Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.     

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb3, and Gb4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB3H4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were GM3 and IV3NeuAcnLc4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Role for up-regulated ganglioside biosynthesis and association of Src family kinases with microdomains in retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Mouse F9 embryonal carcinoma cells have been widely used as a model for studying the mechanism of embryonic differentiation, because they are similar to the inner cell mass of early mouse embryos and can differentiate into primitive endoderm (PrE) following retinoic acid (RA) treatment. During F9 cell differentiation, the carbohydrate chains of glycoproteins and their corresponding glycosyltransferases are known to undergo rapid changes. However, there have been no corresponding reports on the expression of gangliosides. We have developed a custom cDNA array that is highly sensitive for the genes responsible for sphingolipid (SL) biosynthesis and metabolism. Using this, we found that, of the 28 selected genes, 26 exhibited increased expression during F9 differentiation into PrE. Although neutral glycosphingolipids (GSLs) were expressed at similar levels before and after differentiation, a greater than 20-fold increase in total ganglioside content was evident in PrE. Glucosylceramide synthase inhibitors (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol [d-PDMP] and its analog) depleted gangliosides and this resulted in delayed expression of Disabled-2 (Dab-2), suggesting the involvement of gangliosides in F9 cell differentiation. Disruption of cholesterol-enriched membrane microdomains by methyl-beta-cyclodextrin (MbetaCD) also delayed differentiation. Both MbetaCD and d-PDMP blocked the accumulation of Src family kinases (SFKs) to microdomains. However, d-PDMP did not block flotillin accumulation, yet MbetaCD did. Additionally, confocal laser microscopy revealed the formation of distinct functional microdomains integrating SFKs with gangliosides and cholesterol during PrE differentiation. Thus, we demonstrate the outstanding up-regulation of ganglioside biosynthesis and its importance in the formation of distinct microdomains incorporating SFKs with gangliosides during RA-induced differentiation of F9 cells.     

Stemming the tide: glycosphingolipid synthesis inhibitors as therapy for storage diseases

Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that targets the major synthesis pathway of GSLs to stem their cellular accumulation.     

Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells

Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs. [BMB Reports 2013; 46(11): 527-532]     

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.     

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.     

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.     

Glycosaminoglycans as regulators of stem cell differentiation

ES (embryonic stem) cell differentiation is dependent on the presence of HS (heparan sulfate). We have demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAG (glycosaminoglycan) expression. For example, different HS epitopes are synthesized during neural or mesodermal lineage formation. Cell lines mutant for various components of the HS biosynthetic pathway are selectively impaired in their differentiation, with lineage-specific effects observed for some lines. We have also observed that the addition of soluble GAG saccharides to cells, with or without cell-surface HS, can influence the pace and outcome of differentiation, again highlighting specific pattern requirements for particular lineages. We are combining this work with ongoing studies into the design of artificial cell environments where we have optimized three-dimensional scaffolds, generated by electrospinning or by the formation of hydrogels, for the culture of ES cells. By permeating these scaffolds with defined GAG oligosaccharides, we intend to control the mechanical environment of the cells (via the scaffold architecture) as well as their biological signalling environment (using the oligosaccharides). We predict that this will allow us to control ES cell pluripotency and differentiation in a three-dimensional setting, allowing the generation of differentiated cell types for use in drug discovery/testing or in therapeutics.     

Distinctive patterns of cAMP-binding proteins and their relationship to hemopoietic cellular differentiation

In order to clarify distinctions among cAMP-binding protein patterns present in hemopoietic cells of the various major lineages, we have studied extracts from 18 human cell lines by photoaffinity labelling with 8-azido [32P]cAMP, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Four major cAMP-binding protein bands were noted. These occurred in five recognizable patterns of combinations, each of which was restricted to cells of particular lineages. A distinctive pattern was found in the pluripotent stem cell line K562, confirming its unusual nature compared with other lines committed to myeloid or lymphoid differentiation. Three patterns were noted among the pre-B and early and late B cell lines studied, which may thus define sequential stages of differentiation of this series. These studies indicate the utility of cAMP-binding proteins as a biochemical differentiation marker system. The variety of phenotypes noted further suggests a role for them in the development or expression of specialized cellular functions.     

Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and-?4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.     

A hemopoietic cell line dependent upon a factor in pokeweed mitogen-stimulated spleen cell conditioning medium

A continously growing cell line (FMP1.1) has been isolated, which is dependent upon a factor in PWCM for both growth and survival. FMP1.1 appears to be a mast cell, since IgE receptors are present and their graunles react specifically with a mast cell granule stain. The factor in PWCM may be a glycoprotein and has biochemical properties in common with PWCM factors, which stimulate at least four other lineages of hemopoietic colonyforming cell. This line provides a pure population of cells as a model for studying molecular events of hemopoietic precursor cell growth and differentiation.     

Gangliosides are essential for bovine adeno-associated virus entry

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. We have recently identified a bovine adeno-associated virus (BAAV) that demonstrates unique tropism and transduction activity compared to primate AAVs. To better understand the entry pathway and cell tropism of BAAV, we have characterized the initial cell surface interactions required for transduction with BAAV vectors. Like a number of AAVs, BAAV requires cell surface sialic acid groups for transduction and virus attachment. However, glycosphingolipids (GSLs), not cell surface proteins, were required for vector entry and transduction. Incorporation of gangliosides, ceramide-based glycolipids containing one or more sialic acid groups, into the cytoplasmic cell membranes of GSL-depleted COS cells partially reconstituted BAAV transduction. The dependency of BAAV on gangliosides for transduction was further confirmed by studies with C6 cells, a rat glioma cell line that is deficient in the synthesis of complex gangliosides. C6 cells were resistant to transduction by BAAV. Addition of gangliosides to C6 cells prior to transduction rendered the cells susceptible to transduction by BAAV. Therefore, gangliosides are a likely receptor for BAAV.     

Regulation of blood cell diferentiation.

The hemopoietic (blood forming) system contains pluripotent stem cells able to give rise to a variety of differentiated progeny, including erythrocytes, granulocytes, megakaryocytes, monocytes, macrophages, and possible other cell types. Although a good deal is known about cell lineage relationships in the hemopoietic system, only limited information is available about the mechanisms regulating the proliferation and differentiation of the stem cells and their progeny. An approach to this latter problem has been provided by the develoment of new techniques for the cultivation of hemopoietic cells in short-term cultures. In such cultures, the proliferation and differentiation of hemopoietic cells can be studied under controlled conditions. Two areas of investigation show particular promise: elucidation of the role of the cell surface membrane in regulation; and the possible development, through a detailed investigation of the properties of leukoviruses, of new methods for the genetic analysis of hemopoietic cells.     

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.     

Ikaros isoform x is selectively expressed in myeloid differentiation

The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are expressed in early human hematopoietic cells (Lin(-)CD34(+)). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin(-)CD34(+) cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating lineage commitment decisions in mice and humans.     

Flexible association of hemopoietic differentiation programs in multilineage colonies

Pluripotent hemopoietic progenitors lose potentialities during the process of differentiation. We have examined events that lead to lineage restriction by determining the cellular composition of 785 multilineage colonies grown from peripheral blood samples of glucose-6-phosphate-dehydrogenase (G-6-PD) heterozygous volunteers. Of these colonies, 762 contained only one isoenzyme type and were considered to be of clonal origin. A considerable heterogenity was observed. Some colonies were composed of cells belonging to two different lineages, while other colonies contained three or more different cell types. A small number of colonies consisted—in addition to myeloid cells—of T-lymphocytes. The variable association within individual colonies of members belonging to different hemopoietic lineages suggests a flexible determination and expression of differentiation programs by early progenitors.     

Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas

There have been a number of studies on tumor-specific glycolipid antigens. In particular, neuroectoderm-derived cancers express characteristic ganglioside antigens, some of them have been used as tumor markers and/or target of immunotherapy. Molecules in the signaling pathway activated by gangliosides have been analyzed. Here, we reported results on the functions of molecules involved in the signaling pathway to enhance malignant properties of human melanomas under GD3 expression, and emphasized that those molecules including tumor-associated antigens can be targets of therapeutics for malignant melanomas.