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1.
Haematologica ; 86(1): 17-23, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11146565

ABSTRACT

BACKGROUND AND OBJECTIVES: Megakaryocytes undergo a unique cell cycle by which they replicate their complete genome many times in the absence of cytokinesis. In the search for regulators of the endomitotic cell cycle, we previously produced mice transgenic for cyclin D3 to identify this cyclin as able to enhance ploidy and to increase the number of differentiated cells in the megakaryocytic lineage. Of the D-type cyclins, cyclin D3 and to a much lesser extent cyclin D1, are present in megakaryocytes undergoing endomitosis and these cyclins are, respectively, markedly and moderately upregulated following exposure to the ploidy-promoting factor, Mpl-ligand. Our objective was to explore whether cyclin D1 can mimic the effect of cyclin D3 on ploidy in megakaryocytes. DESIGN AND METHODS: We generated transgenic mice overexpressing cyclin D1 in megakaryocytes and analyzed megakaryocyte ploidy, number and platelet levels in these mice and control mice. RESULTS: We show that transgenic mice in which cyclin D1 is overexpressed in megakaryocytes display higher ploidy level than the control mice, with no change in the number of differentiated cells of the megakaryocytic series, or in platelet level. INTERPRETATION AND CONCLUSIONS: Our models support a key role for D-type cyclins in the endomitotic cell cycle, and also indicate that although cyclin D3, from among the D cyclins, is unique in its high levels of expression in megakaryocytes, it is not unique in its ability to promote polyploidization.


Subject(s)
Cyclin D1/pharmacology , Megakaryocytes/drug effects , Ploidies , Animals , Cell Count , Cyclin D1/genetics , Cyclin D1/metabolism , Gene Expression , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice , Mice, Transgenic , Platelet Count
2.
Stem Cells ; 16 Suppl 2: 97-106, 1998.
Article in English | MEDLINE | ID: mdl-11012182

ABSTRACT

The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity. p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.


Subject(s)
Cell Differentiation/genetics , Cyclins/genetics , Cyclins/metabolism , Megakaryocytes/metabolism , Phenotype , Transgenes/physiology , Animals , Cell Cycle/physiology , Cells, Cultured , Cyclin D3 , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , Megakaryocytes/cytology , Mice , Mice, Transgenic , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Polyploidy , Transcription Factors/genetics
3.
Mol Cell Biol ; 17(12): 7248-59, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9372957

ABSTRACT

Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration.


Subject(s)
Cell Cycle/physiology , Cyclins/physiology , Mitosis/physiology , Animals , Blood Platelets/cytology , Blood Platelets/drug effects , Blood Platelets/physiology , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Cycle/drug effects , Cyclin D3 , Cyclins/genetics , DNA/metabolism , Megakaryocytes/cytology , Megakaryocytes/drug effects , Megakaryocytes/physiology , Mice , Mice, Transgenic , Microscopy, Electron , Mitosis/drug effects , Platelet Factor 4/genetics , Polyethylene Glycols/pharmacology , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Spleen/cytology , Spleen/drug effects , Spleen/metabolism , Thrombopoietin/pharmacology , Up-Regulation/drug effects
4.
J Immunol ; 157(6): 2299-309, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8805627

ABSTRACT

The regulation of the STAT1 alpha transcription factor was assessed during B cell activation induced by cross-linking the surface IgM Ag receptor. Surface Ig ligation or pharmacologic stimulation with PMA and ionomycin resulted in the delayed (2-3 h after stimulation) nuclear appearance of tyrosine-phosphorylated STAT1 alpha, in contrast to the rapid induction that follows cytokine treatment. Nuclear expression of phosphorylated STAT1 alpha was abrogated by co-incubation of anti-Ig-treated B cells with the protein synthesis inhibitor cycloheximide (CHX), with the protein kinase inhibitor H-7, or with the immunosuppressive drug rapamycin. Tyrosine-phosphorylated STAT1 alpha was found to be recruited to the STAT binding site of the IFN regulatory factor-1 (IRF-1) gene promoter only after 2 to 3 h, and this association was also inhibitable by CHX and rapamycin. The arrival of STAT1 alpha coincided with attenuation of anti-Ig-induced STAT-binding activity specific for the IRF-1 promoter site, and both rapamycin and CHX treatment counteracted the loss of this activity. Furthermore, basal transcription of the endogenous IRF-1 gene was decreased as a result of anti-Ig treatment, and this effect of anti-Ig was blocked by co-incubation with rapamycin. Thus, STAT1 alpha plays a dynamic role in the composition of IRF-1 promoter-specific DNA binding complexes stimulated by B cell Ag receptor ligation, and nuclear expression of phosphorylated STAT1 alpha is regulated in a unique fashion by Ag receptor engagement. In addition, surface Ig cross-linking imparts negative regulatory control of IRF-1 gene expression, possibly through activation of STAT1 alpha.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Nucleus/metabolism , DNA-Binding Proteins/biosynthesis , Lymphocyte Activation , Receptors, Antigen, B-Cell/physiology , Trans-Activators/biosynthesis , Tyrosine/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , 3T3 Cells , Animals , Antibodies, Anti-Idiotypic/pharmacology , Down-Regulation/immunology , Interferon Regulatory Factor-1 , Mice , Mice, Inbred BALB C , Nucleoproteins/biosynthesis , Phosphoproteins/biosynthesis , Phosphorylation , Polyenes/pharmacology , Protein Biosynthesis , Protein Serine-Threonine Kinases/drug effects , Protein Serine-Threonine Kinases/physiology , STAT1 Transcription Factor , Sirolimus , Transcription Factors/biosynthesis
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