技术资料

The metabolism of oxygen,although central to life,produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer,cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny,and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably,subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing,CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that,similar to normal tissue stem cells,subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny,which may contribute to tumour radioresistance. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. 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

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. View Publication

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells,they are unlike any normal cells of that lineage. Moreover,the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago,the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation,however,has never been corroborated,and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain,the memory B-cell antigen CD27,and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells,sharing immunoglobulin gene rearrangements with lymph node HRS cells,were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear,these data suggest they may be the initiating cells for HL. View Publication

PURPOSE: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here,we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). EXPERIMENTAL DESIGN: To identify the source of cell-free DNA in blood,plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs,and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). RESULTS: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0,n = 69) and metastasized prostate cancer (stage M1,n = 12; P = 0.03) and with the tumor stage of these patients (P textless 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients,respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood,respectively. The occurrence of CTCs correlated with tumor stage (P textless 0.03) and increasing Gleason scores (P = 0.04). Notably,significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03),D9S171 (P = 0.04),and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin,the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1,respectively,were observed. CONCLUSIONS: These findings show,for the first time,a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood,which,therefore,might become a valuable new source for monitoring metastatic progression in cancer patients. 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

BACKGROUND: Multiple measures of endothelial progenitor cells (EPCs) have been described,but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies. METHODS: We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients,EPCs were also identified on the basis of cell surface marker expression (CD133,CD34,and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity. RESULTS: Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however,endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133,CD34,CD133/CD34,and ALDH activity correlated with each other,but not with VEGFR-2(+) cells. CONCLUSIONS: Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells,which give rise to endothelial CFUs,and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research. View Publication

BACKGROUND: Interest in the biology of endogenous progenitor cells (EPCs) continues to grow as evidence of their role in vascular repair mounts. EPC enumeration requires specialized laboratory techniques and is performed immediately after sample acquisition,limiting the clinical contexts in which EPC enumeration can be performed and the ability to increase sample sizes through multi-center participation. METHODS: We compared the numbers of EPCs enumerated in samples processed immediately after acquisition (n = 36) with EPCs enumerated in specimens stored for 24 hours or after cryopreservation of mononuclear cells (MNC) using two EPC identification strategies: cell surface marker expression (CD133/CD34) and aldehyde dehydrogenase activity (ALDH(br) cells). RESULTS: EPCs assessed in fresh samples correlated with EPCs enumerated after whole blood storage (r = 0.699 for CD133(+)CD34(+) cells,r = 0.880 for ALDH(br) cells,P textless 0.005 and P textless 0.0001,respectively) or mononuclear cryopreservation (r = 0.590 for CD133(+)CD34(+) cells,r = 0.894 for ALDH(br) cells,P textless 0.0001 for each); however,correlation based on assessment of ALDH(br) cells was higher (P textless 0.0003 for comparison of correlation coefficients). Initial results from a multi-site clinical trial suggest that EPC enumeration after mononuclear cell cryopreservation is feasible. CONCLUSION: EPC analysis based on ALDH activity is reproducible,even after extended whole blood storage or MNC cryopreservation. 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