技术资料

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian,Botryllus schlosseri,vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues,gametes,or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent,self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates,but not to both,is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele,these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance. View Publication

CD154 (CD40 ligand) expression on CD4 T cells is normally tightly controlled,but abnormal or dysregulated expression of CD154 has been well documented in autoimmune diseases,such as systemic lupus erythematosus. Beyond regulation by NFAT proteins,little is known about the transcriptional activation of the CD154 promoter. We identified a species-conserved purine-rich sequence located adjacent to the CD154 transcriptional promoter proximal NFAT site,which binds early growth response (Egr) transcription factors. Gel shift assays and chromatin immunoprecipitation assays reveal that Egr-1,Egr-3,and NFAT1 present in primary human CD4 T cells are capable of binding this combinatorial site in vitro and in vivo,respectively. Multimerization of this NFAT/Egr sequence in the context of a reporter gene demonstrates this sequence is transcriptionally active upon T cell activation in primary human CD4 T cells. Overexpression of Egr-1,but not Egr-3,is capable of augmenting transcription of this reporter gene as well as that of an intact CD154 promoter. Conversely,overexpression of small interfering RNA specific for Egr-1 in primary human CD4 T cells inhibits CD154 expression. Similarly,upon activation,CD154 message is notably decreased in splenic CD4 T cells from Egr-1-deficient mice compared with wild-type controls. Our data demonstrate that Egr-1 is required for CD154 transcription in primary CD4 T cells. This has implications for selective targeting of Egr family members to control abnormal expression of CD154 in autoimmune diseases such as systemic lupus erythematosus. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

Many tumors,including Hodgkin's lymphoma,are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)),a known inhibitor of CD4+ T cell activation,suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells,we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed,PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence,PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly,CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma. View Publication

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development,molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells,indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame,suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast,in alphabeta cells,the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were,however,depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell,suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34,and plateaued at the early double positive stage. Surprisingly,however,some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway,an instructional role for the TCR in the alphabeta/gammadelta lineage choice,and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci,the timing of beta selection,and maintenance of gammadelta potential through the early double positive stage of development. View Publication