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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

In the past,the analysis of primitive human hematopoietic progenitor cells with repopulating activity was limited by lack of appropriate in vitro assay systems. It was recently shown that cobblestone area-forming cells (CAFC) giving rise to cobblestone areas after 5 weeks in long-term marrow cultures (LTMC) represent a population of pluripotent progenitor cells with long-term marrow-repopulating activity. We have used a microtiter limiting dilution-type human LTMC system to quantitate the frequency of CAFC (week 5) in aplastic anemia (AA). In bone marrow mononuclear cells (BM-MNC) of healthy donors (n = 36) we observed a mean frequency of 84.4 CAFC per 10(5) BM-MNC (95% confidence interval limits,66.4 to 102.4). The mean frequency of CAFC in BM of 31 AA patients was 6.6 per 10(5) BM-MNC (95% confidence interval limits,5.3 to 7.9; n = 47). This frequency is significantly lower as compared with controls (P textless .0001). The frequency of CAFC was reduced not only in pancytopenic AA patients (6.2 per 10(5) BM-MNC; P textless .0001 v control),but also in patients in remission after immunosuppression (7.6; P textless .0001 v control; P = .1 v pancytopenic AA patients). The CAFC frequency did not correlate with the severity or duration of the disease and did not predict response to immunosuppressive treatment. In summary,the frequency of primitive hematopoietic progenitor cells,as measured by the CAFC assay,is significantly reduced in AA. CAFC remain severely reduced even after hematologic recovery after immunosuppressive treatment. The low frequency of CAFC in remission patients is in keeping with other data pointing to a persisting defect of hematopoiesis in patients in remission after immunosuppressive treatment. View Publication

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2),IL-7,and IL-15 cytokines,which share gamma c receptor subunit,in NK cell differentiation,we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF),IL-2,and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells,while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors,it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients. View Publication

We describe a new procedure for large-scale CB processing in the collection bag,thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C,the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal,the cell pellet was resuspended and the percent recovery of total WBC,CD34+ progenitor cells,CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300,600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC,CFU,CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy,effective and particularly suitable for large-scale banking under GMP conditions. View Publication

The immunosuppressant,rapamycin,inhibits cell growth by interfering with the function of a novel kinase,termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein,PHAS-1,in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line,which was selected for resistance to the growth-inhibitory action of rapamycin,was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast,the PI 3-kinase inhibitor,wortmannin,reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM),wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor,LY294002,at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR,and potentially those of other high molecular weight PI 3-kinase homologs,are directly affected by cellular treatment with wortmannin or LY294002. View Publication