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  1. Article ; Online: Erythroblast enucleation.

    Keerthivasan, Ganesan / Wickrema, Amittha / Crispino, John D

    Stem cells international

    2011  Volume 2011, Page(s) 139851

    Abstract: Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude ... ...

    Abstract Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude their nucleus prior to entering circulation, likely to impart flexibility and improve the ability to traverse through capillaries that are half the size of erythrocytes. Recently, there have been many advances in our understanding of the mechanisms underlying mammalian erythrocyte enucleation. This review summarizes these advances, discusses the possible future directions in the field, and evaluates the prospects for improved ex vivo production of red blood cells.
    Language English
    Publishing date 2011-10-05
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2573856-2
    ISSN 1687-9678 ; 1687-966X
    ISSN (online) 1687-9678
    ISSN 1687-966X
    DOI 10.4061/2011/139851
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Erythroblast Enucleation

    Ganesan Keerthivasan / Amittha Wickrema / John D. Crispino

    Stem Cells International, Vol

    2011  Volume 2011

    Abstract: Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude ... ...

    Abstract Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude their nucleus prior to entering circulation, likely to impart flexibility and improve the ability to traverse through capillaries that are half the size of erythrocytes. Recently, there have been many advances in our understanding of the mechanisms underlying mammalian erythrocyte enucleation. This review summarizes these advances, discusses the possible future directions in the field, and evaluates the prospects for improved ex vivo production of red blood cells.
    Keywords Internal medicine ; RC31-1245
    Language English
    Publishing date 2011-01-01T00:00:00Z
    Publisher Hindawi Publishing Corporation
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: A novel role for survivin in erythroblast enucleation.

    Keerthivasan, Ganesan / Liu, Hui / Gump, Jacob M / Dowdy, Steven F / Wickrema, Amittha / Crispino, John D

    Haematologica

    2012  Volume 97, Issue 10, Page(s) 1471–1479

    Abstract: Background: Nucleus free red blood cells are unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. We previously found that survivin, a member of the chromosomal passenger complex ... ...

    Abstract Background: Nucleus free red blood cells are unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. We previously found that survivin, a member of the chromosomal passenger complex that is required for cytokinesis, is highly expressed in late non-dividing cells. The role of survivin in enucleating erythroblasts is not known.
    Design and methods: In order to identify the role of survivin in these late erythroblasts, we performed proteomic analysis on survivin-bound protein complexes purified from murine erythroleukemia cells. Various molecular and cell biological techniques were used to confirm the presence and function of this novel complex. Furthermore, we used survivin(fl/fl) mice to study the effect of loss of survivin in enucleating erythroblasts.
    Results: We found that survivin failed to co-localize with its known partners' inner centromere protein or Aurora-B in enucleating erythroblasts but rather exists in a multi-protein complex with epidermal growth factor receptor substrate15 and clathrin, two proteins that mediate endocytic vesicle trafficking. As evidence for a direct role of this latter complex in enucleation, we found that knockdown of the genes reduced the efficiency of enucleation of primary human erythroblasts. We also observed that loss of survivin in murine erythroblasts inhibited enucleation and that survivin-deficient cells harbored smaller cytoplasmic vacuoles. Interestingly, vacuolin-1, a small molecule that induces vacuole fusion, rescued the defective enucleation caused by survivin deficiency.
    Conclusions: This study identified a novel role for survivin in erythroblast enucleation through previously unknown protein partners.
    MeSH term(s) Adaptor Proteins, Signal Transducing/metabolism ; Animals ; Cell Differentiation/genetics ; Cell Line ; Cell Nucleus/metabolism ; Clathrin/metabolism ; Coated Pits, Cell-Membrane/metabolism ; Erythroblasts/cytology ; Erythroblasts/metabolism ; Gene Expression ; Gene Knockout Techniques ; Heterocyclic Compounds, 4 or More Rings/metabolism ; Humans ; Inhibitor of Apoptosis Proteins/genetics ; Inhibitor of Apoptosis Proteins/metabolism ; Mice ; Protein Binding ; Protein Transport ; Repressor Proteins/genetics ; Repressor Proteins/metabolism ; Survivin ; Vacuoles/metabolism
    Chemical Substances Adaptor Proteins, Signal Transducing ; BIRC5 protein, human ; Birc5 protein, mouse ; Clathrin ; Heterocyclic Compounds, 4 or More Rings ; Inhibitor of Apoptosis Proteins ; Repressor Proteins ; Survivin ; vacuolin-1
    Language English
    Publishing date 2012-04-04
    Publishing country Italy
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2333-4
    ISSN 1592-8721 ; 0017-6567 ; 0390-6078
    ISSN (online) 1592-8721
    ISSN 0017-6567 ; 0390-6078
    DOI 10.3324/haematol.2011.061093
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Vesicle trafficking plays a novel role in erythroblast enucleation.

    Keerthivasan, Ganesan / Small, Sara / Liu, Hui / Wickrema, Amittha / Crispino, John D

    Blood

    2010  Volume 116, Issue 17, Page(s) 3331–3340

    Abstract: Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of ...

    Abstract Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.
    MeSH term(s) Animals ; Cells, Cultured ; Clathrin/genetics ; Clathrin/metabolism ; Cytokinesis/drug effects ; Endocytosis ; Erythroblasts/cytology ; Erythroblasts/drug effects ; Erythropoiesis ; Gene Knockdown Techniques ; Heterocyclic Compounds, 4 or More Rings/metabolism ; Heterocyclic Compounds, 4 or More Rings/pharmacology ; Humans ; Liver/cytology ; Mice ; Monensin/pharmacology ; Nocodazole/pharmacology ; Spleen/cytology ; Vacuoles/drug effects ; Vacuoles/metabolism
    Chemical Substances Clathrin ; Heterocyclic Compounds, 4 or More Rings ; vacuolin-1 ; blebbistatin (20WC4J7CQ6) ; Monensin (906O0YJ6ZP) ; Nocodazole (SH1WY3R615)
    Language English
    Publishing date 2010-07-19
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80069-7
    ISSN 1528-0020 ; 0006-4971
    ISSN (online) 1528-0020
    ISSN 0006-4971
    DOI 10.1182/blood-2010-03-277426
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis.

    Zhao, Baobing / Keerthivasan, Ganesan / Mei, Yang / Yang, Jing / McElherne, James / Wong, Piu / Doench, John G / Feng, Gang / Root, David E / Ji, Peng

    Haematologica

    2014  Volume 99, Issue 7, Page(s) 1157–1167

    Abstract: Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional ... ...

    Abstract Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional screening strategy, we identified a critical role of pleckstrin-2 in actin dynamics and protection of early stage terminal erythroblasts from oxidative damage. Knockdown of pleckstrin-2 in the early stage of terminal erythropoiesis disrupted the actin cytoskeleton and led to differentiation inhibition and apoptosis. This pro-survival and differentiation function of pleckstrin-2 was mediated through its interaction with cofilin, by preventing cofilin's mitochondrial entry when the intracellular level of reactive oxygen species was higher in the early stage of terminal erythropoiesis. Treatment of the cells with a scavenger of reactive oxygen species rescued cofilin's mitochondrial entry and differentiation inhibition induced by pleckstrin-2 knockdown. In contrast, pleckstrin-2 knockdown in late stage terminal erythroblasts had no effect on survival or differentiation but blocked enucleation due to disorganized actin cytoskeleton. Thus, our study identified a dual function of pleckstrin-2 in the early and late stages of terminal erythropoiesis through its regulations of actin dynamics and cofilin's mitochondrial localization, which reflects intracellular level of reactive oxygen species in different developmental stages.
    MeSH term(s) Actin Depolymerizing Factors/metabolism ; Actins/genetics ; Actins/metabolism ; Animals ; Apoptosis/genetics ; Cell Differentiation/genetics ; Erythroblasts/cytology ; Erythroblasts/metabolism ; Erythropoiesis/genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Genes, Reporter ; Membrane Proteins/genetics ; Membrane Proteins/metabolism ; Mice ; Mitochondria/genetics ; Mitochondria/metabolism ; Protein Binding ; RNA Interference ; RNA, Small Interfering/genetics
    Chemical Substances Actin Depolymerizing Factors ; Actins ; Membrane Proteins ; Plek2 protein, mouse ; RNA, Small Interfering
    Language English
    Publishing date 2014-04-18
    Publishing country Italy
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2333-4
    ISSN 1592-8721 ; 0017-6567 ; 0390-6078
    ISSN (online) 1592-8721
    ISSN 0017-6567 ; 0390-6078
    DOI 10.3324/haematol.2014.105809
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article: Differential requirements for survivin in hematopoietic cell development.

    Gurbuxani, Sandeep / Xu, Yanfei / Keerthivasan, Ganesan / Wickrema, Amittha / Crispino, John D

    Proceedings of the National Academy of Sciences of the United States of America

    2005  Volume 102, Issue 32, Page(s) 11480–11485

    Abstract: Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths; erythroid cells undergo cell-cycle exit and enucleation, whereas megakaryocytes continue to progress through the cell ... ...

    Abstract Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths; erythroid cells undergo cell-cycle exit and enucleation, whereas megakaryocytes continue to progress through the cell cycle but skip late stages of mitosis to become polyploid cells. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis family that also has an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, whereas megakaryocytes express approximately 4-fold lower levels of survivin mRNA and no detectable protein. To investigate the role of survivin in these lineages, we overexpressed or knocked down survivin from mouse bone marrow cells and then examined erythroid and megakaryocyte development. These studies revealed that overexpression of survivin antagonized megakaryocyte growth, maturation, and polyploidization but had no effect on erythroid development. This block in polyploidization was accompanied by increased expression of p21 and decreased expression of megakaryocyte genes such as von Willebrand factor and beta(1)-tubulin. In contrast, a reduction in survivin expression interfered with the formation of erythroid cells but not megakaryocytes. Last, consistent with the requirement for survivin in the survival of proliferating cells, survivin-deficient hematopoietic progenitors failed to give rise to either erythroid or megakaryocytic colonies. Together, these studies show that whereas survivin expression is essential for megakaryocyte and erythroid progenitors, its down-regulation is required for terminal differentiation of megakaryocytes.
    MeSH term(s) Animals ; Blotting, Western ; Cells, Cultured ; Cloning, Molecular ; Colony-Forming Units Assay ; Erythrocytes/cytology ; Erythrocytes/metabolism ; Flow Cytometry ; Gene Expression Regulation ; Green Fluorescent Proteins ; Hematopoiesis/physiology ; Humans ; Inhibitor of Apoptosis Proteins ; Megakaryocytes/cytology ; Megakaryocytes/metabolism ; Mice ; Microtubule-Associated Proteins/metabolism ; Neoplasm Proteins/metabolism ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction
    Chemical Substances BIRC5 protein, human ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; Neoplasm Proteins ; Green Fluorescent Proteins (147336-22-9)
    Language English
    Publishing date 2005-08-09
    Publishing country United States
    Document type Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.0500303102
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS.

    Keerthivasan, Ganesan / Mei, Yang / Zhao, Baobing / Zhang, Ling / Harris, Chad E / Gao, Juehua / Basiorka, Ashley A / Schipma, Matthew J / McElherne, James / Yang, Jing / Verma, Amit K / Pellagatti, Andrea / Boultwood, Jacqueline / List, Alan F / Williams, David A / Ji, Peng

    Blood

    2014  Volume 124, Issue 5, Page(s) 780–790

    Abstract: The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. ... ...

    Abstract The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS.
    MeSH term(s) Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/immunology ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; Bone Marrow Cells/immunology ; Bone Marrow Cells/metabolism ; Bone Marrow Cells/pathology ; Carrier Proteins/genetics ; Carrier Proteins/immunology ; Carrier Proteins/metabolism ; Chromosome Deletion ; Chromosomes, Human, Pair 5 ; Female ; Formins ; Gene Expression Regulation ; Granulocytes/immunology ; Granulocytes/metabolism ; Granulocytes/pathology ; Heterozygote ; Humans ; Immunity, Innate ; Lipopolysaccharide Receptors/biosynthesis ; Lipopolysaccharide Receptors/genetics ; Lipopolysaccharide Receptors/immunology ; Lipopolysaccharides/pharmacology ; Male ; Mice ; Mice, Knockout ; Myelodysplastic Syndromes/genetics ; Myelodysplastic Syndromes/immunology ; Myelodysplastic Syndromes/metabolism ; Myelodysplastic Syndromes/pathology ; RNA, Messenger/genetics ; RNA, Messenger/immunology ; RNA, Messenger/metabolism ; Toll-Like Receptor 4/agonists ; Toll-Like Receptor 4/genetics ; Toll-Like Receptor 4/immunology ; Toll-Like Receptor 4/metabolism
    Chemical Substances Adaptor Proteins, Signal Transducing ; Carrier Proteins ; DIAPH1 protein, human ; Diap1 protein, mouse ; Formins ; Lipopolysaccharide Receptors ; Lipopolysaccharides ; RNA, Messenger ; TLR4 protein, human ; Tlr4 protein, mouse ; Toll-Like Receptor 4
    Language English
    Publishing date 2014-06-02
    Publishing country United States
    Document type Clinical Trial ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80069-7
    ISSN 1528-0020 ; 0006-4971
    ISSN (online) 1528-0020
    ISSN 0006-4971
    DOI 10.1182/blood-2014-01-552463
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Targeting survivin overcomes drug resistance in acute lymphoblastic leukemia.

    Park, Eugene / Gang, Eun Ji / Hsieh, Yao-Te / Schaefer, Paul / Chae, Sanna / Klemm, Lars / Huantes, Sandra / Loh, Mignon / Conway, Edward M / Kang, Eun-Suk / Hoe Koo, Hong / Hofmann, Wolf-Karsten / Heisterkamp, Nora / Pelus, Louis / Keerthivasan, Ganesan / Crispino, John / Kahn, Michael / Müschen, Markus / Kim, Yong-Mi

    Blood

    2011  Volume 118, Issue 8, Page(s) 2191–2199

    Abstract: Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that ... ...

    Abstract Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.
    MeSH term(s) Animals ; Combined Modality Therapy ; Drug Resistance, Neoplasm/genetics ; Gene Expression ; Gene Targeting ; Humans ; Inhibitor of Apoptosis Proteins/antagonists & inhibitors ; Inhibitor of Apoptosis Proteins/deficiency ; Inhibitor of Apoptosis Proteins/genetics ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Neoplasm, Residual ; Oligonucleotides/genetics ; Oligonucleotides/therapeutic use ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy ; RNA, Small Interfering/genetics ; Repressor Proteins/deficiency ; Repressor Proteins/genetics ; Survivin ; Tumor Stem Cell Assay ; Xenograft Model Antitumor Assays
    Chemical Substances BIRC5 protein, human ; Birc5 protein, mouse ; EZN 3042 ; Inhibitor of Apoptosis Proteins ; Oligonucleotides ; RNA, Small Interfering ; Repressor Proteins ; Survivin
    Language English
    Publishing date 2011-06-28
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80069-7
    ISSN 1528-0020 ; 0006-4971
    ISSN (online) 1528-0020
    ISSN 0006-4971
    DOI 10.1182/blood-2011-04-351239
    Database MEDical Literature Analysis and Retrieval System OnLINE

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