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BCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types, and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin, little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class I(A) PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition, analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes.

The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.

Granulomas are innate sequestration responses that can be modified by superimposed acquired immune mechanisms. The present study examined the innate stage of pulmonary granuloma responses to bead-immobilized Th1- and Th2-inducing pathogen antigens (Ags), Mycobacteria bovis purified protein derivative (PPD) and Schistosoma mansoni soluble egg Ags (SEA). Compared to a nonpathogen Ag, PPD and SEA bead elicited larger lesions with the former showing accelerated inflammation. Temporal analyses of cytokine and chemokine transcripts showed all Ag beads induced tumor necrosis factor-alpha mRNA but indicated biased interleukin (IL)-1, IL-6, and IL-12 expression with PPD challenge. All beads elicited comparable levels of CXCL9, CXL10, CCL2, CCL17, and CCL22 mRNA, but PPD beads caused biased CXCL2 CXCL5, CCL3, and CCL4 expression whereas both pathogen Ags induced CCL7. Immunohistochemical, electron microscopic, and flow cytometric analyses showed that Ag beads mobilized CD11c+ dendritic cells (DCs) of comparable maturation. Transfer of DCs from PPD Ag-challenged lungs conferred a Th1 anamnestic cytokine response in recipients. Surprisingly, transfer of DCs from the helminth SEA-challenged lungs did not confer the expected Th2 response, but instead rendered recipients incapable of Ag-elicited IL-4 production. These results provide in vivo evidence that lung DCs recruited under inflammatory conditions favor Th1 responses and alternative mechanisms are required for Th2 commitment.

The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S1PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization, and substantially increased SDF-1-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1.

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver, very few HSCs efflux Hoechst 33342 efficiently, and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment�

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists inhibit vascular smooth muscle proliferation and migration and improve endothelial function. It is unknown whether PPAR-gamma agonists favorably modulate bone marrow (BM)-derived angiogenic progenitor cells (APCs) to promote endothelial lineage differentiation and early reendothelialization after vascular intervention. METHODS AND RESULTS: C57/BL6 mice, treated with or without rosiglitazone (8 mg/kg per day), a PPAR-gamma agonist, underwent femoral angioplasty. Rosiglitazone treatment attenuated neointimal formation (intima/media ratio: 0.98+/-0.12 [rosiglitazone] versus 3.1+/-0.5 [control]; Ptextless0.001; n=10 per group). Using a BM transplantation model, we identified that 58+/-12% of the cells within the neointima at 4 weeks were derived from the BM. Pure endothelial marker-positive, pure alpha-smooth muscle actin (alphaSMA)-positive, or double-positive APCs could be found both in mouse BM and in human peripheral blood after culture in conditional medium enriched with vascular endothelial growth factor. Rosiglitazone caused a 6-fold (Ptextless0.001) increase in colony formation by human endothelial progenitor cells, promoted the differentiation of APCs toward the endothelial lineage in mouse BM in vivo (0.66+/-0.06% [control] to 0.95+/-0.08% [rosiglitazone]; Ptextless0.05) and in human peripheral blood in vitro (13.2+/-1.5% [control] to 28.4+/-3.3% [rosiglitazone]; Ptextless0.05), and inhibited the differentiation toward the smooth muscle cell lineage. Within the neointima, rosiglitazone also stimulated APCs to differentiate into mature endothelial cells and caused earlier reendothelialization compared with controls (31+/-5 versus 8+/-2 CD31-positive cells per millimeter of neointimal surface on day 14; Ptextless0.01). CONCLUSIONS: Similar to embryonic stem cell-derived progenitors, the adult BM and peripheral blood harbor APCs that are at least bipotential and able to differentiate into endothelial and smooth muscle lineages. The PPAR-gamma agonist rosiglitazone promotes the differentiation of these APCs toward the endothelial lineage and attenuates restenosis after angioplasty.

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

The myeloproliferative disorder of mice lacking the Src homology 2 (SH2)-containing 5' phosphoinositol phosphatase, SHIP, underscores the need for closely regulating phosphatidylinositol 3-kinase (PI3K) pathway activity, and hence levels of phosphatidylinositol species during hematopoiesis. The role of the 3' phosphoinositol phosphatase Pten in this process is less clear, as its absence leads to embryonic lethality. Despite Pten heterozygosity being associated with a lymphoproliferative disorder, we found no evidence of a hematopoietic defect in Pten(+/-) mice. Since SHIP shares the same substrate (PIP(3)) with Pten, we hypothesized that the former might compensate for Pten haploinsufficiency in the marrow. Thus, we examined the effect of Pten heterozygosity in SHIP(-/-) mice, predicting that further dysregulation of PIP(3) metabolism would exacerbate the pheno-type of the latter. Indeed, compared with SHIP(-/-) mice, Pten(+/-)SHIP(-/-) animals developed a myelodysplastic phenotype characterized by increased hepatosplenomegaly, extramedullary hematopoiesis, anemia, and thrombocytopenia. Consistent with a marrow defect, clonogenic assays demonstrated reductions in committed myeloid and megakaryocytic progenitors in these animals. Providing further evidence of a Pten(+/-)SHIP(-/-) progenitor abnormality, reconstitution of irradiated mice with marrows from these mice led to a marked defect in short-term repopulation of peripheral blood by donor cells. These studies suggest that the regulation of the levels and/or ratios of PI3K-derived phosphoinositol species by these 2 phosphatases is critical to normal hematopoiesis.

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE x CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.