Kinetics of endomitosis in primary murine megakaryocytes

Carow, Catherine E.; Fox, Norma E.; Kaushansky, Kenneth

doi:10.1002/jcp.1120

Cited by 30 publications

(36 citation statements)

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“…42,43 Cyclin B1 reduction has been associated with the switch to polyploidization in megakaryocytic cell lines 23,24 ; it was reported that in polyploid megakaryocytes cyclin B1 expression oscillates normally with cell-cycle phase. 28,29 Our results indicated that in CB-derived MKs cyclin B1 expression is present, prevalently at nuclear level, during the whole culture time, correlating with the high cycling activity of these cells. In PB-MKs cyclin B1 expression decreased at days 9 to 12 of the culture.…”

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confidence: 67%

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Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

Mattia

Vulcano

Milazzo

et al. 2002

Blood

230

174

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Ploidy could be the key to understanding megakaryocyte (MK) biology and platelet production. Human CD34 ؉ cells purified from umbilical cord blood (CB) and peripheral blood (PB) were investigated on their capability to give rise, in a serumfree medium containing thrombopoietin, to MKs and platelets. CB-MKs showed reduced polyploidization and platelet number compared with PB-MKs, but a similar membrane phenotype. Most CBMKs showed a 2N content of DNA (ϳ80%) and only 2.6% had 8N, whereas 40% of the PB cells had 8N or more. Platelets were substantially released in PB culture from day 12; at day 14 the CB-derived MKs were able to release platelets although at a reduced level (ϳ35%), correlating with their reduced size. A direct correlation was demonstrated by sorting polyploid cells from PB-MKs and evaluating the platelets released in the supernatant. Furthermore, the study analyzed the expression and distribution of cyclin D3 and cyclin B1. Cyclin D3 protein was increased in PB in comparison to CB-MKs; in PB culture most cells rapidly became positive, whereas in CB-derived cells cyclin D3 expression was evident only from day 9 and in a reduced percentage. Cyclin B1 was essentially localized at the nuclear level in the CB and was expressed during the whole culture. In PB-MKs, at day 9, a reduction was observed, correlating with an advanced ploidy state. The data indicate the inability of the CB-MKs to progress in the endomitotic process and a direct correlation between DNA content and platelet production. IntroductionThe main feature of megakaryocyte (MK) maturation is the development of a single, large, lobulated, polyploid nucleus; the mature MKs cease to proliferate but continue to increase their DNA content without undergoing late stages of mitosis. [1][2][3][4] Increase in megakaryocytic ploidy is associated with increase in megakaryocytic volume; the large size and abundant cytoplasm allow MKs to produce several thousand platelets per cell. 3 It was presumed that higher-ploidy cells could produce more platelets than lower-ploidy cells or that production and release is more efficient from a single large cell than from several smaller ones, but none of these suppositions has been proven. 5 Peripheral blood (PB)-mobilized CD34 ϩ cells induced to differentiate into megakaryocytic lineage gave rise to 3-fold augmentation of platelets compared with bone marrow (BM) CD34 ϩ cells, although the proportion of proplateletdisplaying MKs were similar. 6 Choi et al 7 reported the functionality of the platelets released in vitro from CD34 ϩ cells derived from PB stimulated to form MKs.The generation of large numbers of megakaryocytes became possible by the identification and cloning of thrombopoietin (TPO), the key regulatory cytokine of megakaryocytopoiesis. [8][9][10][11] Then several culture systems have been developed permitting all stages of megakaryocytopoiesis until platelet formation. 6,[12][13][14][15] TPO was shown to induce endomitosis and consequently to increase the polyploidy state of MKs in a significant meas...

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confidence: 67%

“…section 1734. 29 We used CD34 ϩ progenitor cells, purified from human PB and CB, induced to differentiate into the megakaryocytic lineage in the presence of TPO alone to evaluate the differences in the MK differentiation/maturation process, including the polyploidization state and platelet release. …”

mentioning

confidence: 99%

Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

Mattia

Vulcano

Milazzo

et al. 2002

Blood

230

174

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“…Endomitosis is a form of endoreplication in which the cell undergoes early mitotic events such as chromosome condensation. Molecular candidates for the endomitotic switch include cyclin B1 and cyclin E (Carow et al, 2001;Bermejo et al, 2002;Eliades et al, 2010). In addition, live-imaging experiments were performed using the histone2B (H2B)-GFP or YFP-tubulin marker to monitor how mitosis aborted (Geddis et al, 2007;Papadantonakis et al, 2008).…”

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confidence: 99%

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

Sakaue-Sawano

Hoshida

et al. 2013

Development

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The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in ‘licensing’ of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APCCdh1 and SCFSkp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APCCdh1 and SCFSkp2. By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

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“…CD41-positive cells were isolated from mouse marrow by lineage depletion and FACS and were sorted in to 2N and 4N populations based on Hoechst staining. Negative selection for Gr-1 improves ploidy distribution by excluding MKs with adherent neutrophils (62). Diploid and 4N CD41ϩ cells were cultured in IMDM/10%FCS with 10 ng/ml rhTPO and 16 M LY294002 or Me 2 SO for 12 h. Cells were assayed for BrdUrd uptake Apotag positivity, as described above for BaF3/ Mpl cells.…”

Section: Tpo Stimulates Cell Cycling In Baf3/mpl Cells and In Primarymentioning

confidence: 99%

Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors

Geddis¹,

Fox²,

Kaushansky³

2001

Journal of Biological Chemistry

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Mature megakaryocytes are large, polyploid bone marrow cells that give rise to circulating platelets (1). Thrombopoietin (TPO) 1 has been characterized as the primary hematopoietic cytokine regulating normal megakaryocyte (MK) development. The receptor for TPO is encoded by the c-mpl proto-oncogene, a member of the hematopoietic growth factor receptor family, which in altered form causes a myeloproliferative syndrome in mice (2). In addition to its effects on MK development, TPO has been found to play a significant role in promoting stem cell survival, and in conjunction with other cytokines, it can support stem cell expansion (3-5). Characterization of the pathways by which TPO signals to promote survival and proliferation in stem cells and developing MKs is critical to a better understanding of the physiologic, pathologic, and potentially therapeutic roles of the cytokine in stem cell expansion, myeloproliferative disorders, and states of bone marrow failure.Many of the effects of TPO on cell survival and proliferation have been ascribed to activation of the Jak/STAT and Ras/Raf/ MAPK pathways (reviewed in Ref. 6). Activation of Jak2 leads to tyrosine phosphorylation of Mpl as well as STAT3 and STAT5 (7,8), and the phosphorylated STATs dimerize and translocate to the nucleus, where they stimulate transcription. In addition, phosphorylation of distal tyrosine residues on Mpl creates docking sites for SHC, which undergoes phosphorylation and then recruits Grb2/SOS, thus activating the Ras/Raf/ MAPK pathway (8 -10). Activation of the Ras/Raf/MAPK pathway can also occur by an alternative, as yet undetermined mechanism, independent of the distal receptor phosphotyrosine residues (11).Although these studies and others have shown that activation of the Jak/STAT and Ras/Raf/MAPK pathways is important for signaling by Mpl, other pathways likely contribute to the cellular response to TPO. One such pathway that may contribute to Mpl signaling is phosphatidylinositol 3-kinase (PI3K). PI3K has been shown to be activated by many growth factors involved in hematopoiesis, including stem cell factor, platelet-derived growth factor, erythropoietin, interleukin 3 (IL-3), IL-2, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, insulin-like growth factor 1, and TPO (12-16). It has been well established that activation of PI3K plays an important role in promoting cell survival (17-19). However, although there are ample data supporting a requirement for PI3K in proliferation of nonhematopoietic cells (20 -22), data showing specific requirements for PI3K in the cytokine-dependent proliferation of hematopoietic cells are inconclusive. Studies in cell lines with IL-2, IL-3, and erythropoietin have supported a role for PI3K in the proliferative response (23-26); however, studies with Flt-3L have not (27). Importantly, the conclusions from studies performed in primary cells often differ from those performed in cell lines. For example, data from cell lines support the role of PI3K in erythropoietin-...

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Kinetics of endomitosis in primary murine megakaryocytes

Cited by 30 publications

References 86 publications

Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors

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