Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A 2B adenosine receptor-knockout/reporter gene-knock-in (A 2B AR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-a, and a consequent downregulation of IkB-a are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A 2B AR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A 2B AR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A 2B AR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A 2B ARs regulate these processes. Hence, we identify the A 2B AR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

High expression of Aurora-B has been observed in various cancers, and inhibition of this kinase has been shown to halt cellular proliferation. However, the mechanism of effect of Aurora-B on cellular transformation has not been fully explored. Here, we show that overexpression of Aurora-B in murine epithelial cells promotes generation of tetraploids. In search of a related mechanism, spectral karyotyping was carried out, showing premature chromatid separation (PCS). Of interest, PCS is a hallmark of Robert's syndrome, which also involves cellular polyploidy and aneuploidy. Sorted tetraploid Aurora-B-overexpressing cells promoted significant mammary epithelial cancers when injected into nude mice, as compared to injection of nonfractionated cells, suggesting that tetraploidy is an important mediator of Aurora-B-induced tumorigenesis. Comparative chromosome hybridization performed on DNA derived from tetraploid cell-induced tumors indicates amplifications and deletions of regions throughout the genome, which include tumor-promoting or tumor-suppressing genes, respectively. Thus, sustained expression of Aurora-B induces tetraploidy, which, in turn, facilitates genomic instability and tumor development in a xenograft model.

IntroductionMegakaryocytes (MKs) are platelet precursors in the bone marrow (BM) that undergo a complex maturation process called megakaryopoiesis. A hallmark of MK development occurs after their commitment to the MK lineage, in which MKs go through iterative rounds of endomitosis to become large polyploid cells. These cells can contain DNA content of up to and greater than 128N, with 2N being normal diploid DNA content. 1 The primary stimulus for progression of megakaryopoiesis is known to stem from thrombopoietin (TPO) signaling through the c-Mpl receptor 2 ; however, despite this being the focus of many studies, much of TPO signaling in MKs remains unclear.Recently, investigations of reactive oxygen species (ROS), their generation, and downstream signaling effects have shown that low physiologic levels of ROS play a role in promoting cell proliferation in many cells types, such as fibroblast, prostate, macrophage, endothelial, and vascular smooth muscle cells (VSMCs). 3-7 ROSproducing enzymes, such as a member of the NADPH oxidase (Nox) family, Nox1, have been described to be activated by mitogenic stimuli 8 and to produce ROS that have been shown to induce and maintain G 1 phase cyclin D1 expression in mouse lung epithelial cells. 9 Similarly, Havens et al 10 showed that ROSs increase in a cell cycle-dependent manner and oscillate with every cell division in human T98G glioblastoma cells, human leukemic T cells, and NIH 3T3 cells. Although, these investigators did not identify the source of ROS in these cells, they did show that on treatment with various antioxidants, cells undergo transient arrest and fail to make a timely G 1 -S phase progression. [11][12][13] In addition, Havens et al 10 described redox control over the activity of the anaphase promoting complex (APC) in association with Cdh1, mediating cyclin A degradation, a regulator of the G 1 /S transition. In the MK endomitotic cell cycle, cyclin D3 has been described as the predominant D-type cyclin that is up-regulated by TPO stimulation and induces high ploidy levels in cyclin D3 transgenic mice. 14-16 Ablation of cyclin D3 in cultured MKs results in a notable decrease in MK ploidy level, 16 although in vivo studies show a less dramatic effect on ploidy possibly because of compensation by other D cyclins. 17 Cyclin E is another G 1 phase cell-cycle regulator that has been suggested to play a role in MK ploidy promotion, notably inducing nonendomitotic megakaryoblastic K562 cells to undergo re-replication cycles. 18 The importance of cyclin E in promotion of ploidy is also supported by evidence from the cyclin E1 Ϫ/Ϫ E2 Ϫ/Ϫ double knockout (KO) mice produced with the use of the tetraploid complementation rescue method. 19 Interestingly, these mice developed normally with the exception of ploidy promotion in trophoblasts and MKs, showing a marked reduction in polyploid MKs even when cultured with TPO.To date, investigation of ROS in MKs has been limited to studies that used human megakaryocytic leukemic cell lines, such as MO7e and B1647 cells. 11,...

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before rejoining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally overexpress genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic ␤-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by ␤-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4 - IntroductionMegakaryocytes (MKs) progress through distinct developmental stages, including lineage commitment, expansion, polyploidization, maturation, and fragmentation into platelets. 1,2 To investigate mechanisms that control megakaryocytic development, gene targeting is required to manipulate levels of a potential regulatory protein of interest. More than a decade ago, the rat platelet factor 4 (PF4) gene promoter 3 was used as a tool to specifically overexpress genes in early committed megakaryocytes in vivo. Since then, PF4 and other related promoters have been used to examine effects of deregulating the expression of cell-cycle genes, antiapoptotic proteins, receptors, thrombosis-modulating proteins, and others on megakaryocyte/platelet development and/or activity. 4-14 PF4-driven constitutive expression is limited, depending on the transgene used, due to occasional lethality during development or adverse effects during adulthood. For instance, Kufrin et al demonstrated that PF4-driven expression of urokinase-type plasminogen activator in mice exhibited a bleeding diathesis, which may have limited the potential number of founder lines that were available for analysis. 10 In addition, if a given transgene is not expressed due to regulatory elements within the site of genomic integration, increased numbers of founders need to be generated. 13 Tetracycline-inducible promoters and transactivating mouse lines have been used throughout the literature to circumvent such limitations. A successful application of this system was reported for targeted expression in myeloid cells and hematopoietic progenitors in vivo, [15]...