JARID2 Functions as a Tumor Suppressor in Myeloid Neoplasms by Repressing Self-Renewal in Hematopoietic Progenitor Cells

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

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Activation of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 Receptor Subunits in a Multipotential Hematopoietic Progenitor Cell Line Leads to Differential Effects on Development

Blood ◽

10.1182/blood.v94.5.1504.417a27_1504_1514 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1504-1514 ◽

Cited By ~ 3

Author(s):

Caroline A. Evans ◽

Andrew Pierce ◽

Sandra A. Winter ◽

Elaine Spooncer ◽

Clare M. Heyworth ◽

...

Keyword(s):

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor Cell ◽

Colony Stimulating Factor ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Interleukin 3 ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Activation of specific cytokine receptors promotes survival and proliferation of hematopoietic progenitor cells but their role in the control of differentiation is unclear. To address this issue, the effects of human interleukin-3 (hIL-3) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) on hematopoietic development were investigated in hematopoietic progenitor cells. Murine multipotent factor-dependent cell-Paterson (FDCP)-mix cells, which can self-renew or differentiate, were transfected with the genes encoding the unique  and/or shared βc human hIL-3 receptor (hIL-3 R) or hGM-CSF receptor (hGM R) subunits by retroviral gene transfer. Selective activation of hIL-3 R,βc or hGM R,βc transfects by hIL-3 and hGM-CSF promoted self-renewal and myeloid differentiation, respectively, over a range of cytokine (0.1 to 100 ng/mL) concentrations. These qualitatively distinct developmental outcomes were associated with different patterns of protein tyrosine phosphorylation and, thus, differential signaling pathway activation. The cell lines generated provide a model to investigate molecular events underlying self-renewal and differentiation and indicate that the  subunits act in combination with the hβc to govern developmental decisions. The role of the  subunit in conferring specificity was studied by using a chimeric receptor composed of the extracellular hIL-3 R and intracellular hGM R subunit domains. This receptor promoted differentiation in response to hIL-3. Thus, the  subunit cytosolic domain is an essential component in determining cell fate via specific signaling events.

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Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis

Blood ◽

10.1182/blood-2003-10-3423 ◽

2004 ◽

Vol 104 (3) ◽

pp. 675-686 ◽

Cited By ~ 99

Author(s):

Wolfgang Wagner ◽

Alexandra Ansorge ◽

Ute Wirkner ◽

Volker Eckstein ◽

Christian Schwager ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Profiles ◽

Hematopoietic Progenitor Cells ◽

Molecular Evidence ◽

Cdna Clones ◽

Human Umbilical Cord ◽

Primitive Function ◽

Hematopoietic Progenitor ◽

Self Renewal

AbstractThe molecular mechanisms that regulate asymmetric divisions of hematopoietic progenitor cells (HPCs) are not yet understood. The slow-dividing fraction (SDF) of HPCs is associated with primitive function and self-renewal, whereas the fast-dividing fraction (FDF) predominantly proceeds to differentiation. CD34+/CD38– cells of human umbilical cord blood were separated into the SDF and FDF. Genomewide gene expression analysis of these populations was determined using the newly developed Human Transcriptome Microarray containing 51 145 cDNA clones of the Unigene Set-RZPD3. In addition, gene expression profiles of CD34+/CD38– cells were compared with those of CD34+/CD38+ cells. Among the genes showing the highest expression levels in the SDF were the following: CD133, ERG, cyclin G2, MDR1, osteopontin, CLQR1, IFI16, JAK3, FZD6, and HOXA9, a pattern compatible with their primitive function and self-renewal capacity. Furthermore, morphologic differences between the SDF and FDF were determined. Cells in the SDF have more membrane protrusions and CD133 is located on these lamellipodia. The majority of cells in the SDF are rhodamine-123dull. These results provide molecular evidence that the SDF is associated with primitive function and serves as basis for a detailed understanding of asymmetric division of stem cells.

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Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment

Blood ◽

10.1182/blood-2006-02-001560 ◽

2006 ◽

Vol 108 (6) ◽

pp. 2087-2094 ◽

Cited By ~ 45

Author(s):

Gabriel Ghiaur ◽

Andrew Lee ◽

Jeff Bailey ◽

Jose A. Cancelas ◽

Yi Zheng ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Rho Gtpases ◽

Rho Gtpase ◽

Dominant Negative ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Hematopoietic Stem ◽

Rhoa Activity

AbstractRas-related Rho GTPases regulate actin cytoskeletal organization, adhesion, gene transcription, and cell-cycle progression. The Rac subfamily of Rho GTPases and Cdc42 has been shown to play essential roles in hematopoietic stem cell (HSC) engraftment and mobilization. Here, we study the role of RhoA, a related Rho GTPase, in HSC functions. Using retrovirus-mediated gene transfer of a dominant-negative (DN) mutant of RhoA (RhoAN19), we demonstrate that down-regulation of RhoA activity resulted in increased HSC engraftment and self-renewal as measured by competitive repopulation and serial transplantation assays. However, overexpression of RhoAN19 resulted in decreased migration toward SDF-1α and α4β1- and α5β2-integrin–mediated adhesion of hematopoietic progenitor cells in vitro. Low RhoA activity was associated with higher proliferation rate of hematopoietic progenitor cells and increased cells in active phases of cell cycle, most likely via decreasing p21Cip/Waf expression and increasing cyclin D1 levels. Thus, reducing RhoA activity by optimizing the balance between adhesion/migration and proliferation/self-renewal results in a net increase in HSC engraftment. This mechanism could provide a novel therapeutic target to enhance HSC therapies.

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Chelatable cellular copper modulates differentiation and self-renewal of cord blood–derived hematopoietic progenitor cells

Experimental Hematology ◽

10.1016/j.exphem.2005.06.015 ◽

2005 ◽

Vol 33 (10) ◽

pp. 1092-1100 ◽

Cited By ~ 53

Author(s):

Toni Peled ◽

Elina Glukhman ◽

Nira Hasson ◽

Sophie Adi ◽

Harel Assor ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Cellular Copper

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The influence of INK4 proteins on growth and self-renewal kinetics of hematopoietic progenitor cells

Blood ◽

10.1182/blood.v97.9.2604 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2604-2610 ◽

Cited By ~ 31

Author(s):

John L. Lewis ◽

Wimol Chinswangwatanakul ◽

Bo Zheng ◽

Stephen B. Marley ◽

Dao X. Nguyen ◽

...

Keyword(s):

Growth Rate ◽

Progenitor Cells ◽

Cell Lines ◽

Bone Marrow Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Ink4 Proteins ◽

Kinetics Of ◽

Marrow Cells

Abstract This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16INK4aexpression in the p16INK4a-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16INK4a and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34+ bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells fromp16INK4a−/− mice and wild-type mice. The results from p16INK4a−/−marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT ofp16INK4a. Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow ofp16INK4a−/−mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16INK4a−/−cell lines readily became growth factor–independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16INK4a, increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.

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The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.

Blood ◽

10.1182/blood.v104.11.796.796 ◽

2004 ◽

Vol 104 (11) ◽

pp. 796-796 ◽

Cited By ~ 1

Author(s):

Windy D. Berkofsk-Fessler ◽

Jonathan D. Licht ◽

Melanie-Jane McConnell ◽

Donna S. Neuberg ◽

Timothy S. Bowler ◽

...

Keyword(s):

Tumor Suppressor ◽

Polycythemia Vera ◽

Progenitor Cells ◽

Tumor Suppressor Genes ◽

Hematopoietic Progenitor Cells ◽

Multiple Testing Correction ◽

Hematopoietic Progenitor ◽

Suppressor Genes ◽

Putative Tumor Suppressor ◽

Cd34 Cells

Abstract Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.

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Telomerase Promotes the Self-Renewal of Human Hematopoietic Progenitor Cells but Does Not Maintain Telomere Length.

Blood ◽

10.1182/blood.v106.11.647.647 ◽

2005 ◽

Vol 106 (11) ◽

pp. 647-647 ◽

Cited By ~ 1

Author(s):

Junping Wei ◽

Mark Wunderlich ◽

James C. Mulloy

Keyword(s):

Telomere Length ◽

Progenitor Cells ◽

Molecular Mechanisms ◽

Critical Role ◽

Telomerase Activity ◽

The Self ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Telomerase is a reverse transcriptase that adds telomeric sequences onto chromosome ends. During hematopoiesis, telomerase activity is high in proliferating hematopoietic stem and progenitor cells, but decreases sharply as cells differentiate. Approximately 90% of all human cancers are telomerase positive, including samples from AML patients. To understand the role of telomerase in human hematopoietic stem and progenitor cell proliferation and differentiation, and in the development of leukemia, we established stable long-term cultures expressing high levels of telomerase by retrovirus-mediated transduction of the h-TERT (catalytic subunit of telomerase) gene into AML1-ETO-expressing CD34+ (AE) cells. We have previously shown that these AE cells maintain CD34 expression and proliferate for 7 months in culture but gradually lose telomere length and are not immortal, providing a good model for studying the molecular mechanisms involved in telomere shortening. Strikingly, telomerase expression enhanced the self-renewal ability of the human hematopoietic progenitor cells, as shown by continuous replating ability in methylcellulose assays. We also detected a pronounced delay in the differentiation of the progenitor cells upon telomerase expression, and an expansion of the progenitor pool. Telomerase expression promoted proliferation due to increased cell cycle progression as well as a survival advantage. At the same time, these cells demonstrated a progressive decline in telomere length, despite telomerase activity equivalent to that detected in leukemia cell lines. We conclude that expression of hTERT expands the pool of hematopoietic progenitors but does not maintain long telomeres in human CD34+ cells. hTERT plays a critical role not only in telomere homeostasis for genetic stability but also in cellular proliferation, differentiation, and self-renewal, functions that may be part of hTERT involvement in leukemogenesis. These data open an important debate regarding the specific contribution of telomerase expression to the leukemic phenotype and the potential timing of this essential hit in the progression of the disease.

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