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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

MUC-1 mucin is considered to be aberrantly glycosylated in breast,ovary,and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation,we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1,MTSV1-7,and HB-2) derived from cells cultured from human milk,with three breast cancer cell lines (MCF-7,BT-20,and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1,and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern,with NeuAcalpha2-3Galbeta1-3GalNAc-ol,Galbeta1-3GalNAc-ol,and GalNAc-ol predominating; in contrast,MUC-1 from MTSV1-7 had more complex structures,including a number of disialo,core 2 species,i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler,and fewer,carbohydrate chains than MUC-1 from normal breast epithelial cells,and that these differences,combined or separately,explain the differential tumor specificity of some MUC-1 antibodies and T cells. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. 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