scholarly journals Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 651-657 ◽  
Author(s):  
Xingwei Sui ◽  
Sanford B. Krantz ◽  
Zhizhuang Zhao
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Normal Cells ◽  
Protein Tyrosine ◽  
Erythroid Progenitor Cells

Abstract Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

scholarly journals Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 651-657 ◽  
Author(s):  
Xingwei Sui ◽  
Sanford B. Krantz ◽  
Zhizhuang Zhao
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Normal Cells ◽  
Protein Tyrosine ◽  
Erythroid Progenitor Cells

Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

scholarly journals A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277 ◽  
Author(s):  
G D Longmore ◽  
P N Pharr ◽  
H F Lodish
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Active Form ◽  
Erythropoietin Receptor ◽  
Mutant Form ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

scholarly journals Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
M Silva ◽  
D Grillot ◽  
A Benito ◽  
C Richard ◽  
G Nunez ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Survival Factor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

Setaria cervidual specific phosphatase: characterization and its effect on eosinophil degranulation

Parasitology ◽  
2009 ◽  
Vol 136 (8) ◽  
pp. 895-904 ◽  
Author(s):  
S. RATHAUR ◽  
R. RAI ◽  
E. SRIKANTH ◽  
S. SRIVASTAVA
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Specific Protein ◽  
Enzyme Concentration ◽  
Cross Reactivity ◽  
Filarial Parasite ◽  
Significant Activity ◽  
Protein Tyrosine

SUMMARYSetaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16·66 mM, 25·0 μM/ml/min with pNPP; 20·0 mM, 40·0 μM/ml/min with phospho-L-tyrosine and 27·0 mM, 25·0 μM/ml/min with phospho-L-serine. KIwith pNPP and sodium orthovanadate (IC5033·0 μM) was calculated to be 50·0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity withW. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Underin vitroconditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest thatS. cerviDSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

PU.1 determines the self-renewal capacity of erythroid progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3615-3623 ◽  
Author(s):  
Jonathan Back ◽  
Andrée Dierich ◽  
Corinne Bronn ◽  
Philippe Kastner ◽  
Susan Chan
Keyword(s):  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Self Renewal ◽  
Adult Mice ◽  
Erythroid Progenitor Cells ◽  
Low Levels ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

Abstract PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation—it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1-deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

scholarly journals Loss of Protein-tyrosine Phosphatase α (PTPα) Increases Proliferation and Delays Maturation of Oligodendrocyte Progenitor Cells

2012 ◽  
Vol 287 (15) ◽  
pp. 12529-12540 ◽  
Author(s):  
Pei-Shan Wang ◽  
Jing Wang ◽  
Yi Zheng ◽  
Catherine J. Pallen
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Oligodendrocyte Progenitor Cells ◽  
Cell Cycle Exit ◽  
Oligodendrocyte Progenitor ◽  
Protein Tyrosine ◽  
Neural Stem Progenitor Cells

Tightly controlled termination of proliferation determines when oligodendrocyte progenitor cells (OPCs) can initiate differentiation and mature into myelin-forming cells. Protein-tyrosine phosphatase α (PTPα) promotes OPC differentiation, but its role in proliferation is unknown. Here we report that loss of PTPα enhanced in vitro proliferation and survival and decreased cell cycle exit and growth factor dependence of OPCs but not neural stem/progenitor cells. PTPα−/− mice have more oligodendrocyte lineage cells in embryonic forebrain and delayed OPC maturation. On the molecular level, PTPα-deficient mouse OPCs and rat CG4 cells have decreased Fyn and increased Ras, Cdc42, Rac1, and Rho activities, and reduced expression of the Cdk inhibitor p27Kip1. Moreover, Fyn was required to suppress Ras and Rho and for p27Kip1 accumulation, and Rho inhibition in PTPα-deficient cells restored expression of p27Kip1. We propose that PTPα-Fyn signaling negatively regulates OPC proliferation by down-regulating Ras and Rho, leading to p27Kip1 accumulation and cell cycle exit. Thus, PTPα acts in OPCs to limit self-renewal and facilitate differentiation.

Proliferative Defect but Normal Transcriptional Response to Erythropoietin in Diamond Blackfan Anemia Erythroid Progenitor Cells in 2-Phase Erythroid Culture.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1309-1309
Author(s):  
Carine Marius ◽  
Yaw Ohene-Abuakwa ◽  
Ken Laing ◽  
Sarah Ball
Keyword(s):  
Progenitor Cells ◽  
Alternative Hypothesis ◽  
Transcriptional Response ◽  
Fold Increase ◽  
Cell Number ◽  
Proliferation Rate ◽  
High Rate ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Abstract Diamond Blackfan anaemia (DBA) is a rare congenital red cell aplasia, characterised by a steroid responsive intrinsic erythropoietic failure. Mutations affecting RPS19 are found in up to 25% of DBA, but the pathophysiological link between ribosomal gene haploinsufficiency and the specific erythroid phenotype of DBA remains elusive. One potential mechanism is that ribosomal insufficiency might affect the level of expression of critical erythroid proteins, resulting in a block in terminal erythroid differentiation and expansion. The aim of this study was to elucidate the cellular and molecular events occurring in response to erythropoietin (EPO) in DBA. We applied a 2-phase erythroid culture system in which we have previously demonstrated a consistent and profound failure of erythroid expansion in DBA cultures, localised to early in the second EPO-dependent phase of culture. The timing of the onset of proliferation in response to EPO was studied by tetrazolium dye assay of enriched erythroid progenitor cells in phase II of culture. An increase in cell number was detectable following 72hrs of EPO exposure in DBA (n=5) as well as control cultures, but with a subsequently slower proliferation rate in DBA. The proliferation rate of both DBA and control cells was enhanced by the addition of dexamethasone. Transcriptional profiles of control and DBA erythroid progenitors were then compared by microarray analysis after 24hrs exposure to EPO. Cy3/Cy5 labelled aRNA was competitively hybridised on an in-house array containing 395 transcript exon identifiers in ENSEMBL. The results showed little difference in expression between control and DBA after 24hrs of EPO exposure, the only significant changes being in the expression of just 4 of the 251 genes expressed above background (CDKN2D, TNFRSF6, MAPKK and CXCR), which were upregulated in DBA (p<0.0001 on t-test analysis). The gene expression profile from 0–96 hrs following EPO exposure was then studied in control and DBA (n=3) erythroid progenitors by real time PCR. There was little change in the first 48hr of exposure to EPO. At 72hrs the expression of both EPOR and α-GLOBIN was increased 3–6 fold in both DBA and controls in comparison with pre-EPO, confirming the occurrence of an erythroid-specific transcriptional response to EPO in DBA erythroid progenitors. These results are consistent with a qualitatively normal onset of terminal erythroid maturation in DBA, rather than a block in differentiation, and tend to favour an alternative hypothesis that the erythroid failure in DBA is the result of ribosomal insufficiency at the simultaneous onset of both rapid cell proliferation and a high rate of globin synthesis. In keeping with this, the expression of RPS19 showed 1.5–2 fold increase at 72hrs after EPO exposure, with 3.5 fold increase in the presence of dexamethasone, with equivalent results in both DBA and controls. We now aim to study translational and cell cycle events during this important wave of erythroid proliferation to further elucidate the molecular basis for the specific erythroid defect in DBA, despite the ubiquitous cellular requirement for ribosomal biogenesis.

Therapeutic Potential of a Desert Plant Triterpenoid Molecule for Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3635-3635 ◽  
Author(s):  
Amos S. Gaikwad ◽  
Jordan U. Gutterman ◽  
Josef T. Prchal
Keyword(s):  
Clinical Trials ◽  
Growth Inhibition ◽  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Therapeutic Efficacy ◽  
Jak2 V617f ◽  
Malignant Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Polycythemia vera (PV) is an acquired, clonal stem cell, myeloproliferative, hematological disorder with variable increase in erythrocytes, neutrophils and granulocytes. PV causes significant morbidity and mortality from thrombotic and hemorrhagic complications and has a propensity for leukemic transformation. Phlebotomy, interferon a and myelosuppressive chemotherapy have been the cornerstones of treatment to date. With no specific drugs to treat PV effectively, the development of new therapeutic modalities is important. A somatic mutation in the JAK2 tyrosine kinase (V617F) causing constitutive activation of the JAK/STAT pathway was recently reported in over 80% of PV patients. Based on this observation, we explored the therapeutic efficacy of tyrosine kinase inhibitors (TKIs) such as imatinib (Gaikwad A et al, Blood106: 2601, 2005) and AMN107 for PV. These TKIs showed marginal efficacy in vitro. Research using natural sources has led to important pharmacological targets for cancer therapy. Several plant molecules have either been introduced to the US market or are in late-phase clinical trials. We focused on a natural product called “Avicin D,” which is a plant derived triterpene electrophilic molecule with cyto-protective and anti-inflammatory functions (Haridas V et al, J. Clin. Invest113: 65, 2004; Haridas V et al, PNAS, 98:11557, 2001). Avicin D is reported to initiate selective pro-apoptotic activity in malignant cells (Mujoo K, et al. Cancer Res.61: 5486, 2001) by direct perturbation of mitochondria (Haridas V, et al. PNAS, 98: 5821, 2001). It has also been shown to down-regulate a group of pro-survival, anti-apoptotic proteins that act downstream of cytochrome c release, including HSP70, HSP90 and XIAP (Gaikwad A, et al. Clin. Cancer Res. 11: 1953Gaikwad A, et al. Clin. Cancer Res. 11: 2005). In addition, Avicin D also blocks glycolysis, a key metabolic process that malignant cells exploit for their proliferation and survival (Warburg phenomenon). To examine Avicin D’s therapeutic efficacy for PV, we utilized mouse reporter cells expressing the JAK2 V617F protein. Avicin D inhibited the growth of these cells with an IC50 of 2μM. Interestingly, in cells carrying wild-type JAK2 there was no significant inhibitory effect. In addition, Avicin D showed marked growth inhibition of human erythroleukemic cells (HEL) that harbor the JAK2 V617F mutation at an IC50 of 2μM. We then examined Avicin D’s effect on in vitro expanded native human erythroid progenitor cells. Avicin D showed specific growth inhibition of erythroid progenitor cells from PV patients (IC50 ~3μM) with no significant effect on the normal progenitors. We conclude that Avicin D should be a promising candidate for clinical trials of PV and other disorders that are associated with JAK2 V617F somatic mutations.

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