Proc. Nati. Acad. Sci.

USA

Vol. 83, pp. 782-786, February 1986 Medical Sciences

Ganglioside GM3: An acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiation of human myeloid and monocytoid leukemic cell lines HL-60 and U937
(ganglioside/differentiation-induction/leukemia)

HISAo NoJiiu*, FUMIMARO TAKAKUt, YASUHITO TERUI*, YASUSADA MIURAt, AND MASAKI SAITO*
*Division of Hemopoiesis, Institute of Hematology and tDepartment of Hematology, Jichi Medical School, Minami-kawachi-machi, Kawachi-gun, Tochigi-ken, Japan 329-04; and tThird Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan 113

Communicated by Eugene P. Cronkite, September 16, 1985

ABSTRACT When human myeloid and monocytoid leukemic cell lines HL-60 and U937, respectively, were treated with an exogenous sialoglycosphingolipid, ganglioside GM3, in serum-free medium, cell growth was markedly inhibited, and their morphological maturation along a monocytic lineage was observed. In addition to a significant increase in phagocytic and nonspecific esterase activities, marked increase of monocytespecific surface antigens detectable with monoclonal antibodies such as OKM1 and OKM5 was observed in GM3-fed cells. Other sialoglycosphingolipids with the carbohydrate structure belonging to ganglio-series oligosaccharide, ganglioside GM1 and a brain ganglioside mixture, had no effect on the cell differentiation, showing instead stimulatory actions on the growth of these cell lines. We recently demonstrated that the ganglio-series ganglioside GM3 characteristically increased during macrophage-like cell differentiation of these cell lines. The present results indicate that ganglioside molecular species that specifically increase during monocytic cell differentiation of human myeloid and monocytoid leukemic cell lines may play, in turn, an important role in the differentiation-nduction of these cell lines along a monocytic cell lineage.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have been shown to be located almost exclusively at the outer leaflet of plasma membranes (1). Dramatic changes in GSL composition and metabolism have been observed during ontogenesis, differentiation, and oncogenic transformation, suggesting a specific role of membrane GSLs in regulation of cell growth and cellular interaction (2). GSLs are classified into three major series-i.e., ganglio-, globoand lacto-series, according to their carbohydrate structures. Human promyelocytic leukemia cell line HL-60 cells (3) have been induced to differentiate into mature granulocytes (4, 5) and macrophage-like cells (6) and consist of stem-like cells that are bipotent with respect to myeloid or macrophage differentiation (7). We recently demonstrated that HL-60 cells expressed distinct GSL profiles for two separate pathways of cell differentiation; (i) the lacto-series acidic sialoGSLs, or lacto-series gangliosides, having longer sugar moieties such as sialosylnorhexaosylceramide, increased with a concomitant decrease in the ganglio-series acidic sialo-GSL, or ganglio-series ganglioside, GM3 during granulocytic differentiation; and (ii) in marked contrast to such changes, ganglio-series ganglioside GM3 markedly increased with a concurrent decrease in lacto-series gangliosides in the process of macrophage-like cell differentiation (8). A characteristic increase in ganglio-sefies ganglioside GM3 was also
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observed during macrophage-like cell differentiation of other human myeloid and monocytic leukemic cell lines such as K562 (9), KG-1 (10), ML-1 (11), and U937 (12) (unpublished data). GSLs exogenously added to the cell culture medium are incorporated into plasma membranes, resulting in enrichment of a particular molecular species of GSL (13-15). Although some of the GSLs could be released by trypsin treatment (16), considerable evidence suggests that the ceramide moiety of the added GSLs is inserted in the lipid layer (17, 18). A recent study involving electron spin resonance measurements supported this organizational notion (19). In the present study, we investigated whether GSL molecules that specifically increased during cell differentiation exhibit any physiological functions in cell differentiation processes with this approach, and we demonstrated that exogenous ganglioside GM3 was highly potent for differentiation-induction of human myeloid and monocytoid leukemic cell lines HL-60 and U937 along a monocytic lineage. It is noteworthy that the ganglioside molecular species that characteristically increased during monocytic cell differentiation of human myeloid and monocytic leukemic cell lines in turn could specifically trigger cell differentiation of the same cell lines along the monocytic cell lineage.

MATERIALS AND METHODS

Ganglioside Preparation. GM3 and GM1 were prepared from dog erythrocytes and bovine brain, respectively, by solvent partition and DEAE-Sephadex chromatography in our laboratory by the method of Momoi et al. (20). Each ganglioside was found to be homogenous on silica gelprecoated high-performance TLC plates (Merck) after development with chloroform/methanol/0.5% CaCl2 (55:45:10, vol/vol/vol). The high-performance TLC plates were sprayed with resorcinol/HCl reagent, and gangliosides were visualized by heating the plates at 95C and were determined quantitatively by densitometric scanning as described (8). The lipid-bound sialic acid of gangliosides was estimated by the resorcinol/HCl method as modified by Suzuki (21). A bovine brain ganglioside mixture (Sigma, type II) and Nacetylneuraminic acid (from Escherichia coli) were purchased from Sigma. The bovine brain ganglioside mixture consisted of 7.5% GM1, 2.6% GD3, 56.7% GD1a, 11.5% GD1b, and 21.7% GT1b.
Abbreviations: GSL, glycosphingolipid; mAb, monoclonal antibody; NapBtOEase, a-napthyl butyrate esterase. The glycolipid nomenclature and symbols used are from an International Union of Biochemistry document [(1977) Eur. J. Biochem. 79, 11-21]. To whom reprint requests should be addressed.
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Cell Culture. Cells of human promyelocytic leukemia cell line HL-60 (3) and human monocytic leukemic cell line U937 (12) were grown in Falcon 3024 tissue culture flasks (Becton Dickinson Labware, Oxnard, CA) in a serum-free synthetic medium composed of equal volumes of Dulbecco's modified Eagle's minimal essential medium and Ham's F-12 medium supplemented with 30 nM selenium dioxide, 1.2 g of sodium bicarbonate per liter, 15 mM Hepes (pH 7.2), 5 Ug of insulin per ml (Sigma), and 5 pug of transferrin (Sigma) per ml (DME/F12 medium) according to the method described by Breitman et al. (22) at 370C in humidified 5% carbon dioxide/95% air. Cell counts were made with a hemocytometer, and viability was assessed by erythrosine B dye exclusion. Purified gangliosides were added to the cell culture medium essentially as described by Bremer et al. (23). Briefly, a ganglioside was dissolved in an appropriate volume of water with the aid of sonication, sterilized by passage through a sterilized 0.22-,um Millipore filter, and quantitated by the resorcinol HCl method. This solution was diluted with an equal volume of DME/F12 medium of 2-fold concentration to 1 mM and was added to the culture medium to the desired ganglioside concentration. Evaluation of Cell Differentiation. Cell differentiation of HL-60 and U937 cells treated with exogenously added gangliosides was assessed morphologically, cytochemically, and functionally. For morphological assessment of the cells, Cytospin slide preparations were prepared using a Shandon Cytospin centrifuge (Shandon Southern Products, Cheshire, England) and stained with Wright-Giemsa staining solution. For cytochemical assessment of cell differentiation, nonspecific esterase activity was determined cytochemically by the esterase double-staining method for a-naphthyl butyrate esterase (NapBtOEase) and naphthol AS-D-chloroacetate esterase as described by Li et al. (24). Functional differentiation was assessed on the basis of phagocytic activity of the cells. Phagocytic activity of undifferentiated and differentiation-induced cells was measured by counting the number of cells that phagocytosed latex or yeast particles as described (25). Polystyrene latex particles (Dow Chemical, Indianapolis; 1.09-,um diameter) were added to the cell suspension in RPMI 1640 medium and were left in contact with the cells for 60 min at 37C. The final concentrations were 1 x 1010 latex particles per ml and 0.5-1 x 106 cells per ml, respectively. Cells ingesting more than five latex particles were considered to be cells capable of phagocytosis, and at least 200 viable cells were examined in each experimental group. For the yeast-phagocytosis assay, yeast particles (Saccharomyces cerevisiae; Sigma) were added to the cell suspension in Hanks' balanced salt solution (HBSS) containing 5% fresh human serum and were left in contact with the cells for 30 min at 37C. The final concentrations were 6.25 x 107 yeast particles per ml and 2.5 x 106 cells per ml, respectively. After washing out the remaining yeast particles, the cells were stained with Ziehl's carbolfuchsin solution: 1 vol of a 11% fuchsin solution in ethanol and 10 vol of 5% phenol in distilled water were mixed and then diluted 1:10 with HBSS to obtain working Ziehl's carbolfuchsin solution. The yeast particles outside the cells were stained red, and the yeast particles completely ingested by the cells were prevented from taking up the stain. The cells containing unstained yeast particles were considered to be cells capable of phagocytosis, and at least 200 viable cells were examined in each experimental group. Reactivity with Monoclonal Antibodies. Reactivity of the untreated and GM3-treated cells with various monoclonal antibodies (mAbs) was investigated by flow cytometry. The mAbs used were: OKB2 (26), an IgG1 antibody that reacts with human granulocytes and B lymphocytes; OKM1 (27), an IgG2b antibody that reacts with human blood monocytes and granulocytes; and OKM5 (28), an IgG1 antibody that reacts

with human monocytes and platelets (Ortho Diagnostics). Antigen expression with mAbs was quantified by the indirect immunofluorescence method with cytofluorometry in Ortho Spectrum III (Ortho Diagnostics) as described by Talle et al.

(28).

RESULTS Effect of Exogenously Added Gangliosides on Cell Growth and Cell Differentiation of HL-60 and U937 Cells. The effect of exogenously added gangliosides on the growth of HL-60 and U937 cells was examined. When HL-60 cells were cultured with 50 nmol of exogenous ganglioside GM3 per ml, cell growth was completely inhibited. However, other ganglio-series gangliosides, GM1 and a bovine brain ganglioside mixture, showed instead a stimulatory action on cell growth, and free N-acetylneuraminic acid had no effect on cell growth at the same concentration (Fig. 1 Left). Similar effects of exogenous gangliosides were observed on U937 cells (Fig. 1 Right). The inhibition of growth of HL-60 cells by GM3 was accompanied by striking changes in the morphological characteristics of monocytic differentiation. After 6 days of culture, Wright-Giemsa staining of Cytospin cell preparations revealed the loss of cytoplasmic basophilia, lobulation of nuclei, and somewhat vacuolated cytoplasms with ruffled surface membranes, suggesting the differentiation into monocytic mature cells (Fig. 2B). About 20% of the untreated cells cultured in serum-free medium exhibited weak NapBtOEase activity, although only 3% of the original cells cultured in RPMI 1640 medium supplemented with 1o heat-inactivated fetal calf serum did so (Fig. 2C). When HL-60 cells were cultured with exogenously added GM3, the number of NBE-positive cells remarkably increased in a time-dependent manner, and the degree of NapBtOEase staining was much more intensive (Figs. 2D and 3). This esterase activity was completely inhibited by NaF, indicating the specificity for the monocytic lineage. However, other ganglio-series gangliosides and free N-acetylneuraminic acid had no effect on the number of NapBtOEase-positive cells (Fig. 3). The inhibition of growth of U937 cells by GM3 was also accompanied by morphological changes consistent with
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FIG. 1. Effect of exogenously added gangliosides on cell growth of HL-60 and U937 cells. HL-60 (Left) and U937 (Right) cells were seeded into Falcon 3024 tissue culture flasks at an initial concentration of 1.5 x 105 cells per ml and grown in serum-free hormonally defined DME/F12 medium containing no gangliosides (o), 50 1uM N-acetylneuraminic acid (e), 50 ,uM GM3 (A), 50 ,gM GM1 (A), or 50 ,uM bovine brain ganglioside mixture (c). Cell counting was performed with a hemocytometer, and viability was checked by the erythrosine B dye-exclusion test. Each data point represents the mean of three determinations. Standard deviations were <10%o. The viability was >90%o in each group throughout the culture periods.

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Proc. Natl. Acad. Sci. USA 83 (1986)

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tured in the serum-free medium with 50 AM exogenously added GM3 for 6 days. (Wright-Giemsa stain.) (C) NapBtOEase staining of untreated HL-60 cells cultured in the serum-free medium for 6 days. (D) NapBtOEase staining of HL-60 cells treated with 50 AiM
exogenously added GM3 for 6

days. free medium for 6HL-60 (WrightGiemsa stain.) (B) cells cul-

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the induction of a monocyte-macrophage phenotype (Fig. 4). GM3-treated U937 cells were characterized by an increase in cell size, a decrease in the nuclear/cytoplasmic ratio, a paler cytoplasm, more prominent granules, and a greater degree of vacuolization in the cytoplasm (Fig. 4B). Although U937 cells were originally positive as to NapBtOEase staining, the GM3-treated cells were stained more strongly for NapBtOEase activity (Figs. 4 C and D). This NapBtOEase activity was also completely inhibited by NaF. Other ganglio-series gangliosides and free N-acetylneuraminic acid had no effect on the morphology or NapBtOEase staining intensity. In addition to morphological differentiation, HL-60 and U937 cells treated with GM3 showed functional differentiationi.e., a significant increase in the capacity to phagocytose latex or yeast particles (Fig. 5). Changes of Cell-Surface Phenotypes Induced by Exogenous Gangliosides. Changes of the cell-surface antigens on HL-60
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and U937 cells caused by exogenously added gangliosides were evaluated by cytofluorography. The treatment of HL-60 cells with 50 ,uM exogenous GM3 for 6 days resulted in a marked increase in monocyte-specific antigens detectable with mAbs such as OKM1 and OKM5 with a concomitant decrease in granulocyte-specific epitope recognized by the mAb OKB2 (Table 1). Marked increases in the reactivity with monocyte-specific mAbs OKM1 and OKM5 and the mean fluorescent intensity of the positive cells were also observed in GM3-treated U937 cells (Table 1). No significant changes in the cell-surface phenotypes were observed in HL-60 and U937 cells treated with other ganglio-series gangliosides or free N-acetylneuraminic acid. Dose-Dependent Effect of Exogenous GM3 on Cell Growth and Cell Differentiation of HL-60 Cells. When HL-60 cells were cultured with various concentration of GM3, cell growth was suppressed in a dose-dependent manner (Fig. 6 Left). With 50 ,M GM3, cell growth was completely inhibited. After 6 days of incubation with various concentrations of exogenous GM3, the percentage of NapBtOEase-positive cells also varied in a dose-dependent manner, reaching a maximum of 84.8% at 50 ,AM (Fig. 6 Right). During the culture period, HL-60 cells constantly exhibited a viability of >90%o, indicating that the suppressive effect of GM3 on cell growth was not due to a cytotoxic effect but was due to an inhibition of the proliferation of the leukemic cells by the enforced differentiation to postmitotic cells. The high percentage of viable cells also eliminated the possibility that all or major part of the GM3-induced differentiation was a result of selective enrichment for differentiated cells.

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FIG. 3. Effect of exogenously added gangliosides on cell differentiation of HL-60 cells. HL-60 cells were seed-d at an initial concentration of 1.5 x 10i cells per ml and grown i the serum-free DME/F12 medium containing no gangliosides (o), 50 ,uM N-acetylneuraminic acid (e), 50 ,uM GM3 (A), 50 1uM GM1 (A), or 50 IAM bovine brain ganglioside mixture (n). Differentiation was determined on the basis of the induction of NapBtOEase activity. The number of NapBtOEase-positive cells was determined on days 3 and 6 of culture. Each point represents the average of three determinations, and vertical bars show standard deviations.

DISCUSSION As to the GSL composition of human myeloid leukemic cells, we have reported as follows: (i) leukemic blast cells exhibit simple GSL profiles with short-sugar-chain GSL species, such as ceramide monohexoside and ganglioside GM3, as major components, (ii) the major GSL component of normal mature cells was considerably reduced in the leukemic cells; and (iii) the GSL content of the leukemic cells was much lower than that of normal mature cells (8, 29). Similar results also have been reported by Macher et al. (30). When leukemic cells were induced to differentiate into mature cells, a drastic change in the GSL composition was observed, indicating that the GSL-profiles were differentiation-asso-

Medical Sciences:

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Proc. Natl. Acad. Sci. USA 83 (1986)

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FIG. 4. Differentiation-induction of U937 cells into monocytic mature cells with exogenously added GM3. (A) Untreated U937 cells cultured in the serumfree medium for 6 days. (Wright-Giemsa stain.) (B) U937 cells cultured in the serum-free medium with 50 uM exogenously added GM3 for 6 days. (WrightGiemsa stain.) (C) NapBtOEase staining of untreated U937 cells cultured in the serum-free medium for 6 days. (D) NapBtOEase staining of U937 cells treated with 50 ,iM GM3 for 6 days. (Original magnification x 500 for A and B and x 250 for C and D; reproduced at 75% of

original.)

ciated phenotypes in leukemic cells. HL-60 cells, which are bipotent with respect to the myeloid or monocytic pathway of differentiation, exhibited distinct quantitative changes in the GSL composition, depending not only on the differentiation stage but also on the differentiation direction (8). A remarkable increase in ganglio-series ganglioside GM3 with a concomitant decrease in lacto-series gangliosides was observed in the process of macrophage-like cell differentiation (8). A marked increase in ganglioside GM3 was also observed during macrophage-like cell differentiation of other human nonlymphoid leukemia cell lines, K562, KG-1, ML-1, and U937 (unpublished data), suggesting the presence of a common mechanism for macrophage-like cell differentiation of human nonlymphoid leukemia cell lines. However, the actual
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biological implications of glycolipid changes in association with hematopoietic cell differentiation have remained unclear. It has been reported that GSLs exogenously added to the cell culture medium were incorporated into plasma membranes, resulting in enrichment of a specific type of membrane glycolipid (13-15). On the basis of the fact that changes in the GSL composition are closely associated with the induction of leukemic cell differentiation, our main aim in the present study was to investigate whether leukemic cells would differentiate into functional mature cells when GSL molecular species that are reduced in leukemic cells were supplemented. In the present study, we demonstrated that HL-60 and U937 cells were specifically induced by exogenously added GM3 to differentiate morphologically and functionally into monocytic mature cells, their growth being markedly suppressed, and that other ganglio-series gangliosides and free N-acetylneuraminic acid were inactive as to the monocytic differentiation. These findings are of great interest because the ganglioside molecular species that specifically increases during macrophage-like cell differentiation of HL-60 and U937 cells induced by chemical agents can trigger the monocytic cell differentiation of these cell lines. The present results suggest that such a marked increase of GM3 constitutes not merely the monocytic cell differentiation-associated phenotype but also may play an important
Table 1. Expression of cell surface markers on HL-60 and U937 cells after treatment with GM3 GM3 treat% positive cells OKM5 ment OKB2 OKM1 Cells 1.9 (7.0) HL-60 31.3 (61.2) 10.6 (26.2) None 11.7 (14.3) 35.7 (57.8) 50 tM 1.3 (18.1) 76.9 (91.9) 1.7 (14.6) 18.5 (32.1) U937 None 97.0 (189.6) 50 ,uM 3.2 (21.3) 49.3 (54.0) HL-60 and U937 cells were seeded at 2.0 x 10W cells per ml and grown in serum-free DME/F12 medium containing 50 ,uM GM3 for 6 days and then were assayed for antigen expression by cytofluorometry with Ortho Spectrum III. Cells were stained for indirect immunofluorescence. Values in parentheses denote mean fluorescent intensity as measured by cytofluorography of positive cells. Values represent means of duplicate experiments, each of which included triple replicates. Standard deviations were <10%.

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FIG. 5. Effect of exogenously added gangliosides on phagocytic activity of HL-60 (m) and U937 (m) cells. HL-60 and U937 cells (2.0 x 10- cells per ml) in DME/F12 serum-free medium were incubated with or without 50 ,uM exogenous gangliosides for 6 days and then were assayed for phagocytic activity against latex (Left) and yeast (Right) particles. Phagocytic activity is expressed as the percentage of cells ingesting latex or yeast particles. Columns represent means of duplicate experiments, each of which included triple replicates. Vertical bars on columns show standard deviations, which were <10%o. BBGM, bovine brain ganglioside mixture; NeuAc, N-acetylneuramiic acid.

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supplemented with 10% heat-inactivated fetal calf serum). Further studies on the mechanisms of differentiation-induction with GM3 and on whether the lacto-series gangliosides, which having longer sugar-moieties and characteristically increase during granulocytic cell differentiation of HL-60 cells, can induce granulocytic cell differentiation are needed.
We thank Drs. T. Suda, M. Ohta, M. Akashi, Y. Furukawa, and K. Motoyoshi (Division of Hemopoiesis, Institute of Hematology, Jichi Medical School) for the invaluable advice during this work. We also thank Mr. N. Yahagi (Ortho Diagnostics) for the cytofluorometric analysis. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan.
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FIG. 6. Effect of various concentrations of exogenously added GM3 on cell growth and differentiation of HL-60 cells. (Left) Dose-dependent effect of GM3 on cell growth of HL-60 cells. HL-60 cells were seeded at an initial concentration of 2.0 x 10 cells per ml and grown in the serum-free DME/F12 medium with various concentrations of GM3. Cells were counted in a hemocytometer, and various concentrations of GM3. Cells were counted in a hemocytometer, and viability was checked by the erythrosine B dyeexclusion test. Each data point represents the mean of three determinants. Standard deviations were <10%o. The viability was >90% in each group throughout the culture periods. o, Without GM3;e, with 1 AM GM3; A, with 10 AM GM3; A, with 25,uM GM3; with 50 IiM GM3. (Right) Dose-dependent effect of GM3 on cell differentiation of HL-60 cells. Cells were seeded at 2.0 x 105 cells per ml. Differentiation was determined by NapBtOEase staining after 6 days of growth with the indicated concentration of GM3. Assessment of NapBtOEase-positive cells was performed for a minimum of 200 cells by light microscopy. Each point represents the mean of three determinations, and vertical bars show standard deviations.
n,

role as the primary trigger in the differentiation-induction of human myeloid leukemic cell lines along a monocytic cell lineage. Accumulation of particular GSL molecules in the plasma membrane and the resulting membrane constitutional change may trigger cell differentiation. It has been reported that exogenously added GSLs greatly reduced the growth of transformed cells (13, 15, 31). Recently, Bremer et al. reported that the same ganglioside (GM1 or GM3) that inhibited cell growth, inhibited platelet-derived growth factor (PDGF)-dependent DNA synthesis, altered PDGF binding, and reduced PDGF-stimulated protein phosphorylation was also the same ganglioside that was greatly reduced upon oncogenic transformation of Swiss 3T3 cells (23). Together with their results, our present report suggests that a state of decarcinogenesis might be induced in transformed or tumor cells on the supply to the plasma membranes of a particular species of GSLs, which is greatly reduced in the tumor cells. As for the specific functions of gangliosides, Tsuji et al. (32) recently reported that ganglioside GQ1b specifically exhibited nerve growth factor-like activity. It should be emphasized that a particular ganglioside molecular species, ganglioside GM3, which characteristically increased during monocytic cell differentiation of human myeloid and monocytic leukemic cell lines, not only specifically suppressed cell growth but also functioned as a highly potent inducer for the monocytic cell differentiation of these cell lines. In preliminary experiments, we have observed that fresh leukemic cells obtained from patients with acute promyelocytic leukemia also showed a tendency to differentiate along the monocytic lineage when treated with 50 AM GM3 in a serum-containing medium (RPMI 1640 medium

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