bonds, according to an order posted on the court's website.
A spokesman for Sahara, one of India's biggest business groups and a household name through its sponsorship of the national cricket team, was not immediately available for a comment.
(Also read: Sahara - massive, splashy ... and mysterious, click here)
(Reporting by Devidutta Tripathy; Editing by Mark Potter)
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Fuelling Change
Falling global crude prices bolster economic outlook
As well as making the lives of millions of middle class Indians easier, the sharp drop in Brent crude prices since June is a boon for Prime Minister Narendra Modi in his fight to revive an economy growing at its slowest rate since the 1980s. Full Article


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Anybody who believes rapper Drake gets everything he wants must not have heard about his desire to collaborate with Sade for his upcoming album Thank Me Later. Continue
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DVD Extra
Is that DVD you've got your eye on really worth the investment? Should you wait a few months until the "Special Edition" hits shelves? DVD Extra lights the way.
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Among my litany of associates, I know of only one who believes the findings reported by the Warren Commission in September 1964. Chaired by Supreme Court Chief Justice Earl Warren, the commission had been charged by President Lyndon B. Johnson with investigating the assassination in Dallas of his predecessor, John F. Kennedy, on Nov. 22, […] [Read More]
'The Eagle Has Landed': A WWII tale from the German viewpoint
In the 1970s, the Second World War wasn't some distant conflict relegated to the history books. The people who'd been involved in it were very much a part of everyday life — in fact, they pretty much ran things. Hence, it's logical that some of the biggest movies of the '70s — "Patton," "Tora, Tora, Tora," […] [Read More]
'Behind the Candelabra' sheds light on Liberace
As odd as it might seem now, a favorite guessing game in the 1970s and early '80s was attempting to answer the question: Is Liberace homosexual? (At the time, the term "gay" was not in widespread usage regarding sexual orientation.) Generally, older people, who'd became familiar with the colorful pianist via TV in the relatively […] [Read More]
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» Business
Thai Oil keen on refinery projects in Indonesia, Myanmar
Publication Date : 16-08-2014
Thai Oil is interested in taking part in oil-refinery projects in Indonesia and Myanmar, according to chief executive officer Veerasak Kositapaisal.
Indonesia's state-owned oil and gas company Pertamina recently approached PTT group on jointly investing in a project to expand the oil-refining output there. Pertamina currently runs six oil refineries across the country, with a combined capacity of about a million barrels per day.
Veerasak said that since he had received few details about the project, it was too early to discuss whether Thai Oil, a listed oil-refining subsidiary of PTT, would join in the project. Nevertheless, Indonesia, he pointed out, currently has to import as much as 500,000 barrels of oil per day to satisfy its current demand of 1.2 million barrels per day.
Earlier, Thai Oil signed a heads of agreement with Pertamina jointly to undertake a detailed study on a project to produce paraffin wax. He said Thai Oil expected to decide whether to invest in the project, which would use raw materials from Pertamina's Cilacap refinery in Central Java, by the end of this year. Paraffin wax is a raw material for the manufacturing of various products such as batik fabric, automotive tyres, particleboard, furniture, shoe polish and candles.
As for Myanmar, Veerasak said Thai Oil regarded it as a "focused country" where it would like to expand its investments outside Thailand. He said the company, together with the parent firm PTT, would submit a proposal to upgrade the Thanlyin oil refinery near Yangon. The Myanmar Petrochemical Enterprise last month issued an invitation to tender for the rehabilitation of the Thanlyin refinery under a joint-venture scheme. The deadline for submitting bids is October 13.
An initial plan would involve an upgrade of the Thanlyin refinery to a minimum capacity of 15,000bpd, while building new oil refineries and petrochemical plants would be carried out over the long term. Myanmar currently has to import all refined oil products to meet its annual consumption of 70,000bpd, which could easily go up to 150,000-200,000bpd in a near future, he said.
Thai Oil expects gross margin of its key products - refined oil, paraxylene, and lubricating oil - in the current half of this year to change little from the first half. Nevertheless, the oil-refining business will lose a month of income this third quarter due to the 46-day maintenance shutdown that started on June 15 and finished at the end of last month. Overall, the refinery's utilisation rate is expected at 94 per cent of its designed capacity this year, down from 102 per cent in 2013.
Thai Oil's gross margin, excluding accounting impacts from oil inventories, stood at 5.1 per cent in the second quarter, down from 6.1 per cent in the first quarter and 5.6 per cent booked a year ago. During the first half of 2014, the company reported net profit of 4.63 billion baht, up from 1.68 billion baht recorded during the same period of last year. Earnings before interest, taxes, depreciation and amortisation increased from 7.63 billion baht to 8.57 billion baht during the same period.
Meanwhile, Veerasak said a 12-billion-baht linear alkyl benzene (LAB) project, undertaken through a joint venture between its subsidiary Thai Paraxylene and Mitsui & Co, was 52 per cent complete would commence commercial operation in the fourth quarter of 2015 as scheduled. The plant will supply its products mainly to detergent producers in Thailand and other Southeast Asia countries.
US$1 = 31.82 baht
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Awesome Stuff Around The Internet
October 22, 2014 | No Comments » | Topics: Links |
18 Epiphanies That Will Change The Way You Look At The World – Ned Hardy
This Post Is Depressing…22 Pictures That Will Make You Feel Old – Caveman Circus
14 Difficulties Of Wanting To Be Outgoing But Having Social Anxiety – Thought Catalog
Selena Gomez in some booty shorts – Celeb Jihad
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Alessandra Ambrosio takes awesome selfie in a bikini – Drunken Stepfather
12 Women JFK Allegedly Banged Over The Years… Besides His WIfe – Radass
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Colleen Shannon is in a bikini – Celeb Slam
32 Cities Want to Challenge Big Telecom, Build Their Own Gigabit Networks – Motherboard
And you thought YOUR day was pretty sh!tty…think again my friends (37 Photos) – Bad Sentinel
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10 Brutally Honest Facts of Life You Need to Be Reminded Of – Chad Howse
No Comments »
n. the inexplicable urge to push people away, even close friends who you really like—as if all your social tastebuds suddenly went numb, leaving you unable to distinguish cheap politeness from the taste of genuine affection, unable to recognize its rich and ambiguous flavors, its long and delicate maturation, or the simple fact that each tasting is double-blind.
The Dumping Grounds
October 22, 2014 | No Comments » | Topics: main, The Dumping Grounds |
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October 22, 2014 | 1 Comment » | Topics: Art |
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Music Of The Week Archive
Jonpaul Douglass Is Pushing The Boundaries Of The Pizza Movement With His 'Pizza In The Wild' Photo Series
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Is It Just Me Or Is Steven Tyler Starting To Look Like An Old Woman??? – Caveman Circus
Nina Dobrev In A Bikini On 'The Vampire Diaries' - Celeb Jihad
22 Ways to Get a Better Night's Sleep – The Art Of Manliness
Girls Showing Sideboob = Hot – Linkiest
Jehane "Gigi" Paris and Her Thong Swimwear Photoshoot – G-Celeb
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The 50 Greatest Perfectly Timed Pictures Of All Time – World Wide Interweb
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Miss Virginia got hacked – Celeb Slam
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A 'cure for paralysis': World first as man with completely severed spine walks again – Independent
Things That Make Us LOL! – OMG Cute Things


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BKD adds accountants
by MBJ Staff
Published: November 16,2012
Tags: BKD LLP, Calvin Thigpen, Chandler Croom
BKD, LLP has added Calvin Thigpen Jr. and Chandler Croom as staff accountants in the accounting firm's Jackson office. Thigpen joins BKD in the audit department with more than 16 years of experience in the banking industry. Before joining BKD, he served as a branch manager for Trustmark National Bank in Jackson. Thigpen is a recent graduate of Mississippi College where he earned a bachelor's degree in accounting in May 2012. In 1996, he earned a bachelor's degree in business administration from Mississippi Valley State University. Additionally, he is a 2004 graduate from the University of Mississippi's School of Banking and a 2010 graduate from Louisiana State University's School of Banking.
Croom is a graduate from the University of Mississippi where he earned a bachelor's degree in accountancy in 2011 and a master of accountancy in 2012, and begins his career in the audit department. Croom interned at BKD in the spring of 2011 in the Jackson office's audit department.
To sign up for Mississippi Business Daily Updates, click here.
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Former OU QB Jack Mitchell had profound impact
uot;I liked Jack," Switzer said. "He could get a team ready to play. Very animated." Mitchell grew up in Arkansas City, Kan., but went to the University of Texas. He entered the military during World War II, and when Mitchell returned to Ark City after the war, new OU coach Jim Tatum talked Mitchell into coming to Norman for the historic 1946 spring practice that included so many war veterans. Mitchell took control of the team, became a master optioneer under Wilkinson after Tatum's departure and in 1948 OU's first All-America quarterback. "Jack was an exceptional runner," Arnold said. "Really tricky. He was great for the split-T. We didn't throw the ball hardly at all. But his personality was just dynamite." Mitchell's career punt return average of 23.6 yards is the NCAA record.
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OK, now that we're looking at the Mormon of the Year, I'd also like to look at what the big news stories were for the year. In a lot of ways its been a very busy news year, with, by my count, three big stories dominating: Mitt Romney's presidential candidacy The confusion of the LDS Church with the FLDS Church in the news The Mormon role in the successful effort to pass Proposition 8. But there were also smaller, important stories that happened during the year, especially if you include in News about Mormonism news about people who are... Read more »
Times and Seasons is a place to gather and discuss ideas of interest to faithful Latter-day Saints.
Recent Comments
- The Body of Christ
- gst: "Yeah, the Bible says a lot of stuff." –Reverend Lovejoy.
- Cameron N.: Well, Jesus also said "the only true and living church upon the face of...
- Terry H: @thabermayer. I concur in the previous posts on VSI, but the Jesus one by Richard...
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Notes From All Over
The more that changes the more it stays the same. Or something. ... See MoreSee LessView on Facebook
Read the entry for 2014 re whether the Women's Meeting is part of General Conference.(Julie) ... See MoreSee LessView on Facebook
"While the women's meetings have long been an important part of General Conference week, they are not usually referred to as a session of General Conference" (Marc) ... See MoreSee LessView on Facebook
New York Times offers rare warning for why Mormon movie is PG (Marc) ... See MoreSee LessView on Facebook
Brian C. Hales publishes a response to Grant Palmer's latest (Marc) ... See MoreSee LessView on Facebook
LDS Newsroom
- Indianapolis Indiana Temple Topped With Angel Moroni Statue October 17, 2014
- Broadway Star Santino Fontana and 'Sesame Street' Muppets™ Headline Christmas Concerts October 16, 2014
- Apostle Speaks on 50th Commemoration of 1964 New York World's Fair October 15, 2014
- 'Meet the Mormons' Pulls in Audiences Nationwide October 11, 2014
- Photo Essay: Prominent Figures Gather for the 'Meet the Mormons' Movie Premiere October 10, 2014
- Church Partners With Other Faith Communities in "Faith Counts" Initiative October 10, 2014
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Posts Tagged ' Fundamentalist LDS Church '
Reviewing News about Mormonism for the Year
December 29, 2008 | 14 commentsBy Kent Larsen


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About Us
Action Plan
-End-of-Year Report
-Promotional Requirements
School Activities
-Activity Code of Conduct
-After the Bell
-Athletics (8th Only)
Parent Group
Contact Us
District Boundary Study
This year, the West Des Moines Community Schools will study trends in student enrollment and review the location of school boundaries. The last boundary/enrollment study was completed by the district in 2007.
The boundary committee - made up of parents, staff, administrators and community partners - has been charged with sending a recommendation to the school board for its Dec. 8 meeting. Throughout the process you can stay informed through public forums (dates to be announced), emails, the Informaline, social media and on the district website. CLICK HERE FOR MORE INFORMATION
Stilwell Announcements, October 20, 2014 Day 2
Building Safety- Check In Procedures
Parents entering the building need to always check in the office first.
Parents Visiting A Classroom or Teacher
Please talk to an administrator to set a time up to meet with a teacher. Those kind of appointments need to be scheduled.
Parking Lot Safety
For the safety of our staff and students please slow down when you enter and exit the Stilwell parking lot.
Dismissal Info: Weather Related
With Iowa's ever changing weather conditions, please discuss with your child a contingency plan if activities are cancelled or there is early dismissal – please let your child know if they should get on the bus or wait for a parent.
Parents-Important to Note
If your student is going to be leaving early from school, please send a note with your student and they can come into the office at 7:25 a.m. to get a pass to leave when needed. This helps so we do not have to call into a classroom and disrupt learning time. If your student is coming in LATE, we do not answer phones during that time. Please send a note with them.
Stilwell's math competition team wants YOU! Please come to Miss Werger's room on Tuesday, October 21st at 2:45 to find out what this club is all about!"
Today we begin kickball for intramurals at 2:45
I-CARE - Look for you name in the cafeteria, check in with your teacher, then meet Mrs Davis by the front doors
Cross Country
Some members of the Cross Country team ran at the State JH Cross Country meet in Ankeny on Saturday. Despite the cold and windy weather, we had many great performances. The boys finished 14th place and the girls finished 7th place overall. The Boys runners were; Evan Armitage (16th), Ryan Staub (61st), Nick Songer (82nd), Mike Maigaard (120th) and Carter Owen- 3rd in 7th grade race. Emily Ash finished 7th in the girls race.
Student Council
Reminder to Student Council members to meet in the gym tomorrow – that's Tuesday – morning at 7:15. Thank you!
Microsoft, partnering with the Technology Association of Iowa , invites girls from 8th – 12th grade to attend a FREE DigiGirlz event, which will be held November 11th at the Prairie Meadows Events Center , in Altoona. This event is prior to the Iowa Women of Innovation awards dinner that elevates and celebrates today's extraordinary women in the fields of Science, Technology, Engineering and Math.
During the event, the girls will interact with women in technology executives, participate in technology sessions and demonstrations, network, and learn with hands-on experience.
8th grade girls interested in attending this event should see Mrs. Henderson in room 1106 as soon as possible as space is limited.
Valley Volleyball
The Valley volleyball varsity coaching staff will be running a clinic to help players (ages 10-18) prepare for upcoming club tryouts. This clinic will overview the club tryout process, focusing on drills, tips for making a team, and a time to answer any questions about the tryout process. The clinic will be held on Saturday, October 25th (9:00-10:30 for ages 10-14, 11:00-noon for ages 15-18) and the cost is $10. For more information and to register, please visit www.valleyvolleyball.com or email Coach Ross ( email@example.com ).
Archery Club
Valley Archery Club: If you want to be part of one of the coolest sports ever, please join Valley Archery Club. Please pick up registration packets from the office or Coach McConnell . Please turn all info into Coach McConnell.
Forms 2014/15
School Supply Lists
Please see the below links for the school supply lists for your team.
Understanding and Preventing Suicide
Please find included a link to a website that gives valuable information about understanding and preventing suicide: http://www.helpguide.org/mental/suicide_prevention.htm#tip2
Valley Southwoods Curriculum Catalog - Coming Soon!
Valley High School Curriculum Catalog
Magazine Renewal Program 2013
Visit www.gaschoolstore.com & Login
Register using our school online ID# : 818286
Questions on magazines ordered? Call
or email email@example.com
Stilwell School Play
Nov. 9
1:00 p.m.
View the Stilwell
Daily Announcements Here
Eric Boyle, Principal
Brady Weber, Assistant Principal
Kathryn Zaun, A-R
Brady Johnson , S-Z
Karen vanOostenrijk
Ann Stover
To email the teams:
T - firstname.lastname@example.com
I - firstname.lastname@example.com
G - firstname.lastname@example.com
E - firstname.lastname@example.com
R - firstname.lastname@example.com
S - email@example.com


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After a shootout in Watertown, Mass., late Thursday night, a day-long manhunt and a tense evening standoff, one Boston Marathon bombing suspect is dead. The second is seriously injured and in police custody.
Article ID:
(David R. Jennings / Daily Camera)
Football: Fairview wins thriller at Legacy
Gutsy two-point conversion in final seconds gives Knights FRL crown
When the Fairview football team had the chance, just like Fossil Ridge against them last Friday night, Tom McCartney wasn't going to shy away. Full Story
(Cliff Grassmick / Daily Camera)
Men's basketballCU men's basketball: Buffs, Utes make life difficult on the road for Pac-12 foes
Conference newcomers considered among league's elite teams
SAN FRANCISCO — Just three years ago, Pac-12 coaches may have viewed the new mountain trip as a cake walk. Full Story
(Paul Aiken / Daily Camera)
Second Story Garage: Strange Americans
It's refreshing when bands don't have to tie themselves in knots when describing their music. Strange Americans play rock 'n' roll. That's what they say and that's who they are, and that's what they did when they played the garage. Full Story


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mindy project
Results: 1 - 2 of 2
The GQ Eye: GQ on Style
Your Daily Eye Queue: The Real-Life Work of Danny Castelano, Sustainable Style, and More...
Film Chris Messina (aka Danny Castelano) debuts his first movie, Alex of Venice, at Tribeca. (Huffington Post) Celebrity Johnny Weir and Tara Lipinski take their Olympic routine to the Kentucky Derby. (The Cut) Style... read more
Tuesday, 4/22/2014 at 4:30PM
- Tags:
- Denim,
- Earth Day,
- James Bond,
- Mindy Project,
- Nike
The Feed
The Mindy Project's Morgan Tookers on His Summer Vacation
Summer is unofficially over—which means, so is summer vacation. It also means that plenty of your favorite TV shows are back. Exhibit A: The Mindy Project's season premiere tomorrow night on FOX. We assigned... read more
Monday, 9/16/2013 at 7:00AM
- Tags:
- Hollywood,
- Humor,
- Mindy Project,
- Morgan Tooker,
- Summer
Results: 1 - 2 of 2
- New Arrivals: 13 Things We Want to Buy This Week
- Your Daily Eye Queue: The Sweatshirt Kanye Couldn't Pass Up, Hood By Air's Party at MoMa, and More...
- Introducing: The Style Chatroom: Creepwave, '70s Style, and Halloween
- Suited, Booted, and Silver: Russell Westbrook and Kevin Durant Open the Season in Style
- The Complete Lazy Man's Guide to a Killer Halloween Costume
- GQ's Fall 2014 Boot Guide: The F#@k Winter Boot
News + Politics
- GQ Style: Wes Anderson's Highly Influential, Slightly Tweaked Take on Reality
- Jerry Lee Lewis Will Not go to Hell
- Matthew McConaughey: America's Space Cowboy
- Logan Lerman Went to Bootcamp with Brad Pitt for Fury
- Under the Influence: How Def Jam's Early Days Inspired this Season's Look
- Outlaw, Artist, Wild Man, Style Icon: How Dennis Hopper Inspired this Season's Look
How To
Food + Travel
- David Chang's Kitchen: Don't Get All "Fresh" with Me
- David Chang's Kitchen: Your Idea of Bologna is Total Baloney
- Chart: The Family Tree of Bourbon Whiskey
- Recipe: Semi-Summer Salad - Chef Jeff Taibe of Oak + Almond
- The Great Franco-Italian Pasta Experiment
- How to Pick from an Overpriced Restaurant Wine List


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The Top 10 In Action Sports
01Attack Of The Drones: This Star Wars Style Drone Racing Is Outrageously Cool
02These Incredible Images From This Glow-In-The-Dark Ski Movie Will Blow Your Mind
03If You Only Watch One Snowboard Video This Week, This Should Be It
04This Guy Is Sailing Around The World on a Tiny Catamaran, With No GPS and No Cabin
05Strange Style And Shit Philosophy: This Shaun White Twitter Parody Is Absolutely Hilarious...
06Inside A Dishwasher, Toilet Plunging & Extreme Tea Making: These GoPro Videos Should Be Mundane But Are Actually Fascinating
07James Bond's Submarine-Car Has Been Listed On Ebay… For $1,000,000
08These Guys Have Invented A Wireless GoPro Charger That Works Through The Case
09Heading To The Mountains This Winter? Here Are The 8 Best Festivals Being Held On The Slopes...
10"I Get Called Crazy Pretty Much Every Day...."
Peeping Bear Productions 2011 Best of
Here is the final instalment of Peeping Bear Productions 2011 featuring some of the best parts of each episode and more of the year. A long with that is a cheeky preview for the logo for Peeping Bear Productions 2012 at the end.
What Do You Think?
Get the best in action sports straight to your inbox


text:
Loved it. Written for fun and gifted with artistic integrity. (Report) Reply
Stanley Cooper
Artistic Integrity
To artistic integrity
I make no claim
Rhyming eccentricity
Is more my game
What pops into my head
Is written down
Never sure what's ahead
Be it verb, be it noun
I've been laden with doubt
By critic dissenters
They are so devout
These nitpick tormentors
When my poem is all done
Not completely a sham
I've written my fun
Artistic integrity be damned
Stanley Cooper's Other Poems
Read this poem in other languages
This poem has not been translated into any other language yet.
I would like to translate this poem »
Comments about this poem (Artistic Integrity by Stanley Cooper )
Savita Tyagi (1/8/2014 9:21:00 AM)
* Sunprincess * (1/8/2014 7:37:00 AM)
Alistair Graham (1/8/2014 2:51:00 AM)
Poetry Shelf
In the main room of every home
at arm's length of everyone
A shelf with screws and plugs
should hug the wall
And on this shelf the paperbacks
from left to right and pamphlets
the spices in your life,
may you grow tall
The poet speaks when others won't
with words in ways that others can't
the like of which you won't
have heard til now
Surprise yourself, buy a new book
surprise your family or even look
to buy a book
for everyone you know (Report) Reply
Gajanan Mishra (1/8/2013 4:46:00 AM)
A rhymer with a smile. If you wanted a tighter syllable count in the last line, you could simply go with 'Integrity be damned'. You've already used artistic integrity 2x before so point would already be made. Thanks for the enjoyable read. (Report) Reply
Chuck Audette (1/5/2006 11:08:00 AM)
A 10 from me, too! Damn the syllables, full speed ahead! and rhymes are like landmines, as soon as you find one, you realize you're surrounded, and good luck getting out alive! -chuck (with a vaguely similar poem 'Poetry Hell') (Report) Reply
Faith Elizabeth Brigham (12/30/2005 8:49:00 PM)
i loved this...nothing short of a 10 would befit this...you got one from me...excellent job! (Report) Reply
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Jason A Facebook: https://www.facebook.com/pages/End-Times-Watch/261386594016408 MARTIAL LAW drills across the country, liberties being stripped away daily, ...
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The Doors - The End (original)
Doors song The End full and uncensored.
- 6cath6arsis6
- Length: 11:48
- Tags:
When Will The World End?
When Will The World End? Ever wondered what the end of the world will be like? Be a nuclear annihilation or asteroid apocalypse, learn some scary stuff about the end of the world! Subscribe...
- Alltime Numbers
- Length: 3:38
- Tags:
Linkin Park - In The End | Ten Second Songs 20 Style Cover
My name is Anthony Vincent and I'm the voice of Ten Second Songs, custom songs made in ANY style. Make sure to subscribe to my channel to see/hear some more ...
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INSANE!! WHEN WILL IT END!!? - FIFA 15 Ultimate Team Pack Opening
My pack luck in FIFA 15 Ultimate Team Pack Opening.... ▻ BUY CHEAP FIFA 15 COINS HERE: ▻ http://www.buycheapfifacoins.com ▻ USE DISCOUNT CODE 'TWOSYNC' FOR 5% OFF! Follow Us ...
- Length: 4:57
- Tags:
The End Is Never The End Is Never The End | The Stanley Parable | Part 6
The Stanley Parable: Today Hank Green finds out the end is never the end is never the end is never the end is never the end is never the end is never the end is never the end is never the end...
- GamesWithHank
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- Tags:
Alien Isolation Walkthrough Part 10 - The End
Alien Isolation Walkthrough Part 10 - The End! Will she make it out alive? Ripley communicates with the Torrens and attempts to evacuate from the doomed Sevastopol Station... Playlist: http://goo.g...
- jackfrags
- Length: 52:52
- Tags:
The End | Danny Edge
Fellow YouTuber Jack Harries recently did a poem video to get his message across, so I decided to use a poem to get across something that I've been trying to say for months.. Danny's Twitter:...
- ExclamationPointYT
- Length: 2:29
- Tags:
Destiny - NEW Exotic Weapons, Exotic Core Shards, & Crota's End Raid! (Destiny Legendary Rewards)
NEW INFO ABOUT WEAPONS, SHIP SHADERS, LEGENDARY AND EXOTIC ARMOR, EXOTIC CORES AND SHARDS, AND MORE! In this video, I talk about all the new DLC items coming in "The ...
- NoahJ456
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Janji ft. T.R. - End Of Summer
Artist : Janji, T.R. Song : End Of Summer Genre : Saxophone / House ♫ Free Download ○ https://soundcloud.com/janjimusic/janji-feat-tr-end-of-summer ☆ Janji Soundcloud → https://sound...
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Why I Think This World Should End
Sorry if this Offends You. Download the Spoken Word Free Here https://princeea.bandcamp.com/track/prince-ea-the-world-is-coming-to-an-end Download the Instru...
- Prince Ea
- Length: 4:14
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