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Hofmann S, Dehn S, Businger R, Bolduan S, Schneider M, Debyser Z, Brack-Werner R, Schindler M. Dual role of the chromatin-binding factor PHF13 in the pre- and post-integration phases of HIV-1 replication. Open Biol 2018; 7:rsob.170115. [PMID: 29021215 PMCID: PMC5666080 DOI: 10.1098/rsob.170115] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 09/08/2017] [Indexed: 12/23/2022] Open
Abstract
Viruses interact with multiple host cell factors. Some of these are required to promote viral propagation, others have roles in inhibiting infection. Here, we delineate the function of the cellular factor PHF13 (or SPOC1), a putative HIV-1 restriction factor. Early in the HIV-1 replication cycle PHF13 increased the number of integrated proviral copies and the number of infected cells. However, after HIV-1 integration, high levels of PHF13 suppressed viral gene expression. The antiviral activity of PHF13 is counteracted by the viral accessory protein Vpr, which mediates PHF13 degradation. Altogether, the transcriptional master regulator and chromatin binding protein PHF13 does not have purely repressive effects on HIV-1 replication, but also promotes viral integration. By the functional characterization of the dual role of PHF13 during the HIV-1 replication cycle, we reveal a surprising and intricate mechanism through which HIV-1 might regulate the switch from integration to viral gene expression. Furthermore, we identify PHF13 as a cellular target specifically degraded by HIV-1 Vpr.
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Affiliation(s)
- Stephan Hofmann
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Virology, Neuherberg, Germany
| | - Sandra Dehn
- Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Ramona Businger
- Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
| | - Sebastian Bolduan
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Virology, Neuherberg, Germany
| | - Martha Schneider
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Virology, Neuherberg, Germany
| | - Zeger Debyser
- Molecular Virology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Ruth Brack-Werner
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Virology, Neuherberg, Germany
| | - Michael Schindler
- Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Virology, Neuherberg, Germany .,Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany
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Payne EH, Ramalingam D, Fox DT, Klotman ME. Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells. J Virol 2018; 92:e01718-17. [PMID: 29093088 PMCID: PMC5752950 DOI: 10.1128/jvi.01718-17] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Accepted: 10/19/2017] [Indexed: 01/24/2023] Open
Abstract
Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G2-arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells.IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish between HIV-positive cell survival and death in a known HIV compartment, as well as pharmacological agents that alter these outcomes.
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Affiliation(s)
- Emily H Payne
- Department of Pathology, Duke University, Durham, North Carolina, USA
| | - Dhivya Ramalingam
- Department of Medicine, Duke University, Durham, North Carolina, USA
| | - Donald T Fox
- Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA
| | - Mary E Klotman
- Department of Pathology, Duke University, Durham, North Carolina, USA
- Department of Medicine, Duke University, Durham, North Carolina, USA
- Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
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Nookala AR, Mitra J, Chaudhari NS, Hegde ML, Kumar A. An Overview of Human Immunodeficiency Virus Type 1-Associated Common Neurological Complications: Does Aging Pose a Challenge? J Alzheimers Dis 2017; 60:S169-S193. [PMID: 28800335 PMCID: PMC6152920 DOI: 10.3233/jad-170473] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
With increasing survival of patients infected with human immunodeficiency virus type 1 (HIV-1), the manifestation of heterogeneous neurological complications is also increasing alarmingly in these patients. Currently, more than 30% of about 40 million HIV-1 infected people worldwide develop central nervous system (CNS)-associated dysfunction, including dementia, sensory, and motor neuropathy. Furthermore, the highly effective antiretroviral therapy has been shown to increase the prevalence of mild cognitive functions while reducing other HIV-1-associated neurological complications. On the contrary, the presence of neurological disorder frequently affects the outcome of conventional HIV-1 therapy. Although, both the children and adults suffer from the post-HIV treatment-associated cognitive impairment, adults, especially depending on the age of disease onset, are more prone to CNS dysfunction. Thus, addressing neurological complications in an HIV-1-infected patient is a delicate balance of several factors and requires characterization of the molecular signature of associated CNS disorders involving intricate cross-talk with HIV-1-derived neurotoxins and other cellular factors. In this review, we summarize some of the current data supporting both the direct and indirect mechanisms, including neuro-inflammation and genome instability in association with aging, leading to CNS dysfunction after HIV-1 infection, and discuss the potential strategies addressing the treatment or prevention of HIV-1-mediated neurotoxicity.
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Affiliation(s)
- Anantha Ram Nookala
- Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA
| | - Joy Mitra
- Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA
| | - Nitish S. Chaudhari
- Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA
| | - Muralidhar L. Hegde
- Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA
- Weill Cornell Medical College of Cornell University, NY, USA
| | - Anil Kumar
- Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA
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Zhang J, Storey KB. Cell cycle regulation in the freeze tolerant wood frog,Rana sylvatica. Cell Cycle 2014; 11:1727-42. [DOI: 10.4161/cc.19880] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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Zhang H, Lee SJ, Richardson CC. Essential protein interactions within the replisome regulate DNA replication. Cell Cycle 2014; 10:3413-4. [DOI: 10.4161/cc.10.20.17523] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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Abstract
To replicate their genomes in cells and generate new progeny, viruses typically require factors provided by the cells that they have infected. Subversion of the cellular machinery that controls replication of the infected host cell is a common activity of many viruses. Viruses employ different strategies to deregulate cell cycle checkpoint controls and modulate cell proliferation pathways. A number of DNA and RNA viruses encode proteins that target critical cell cycle regulators to achieve cellular conditions that are beneficial for viral replication. Many DNA viruses induce quiescent cells to enter the cell cycle; this is thought to increase pools of deoxynucleotides and thus, facilitate viral replication. In contrast, some viruses can arrest cells in a particular phase of the cell cycle that is favorable for replication of the specific virus. Cell cycle arrest may inhibit early cell death of infected cells, allow the cells to evade immune defenses, or help promote virus assembly. Although beneficial for the viral life cycle, virus-mediated alterations in normal cell cycle control mechanisms could have detrimental effects on cellular physiology and may ultimately contribute to pathologies associated with the viral infection, including cell transformation and cancer progression and maintenance. In this chapter, we summarize various strategies employed by DNA and RNA viruses to modulate the replication cycle of the virus-infected cell. When known, we describe how these virus-associated effects influence replication of the virus and contribute to diseases associated with infection by that specific virus.
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Affiliation(s)
- Eishi Noguchi
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania USA
| | - Mariana C. Gadaleta
- Dept of Biochemistry & Molecular Biology, Drexel University College of Medicine, Philadelphia, USA
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Zhang QH, Qi ST, Wang ZB, Yang CR, Wei YC, Chen L, Ouyang YC, Hou Y, Schatten H, Sun QY. Localization and function of the Ska complex during mouse oocyte meiotic maturation. Cell Cycle 2012; 11:909-16. [PMID: 22336914 DOI: 10.4161/cc.11.5.19384] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The Ska (spindle and kinetochore-associated) complex is composed of three proteins: Ska1, Ska2 and Ska3. It is required for stabilizing kinetochore-microtubule (KT-MT) interactions and silencing spindle checkpoint during mitosis. However, its roles in meiosis remain unclear. The present study was designed to investigate the localization and function of the Ska complex during mouse oocyte meiotic maturation. Our results showed that the localization and function of Ska complex in mouse oocyte meiosis differ in part from those in mitosis. Injection of low dose exogenous Myc-Ska mRNA showed that, instead of localizing to the kinetochores (KTs) and mediating KT-MT interactions from pro-metaphase to mid-anaphase stages as in mitosis, the members of the Ska complex were only localized on spindle microtubules from the Pro-MI to MII stages in mouse oocyte meiosis. Time-lapse live imaging analysis showed that knockdown of any member of the Ska complex by Morpholino injection into mouse oocytes resulted in spindle movement defects and enlarged polar bodies. Depletion of the whole Ska complex disrupted the stability of the anaphase spindle and influenced the extrusion of the first polar body. Taken together, these results show that the Ska complex plays an important role in meiotic spindle migration and anaphase spindle stability during mouse oocyte maturation.
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Affiliation(s)
- Qing-Hua Zhang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
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Fritz JV, Briant L, Mély Y, Bouaziz S, de Rocquigny H. HIV-1 viral protein r: from structure to function. Future Virol 2010. [DOI: 10.2217/fvl.10.47] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The viral protein r (Vpr) of HIV-1 binds several host proteins leading to pleiotropic functions, such as G2/M cell cycle arrest, apoptosis induction and gene transactivation. Vpr is encapsidated through the Gag C-terminus into the nascent viral particles, suggesting that Vpr plays several important functions in the early stages of the viral lifecycle. In this regard, Vpr interacts with nucleic acids and membranes to facilitate the preintegration complex migration and incorporation into the nucleus of nondividing cells. Thus, Vpr has to recruit several host and viral factors to promote its functions during HIV-1 pathogenesis. This article focuses on its interacting partners by giving an overview of the functional outcome of the different Vpr complexes, as well as the structural determinants of Vpr required for its binding properties.
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Affiliation(s)
- Joëlle V Fritz
- Department of Infectious Diseases, Virology, Universitätsklinikum, Im Neuenheimer Feld, 324, D-69120, Heidelberg, Germany
| | - Laurence Briant
- Université Montpellier 1, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, CNRS, UMR 5236, CPBS, F-34965 Montpellier, France
| | - Yves Mély
- Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France
| | - Serge Bouaziz
- Laboratoire de Cristallographie et RMN Biologiques, CNRS UMR8015 UFR des Sciences Pharmaceutiques et Biologiques 4, Avenue de L’observatoire, 75006 Paris, France: Université de Strasbourg, Faculté de Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France
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Li G, Park HU, Liang D, Zhao RY. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R. Retrovirology 2010; 7:59. [PMID: 20609246 PMCID: PMC2909154 DOI: 10.1186/1742-4690-7-59] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2010] [Accepted: 07/07/2010] [Indexed: 01/23/2023] Open
Abstract
Background Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive. Results To examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV. Conclusions These data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.
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Affiliation(s)
- Ge Li
- Department of Pathology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA
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Abstract
Like most viral regulatory proteins, HIV-1 Vpr and homologous proteins from primate lentiviruses are small and multifunctional. They are associated with a plethora of effects and functions, including induction of cell cycle arrest in the G(2) phase, induction of apoptosis, transactivation, enhancement of the fidelity of reverse transcription, and nuclear import of viral DNA in macrophages and other nondividing cells. This review focuses on the cellular proteins that have been reported to interact with Vpr and their significance with respect to the known functions and effects of Vpr on cells and on viral replication.
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Affiliation(s)
- Vicente Planelles
- Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive East #2100-Room 2520, Salt Lake City, Utah 84112, USA.
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TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1. Cell Death Differ 2010; 17:1288-301. [PMID: 20168333 DOI: 10.1038/cdd.2010.9] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers show or acquire resistance to TRAIL. In this study we report that TRAIL activates a TGF-beta-activated kinase 1 --> mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6 --> p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the 'death-inducing signaling complex' (DISC), activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis (IAP) antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and activation of the apoptosome was dispensable. Even after MOMP, however, mitochondrial-generated reactive oxygen species (ROS) activated a secondary signaling pathway, involving c-Jun N-terminal kinases (JNKs), that similarly upregulated MCL-1 expression and partially rescued some cells from death. Thus, stress kinases activated at distinct steps, before and after mitochondrial injury, mediate TRAIL resistance through maintenance of MCL-1 expression.
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Abstract
Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.
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Affiliation(s)
- Mira S. Chaurushiya
- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
- Graduate Program, Division of Biology, University of California, San Diego, CA 92093, USA
| | - Matthew D. Weitzman
- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
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Kamata M, Susanto MT, Chen ISY. Enhanced transthyretin tetramer stability following expression of an amyloid disease transsuppressor variant in mammalian cells. J Gene Med 2009; 11:103-11. [PMID: 19065606 DOI: 10.1002/jgm.1276] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The transthyretin (TTR) amyloidosis is an incurable fatal inherited disease that is characterized by progressive peripheral and autonomic neuropathy. It is caused by missense amyloidogenic mutations in the TTR gene that destabilize the native tetrameric state and lead to the cytotoxic misfolded monomeric state. One interesting variant (T119M) stabilizes heterotetramers with amyloidogenic TTR and, in the reported heterozygous individuals, protects the carriers from disease. In the present study, we characterize in vitro and in vivo the ectopic expression of the human T119M mutant, termed a transsuppressor for TTR amyloid disease. METHODS Lentiviral vectors encoding wild or mutant forms of human TTR were constructed and transduced to the human hepatocellular carcinoma cell line, HepG2, or mice. Heterooligomerization between T119M TTR and amyloidogenic variants was analysed by immunoprecipitation following western blotting. RESULTS T119M TTR was stably expressed in transduced HepG2 cells and was secreted as an oligomer that can interact with its native partner, retinol-binding protein. Importantly, the T119M TTR formed secreted heterooligomers with amyloidogenic TTR variants, V30M, L55P and V122I, in HepG2 cells that were more stable than the homooligomers of the same amyloidogenic TTR variants. Human T119M TTR also formed heterooligomers with V30M TTR in transduced mice. CONCLUSIONS The results obtained in the present study demonstrate the stabilization of heterotetramers by T119M TTR in human cells and suggest that gene transfer of T119M TTR may have potential as a gene therapy for TTR amyloidosis.
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Affiliation(s)
- Masakazu Kamata
- Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA
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Andersen JL, Le Rouzic E, Planelles V. HIV-1 Vpr: mechanisms of G2 arrest and apoptosis. Exp Mol Pathol 2008; 85:2-10. [PMID: 18514189 PMCID: PMC2610539 DOI: 10.1016/j.yexmp.2008.03.015] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2007] [Accepted: 03/04/2008] [Indexed: 02/05/2023]
Abstract
Since the first isolation of HIV-1 from a patient with generalized lymphadenopathy in 1983, great progress has been made in understanding the viral life cycle and the functional nuances of each of the nine genes encoded by HIV-1. Considerable attention has been paid to four small HIV-1 open reading frames, vif, vpr, vpu and nef. These genes were originally termed "accessory" because their deletion failed to completely disable viral replication in vitro. More than twenty years after the cloning and sequencing of HIV-1, a great deal of information is available regarding the multiple functions of the accessory proteins and it is well accepted that, collectively, these gene products modulate the host cell biology to favor viral replication, and that they are largely responsible for the pathogenesis of HIV-1. Expression of Vpr, in particular, leads to cell cycle arrest in G(2), followed by apoptosis. Here we summarize our current understanding of Vpr biology with a focus on Vpr-induced G(2) arrest and apoptosis.
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Affiliation(s)
- Joshua L. Andersen
- Center for the Study of Aging and Human Development, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710
| | - Erwann Le Rouzic
- Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France
- Inserm, U567, Paris, France
| | - Vicente Planelles
- Division of Cellular Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132
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Abstract
The integrity of genomic DNA is challenged by genotoxic stress originating during normal cellular metabolism or by external insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, damage repair, apoptosis or cellular senescence. The loss or alterations of genes involved in the damage response pathways have been reported in many cancer susceptibility syndromes and in sporadic tumors. Furthermore, this surveillance pathway is activated during early tumourigenesis presumably due to uncontrolled replicative cycles and has been recognized as one of the main barriers against the development of cancer. This review discusses the relevance of prostatic epithelial cells in prostate tumourigenesis and highlights common molecular changes associated with prostate cancer. Furthermore, DNA damage responses of primary cultures of human prostatic epithelial cells and fresh human prostate tissues are discussed providing evidence for alterations in crucial DNA damage checkpoint molecules. New insights connecting prostate tumourigenesis to alterations and defects in the pathways maintaining genomic integrity will be discussed.
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Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2. J Virol 2008; 82:5672-82. [PMID: 18385244 DOI: 10.1128/jvi.01330-07] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.
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Huard S, Elder RT, Liang D, Li G, Zhao RY. Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25. J Virol 2008; 82:2904-17. [PMID: 18160429 PMCID: PMC2259012 DOI: 10.1128/jvi.01098-07] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2007] [Accepted: 11/28/2007] [Indexed: 02/06/2023] Open
Abstract
Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.
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Affiliation(s)
- Sylvain Huard
- Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, MSTF700A, Baltimore, MD 21201, USA
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Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1. Proc Natl Acad Sci U S A 2007; 104:19867-72. [PMID: 18056802 DOI: 10.1073/pnas.0709879104] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Inactivation of maturation-promoting factor [(MPF) Cdk1/Cyclin B] is a key event in the exit from mitosis. Although degradation of Cyclin B is important for MPF inactivation, recent studies indicate that Cdk1 phosphorylation and inactivation occur before Cyclin B degradation and, therefore, also may be important steps in the exit from mitosis. Cdk1 activity is controlled by the Cdc25C phosphatase, which is turned on at the G(2)/M transition to catalyze Cdk1 activation. PP2A:B56delta is a negative regulator of Cdc25C during interphase. We show here that PP2A:B56delta also regulates Cdc25C at mitosis. Failure of PP2A:B56delta to dephosphorylate Cdc25C at mitosis results in prolonged hyperphosphorylation and activation of Cdc25C, causing persistent dephosphorylation and, hence, activation of Cdk1. This constitutive activation of Cdc25C and Cdk1 leads to a delayed exit from mitosis. Consistent with Cdk1 as a major biological target of B56delta, stable knockdown and germ-line mouse KO of B56delta leads to compensatory transcriptional up-regulation of Wee1 kinase to oppose the Cdc25C activity and permit cell survival. These observations place PP2A:B56delta as a key upstream regulator of Cdk1 activity upon exit from mitosis.
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19
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Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S. Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages. Retrovirology 2007; 4:84. [PMID: 18039376 PMCID: PMC2211753 DOI: 10.1186/1742-4690-4-84] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2007] [Accepted: 11/26/2007] [Indexed: 12/23/2022] Open
Abstract
Background HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages. Results In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first α-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first α-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors. Conclusion These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.
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Affiliation(s)
- Guillaume Jacquot
- Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.
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Davy C, Doorbar J. G2/M cell cycle arrest in the life cycle of viruses. Virology 2007; 368:219-26. [PMID: 17675127 PMCID: PMC7103309 DOI: 10.1016/j.virol.2007.05.043] [Citation(s) in RCA: 119] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2007] [Revised: 03/29/2007] [Accepted: 05/23/2007] [Indexed: 11/30/2022]
Abstract
There is increasing evidence that viral infection, expression of viral protein or the presence of viral DNA causes the host cell cycle to arrest during G2/M. The mechanisms used by viruses to cause arrest vary widely; some involve the activation of the cellular pathways that induce arrest in response to DNA damage, while others use completely novel means. The analysis of virus-mediated arrest has not been proven easy, and in most cases the consequences of arrest for the virus life cycle are not well defined. However, a number of effects of arrest are being investigated and it will be interesting to see to what extent perturbation of the G2/M transition is involved in viral infections.
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Affiliation(s)
| | - John Doorbar
- Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
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21
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Dehart JL, Planelles V. Human immunodeficiency virus type 1 Vpr links proteasomal degradation and checkpoint activation. J Virol 2007; 82:1066-72. [PMID: 17855541 PMCID: PMC2224437 DOI: 10.1128/jvi.01628-07] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Affiliation(s)
- Jason L Dehart
- Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive East #2100, Room 2520, Salt Lake City, UT 84112, USA
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22
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Park EY, Wilder ET, Lane MA. Retinol inhibits the invasion of retinoic acid-resistant colon cancer cells in vitro and decreases matrix metalloproteinase mRNA, protein, and activity levels. Nutr Cancer 2007; 57:66-77. [PMID: 17516864 DOI: 10.1080/01635580701268238] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Retinol inhibits the growth of all-trans-retinoic acid (ATRA)-resistant human colon cancer cell lines through a retinoic acid receptor (RAR)-independent mechanism. The objectives of the current study were to determine if retinol inhibited the invasion of ATRA-resistant colon cancer cells independent of RAR and the effects of retinol on matrix metalloproteinases (MMPs). Retinol inhibited the migration and invasion of two ATRA-resistant colon cancer cell lines, HCT-116 and SW620, in a dose-dependent manner. To determine if transcription, particularly RAR-mediated transcription, or translation of new genes was required for retinol to inhibit cell invasion, cells were treated with retinol and cycloheximide, actinomycin D, or an RAR pan-antagonist. Treatment of cells with retinol and cycloheximide, actinomycin D, or an RAR pan-antagonist did not block the ability of retinol to inhibit cell invasion. In addition, retinol decreased MMP-1 mRNA levels in both cell lines, MMP-2 mRNA levels in the SW620 cell line, and MMP-7 and -9 mRNA levels in the HCT-116 cell line. Retinol also decreased the activity of MMP-2 and -9 and MMP-9 protein levels while increasing tissue inhibitor of MMP-1 media levels. In conclusion, retinol reduces the metastatic potential of ATRA-resistant colon cancer cells via a novel RAR-independent mechanism that may involve decreased MMP mRNA levels and activity.
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Affiliation(s)
- Eun Young Park
- Department of Human Ecology, Institute of Cellular and Molecular Biology, The University of Texas at Austin 78712, USA
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23
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Andersen JL, DeHart JL, Zimmerman ES, Ardon O, Kim B, Jacquot G, Benichou S, Planelles V. HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT. PLoS Pathog 2007; 2:e127. [PMID: 17140287 PMCID: PMC1665652 DOI: 10.1371/journal.ppat.0020127] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2006] [Accepted: 10/25/2006] [Indexed: 11/18/2022] Open
Abstract
The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage–signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45α was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4–3vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked. HIV-1 encodes a small gene known as vpr (viral protein regulatory) whose product is a 96–amino acid protein. HIV-1 infects cells of the immune system, such as CD4-positive lymphocytes. When cells become infected with HIV-1, two deleterious effects result from expression of the vpr gene. One effect of vpr is to manipulate the cell cycle by blocking the cells in G2 (the phase of the cell cycle immediately preceding mitosis). Thus, cells infected with HIV-1 cease to proliferate, due to the action of vpr. A second effect of vpr is the induction of cell death by a process known as apoptosis or programmed cell death. When cells die by apoptosis, they do so following activation of a cellular set of genes and proteins whose primary function is to inactivate various cellular functions that are needed in order to maintain cellular viability. In this study, Andersen et al. demonstrate that the above two effects of vpr are linked. In particular, the authors show that the blockade in cell proliferation in G2 is a requirement toward the onset of programmed cell death. Programmed cell death can be accomplished by a number of cellular proteins known as “executioners.” Various executioner proteins reside on the mitochondrial membranes and may trigger release of factors from the mitochondria, which in turn will precipitate the onset of apoptosis. In this work Anderson et al. identify the mitochondrial protein, Bax, as the key executioner of apoptosis in the context of HIV-1 vpr. The authors' findings provide important mechanistic understanding of how the vpr gene contributes to HIV-1–induced cell death.
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Affiliation(s)
- Joshua L Andersen
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
| | - Jason L DeHart
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
| | - Erik S Zimmerman
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
| | - Orly Ardon
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
| | - Baek Kim
- Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
| | - Guillaume Jacquot
- Departement de Maladies Infectieuses, Institut Cochin, Institut National de la Santé et de la Recherche Médicale, Paris, France
| | - Serge Benichou
- Departement de Maladies Infectieuses, Institut Cochin, Institut National de la Santé et de la Recherche Médicale, Paris, France
| | - Vicente Planelles
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
- * To whom correspondence should be addressed. E-mail:
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24
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Alli E, Yang JM, Ford JM, Hait WN. Reversal of stathmin-mediated resistance to paclitaxel and vinblastine in human breast carcinoma cells. Mol Pharmacol 2007; 71:1233-40. [PMID: 17272681 DOI: 10.1124/mol.106.029702] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Antimicrotubule agents are commonly used chemotherapy drugs for the treatment of breast and other cancers. However, these agents have variable activity partly because of microtubule regulatory proteins. Stathmin, an 18-kDa phosphoprotein that promotes microtubule depolymerization, was found to be frequently overexpressed in breast cancer. We previously identified stathmin-mediated mechanisms of resistance to antimicrotubule agents, including altered drug binding and delayed transit from G(2) into M phase, where these agents are effective in disrupting microtubule dynamics. We hypothesized that by reversing stathmin-mediated depolymerization of microtubules or by promoting entry into mitosis, this could increase sensitivity to antimicrotubule agents in human breast cancer cells overexpressing stathmin. We found that targeting stathmin or wee-1 expression with RNA interference can induce microtubule polymerization and promote G(2)/M progression, respectively, and sensitize stathmin-overexpressing breast cancer cells to paclitaxel and vinblastine. Furthermore, targeting wee-1 led to the phosphorylation of stathmin, which is known to attenuate its activity. Therefore, these data suggest a novel approach to improving the efficacy of certain antimicrotubule agents against breast cancer by regulating the function of stathmin.
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Affiliation(s)
- Elizabeth Alli
- The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Department of Pharmacology, New Brunswick, NJ 08901, USA
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25
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Massaro D, Alexander E, Reiland K, Hoffman EP, Massaro GD, Clerch LB. Rapid onset of gene expression in lung, supportive of formation of alveolar septa, induced by refeeding mice after calorie restriction. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1313-26. [PMID: 17237152 DOI: 10.1152/ajplung.00146.2006] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration.
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Affiliation(s)
- Donald Massaro
- Lung Regeneration Laboratory, Department of 1Medicine, Georgetown University School of Medicine, Children's National Medical Center, Washington, District of Columbia 20057-1481, USA.
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26
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Li G, Elder RT, Qin K, Park HU, Liang D, Zhao RY. Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1. J Biol Chem 2007; 282:7287-98. [PMID: 17210576 DOI: 10.1074/jbc.m607951200] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser(345) phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser(345) phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G(2) arrest and Cdk1-Tyr(15) phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic beta-isoform PP2A(Cbeta) and the A regulatory alpha-isoform PP2A(Aalpha) are involved in the G(2) induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr(15) and Chk1-Ser(345). In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser(345) phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated gammaH2AX-Ser(139) phosphorylation and foci formation, but down-regulation of PP2A(Aalpha) or PP2A(Cbeta) did not decrease gammaH2AX-Ser(139) phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aalpha/Cbeta)-mediated G(2) arrest and Chk1-Ser(345) phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cbeta) or PP2A(Aalpha) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aalpha/Cbeta) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.
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Affiliation(s)
- Ge Li
- Department of Pathology, Department of Microbiology-Immunology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA
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27
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Zhao RY, Elder RT, Bukrinsky M. Interactions of HIV-1 viral protein R with host cell proteins. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 2007; 55:233-60. [PMID: 17586317 DOI: 10.1016/s1054-3589(07)55007-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Richard Y Zhao
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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28
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Nakai-Murakami C, Shimura M, Kinomoto M, Takizawa Y, Tokunaga K, Taguchi T, Hoshino S, Miyagawa K, Sata T, Kurumizaka H, Yuo A, Ishizaka Y. HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination. Oncogene 2006; 26:477-86. [PMID: 16983346 DOI: 10.1038/sj.onc.1209831] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.
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Affiliation(s)
- C Nakai-Murakami
- Department of Intractable Diseases, International Medical Center of Japan, Shinjuku-ku, Tokyo, Japan
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29
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Matsuda N, Tanaka H, Yamazaki S, Suzuki JI, Tanaka K, Yamada T, Masuda M. HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation. Microbes Infect 2006; 8:2736-44. [PMID: 16968670 DOI: 10.1016/j.micinf.2006.08.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2006] [Revised: 08/07/2006] [Accepted: 08/07/2006] [Indexed: 11/20/2022]
Abstract
Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.
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Affiliation(s)
- Naoto Matsuda
- Department of Microbiology, Dokkyo Medical University School of Medicine, Kita-kobayashi 880, Mibu, Tochigi 321-0293, Japan
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30
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Knight GL, Turnell AS, Roberts S. Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins. J Virol 2006; 80:7416-26. [PMID: 16840322 PMCID: PMC1563741 DOI: 10.1128/jvi.00196-06] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.
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Affiliation(s)
- Gillian L Knight
- Cancer Research UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom
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31
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Kamata M, Wu RP, An DS, Saxe JP, Damoiseaux R, Phelps ME, Huang J, Chen ISY. Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death. Biochem Biophys Res Commun 2006; 348:1101-6. [PMID: 16904642 PMCID: PMC1761125 DOI: 10.1016/j.bbrc.2006.07.158] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2006] [Accepted: 07/25/2006] [Indexed: 11/30/2022]
Abstract
Viral protein R (Vpr), one of the human immunodeficiency virus type 1 (HIV-1) accessory proteins, contributes to multiple cytopathic effects, G2 cell cycle arrest and apoptosis. The mechanisms of Vpr have been intensely studied because it is believed that they underlie HIV-1 pathogenesis. We here report a cell-based small molecule screen on Vpr induced cell death in the context of HIV-1 infection. From the screen of 504 bioactive compounds, we identified damnacanthal (Dam), a component of noni [corrected] as an inhibitor of Vpr induced cell death. Our studies illustrate a novel efficient platform for drug discovery and development in anti-HIV therapy which should also be applicable to other viruses.
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Affiliation(s)
- Masakazu Kamata
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
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32
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Abstract
Hijacking of the host cell’s signal transduction machinery has been increasingly regarded as an important strategy for facilitating virus propagation. The positive-transcription elongation factor (P-TEFb) complex, cyclin-dependent kinase (CDK)9/cyclin T1, is an example of such an attack by HIV. Upon infection of cells, the HIV protein transactivator of transcription (Tat) forms a highly specific complex with the two host cell proteins CDK9 and cyclin T1. This complex ensures phosphorylation of the native CDK9 substrate, RNA polymerase II, leading to productive elongation of viral RNA in the host cell. Although challenging, inhibition of CDK9 activity with small molecules is a therapeutically valid strategy to inhibit HIV replication. Other than direct antiviral agents, that inhibit HIV replication through a direct interaction with viral proteins, CDK9 inhibitors might not suffer from the emergence of resistant virus strains. This review outlines the advantages and prospects of selective CDK9 inhibitors in the management of HIV infections.
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Affiliation(s)
- Bert M Klebl
- GPC Biotech AG, Fraunhoferstr. 20, D-82152 Martinsried, Germany
| | - Axel Choidas
- GPC Biotech AG, Fraunhoferstr. 20, D-82152 Martinsried, Germany
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33
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Bernstein H, Holubec H, Bernstein C, Ignatenko N, Gerner E, Dvorak K, Besselsen D, Ramsey L, Dall'Agnol M, Blohm-Mangone KA, Padilla-Torres J, Cui H, Garewal H, Payne CM. Unique dietary-related mouse model of colitis. Inflamm Bowel Dis 2006; 12:278-93. [PMID: 16633050 DOI: 10.1097/01.mib.0000209789.14114.63] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND A high-fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high-fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD. METHODS Wild-type B6.129 mice were fed an AIN-93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses. RESULTS Mice fed the DOC-supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin-proteasome pathway showed altered expression in the DOC-fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding. CONCLUSIONS The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC-fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.
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Affiliation(s)
- Harris Bernstein
- Department of Cell Biology and Anatomy, College of Medicine, Tucson, AZ 85724, USA.
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Lin JP, Yang JS, Lee JH, Hsieh WT, Chung JG. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line. World J Gastroenterol 2006; 12:21-8. [PMID: 16440412 PMCID: PMC4077487 DOI: 10.3748/wjg.v12.i1.21] [Citation(s) in RCA: 109] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/05/2005] [Revised: 07/08/2005] [Accepted: 07/20/2005] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action. METHODS The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting. RESULTS For SNU-5 cell line, the IC50 was found to be 48 micromol/L of berberine. In SNU-5 cells treated with 25-200 micromol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25-200 micromol/L berberine-treated cells by monitoring the apoptotic pathway. Apoptosis was identified by sub-G0 cell population, and upregulation of Bax, downregulation of Bcl-2, release of Ca2+, decreased the mitochondrial membrane potential and then led to the release of mitochondrial cytochrome C into the cytoplasm and caused the activation of caspase-3, and finally led to the occurrence of apoptosis. CONCLUSION Berberine induces p53 expression and leads to the decrease of the mitochondrial membrane potential, Cytochrome C release and activation of caspase-3 for the induction of apoptosis.
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Affiliation(s)
- Jing-Pin Lin
- School of Chinese Medicine, China Medical University, Taichung City 404, Taiwan. China
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Abstract
Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.
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Affiliation(s)
- Richard Y Zhao
- Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 2120, USA.
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Wang F, Zhu Y, Huang Y, McAvoy S, Johnson WB, Cheung TH, Chung TKH, Lo KWK, Yim SF, Yu MMY, Ngan HYS, Wong YF, Smith DI. Transcriptional repression of WEE1 by Kruppel-like factor 2 is involved in DNA damage-induced apoptosis. Oncogene 2005; 24:3875-85. [PMID: 15735666 DOI: 10.1038/sj.onc.1208546] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Human Kruppel-like factor 2 (KLF2) is a Cys(2)/His(2) zinc-finger-containing transcriptional factor, which is involved in multiple cellular pathways. Utilizing gene expression profiling to identify aberrantly expressed genes in ovarian cancer, we found that KLF2 was significantly and specifically downregulated in ovarian tumors. After reintroducing KLF2 into ovarian cancer cell lines, we observed decreased cell growth and increased sensitivity to DNA damage-induced apoptosis. Analysis of genes that could be potential targets of KLF2 revealed that KLF2 negatively regulated WEE1 expression. WEE1 encodes a tyrosine kinase that regulates the G2/M cell cycle transition. Expression of KLF2 markedly repressed the transcription of WEE1 by directly binding to an SP1/CPBP motif located between -252 bp and the start codon of the WEE1 promoter. Both activation and zinc-finger domains of KLF2 were required for this suppression of Wee1 expression. In addition, we demonstrated that Wee1 expression prevents cancer cells from undergoing apoptosis in response to DNA damage; however, this resistance was abolished by coexpression of KLF2, which inhibits WEE1 transcription. Thus, the level of WEE1 is regulated by KLF2 and enhanced KLF2 expression sensitizes cells to DNA damage-induced apoptosis.
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Affiliation(s)
- Fang Wang
- Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA
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Groschel B, Bushman F. Cell cycle arrest in G2/M promotes early steps of infection by human immunodeficiency virus. J Virol 2005; 79:5695-704. [PMID: 15827184 PMCID: PMC1082730 DOI: 10.1128/jvi.79.9.5695-5704.2005] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
We have identified four small molecules that boost transduction of cells by human immunodeficiency virus (HIV) and investigated their mechanism of action. These molecules include etoposide and camptothecin, which induce DNA damage by inhibiting religation of cleaved topoisomerase-DNA complexes, taxol, which interferes with the function of microtubules, and aphidicolin, which inhibits DNA polymerases. All four compounds arrest the cell cycle at G2/M, though in addition high concentrations of aphidicolin arrest in G1. We find that early events of HIV replication, including synthesis of late reverse transcription products, two-long terminal repeat circles, and integrated proviruses, were increased after treatment of cells with concentrations of each compound that arrested in G2/M. Stimulation was seen for both transformed cell lines (293T and HeLa cells) and primary cells (IMR90 lung fibroblasts). Arrest in G1 with high concentrations of aphidicolin boosted transduction, though not much as with lower concentrations that arrested in G2/M. Arrest of IMR90 cells in G1 by serum starvation and contact inhibition reduced transduction. Previously, the proteasome inhibitor MG132 was reported to increase HIV infection-here we investigated the effects of combinations of the cell cycle inhibitors with MG132 and obtained data suggesting that MG132 may also boost transduction by causing G2/M cell cycle arrest. These data document that cell cycle arrest in G2/M boosts the early steps of HIV infection and suggests methods for increasing transduction with HIV-based vectors.
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Affiliation(s)
- Bettina Groschel
- University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA
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