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Angiotensin I-converting enzyme (ACE) inhibitors can affect hematopoiesis by several mechanisms including inhibition of angiotensin II formation and increasing plasma concentrations of AcSDKP (acetyl-N-Ser-Asp-Lys-Pro),an ACE substrate and a negative regulator of hematopoiesis. We tested whether ACE inhibition could decrease the hematopoietic toxicity of lethal or sublethal irradiation protocols. In all cases,short treatment with the ACE inhibitor perindopril protected against irradiation-induced death. ACE inhibition accelerated hematopoietic recovery and led to a significant increase in platelet and red cell counts. Pretreatment with perindopril increased bone marrow cellularity and the number of hematopoietic progenitors (granulocyte macrophage colony-forming unit [CFU-GM],erythroid burst-forming unit [BFU-E],and megakaryocyte colony-forming unit [CFU-MK]) from day 7 to 28 after irradiation. Perindopril also increased the number of hematopoietic stem cells with at least a short-term reconstitutive activity in animals that recovered from irradiation. To determine the mechanism of action involved,we evaluated the effects of increasing AcSDKP plasma concentrations and of an angiotensin II type 1 (AT1) receptor antagonist (telmisartan) on radioprotection. We found that the AT1-receptor antagonism mediated similar radioprotection as the ACE inhibitor. These results suggest that ACE inhibitors and AT1-receptor antagonists could be used to decrease the hematopoietic toxicity of irradiation. View Publication

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly,purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic,adipocytic,and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore,mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models. View Publication

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment,and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells,viral gene expression was undetectable by 10 days after infection. Importantly,viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly,a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency. View Publication

The fate of pluripotent stem cells is tightly controlled during early embryonic development. Both the derivation and the maintenance of embryonic stem cells (ES cells) in vitro depend on feeder cell-derived growth factors that are largely unidentified. To dissect the mechanisms governing pluripotency,we conducted a screen to identify factors that are produced by mouse embryonic fibroblast STO cells and are required to maintain the pluripotency of ES cells. One of the factors is bone morphogenetic protein 4 (BMP4). Unexpectedly,the major effect of BMP4 on the self-renewal of ES cells is accomplished by means of the inhibition of both extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways,and inhibitors of ERK and p38 MAPKs mimic the effect of BMP4 on ES cells. Importantly,inhibition of the p38 MAPK pathway by SB203580 overcomes the block in deriving ES cells from blastocysts lacking a functional Alk3,the BMP type IA receptor. These results uncover a paradigm for BMP signaling in the biology of pluripotent stem cells. View Publication

Malignant pleural mesothelioma is an uncommon tumor largely confined to the thoracic cavity,which is resistant to conventional therapies,therefore prompting an intensive search for effective treatment alternatives. This study focuses on dendritic cell (DC) vaccination for malignant pleural mesothelioma and evaluates the in vitro efficacy of antigen-loaded DC-based vaccines for the induction of major histocompatibility complex Class I-restricted antimesothelioma cytotoxic T lymphocyte responses. The source of tumor-associated antigens for HLA-A2(+) DCs from healthy donors was apoptotic HLA-A2(-) mesothelioma cells either lacking or expressing heat shock protein 70 according to whether tumor cells were heat shocked or not before ultraviolet-mediated apoptosis. Our results show that both apoptotic preparations were equivalent regarding the responsiveness of DCs to combined treatment with tumor necrosis factor-alpha and poly(inosinic-cytidylic) acid,as determined by similar increased expression of costimulatory molecules and interleukin-12 production. However,only DCs loaded with apoptotic heat shock protein 70-expressing cells were found to be potent in vitro inducers of cytotoxic T lymphocyte activity against HLA-A2(+) mesothelioma cells. Such elicited cytotoxic T lymphocytes also exhibit cytotoxic activity against an HLA-A2(+) melanoma cell line,suggesting recognition of shared antigens. These findings therefore carry the potential of offering an alternative,promising approach for the therapy of patients with malignant pleural mesothelioma. View Publication

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere,flatten,and spread underwent osteogenesis,while unspread,round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes,while constitutively active RhoA caused osteogenesis. However,the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape,respectively,while constitutive activation of the RhoA effector,ROCK,induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts,embodied by cell shape,cytoskeletal tension,and RhoA signaling,are integral to the commitment of stem cell fate. View Publication

Loss-of-function mutations in the murine dominant white spotting/c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes,including severe anemia,defective pigmentation,sterility,mast cell deficits,a lack of interstitial cells of Cajal,spatial learning memory deficits,and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development,but are dispensable for the normal development of erythroid,interstitial cells of Cajal and germ cells. Furthermore,adult mice lacking both tyrosines exhibit splenomegaly,dysregulation of B-cell and megakaryocyte development,and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together,these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase. View Publication

Point mutations constitute a major mode of oncogenic activation of the Met receptor tyrosine kinase. Met is aberrantly activated in many types of human malignancies and its deregulated activity is correlated with aggressive tumor traits such as abnormal proliferation and survival,leading to tumor growth,local invasion and metastasis. Here we report that the Met kinase inhibitor SU11274 differentially affects the kinase activity and subsequent signaling of various mutant forms of Met. Two Met variants tested,M1268T and H1112Y,were potently inhibited by 2 microM SU11274,while two other variants,L1213V and Y1248H,remained resistant under similar experimental conditions. Inhibition of the kinase altered cell proliferation,morphology and motility,while cells containing resistant mutants appeared unaffected by the compound. The basis for the sensitivity or resistance to SU11274 is discussed in terms of the position of the mutations predicted from a homology model. View Publication

Erythropoietin (EPO) is the hormone necessary for development of erythrocytes from immature erythroid cells. EPO activates Jun N-terminal kinase (JNK),a member of the mitogen-activated protein kinase (MAPK) family in the EPO-dependent murine erythroid HCD57 cells. Therefore,we tested if JNK activity supported proliferation and/or survival of these cells. Treatment with the JNK inhibitor SP600125 inhibited JNK activity and EPO-dependent proliferation of HCD57 cells and the human EPO-dependent cell lines TF-1 and UT7-EPO. SP600125 also increased the fraction of cells in G2/M. Introduction of a dominant-negative form of JNK1 inhibited EPO-dependent proliferation in HCD57 cells but did not increase the fraction of cells in G2/M. Constitutive JNK activity was observed in primary murine erythroid progenitors. Treatment of primary mouse bone marrow cells with the SP600125 inhibitor reduced the number of erythroid burst-forming units (BFU-e's) but not the more differentiated erythroid colony-forming units (CFU-e's),and SP600125 protected the BFU-e's from apoptosis induced by cytosine arabinoside,demonstrating that the SP600125 inhibited proliferation of the BFU-e's. Therefore,JNK activity appears to be an important regulator of proliferation in immature,primary erythroid cells and 3 erythroid cell lines but may not be required for the survival or proliferation of CFU-e's or proerythroblasts. View Publication

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. View Publication

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. View Publication

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. View Publication