技术资料

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified,but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved,we performed Northern analyses on RNA extracted from human marrow LTC adherent layers,or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta),IL-6,and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF,GM-CSF,and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast,transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC,and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures,respectively. Together,these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC,with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF,GM-CSF,and IL-6. Given the similarities between the LTC system and the marrow microenvironment,it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. View Publication

Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells,however,have hardly been investigated. In the present study,the ability of trichostatin A (TSA),a prototype hydroxamate HDI,to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA,it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity,whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1,CYP2B1 and CYP3A11 protein and mRNA levels,respectively,further revealed that TSA acts at the transcriptional level. In addition,protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4alpha) and CCAAT/enhancer binding protein alpha (C/EBPalpha) were accordingly increased by TSA throughout culture time. In conclusion,these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs. View Publication

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined,undifferentiated ESC in culture. In each dataset,we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets,despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest,we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728at,8430410A17Rik,Klf2,Nr0b1,Sox2,Tcl1,and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis,this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types. View Publication

BACKGROUND: We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study,we seek to apply Aldefluor flow cytometry-based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines,which may become a new tool that will facilitate our continued research in this field. EXPERIMENTAL DESIGN: Several established lung cancer cell lines were used,including A549 cell line expressing siRNA against aldehyde dehydrogenase class-1A1 (ALDH1A1). Western blot analysis,spectrophotometry assay,and Aldefluor staining were used to measure protein or enzyme activity in these cell lines. For the purpose of measurement of ALDH activity by Aldefluor in cells with known high ALDH levels,cells were mixed 1:10 with immortalized lung epithelial cell line (Beas-2B),which is known to lack ALDH activity. To delineate dead cells,double staining using Aldefluor and propidium iodide (PI) was done. Double staining was also used to detect changes in ALDH activity in two different cell lines after treatment with 4-hydroperoxycyclophosphamide (4-HC). RESULTS: Our results show a very good correlation between Aldefluor,Western blot,and spectrophotometry assays. Mixing experiments with Beas-2B cells allowed accurate assessment of ALDH activity in A549 cells at baseline and after siRNA expression,thus establishing an approach that facilitates the measurement of very high ALDH using the Aldefluor assay. Aldefluor staining was able to detect heterogeneity in ALDH expression among as well as within the same cell lines and better assess viability after 4-HC treatment when combined with PI. CONCLUSIONS: Aldefluor assay can be adapted successfully to measure ALDH activity in lung cancer cells and may have the advantage of providing real time changes in ALDH activity in viable cells treated with siRNA or chemotherapy. View Publication

In previous reports,we have demonstrated that only direct cell-cell contact with stromal cells,such as the murine stromal cell line AFT024,was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment,we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose,we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours,human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g.,fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC,thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism. View Publication

The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First,modulation of SIRT1 activity affects lipid accumulation in adipocytes,which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second,activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening,the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes,as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents,reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition,the same compounds have anti-inflammatory properties,as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast,a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes,a feature often linked to insulin sensitization. View Publication

The immunosuppressive macrolide rapamycin shows marked structural similarity to FK-506,and like FK-506 inhibits the activation of cultured T and B lymphocytes at concentrations as low as 10(-10) M. However,rapamycin blocks T-lymphocyte proliferation at a much later stage than FK-506. It also inhibits human,porcine and murine T- and B-lymphocyte activation by all pathways tested,including pathways which are insensitive to FK-506,such as interleukin-2 (IL-2)-mediated proliferation of IL-2-dependent T-cell lines,activation of human peripheral blood T lymphocytes by phorbol ester and anti-CD28 and activation of murine B lymphocytes by bacterial lipopolysaccharide. Thus these two macrolides that bind competitively to the same major intracellular receptor protein inhibit T- and B-lymphocyte activation by quite distinct mechanisms. View Publication

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor,which encodes neurofibromin,a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-,beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly,when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells,concomitant loss of beta c-/- reduced the severity of the MPD,but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML,our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor. View Publication

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast,T cells with other antigen specificities from these patients,or autoreactive T cells from healthy individuals and disease controls,express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells,Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2,SAP97,ZIP,p56(lck),and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation,they suppress Ca2+-signaling,cytokine production,and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted. View Publication

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle,SC1,was discovered that allows one to propagate murine ES cells in an undifferentiated,pluripotent state under chemically defined conditions in the absence of feeder cells,serum,and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy,but also provide previously undescribed insights into the complex biology of stem cells. View Publication

A remarkable interdisciplinary effort has unraveled the WNT (Wingless and INT-1) signal transduction cascade over the last two decades. Wnt genes encode small secreted proteins that are found in all animal genomes. Wnt signaling is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues. Germline mutations in the Wnt pathway cause several hereditary diseases,and somatic mutations are associated with cancer of the intestine and a variety of other tissues. View Publication