Cyclin A2 Plays a Critical Role in Proliferation of Lymphoid Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 914-914
Author(s):  
Kentaro Kohno ◽  
Hiromi Iwasaki ◽  
Tadafumi Iino ◽  
Shin-ichi Mizuno ◽  
Peter Sicinski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Cyclin A2

Abstract Abstract 914 Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors. Disclosures: No relevant conflicts of interest to declare.

Cyclin A2 Plays a Critical Role Not Only in Self-Renewal of Hematopoietic Stem Cells, but Also in Non-Self-Renewing Proliferation of Lymphoid Progenitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 381-381 ◽  
Author(s):  
Kentaro Kohno ◽  
Tadafumi Iino ◽  
Kyoko Ito ◽  
Shin-ichi Mizuno ◽  
Piotr Sicinski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Mammalian Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cyclin A2

Abstract Abstract 381 Cyclins are regulatory subunits of cyclin-dependent kinase, and are important components of cell cycle engine. The A-type cyclin is generally the S-phase cyclin. Mammalian cells express two A-type cyclins, including cyclin A1 that is exclusively expressed in the testis, and cyclin A2 whose expression is ubiquitous. We have recently reported that cyclin A2 is not required for fibroblast proliferation but it is indispensable in maintenance of self-renewal of stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here we further assessed the requirement of A-type cyclin in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic stem and progenitor cells, but its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Rag1 expression is initiated at the preproB to the proB stages, and the DN1-DN3 stages in the thymus, while their proliferation is dependent at least upon pre-BCR or pre-TCR signal at these stages. Interestingly, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors in the bone marrow. They, however, displayed severe reduction of T and B cell numbers that were only 1/100 - 1/10 of wild-type controls; the number of common lymphoid progenitor was unchanged, but there were almost complete loss of proB and preB cells. Similarly, all thymic T cell progenitor compartments such as CD4-CD8- double negative, and CD4+CD8+ double positive populations were severely reduced. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewal of HSCs, but also for proliferation of T and B cell progenitors. Disclosures: No relevant conflicts of interest to declare.

Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3357-3357
Author(s):  
Sara Rohrabaugh ◽  
Charlie Mantel ◽  
Hal E. Broxmeyer
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Mitotic Spindle ◽  
Flow Cytometric Analysis ◽  
Spindle Checkpoint ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Mitotic Spindle Checkpoint

Abstract Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

Pum2 Protein Supports Maintenance and Suppresses Differentiation of Multipotent Hematopoietic Progenitors by Regulating the Function and Activation of c-kit Receptor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1692-1692
Author(s):  
Jie Yang ◽  
Danislav S. Spassov ◽  
Ronald G. Nachtman ◽  
Roland Jurecic
Keyword(s):  
Stem Cells ◽  
Rna Binding ◽  
Critical Role ◽  
Multilineage Differentiation ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Constitutive Activation ◽  
Gm Csf ◽  
Kit Receptor

Abstract Intrinsic mechanisms that regulate self-renewal of mammalian stem cells remain largely unknown. Stem cell maintenance and self-renewal in Drosophila and C. elegans are regulated by members of the conserved Pumilio family of RNA-binding proteins. We have previously described cloning and characterization of two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the role of mammalian Pum proteins in HSC, Pum2 was over-expressed in a SCF-dependent multipotent progenitor cell line EML, which has the capacity for multilineage (erythroid, myeloid, B and T lymphoid) differentiation in vitro. In the presence of SCF EML cells undergo SCF-dependent self-renewal, thus remaining undifferentiated and retaining an immature phenotype. When cultured with hematopoietic cytokines (IL-3, GM-CSF, Epo, Tpo) EML cells differentiate into lineage-committed hematopoietic progenitors (e.g. granulocyte/macrophage (CFU-GM), burst-forming unit erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors). Pum2 over-expression leads to uncoupling of the survival and differentiation signals in EML cells, and their SCF-independent maintenance. EML cells over-expressing Pum2 (Pum2-EML cells) also exhibit almost complete block of differentiation into multiple lineages in the absence of SCF. Moreover, although the culture with cytokine cocktail (IL-3, Epo, Tpo and GM-CSF) and retinoic acid enhances differentiation capacity of wild type EML cells, it was not sufficient to overcome the differentiation block in Pum2-EML cells. However, the repression of Pum2-EML cell differentiation is a reversible phenomenon, since the addition of SCF to Pum2-EML cell cultures, for at least 48 hours, restores their capacity to undergo multilineage differentiation and generate hematopoietic colonies. The SCF-independent maintenance of Pum2-EML cells seems to be caused by upregulated expression and constitutive activation of the SCF receptor c-kit, and is accompanied by constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. More importantly, Pum2-EML cells also exhibit upregulated expression and constitutive activation of a novel truncated form of c-kit receptor called tr-kit, which was found previously to be expressed preferentially in HSC. Tr-kit could play a critical role in the SCF-independent activation of the full-length c-kit receptor, leading to SCF-independent maintenance of Pum2-EML cells, and inhibition of their multilineage differentiation. The observation that Pum2-EML cells, maintained with or without SCF, are resistant to treatment with blocking anti-c-kit antibody (ACK2) and c-kit inhibitor STI-571, supports the notion that maintenance and survival of Pum2-EML cells in the presence of SCF is not due to an external activation of c-kit receptor through ligand binding. Taken together, these findings suggest a model in which survival and maintenance of multipotent hematopoietic progenitors are mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. In summary, mouse Pum2 protein could play an important role in supporting maintenance of HSC and multipotent progenitors through regulation of the SCF/c-kit signaling pathway, and could represent a part of the mechanism through which HSCs balance their self-renewal and commitment to differentiation.

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

scholarly journals Cell cycle regulation in hematopoietic stem cells

2011 ◽  
Vol 195 (5) ◽  
pp. 709-720 ◽  
Author(s):  
Eric M. Pietras ◽  
Matthew R. Warr ◽  
Emmanuelle Passegué
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Cycle Regulation ◽  
Critical Role ◽  
Cellular Damage ◽  
Fetal Life ◽  
Extrinsic Factors ◽  
Hematopoietic Stem ◽  
Cycle Regulation

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy.

scholarly journals Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

2018 ◽  
Author(s):  
Tatyana Grinenko ◽  
Anne Eugster ◽  
Lars Thielecke ◽  
Beata Ramazs ◽  
Anja Krueger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Erythroid Progenitors ◽  
Multipotent Stem Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

scholarly journals Toward a better definition of hematopoietic progenitors suitable for B cell differentiation

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243769
Author(s):  
Florian Dubois ◽  
Anne Gaignerie ◽  
Léa Flippe ◽  
Jean-Marie Heslan ◽  
Laurent Tesson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Embryonic Stem ◽  
B Cell Differentiation ◽  
Hematopoietic Progenitors ◽  
Cell Based Therapy ◽  
Definition Of

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34+CD43+ hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.

Genetically-Modified Stem Cell in Regenerative Medicine and Cancer Therapy; A New Era

2021 ◽  
Vol 21 ◽  
Author(s):  
Ali Hassanzadeh ◽  
Somayeh Shamlou ◽  
Niloufar Yousefi ◽  
Marzieh Nikoo ◽  
Javad Verdi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cancer Therapy ◽  
Gene Delivery ◽  
Regenerative Medicine ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
In Vivo Studies ◽  
Cell Potential ◽  
Hematopoietic Stem

: Recently, genetic engineering by various strategies to stimulate gene expression in a specific and controllable mode is a speedily growing therapeutic approach. Genetic modification of human stem or progenitor cells, such as embryonic stem cells (ESCs), neural progenitor cells (NPCs), mesenchymal stem/stromal cells (MSCs), and hematopoietic stem cells (HSCs) for direct delivery of specific therapeutic molecules or genes has been evidenced as an opportune plan in the context of regenerative medicine due to their supported viability, proliferative features, and metabolic qualities. On the other hand, a large number of studies have investigated the efficacy of modified stem cells in cancer therapy using cells from various sources, disparate transfection means for gene delivery, different transfected yields, and wide variability of tumor models. Accordingly, cell-based gene therapy holds substantial aptitude for the treatment of human malignancy as it could relieve signs or even cure cancer succeeding expression of therapeutic or suicide transgene products; however, there exist inconsistent results in this regard. Herein, we deliver a brief overview of stem cell potential to use in cancer therapy and regenerative medicine and importantly discuss stem cells based gene delivery competencies to stimulate tissue repair and replacement in concomitant with their potential to use as an anti-cancer therapeutic strategy, focusing on the last two decades in vivo studies.

Change in SP Distribution with Age Reflects Intrinsic Age-Based Changes in Stem Cell Biology: Implications for the Mechanism of Aging.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4209-4209
Author(s):  
Daniel J. Pearce ◽  
Catherine Simpson ◽  
Kirsty Allen ◽  
Ayad Eddaoudi ◽  
Derek Davies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
The Absolute

Abstract It has been postulated that as we age, accumulated damage causes stem cells to die by apoptosis. This could lead to a diminished stem cell pool and consequently a reduced organ regeneration potential that contributes to somatic senescence. Hematopoietic stem cells have evolved many mechanisms to cope with their exposure to toxins during life. Cell surface transporters and anti-toxic enzymes are highly expressed in hematopoietic stem cells. If toxins do get the opportunity to damage the DNA of stem cells then the cell is more likely to die by apoptosis than attempt DNA repair and risk an error. Summarised below are our results from an investigation of the frequency, phenotype, cell cycle status and repopulation potential (in young recipients) of C57BL6 side population (SP) cells from mice with a range of ages. The absolute frequency of SP cells increases with age (Figure-A). The proportion of the lineage negative, Sca-1+, c-kit+ (KLS) cell population that is an SP stem cell increases from ~1% to over 30% during the murine lifetime (red bars in Figure-B). These SP cells from older mice have a reduced 4-month competitive repopulation potential when compared to SP cells from younger mice but contain a similarly low proportion of phenotypically-defined mature cells (blue bars in Figure-B) and have a similar cell cycle profile and progenitor cell output (2% of 3 x 96 well plates for each). SP cells from older mice contained a higher proportion of SP cells with the highest efflux ability (61 vs 414 days, p=<0.001, n=6) Engrafted cells derived from old SP cells 4 months previously still displayed an increased SP frequency when compared to engrafted cells derived from SP cells of young mice. Hence, more progenitors or committed cells have not gained the SP ability; rather this difference in SP distribution reflects an age-dependent change in hematopoietic stem cell biology that is independent of the microenvironment. Specifically, the proportion of stem and progenitor cells (KLS) that is a stem cell (SP fraction of KLS) increases with age. We hypothesize that this may be a progressive enrichment of primitive cells over time via selection. As we age, accumulative damage to hematopoietic stem and progenitor cells causes more cells to die by apoptosis. It may be that the stem/progenitor cells with the lowest hoechst efflux ability are most susceptible to damage and hence most likely to die by apoptosis. Since the HSCs with the highest efflux of hoechst are thought to be the most primitive, it may be that there is an enrichment of primitive cells. This could account for the increased SP proportion observed within KLS cells. As there may be cells with ABC/G2 activity that is undetectable via the SP technique, selection of cells with a higher pump activity could also explain the increased SP frequency we observed. This hypothetical mechanism would also be independent of microenvirinment. In summary, we surmise that HSCs have a mechanism that copes with cellular damage while compensating for the reduced cellular output of HSCs with age by increasing the absolute number of HSCs. Figure Figure

Nucleophosmin Regulates Differentiation, Cell Cycle Progression, and Stress Response in Hematopoietic Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 312-312
Author(s):  
June Li ◽  
Daniel P. Sejas ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stress Response ◽  
Progenitor Cells ◽  
Cell Cycle Progression ◽  
Proliferative Potential ◽  
Hematopoietic Progenitors ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Empty Vector

Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Strong evidence indicates that NPM is involved in hematopoiesis and leukemic development. Here we report that NPM enhances the proliferative potential of hematopoietic stem/progenitor cells and increases cell survival upon stress challenge. Specifically, lin-Sca1+c-kit+ bone marrow cells transduced with retroviral vector expressing NPM exhibited higher proliferative rates in both short-term liquid culture and clonogenic progenitor cell assays, compared to the cells transduced with empty vector. Interestingly, NPM overexpression appears to inhibit differentiation of myeloid progenitors. Hematopoietic stem/progenitor cells infected with the NPM retrovirus expressed significantly lower levels of mature cell markers Gr-1 and Mac-1 compared to empty vector transduced cells, and majority of the NPM-overexpressing cells remained Sca1+C-Kit+ during the 5-day culture. Bone marrow transplantation experiments demonstrated that NPM overexpression increases long-term multi-lineage repopulating capacity of hematopoietic progenitors. We have not observed any evidence of proliferative disorders or leukemia in recipients transplanted with NPM-expressing progenitors thus far (4 months posttransplantation). Through cell-cycle profile analysis and single-cell division experiments, we showed that NPM overexpression induces rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G0/G1 to S transition. Furthermore, immunocytochemical and Western-blot analyses demonstrated that NPM-transduced cells expressed higher level of cyclin A compared to vector-transduced cells. Finally, overexpression of NPM significantly increased the survival of hematopoietic progenitors exposed to mitomycin C or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of progenitor differentiation, cell cycle progression, and stress response.

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