参考文献

Adult-derived hippocampal progenitors generate neurons,astrocytes,and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal,it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking,we have followed individual cells through the process of proliferative expansion,commitment,and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid,elevated cyclic AMP,or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain. View Publication

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC),and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34+ CD38- normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained,and dependent on the presence of a preformed feeder layer. Interestingly,the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph+) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph+) counterparts. View Publication

Acute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo,we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1-. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1- phenotype. However,significant engraftment was achieved using both CD34+/Thy-1- and CD34- subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients,the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1. View Publication

Previous studies have shown that primitive human hematopoietic cells detectable as long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs) can be amplified when CD34(+) CD38(-) marrow cells are cultured for 10 days in serum-free medium containing flt3 ligand (FL),Steel factor (SF),interleukin (IL)-3,IL-6,and granulocyte colony-stimulating factor. We now show that the generation of these two cell types in such cultures is differentially affected at the single cell level by changes in the concentrations of these cytokines. Thus,maximal expansion of LTC-ICs (60-fold) was obtained in the presence of 30 times more FL,SF,IL-3,IL-6,and granulocyte colony-stimulating factor than could concomitantly stimulate the near-maximal (280-fold) amplification of CFCs. Furthermore,the reduced ability of suboptimal cytokine concentrations to support the production of LTC-ICs could be ascribed to a differential response of the stimulated cells since this was not accompanied by a change in the number of input CD34(+) CD38(-) cells that proliferated. Reduced LTC-IC amplification in the absence of a significant effect on CFC generation also occurred when the concentrations of FL and SF were decreased but the concentration of IL-3 was high (as compared with cultures containing high levels of all three cytokines). To our knowledge,these findings provide the first evidence suggesting that extrinsically acting cytokines can alter the self-renewal behavior of primary human hematopoietic stem cells independent of effects on their viability or proliferation. View Publication

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to textgreater 100 Mk,detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions,the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However,optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors,of which a combination of IL-3,IL-6,GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+� View Publication

The immunosuppressive effects of microcolin A,a lipopeptide extracted from the marine blue green alga Lyngbya majuscula were investigated. Microcolin A suppressed concanavalin A (IC50 = 5.8 nM),phytohemagglutinin (IC50 = 12.5 nM) and lipopolysaccharide (IC50 = 8.0 nM) induced proliferation of murine splenocytes. Mixed lymphocyte reaction (IC50 = 5.0 nM),anti-IgM (mu-chain specific) (IC50 = 10.0 nM),and phorbol 12-myristate 13-acetate plus ionomycin (IC50 = 5.8 nM) stimulation of murine splenocytes were all similarly suppressed by microcolin A. The inhibitory activity of microcolin A was time-dependent and reversible and was not associated with a reduction in cell viability. Moreover,microcolin A not only inhibited IL-2 production and IL-2 receptor expression by concanavalin A activated splenocytes,but also suppressed in vitro antibody responsiveness to keyhole limpet hemocyanin. These results indicate that microcolin A is a potent immunosuppressive and antiproliferative agent. View Publication

It is well established that mitogens inhibit differentiation of skeletal muscle cells,but the insulin-like growth factors (IGFs),acting through a single receptor,stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types,little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway,we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059,an inhibitor of mitogen-activated protein (MAP) kinase kinase activation,inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it,such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly,PD098059 caused a dramatic enhancement of differentiation,evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression,as well as creatine kinase activity). In sharp contrast,LY294002,an inhibitor of phosphatidylinositol 3-kinase,and rapamycin,an inhibitor of the activation of p70 S6 kinase (p70(S6k)),completely abolished IGF stimulation of L6A1 differentiation. We found that p70(S6k) activity increased substantially during differentiation,and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts,while activation of the phosphatidylinositol 3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation. Thus,it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation. View Publication

In a number of cell types,elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells,which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response. View Publication

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently,techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs,and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex),0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid,and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation,as determined by osteoblastic morphology,expression of alkaline phosphatase (APase),reactivity with anti-osteogenic cell surface monoclonal antibodies,modulation of osteocalcin mRNA production,and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex,0.05 mM AsAP,and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity,significantly more mineral was deposited in these cultures,suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore,cultures allowed to concentrate their soluble products in the media produced more mineralized matrix,thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium,dose of physiologic supplements,cell seeding density,and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts,and eventually terminally differentiated osteocytes. View Publication

Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P,the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel,ubiquitously expressed PtdIns 4-kinase (PI4Kbeta). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli,and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kbeta are characteristic of a type III enzyme. Interestingly,both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin,suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi,S.,Catt,J. K.,and Balla,T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,5317-5321). View Publication

Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel,R.,and Futerman,A. H. (1993) J. Biol. Chem. 268,14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz,A.,Rapaport,E.,Hirschberg,K.,and Futerman,A.H. (1995) J. Biol. Chem. 270,10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3,the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments,but fewer actin-rich stress fibers were observed,and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton,cytokinesis,microvilli formation,and the formation of long processes induced by protein kinase inhibitors,were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1,whereas the bioactive intermediates,sphinganine,sphingosine,and ceramide,were without effect. Finally,FB1 blocked cell proliferation and DNA synthesis in a reversible manner,although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together,these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton. View Publication

Resveratrol,a phytoalexin found in grapes and other food products,was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity). In addition,it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. These data suggest that resveratrol,a common constituent of the human diet,merits investigation as a potential cancer chemopreventive agent in humans. View Publication