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Search Results: 1 - 10 of 2844 matches for " Helen Piwnica-Worms "
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Human Cytomegalovirus Protein pUL117 Targets the Mini-Chromosome Maintenance Complex and Suppresses Cellular DNA Synthesis
Zhikang Qian,Van Leung-Pineda,Baoqin Xuan,Helen Piwnica-Worms,Dong Yu
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1000814
Abstract: Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.
Contributions Made by CDC25 Phosphatases to Proliferation of Intestinal Epithelial Stem and Progenitor Cells
Gwanghee Lee,Sofia Origanti,Lynn S. White,Jinwu Sun,Thaddeus S. Stappenbeck,Helen Piwnica-Worms
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015561
Abstract: The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.
A Fluorescence-Coupled Assay for Gamma Aminobutyric Acid (GABA) Reveals Metabolic Stress-Induced Modulation of GABA Content in Neuroendocrine Cancer
Joseph E. Ippolito, David Piwnica-Worms
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088667
Abstract: Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.
Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems
Robin S. Dothager,Reece J. Goiffon,Erin Jackson,Scott Harpstrite,David Piwnica-Worms
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013300
Abstract: Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β+)-emitting radionuclides in small animals and humans. Upon β+ decay, the initial velocity of high-energy β+ particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.
T-cell activation promotes tumorigenesis in inflammation-associated cancer
Dan Rauch, Shimon Gross, John Harding, Sirosh Bokhari, Stefan Niewiesk, Michael Lairmore, David Piwnica-Worms, Lee Ratner
Retrovirology , 2009, DOI: 10.1186/1742-4690-6-116
Abstract: Malignant transformation of the cancer cell is promoted and often preceded by changes in the tumor microenvironment, rich in inflammatory cells, growth factors, and DNA damage promoting agents. A wide range of malignancies are promoted by chronic inflammation associated with chemical, physical, or microbial factors [1-4]. The diversity of oncogenic factors associated with inflammation highlights the importance of characterizing those common to a wide range of malignancies. The cellular effectors, signaling pathways, and secreted regulators involved in chronic inflammation are the soil in which the seeds of these cancers are initiated.T-cells are central regulators of the immune response; T-cells are recruited to sites of chronic inflammation, and the infiltration of T-cells within the tumor is a critical determinant of neoplastic outcome. Na?ve CD4+ T-cells, or T-helper cells, that have not previously encountered an antigen differentiate into one of four committed lineages (TH1, TH2, TH17, Treg) in response to antigen presenting cells [5-10]. Conventionally, TH1 and TH2 cells promote the elimination of intracellular and extracellular pathogens respectively. More recently TH17 cells have been characterized for their ability to promote inflammation by recruiting neutrophils to peripheral tissues to remove extracellular pathogens, while Treg cells repress inflammation to keep immune hyperactivity in check. While there is no question that T-cells are recruited to sites of chronic inflammation, it is unclear whether activated T-cells promote or restrict malignancies in vivo.Molecular pathways often involved in inflammation-associated tumorigenesis include JNK, STAT3, HIF-1, and nuclear factor κB (NFκB) signaling, and generation of reactive oxygen species [1,3,11,12]. These pathways are interrelated and signaling through NFκB serves as a master regulator. NFκB signaling during tumorigenesis prevents apoptosis and promotes proliferation, metastasis, and angiogenesis [13].
Single-Cell Resolution Imaging of Retinal Ganglion Cell Apoptosis In Vivo Using a Cell-Penetrating Caspase-Activatable Peptide Probe
Xudong Qiu, James R. Johnson, Bradley S. Wilson, Seth T. Gammon, David Piwnica-Worms, Edward M. Barnett
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088855
Abstract: Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.
Bioluminescent Imaging Reveals Divergent Viral Pathogenesis in Two Strains of Stat1-Deficient Mice, and in α?γ Interferon Receptor-Deficient Mice
Tracy Jo Pasieka, Lynne Collins, Megan A. O'Connor, Yufei Chen, Zachary M. Parker, Brent L. Berwin, David R. Piwnica-Worms, David A. Leib
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024018
Abstract: Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1?/? and IFNα?γR?/? mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1?/? mice and in IFNα?γR?/? mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1?/? (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNα?γR?/? mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1?/? and IFNα?γR?/? mice, we infected an additional Stat1?/? strain deleted in the DNA-binding domain (129Stat1?/? (DBD)). These 129Stat1?/? (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNα?γR?/? mice. This lethal pattern was also observed when 129Stat1?/? (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1?/? (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1?/? mouse strains. The data are consistent with the hypothesis that Stat1?/? (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons between knockout mouse strains in viral pathogenesis, and may also be relevant to the causation of HSV hepatitis in humans, a rare but frequently fatal infection.
CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma
Shyam Rao, Rajarshi Sengupta, Eun Joo Choe, B. Mark Woerner, Erin Jackson, Tao Sun, Jeffrey Leonard, David Piwnica-Worms, Joshua B. Rubin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033005
Abstract: Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.
Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction
Claus S Sondergaard, David A Hess, Dustin J Maxwell, Carla Weinheimer, Ivana Rosová, Michael H Creer, David Piwnica-Worms, Attila Kovacs, Lene Pedersen, Jan A Nolta
Journal of Translational Medicine , 2010, DOI: 10.1186/1479-5876-8-24
Abstract: Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.Acute myocardial infarction (AMI) and the resulting complications are a leading cause of morbidity and mortality in the Western world. While conventional treatment strategies for AMI may efficiently alleviate symptoms and hinder disease progression, recovery of lost cells and tissue is rarely achievable. Transplantation of primitive progenitor cells of hematopoietic, mesenchymal, and endothelial lineages have, however, been found to enhance endogenous tissue repair in small animal disease models and to improve overall function of the affected tissues in early phase clinical trials[1]. The exact mechanism of repair is not known but may involve paracrine signaling by the donor cells or direct replacement of damaged tissue by donor cells[2].Stem and progenitor cells derived from hematopoietic tissue have attracted much attention as a source of transplantable cells for cell-based regenerative therapy. Hematopoietic, mesenchymal, and endothelial progenitors have been identified in human bone marrow (BM) and umbilical cord blood (UCB) [3-5]. All three progenitor populations can be simultaneously isolated from human BM based on the expression of the cytosolic enzyme aldehyde dehydrogenase (ALDH)[6], although the relative contributions of the different sub-populations and consequently their relative therapeutic contribution may vary between the different cell sources. We and others have found that lineage depleted (Lin-) cells from BM and UCB that express high levels of ALDH (ALDHhiLin) have superior long term repopulating potential in the hematopoietic tissues of NOD/LtSz-scid/scid (NOD/SCID) mice whereas lineage depleted cells that express low levels of ALDH (ALDHloLin-) are virtually devoid of long term repopula
The ARF Tumor Suppressor Regulates Bone Remodeling and Osteosarcoma Development in Mice
Daniel A. Rauch,Michelle A. Hurchla,John C. Harding,Hongju Deng,Lauren K. Shea,Mark C. Eagleton,Stefan Niewiesk,Michael D. Lairmore,David Piwnica-Worms,Thomas J. Rosol,Jason D. Weber,Lee Ratner,Katherine N. Weilbaecher
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015755
Abstract: The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice. Arf-/- mice displayed increased osteoblast (OB) and osteoclast (OC) activity with a significant net increase in trabecular bone volume. The long bones of Arf-/- mice had increased expression of OB genes while Arf-/- OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+Arf-/- mice uniformly developed spontaneous OS by 7 months of age. Tax+Arf-/- tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax+Arf+/- mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+Arf-/- mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS.
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