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Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein, a constitutively active tyrosine kinase. In previous studies, we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study, using UT-7/9 cells, a high level Bcr/Abl transfectant of UT-7 cells, we show that the treatment of Bcr/Abl target by imatinib mesylate (IM), a specific Abl tyrosine kinase inhibitor, hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets, as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor, particularly NKG2D. In addition, we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells, and IM treatment inhibits this activating pathway. Taken together, our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants.

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.

Histone methylation plays a key role in establishing and maintaining stable gene expression patterns during cellular differentiation and embryonic development. Here, we report the characterization of the fungal metabolite chaetocin as the first inhibitor of a lysine-specific histone methyltransferase. Chaetocin is specific for the methyltransferase SU(VAR)3-9 both in vitro and in vivo and may therefore be used to study heterochromatin-mediated gene repression.

Hedgehog (Hh) signaling is an important regulator of embryonic patterning, tissue regeneration, stem cell renewal and cancer growth. A purine derivative named purmorphamine was previously found to activate the Hh pathway and affect osteoblast differentiation through an unknown mechanism. We demonstrate here that purmorphamine directly targets Smoothened, a critical component of the Hh signaling pathway.

NK cell transcript 4 (NK4), now denoted as IL-32, was originally identified as a transcript whose expression was increased in activated NK cells. It has been very recently demonstrated that NK4 is secreted from several cells upon the stimulation of some inflammatory cytokines such as IL-18, IL-1beta, IFN-gamma and IL-12. Furthermore, NK4 induces production of tumor necrosis factor, macrophage inflammatory protein (MIP)-2 and IL-8 in monocytic cell lines, indicating that this factor would be involved in the inflammatory responses. Based on these findings, NK4 was renamed IL-32. However, the biological activities of IL-32 on other cell types remained undetermined. Furthermore, it was still argued whether IL-32 acts on cells from outside or inside the cells. In this article, we first report that expression of IL-32 was up-regulated in activated T cells and NK cells, and that IL-32beta was the predominantly expressed isoform in activated T cells. IL-32 was specifically expressed in T cells undergoing apoptosis and enforced expression of IL-32-induced apoptosis, whereas its down-regulation rescued the cells from apoptosis in HeLa cells. IL-32 existing in the supernatant would be derived from the cytoplasm of apoptotic cells. These results strongly indicated that IL-32 would be involved in activation-induced cell death in T cells, probably via its intracellular actions. Our present findings expand our understanding of the biological function of IL-32 and argue that IL-32 may act on cells, not only from the outside but also from the inside.

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.

Dendritic cell (DC) maturation state is a key parameter for the issue of DC-T cell cognate interaction, which determines the outcome of T cell activation. Indeed, immature DCs induce tolerance while fully mature DCs generate immunity. Here we show that, in the absence of any deliberate activation signal, DCs freshly isolated from mouse spleen spontaneously produce IL-12 and tumor necrosis factor-alpha and up-regulate co-stimulation molecules, even when directly re-injected into their natural environment. Furthermore, after their isolation, these cells acquire the capacity to induce specific T(h)1 responses in vivo. These results demonstrate that the sole isolation of spleen DCs leads to the full maturation of these cells, which therefore cannot be considered as immature DCs. Moreover, we also show that the kinetics of DC activation do not influence the polarization of T(h) response in vivo challenging the idea that exhausted DCs induce preferentially T(h)2 response. Altogether, these observations should be taken into account in all experiments based on the transfer of ex vivo purified DCs.

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice, we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression, colony formation by hematopoietic progenitors, and the peripheral white blood cell count. Menin directly activates Hoxa9 expression, at least in part, by binding to the Hoxa9 locus, facilitating methylation of H3K4, and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin, ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover, Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy.

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agents which targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion, without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly, Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation, followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity, suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly, phosphorylation of extracellular signal-related kinase (ERK) is increased by perifosine; conversely, MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore, perifosine augments dexamethasone, doxorubicin, melphalan, and bortezomib-induced MM cell cytotoxicity. Finally, perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model, associated with down-regulation of Akt phosphorylation in tumor cells. Taken together, our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM.

Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque, often at the site of T cell and macrophage infiltration. Here, we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell-mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2), and anti-TRAIL and anti-DR5 antibodies block T cell-mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs, which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis, a process which may lead to plaque destabilization.

Flt3 ligand (Flt3L) is a nonredundant cytokine in type I interferon-producing cell (IPC) and dendritic cell (DC) development, and IPC and DC differentiation potential is confined to Flt3+ hematopoietic progenitor cells. Here, we show that overexpression of human Flt3 in Flt3- (Flt3(-)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) and Flt3+ (Flt3(+)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) hematopoietic progenitors rescues and enhances their IPC and DC differentiation potential, respectively. In defined hematopoietic cell populations, such as Flt3- megakaryocyte/erythrocyte-restricted progenitors (MEPs), enforced Flt3 signaling induces transcription of IPC, DC, and granulocyte/macrophage (GM) development-affiliated genes, including STAT3, PU.1, and G-/M-/GM-CSFR, and activates differentiation capacities to these lineages. Moreover, ectopic expression of Flt3 downstream transcription factors STAT3 or PU.1 in Flt3- MEPs evokes Flt3 receptor expression and instructs differentiation into IPCs, DCs, and myelomonocytic cells, whereas GATA-1 expression and consecutive megakaryocyte/erythrocyte development is suppressed. Based on these data, we propose a demand-regulated, cytokine-driven DC and IPC regeneration model, in which high Flt3L levels initiate a self-sustaining, Flt3-STAT3- and Flt3-PU.1-mediated IPC and DC differentiation program in Flt3+ hematopoietic progenitor cells.