参考文献

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are,in part,controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis,we have compared gene expression profiles of human erythroblasts,megakaryocytes,B cells,cytotoxic and helper T cells,natural killer cells,granulocytes,and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors,immunoglobulin superfamily members,and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude,ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition,we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg,GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data,which are freely accessible,will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. View Publication

AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4(+)CD7(-) Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1-mediated transformation,microarray analysis was performed to identify differentially expressed genes in AHI-1-suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4(+)CD7(-) Sezary cells from Sezary syndrome patients. HCK and BIN1 emerged as new candidate cooperative genes,with differential protein expression,which correlates with observed transcript changes. Interestingly,changes in HCK phosphorylation and biologic response to its inhibitor,dasatinib,were observed in AHI-1-suppressed or -overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells,which also exhibit differential MYC protein expression. In addition,aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells. View Publication

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes,leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen,the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells,established B-leukemia cell lines,and animal models. In CLL cells,silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration,there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood,silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage,as evidenced by reactive oxygen species generation and membrane depolarization. In vivo,silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias. View Publication

Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3(+) regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However,it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here,we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2,an isoform of Aldh1a,but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2,but not the other known RA-producing enzymes. Employing a flow cytometric method,we detected ALDH activities in 10-30% of PP-DCs and MLN-DCs. They were CD11c(high)CD4(-/low)CD8alpha(intermediate)CD11b(-/low) F4/80(low/intermediate)CD45RB(low)CD86(high)MHC class II(high)B220(-)CD103(+). Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs,however,additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2(+) DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria,whereas IL-4 receptor-mediated signals were dispensable. GM-CSF(+)CD11c(-)F4/80(+) cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA. View Publication

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here,we show that MLL normally regulates expression of mir-196b,a hematopoietic microRNA located within the HoxA cluster,in a pattern similar to that of the surrounding 5' Hox genes,Hoxa9 and Hoxa10,during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage,mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b,while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore,overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival,as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. View Publication

Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a procarcinogenic cytokine,IL-23,while inhibiting a central anticarcinogenic cytokine,IL-12,thereby shifting the balance of tumor immunity toward carcinogenesis. Stat3 induces expression of IL-23,which is mainly produced by tumor-associated macrophages,via direct transcriptional activation of the IL-23/p19 gene. Furthermore,Stat3 inhibits NF-kappaB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Tregs) express IL-23 receptor,which activates Stat3 in this cell type,leading to upregulation of the Treg-specific transcription factor Foxp3 and the immunosuppressive cytokine IL-10. These results demonstrate that Stat3 promotes IL-23-mediated procarcinogenic immune responses while inhibiting IL-12-dependent antitumor immunity. View Publication

Antibody-based therapies,such as rituximab and alemtuzumab,have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas,diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy. View Publication

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans,the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD,which extends over many months,is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC,CD1a(-)CD14(+) DC,and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro,each subset has characteristic properties. After transplantation,both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells,but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells,macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages,although unlikely to initiate alloreactivity,may contribute to GVHD by sustaining the responses of previously activated T cells. View Publication

Alcohol abuse predisposes the host to bacterial infections. In response to bacterial infection,the bone marrow hematopoietic activity shifts toward granulocyte production,which is critical for enhancing host defense. This study investigated the hematopoietic precursor cell response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in BALB/c mice 30 min before initiation of Escherichia coli bacteremia. Bacteremia caused a significant increase in the number of bone marrow lineage (lin(-))-c-kit(+)Sca-1(+) cells. Marrow lin(-)c-kit(+)Sca-1(+) cells isolated from bacteremic mice showed an increase in CFU-granulocyte/macrophage activity compared with controls. In addition to enhanced proliferation of lin(-)c-kit(+)Sca-1(+) cells as reflected by BrdU incorporation,phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells primarily accounted for the rapid increase in marrow lin(-)c-kit(+)Sca-1(+) cells following bacteremia. Bacteremia increased plasma concentration of TNF-alpha. Culture of marrow lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells with murine rTNF-alpha for 24 h caused a dose-dependent increase in conversion of these cells to lin(-)c-kit(+)Sca-1(+) cells. Sca-1 mRNA expression by the cultured cells was also up-regulated following TNF-alpha stimulation. Acute alcohol intoxication inhibited the increase in the number of lin(-)c-kit(+)Sca-1(+) cells in the bone marrow after E. coli infection. Alcohol impeded the increase in BrdU incorporation into marrow lin(-)c-kit(+)Sca-1(+) cells in response to bacteremia. Alcohol also suppressed the plasma TNF-alpha response to bacteremia and inhibited TNF-alpha-induced phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells in vitro. These data show that alcohol inhibits the hematopoietic precursor cell response to bacteremia,which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections. View Publication

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac),a part of the core enzyme,has two isoforms,alpha (PP2Ac alpha) and beta (PP2Ac beta),of which PP2Ac alpha is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases,but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac alpha promoter in human primary T cells. After preparing serially truncated PP2Ac alpha promoter luciferase constructs,we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore,we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phosphorylated CREB and the activity of the PP2Ac alpha promoter. In contrast,the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine,a DNA methyltransferase inhibitor,showed increased expression of PP2Ac alpha mRNA. We propose that conditions associated with hypomethylation of CpG islands,such as drug-induced lupus,permit increased PP2Ac expression. View Publication