技术资料

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

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However,it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3,a small molecule inhibitor of the p53/MDM2 interaction,is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells,but not on normal CD19(+) B lymphocytes,peripheral-blood mononuclear cells,or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined,only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway,nutlin-3 up-regulated the steady-state mRNA levels of PCNA,CDKN1A/p21,GDF15,TNFRSF10B/TRAIL-R2,TP53I3/PIG3,and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover,nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL. View Publication

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice,with a corresponding retention of progenitors in blood. Remarkably,the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells,studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin,studied in parallel. Furthermore,the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies,in contrast to antibodies against alpha4 integrin,did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells. View Publication

Therapies that target signaling pathways critical to the pathogenesis and progression of squamous cell carcinoma of the head and neck (HNSCC) are needed. One such target,phosphatidylinositol 3-kinase,and its downstream target serine/threonine kinase,Akt,are up-regulated in HNSCC. Targeted therapy could consist of inhibitors of these kinases or,alternatively,of inhibitors of the pathways that they regulate. To explore the effect of Akt inhibition on the growth and survival of HNSCC tumors,we evaluated the effect of a novel Akt inhibitor,KP372-1,on the growth,survival,and sensitivity to anoikis of HNSCC cell lines in culture. Using Western blotting of head and neck cancer cell lines and squamous mucosa and carcinoma specimens,we found that Akt was highly phosphorylated in head and neck cancer cell lines and human head and neck squamous carcinoma specimens. Treatment of HNSCC cell lines with KP372-1 blocked the activation of Akt,inhibited head and neck cancer cell proliferation,and induced apoptosis and anoikis in several HNSCC cell lines. Furthermore,KP372-1 decreased the phosphorylation of the S6 ribosomal (Ser240/244) protein,which is a downstream target of Akt. Taken together,these findings indicate that KP372-1 may be a useful therapeutic agent for HNSCC and should be further evaluated in preclinical models of HNSCC. View Publication

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival,proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family,which mediate antiapoptotic signals. We show that three TRK ligands,brain-derived neurotrophic factor,neurotrophin 3 and neurotrophin 4,are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins,TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation. View Publication