IntroductionMicroRNAs (miRNAs) are small (20-24 nt's), noncoding RNAs that function as key regulators of gene expression. By pairing with the transcripts of protein-coding genes, they mediate cleavage of the targeted mRNAs or repression of their productive translation. [1][2][3] Notably, miRNAs exhibit dynamic temporal and spatial expression patterns, disruption of which may be associated with tumorigenesis. [4][5][6][7] The C13orf25/miR-17-92 cluster encodes 6 miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92). The miR-17-92 cluster is known to be overexpressed in a variety of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, follicular lymphoma, and mantle cell lymphoma, 5,[8][9][10][11][12][13] all of which arise through various primary and additional genetic changes, including genomic translocations, losses, and amplifications. [8][9][10][11][12][13] Recurrent overexpression of miR-17-92 in B-cell lymphomas suggests the polycistron possesses tumorigenic potential. Consistent with that idea, He et al demonstrated that forced expression of the miR-17-19b-1 (a miR-17-92 variant) in the Eu-myc transgenic mouse model of B-cell lymphoma accelerated disease onset and progression. 5 This suggests dysregulation of the miR-17-92 polycistron contributes to lymphomagenesis by repressing tumor suppressor gene(s). The most likely target of miR-17-92 in lymphomagenesis is the proapoptotic protein Bim, 14 which is known to be a tumor suppressor. 15 Recently, Xiao et al generated a mouse line that selectively overexpressed miR-17-92 in their lymphocytes. 16 As a result, these mice developed lymphoproliferative disease and died prematurely. Following activation, B and T cells from these transgenic mice showed increased proliferation and survival as a result of dysregulation of the proapoptotic gene Bcl2l11/Bim. In addition, Ventura et al established a strain of miR-17-92 knockout mice, 17 which enabled the researchers to demonstrate that the miR-17-92 cluster is essential for the survival signal that mediates progression from the pro-B-to the pre-B-cell stage. The absence of miR-17-92 led to elevated Bim protein levels and inhibition of B-cell development. Finally, Koralov et al ablated Dicer in early B-cell progenitors by conditionally deleting dcr-1 in the B-cell lineage of mice. The resulting phenotype was similar to that seen with miR-17-92 deletion, which was characterized by a developmental block at the transition from the pro-B-to the pre-B-cell stage. The researchers also found that cells lacking Dicer showed increased Bim transcription and Bim protein production. 18 All of these reports suggest that down-regulation of Bim mRNA and protein induced by miR-17-92 overexpression could contribute lymphomagenesis via antiapoptotic activity. Nonetheless, Bim is an unlikely target of miR-17-92 in some aggressive B-cell lymphomas, despite overexpression of the polycistron. For instance, Jeko-1 cells, which are derived from an aggressive B-cell lymphoma, exhibit both homozygous...

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various celldivision inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34 ؉ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts. IntroductionDuring erythropoiesis, stem cells undergo lineage specific commitment and generate erythroid progenitor cells through cellular division events including nuclear (mitosis) and cytoplasmic (cytokinesis) division. These progenitor cells consist of immature and mature erythroid progenitors, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E), respectively. The BFU-E can be considered as a progenitor of the CFU-E. Indeed, after 6 to 7 days in culture, cells generated from human BFU-E have all the functional characteristics of CFU-E 1 . After an additional 6 to 7 days in culture, human CFU-E proliferate and differentiate into mature erythroblasts. 1 Terminally differentiated erythroblasts in mammals expel their nuclei via a process termed enucleation, becoming reticulocytes and subsequently mature erythrocytes. The nucleus separates from the remainder of the cell and is phagocytosed by reticular cells such as macrophages (for a review, see Chasis et al 2 ).Enucleation of erythroblasts is thought to occur through a process similar to cytokinesis. Several general principles apply to cytokinesis. Firstly, the microtubule cytoskeleton plays an important role in both the choice and positioning of the division site. Once this site is chosen, the local assembly of the actomyosin contractile ring remodels the plasma membrane. Finally, membrane trafficking to, and membrane fusion at the division site result in the physical separation of the daughter cells, a process termed abscission (for reviews, see Barr et al 3 and Glotzer et al 4 ). Although modulation of the actomyosin cytoskeleton is crucial for proper cytokinesis, there is a paucity of information regarding how non-muscle myosin II contributes to enucleation.Several investigations have studied the mol...

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = -0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.

Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units-erythroid (CFU-Es) and mature erythroblasts generated from purified CD34(+) cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein.

How human erythroblasts enucleate remains obscure, and some investigators suspect the effect of mechanical forces on enucleation in vitro. We determined the dynamics of the enucleation process of highly purified human erythroblasts and whether enucleation can occur without external mechanical forces. Highly purified human CD34(+) cells were cultured in liquid phase with interleukin-3, stem cell factor and erythropoietin (EPO) for 7 days and the generated erythroblasts were replaced in the same medium with EPO alone. In some experiments, the enucleating cells were processed without centrifugation and pipette aspiration to avoid physical stress and were directly observed by differential interference contrast (DIC) microscopy. Enucleation initiated at day 12 and the enucleation ratio (percent of enucleated reticulocytes in total cells) reached a maximum at day 14 with a value of 63 +/- 7%. The direct observation by DIC microscopy showed 61 +/- 9% of enucleation ratio at day 14. The human erythroblasts enucleated without contact with macrophage. The time required for enucleation was 8.4 +/- 3.4 min. The enucleation rate was 1.16 +/- 0.42%/h at day 12 and then decreased with a time dependent manner. The expelled nucleus was connected to the reticulocyte through plasma membrane and associated cytoskeletal elements, and spontaneous separation of the extruded nucleus from reticulocyte was extremely rare. In conclusion, human erythroblasts enucleate in a relatively short period without contact with macrophages, but nascent reticulocytes fail to completely separate from nuclei in the absence of macrophages, unless some physical force is applied to them.