[CANCER RESEARCH 51. 1940-1945. April 1. 1991]

Ganglioside GM3 Can Induce Megakaryocytoid Differentiation of HumanLeukemia Cell Line K562 Cells1

Mitsuru Nakamura, Keita Kirito,2 Jinko Yamanoi, Tasuku Wainai, Hisao Nojiri, ' and Masaki Saito4

Division of Hemopoiesis, Institute of Hematology, Jichi Medical School, 331 l-l Yakushiji, Minamikawachi, Tochigi 329-04, Japan

ABSTRACT

The role of acidic glycosphingolipids in cell growth and differentiationwas investigated using the multipotent leukemia cell line KS62. When(¡Miwas added to cell culture media, the growth of K562 cells wasremarkably inhibited and the cells were shown to have megakaryocytoidmorphology. Ultrastructural study demonstrated that KS62 cells treatedwith CM., had platelet peroxidase-positive structures, which were considered to be the specific marker of megakaryocyte. Furthermore, AP-3directed against an epitope present on membrane glycoprotein Ilia reacted with the GMj-treated cells. Free /V-acetylneuraminic acid, GMi,<.M,. (.!),„,and a mixture of bovine brain gangliosides containing (.!>,.,and GTib did not affect growth of K562 cells or show morphologicalchanges. According to chemical analyses, (¡Micontent increased inmegakaryocytoid differentiation induced by tetradecanoylphorbol-13-ace-tate, whereas (.M, decreased in erythroid differentiation induced byhemin. Enzymatic analysis showed that the (.Mi increase during megakaryocytoid differentiation was a result of the sialyltransferase activation.These results indicated that exogenous GM.i induced differentiation ofK562 cells into a "GMj-rich" lineage, i.e., mainly megakaryocytoid

lineage, and that (.M, accumulation in the GM.,-rich lineage was theresult of the activation of GM., synthase. These Findings strongly suggested that (.Mi ganglioside, a minor membrane component, has a crucialrole in not only the differentiation induction but also the determinationof the differentiation direction in pluripotent KS62 cells.

INTRODUCTION

Gangliosides, sialic acid-containing glycosphingolipids(GSLs5), are a structurally varied class of complex carbohy

drates and are ubiquitous membrane components in vertebrates,localized almost exclusively on the outer leaflet of the plasmamembrane (1, 2). They have been proposed to play importantroles in cellular interactions, differentiation, and oncogenesis(3), especially in cell to cell interaction (4), neurite outgrowth(5, 6), and cell growth (7, 8) in various cell systems.

Changes in glycosphingolipids during hematopoietic cell differentiation were also studied using human leukemia cell lineHL-60 cells and were found to be dependent on the directionsof differentiation (9,10). This suggested a specific role of GSLsin differentiation induction of leukemia cells. Previously wedemonstrated that exogenous ganglioside CM, and neolacto-series gangliosides induced monocytic and granulocytic differ-

Received 11/5/90; accepted 1/17/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grant-in-Aid for Scientific Research on Priority AreasNo. 02259101 from the Ministry of Education. Science and Culture. Japan.

2 Present address: Yamanashi-kenritsu Chuo Hospital. Fujimi 1-1-1. Kofu.

Yamanashi 400. Japan.'Present address: Biomembrane Institute. 201 Elliott Ave. W.. Suite 305.

Seattle. WA 98119.4To whom requests for reprints should be addressed.5The abbreviations used are: GSL, glycosphingolipid; LacCer. lactosylceram-

ide; GMj. II3NeuAcLacCer; GD,. Il'(NeuAc)2LacCer; CM,. II3NeuAcGgO4Cer;GDU, IV3NeuAc,II3NeuAcGgOse4Cer; GD,b. II3(NeuAc)2GgOse4Cer; GT,b.IV3NeuAc,II3(NeuAc)2GgOse4Cer; GQ,b. IV3(NeuAc)3,II3(NeuAc)2GgOse4Cer;a2 —»3 sialosylparagloboside, !V3NeuAcLcnOse4Cer; «2—»6 sialosylparaglobo-side, IV*NeuAcLcrtOse4Cer: NeuAc. /V-acetylncuraminic acid; GM3 synthase.CMP-NeuAc:lactosylceramide «2 —»3 sialyltransferase; Gp, glycoprotein;HPTLC, high performance thin layer chromatography; PPO, platelet peroxidase;TPA, 12-O-tetradecanoyl phorbol-13-acetate.

entiation in HL-60 cells, respectively (11, 12). This indicatedthat GSLs may play an important role as a trigger in differentiation induction of HL-60 cells and as a determinant of thedifferentiation direction.

The K562 cell line was established from the pleural effusionof a patient with chronic myelogenous leukemia in terminalblast crisis (13). The K562 cell line was thought to be multi-potent hematopoietic cells and to be capable of differentiatinginto erythrocytic (14-16), monocytic (17), granulocytic (17),and megakaryocytic (18, 19) lineages.

It was of great interest to use pluripotent K562 cells and toanalyze whether exogenous gangliosides could induce differentiation of the cells, in addition to studying oligopotent HL-60cells. In the present study, we analyzed (a) the effect of gangliosides including GM.( on differentiation induction of the cells,(¿>)the ganglioside composition of K562 cells, and (c) theenzymatic basis of GM, synthesis in the cells. We demonstratedthat the hypothesis that glycosphingolipids have crucial rolesin the determination of the differentiation direction as well asdifferentiation induction itself could be applied to this pluripotent hematopoietic cell line.

MATERIALS AND METHODS

Glycolipids and Other Chemicals. GM., was prepared from dog eryth-rocytes, and GM, and GDu from bovine brain, by the method describedelsewhere (20). GM2 was purchased from latron (Tokyo, Japan). Lac-tosylceramide (LacCer), /V-acetylneuraminic acid (from Escherichiacoli), and a bovine brain ganglioside mixture (type III) were obtainedfrom Sigma Chemical Co. (St. Louis, MO). The bovine brain ganglioside mixture consisted of 7.5% GM,, 2.6% GD,, 56.7% GD,a, 11.5%GD,b, and 21.7% GT,b. CMP-NeuAc and Triton CF-54 were also fromSigma. CMP-[sialic-435,6,7,8,9-'4C]NeuAc (11.1 GBq/mmol) was purchased from New England Nuclear (Boston, MA). The reverse-phaseadsorption column Sep-Pak CIS was from Waters (Milford, MA).Silica gel-precoated HPTLC plates were from Merck (Darmstadt,Federal Republic of Germany). Mouse anti-GM, monoclonal antibodyM2590 (21) was a generous gift from Dr. Masaru Taniguchi (ChibaUniversity, Chiba, Japan). Rabbit anti-GM2 and anti-GM, polyclonalantibodies were purchased from latron. All other reagents were ofhighest grade available.

Cell Culture. Cells of the human leukemia cell line K562 (13) weregrown in a serum-free medium composed of equal volumes of Dulbec-co's modified Eagle's minimal essential medium and Ham's F-12

medium supplemented with 30 nM selenium dioxide, 1.2 g of sodiumbicarbonate/liter, 15 mivi A'-2-hydroxyethylpiperazine-A"-2-ethanesul-

fonic acid (pH 7.2), 5 jig/ml of insulin (Sigma), and 5 Mg/mg oftransferrin (Sigma) (22) at 37°Cin humidified 5% carbon dioxide/95%

air. Cell counts were made by erythrosin B dye exclusion. In the studyof differentiation induction by chemical inducers, K562 cells werecultivated at an initial concentration of 1 x IO5cells/ml in RPMI 1640

medium supplemented with 5% fetal calf serum. The cells were treatedwith 6 n\t TPA and 50 /IM hemin (16, 19), harvested on day 3 and day6, respectively, washed with phosphate-buffered saline two times, andstocked at -20°Cuntil use.

Gangliosides were dissolved in an appropriate volume of water withsonication and sterilized by passage through a Q.22-pm Millipore filter,and the lipid-bound sialic acid of gangliosides was quantitated by theresorcinol-HCl method (23). These solutions were diluted to 1 HIMwith

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GANGLIOSIDE GM3 INDUCES K562 CELL DIFFERENTIATION

an equal volume of Dulbecco's modified Eagle's minimal essentialmedium and Hani's F-12 medium of 2-fold concentration and was

added to the culture medium to the desired ganglioside concentration(11).

Evaluation of Cell Differentiation. For morphological assessment ofcell differentiation, cytospin slides were prepared with a Shandoncytospin centrifuge (Shandon Southern Products, Cheshire, England)and stained with Wright-Giemsa staining solutions. Nonspecific esterase activity was determined using «-naphthyl butyrate and naphtholAS-D-chloroacetate as substrates (24). Hemoglobin staining was conducted by the method using o-dianisidine (3,3'-dimethoxybenzidine).

For PPO analysis (25), untreated and ganglioside-treated cells werefixed with a mixture of 4% paraformaldehyde and 1% tannic acid inphosphate buffer (pH 7.4) for l h and then incubated in the mediumcontaining 10 mg of 3-3'-diaminobenzidine tetrahydrochloride in 10

mM Tris-HCl buffer (pH 7.6) with 0.01% hydrogen peroxide for 60min to detect the activity of platelet peroxidase. The specimens werethen washed several times in phosphate buffer (pH 7.4) which contained4% glucose and postfixed for l h in 1% osmium tetroxide. Afterpostfixation, the specimens were dehydrated and embedded in Epoxyresin. Ultrathin sections were examined under an electron microscope.

Untreated K562 cells, chemical inducer-treated cells, and GMj-treated cells were examined for expression of membrane glycoproteinGp Ilia with flow cytometry. an Ortho Spectrum III, using a monoclonal antibody AP-3 (26). which was a generous gift from Dr. Peter J.Newman (The Blood Center of Southeastern Wisconsin, Milwaukee,WI).

Ganglioside Analysis of K562 Cells. Gangliosides from KS62 cellstreated with chemical inducers were prepared by chloroform/methanolextraction and DEAE-Sephadex A-25 chromatography (20) and separated on an HPTLC plate with a solvent system of chloroform/meth-anol/0.5% CaCl2(OH)2 (50:50:10. v/v/v). The HPTLC plates weresprayed with resorcinol/HCl reagent and gangliosides were visualizedby heating the plates at 95°C(27) followed by quantitation using a CS-

9000 flying-spot scanner (Shimadzu, Kyoto, Japan).CM.,, GM2, and GD,a were characterized by the thin layer chroma-

tography-immunostaining method (28). After separation of gangliosides on an HPTLC plate, the plate was coated with 0.4% polyisobu-tylmethacrylate (Aldrich, Milwaukee, WI). The plate was reacted withanti-GM, antibody M2590 or anti-GM2 antibody for CM, and GM2detection, respectively. To characterize the structure of GDla, the platewas first reacted with Clostridium perfringens sialidase and then reactedwith anti-GMi antibody (29). The plate was then coated with [I2'I]-

staphylococcal protein A solution and autoradiographed.CM., Synthetic Activity Assay. K562 cells (5-10 x IO7)treated with

chemical inducers were homogenized in 1-2 ml of 0.25 M sucrose in25 mM sodium cacodylate buffer, pH 6.9, with 1 mM EDTA using aPolytron homogenizer (Kinematika GmbH, Littau-Luzern, Switzerland) with a PTA10S generator for 30 s at a maximum speed. Aftercentrifugation with 700 x g for 10 min. the supernatant was spun at105,000 x g for 60 min using a TL-100 ultracentrifuge (Beckman, PaloAlto, CA). The pellet was resuspended in about 100 /al of homogeni-zation buffer using a Potter-type homogenizer.

GM3 synthetic activity was assayed as follows; 10 nmol of LacCerand 2.5 n\ of CMP-|sialic-4,5,6,7,8,9-'4C]-NeuAc was added in a micro-tube and then dried. CMP-NeuAc (4.8 nmol), sodium cacodylate buffer(pH 6.2. 3.75 /¿mol).Triton CF-54 (75 /*g), and enzyme preparation(100-200 //g protein) were incubated in a total volume of 25 p\ for 3 h.

The radioactive product was separated from other radioactive compounds using reverse-phase adsorption column chromatography asdescribed previously (30). Radioactivity was counted in liquid scintillation spectrometry.

Protein Assay. Protein was determined by an Amido-Schwarz dye-binding method (31) using bovine serum albumin as a standard.

RESULTS

Effect of Exogenous Gangliosides on Cell Growth and CellDifferentiation in K562 Cells. To examine the effect of exogenous gangliosides on growth of K562 cells, gangliosides were

added to the cell culture medium. According to gangliosideanalyses of K562 cells, GM,, GM2, and GDU were found to bethe major components. Therefore, we examined CM.,, GM2,GM|, GDia, a bovine brain ganglioside mixture, and free NeuAcfor their bioactivities. The final concentrations of these gangliosides were adjusted to 50 ^M- As shown in Fig. 1/4, cell growthwas completely inhibited only when treated with exogenousGM,. Other gangliosides and free NeuAc had no effect ongrowth of K562 cells. For morphological examination, K562cells were stained by Wright-Giemsa procedures after 6 days ofincubation with 50 ^M GM,. Changes were found in the morphology of K562 cells treated with GM,. Forty % of GMrtreated cells showed megakaryocytoid cell morphology (Fig.2B) and another 40% showed the morphology, which wasdistinct from megakaryocyte and other hematological cells, withlarge cytoplasma and 4 nuclei (Fig. 2C). Ten % of the cellsshowed macrophage-like morphology (Fig. 2D). Unlike GM3,other ganglio-series gangliosides and free NeuAc had no effecton morphology of K562 cells.

When K562 cells were cultured with various concentrationsof GM i, cell growth was inhibited markedly in a dose-dependentmanner (Fig. \B) and was completely inhibited with 50 ßMGM.,. During the culture period, K562 cells displayed >90%viability. This indicated that the suppressive effect of GM3 oncell growth was not due to a cytotoxic effect or a result ofselective enrichment for differentiated cells but was due to aninhibition of the proliferation of the leukemic cells by theenforced terminal differentiation.

Ultrastructural Morphology. Nontreated K562 cells seemedto have lobulated nucleus with a smooth outline and withnucleoli (Fig. 3A). No chromatin condensation was observed.The cytoplasmic organdíes were not well developed and thecytoplasm contained only a few mitochondria, monosomes, andpolysomes. No rough endoplasmic reticulum was found. Onthe other hand, GM,-treated K562 cells showed a low nuclear/cytoplasmic ratio and their nuclei showed lobular structureswith nuclear clefts and little chromatin condensation (Fig. 3Ä).Rough endoplasmic reticulum and granules in the cytoplasmwere recognized. These results showed that GM.,-treated cellsbecame mature and differentiated. Nontreated K562 cells werenegative for PPO (Fig. 3A). After stimulation with GM,, however, cells with a PPO-positive reaction in rough endoplasmic

Fig. 1. A. effect of exogenous gangliosides upon cell growth of K562 cells.K562 cells were seeded into culture flasks at an initial concentration of I x 10'cells/ml and grown in serum-free medium containing no gangliosides (•).50 /IMA'-acetylneuraminic acid (O). 50 /<MGMj (•).50 »itGM2 (D). 50 JIMGM, (A).

50 /iM GD,, (X). or 50 MMbovine brain ganglioside mixture (V). B, growth ofK562 cells in various concentrations of GMj. K562 cells were seeded at an initialconcentration of 1.0 x 10' cells/ml and grown in serum-free medium containing

no GM., (•).5 pM GMj (O), 10 /IM GM3 (•).25 /JM GM3 (D), or 50 /IM GM3(A). Point, mean of three determinations. SD < 10%.

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GANGLIOSIDE GMj INDUCES K562 CELL DIFFERENTIATION

Fig. 2. Differentiation induction of K562cells with exogenous GM3. A, untreated K562cells cultured in the serum-free medium for 6days (Wright-Giemsa stain). B-l). K562 cellscultured in the scrum-free medium with 50 JJMexogenous GMj for 6 days (\\right-Giemsastaining). Original magnification, x 400.

A B

C D

"W-W*\

x),í-^'**%$''

^y/*.-

%v•-"'**\X.^r *O^*j¿^

Fig. 3. Electron microscopie observation of K562 cells after treatment withexogenous GM,. A, untreated K562 cells cultured in serum-free medium. B, K562cells cultured in the serum-free medium with 50 ^M GM.,: arrows, PPO-positivestructures. Bars, I jjm.

reticulum were detected (Fig. 3#). These were considered typical cells of megakaryocytic origin induced by exogenous GM.i.

Expression of Differentiation Markers in K562 Cells. Markersfor differentiation were analyzed with hemoglobin staining,esterase double staining, and membrane glycoprotein expression. As shown in Table 1, TPA-treated K562 cells expressedGp Ilia recognized by monoclonal antibody AP-3, whereas o-dianisidine-positive cells did not increase during the TPA treatment. o-Dianisidine-positive cells were markedly increased afterhemin treatment, while AP-3-positive cells showed only a littleincrease. TPA induced K562 cells into a megakaryocytic lineageand hemin induced the cells into an erythroid lineage becauseGp Ilia was specific for platelets or megakaryocytoid cells (32)and o-dianisidine reactivity was specific for an erythroid lineageas previously described (16, 18). On the other hand, none ofthe cells was stained by o-dianisidine after GM3 treatment;however, the cells having «-naphthyl butyrate esterase activity,which was completely inhibited by NaF, increased 3-fold asmuch as the control. Furthermore, in GM,-treated K562 cells,Gp Ilia-positive cells increased 2-fold as much as the control.

Analysis of Gangliosides from K562 Cells. We analyzed gan-glioside composition of K562 cells after treatment with chemical inducers. Fig. 4 shows the ganglioside patterns of TPA-and hemin-treated K562 cells as well as nontreated cells. Thecompounds corresponding to GM3, GM2, and GD,a were characterized by the immunostaining method using specific anti-

Table1 Expressionofdifferentiationmarkersin A'562cells

Hemoglobinstaining"

(o-dianisidine)ControlTPAHeminGM,-100*1003100±0050+00920++0010Esterase((v-naphthylbutvrateesterase)4.5N

D'NDf13.2AP-3(anti-GPIlia)25.890.039.158.0

°o-Dianisidine positive cells »ereclassified into three groups, i.e.. ++. stronglystained: +, normally stained; ±.faintly stained; —,not stained.

* % positive cells; mean of two determinations. SD < 10cr.' ND, not done.

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GANGLIOSIDE GM3 INDUCES K562 CELL DIFFERENTIATION

Ml

A. B.IJg lipid-bound sialic acidiIff cells pmollmg protein/hr

0123-1 0 100 200

BFig. 4. Thin layer chromatograms of gangliosides from K562 cells after

treatment with TPA and hemin. Gangliosides from K562 cells treated with 6 nMTPA for 3 days and 50 >J.Mhemin for 6 days were prepared by chloroform/methanol extraction followed by DEAE-Sephadex A-25 chromatography. Developing solvent was chloroform/methanol/0.5% CaCI2(OH)2 (50:50:10, v/v/v).Lane A, authentic standards, GMj, GM2, GM,, and CD,,, in order from solventfront to origin (arrows): lane B, gangliosides from nontreated K562 cells; lane C,gangliosides from the TPA-treated cells; lane D, gangliosides from the hemin-treated cells.

bodies (data not shown) as described in "Materials and Methods." As shown in Fig. 4 and Fig. 5A, GM3 content increased

in TPA-treated K562 cells. On the other hand, GM3 wasmarkedly decreased in hemin-treated K562 cells. The othercharacteristic change in ganglioside composition was an increase in GM2 and GDhl after TPA and hemin treatments.There were two components located between GM2 and GDla,which corresponded to «2—»3 and «2—»6 sialosylparaglobo-sides when judged by their mobilities on HPTLC. These twowere decreased in all treatments.

Enzymatic Basis for GM3 Increase in Megakaryocytoid Differentiation. To assess the enzymatic background of GM3 increase in TPA-treated K562 cells, GM, synthase activities weredetermined. Fig. 5B clearly shows that the sialyltransferaseactivities increased in TPA-treated K562 cells and decreased inhemin-treated cells. GM, synthase activities corresponded wellto GM3 contents in the control K562 cells, TPA-treated cells,and hemin-treated cells. Furthermore, GM3 synthase activitieswere elevated in a time-dependent manner in TPA-treated K562cells, and the radioactive product after the enzymatic reactionwas clearly identified as GM3 on HPTLC plates (data notshown).

DISCUSSION

Ganglioside profiles of leukemia cells showed specific patterns and were thought to be biochemical markers for humanhematopoietic cell lines (33). Moreover, they were dependentupon both differentiation stages and directions of differentiation (9). In the human myelogenous leukemia cell line HL-60,we previously demonstrated that GM, increased during mono-cytic differentiation, while neolacto-series gangliosides characteristically increased during granulocytic differentiation (10).Furthermore, we reported that exogenous ganglioside GM3could induce monocytic differentiation (11) and neolacto-seriesgangliosides could induce granulocytic differentiation (12) ofHL-60 cells. These results indicated that gangliosides in thecell surface membrane may play critical roles in regulation ofthe growth and differentiation of leukemia cells.

Control

TPA

Hemin

Control

ÃŒTA

Hemin

Fig. 5. A, quantitative analysis of gangliosides in K562 cells. The HPTLCplate was analyzed quantitatively by CS-9000 flying-spot scanner (Shimadzu) asdescribed in "Materials and Methods." •.GM3; D, GM2; •,the componentcorresponding to «2—»3 sialosylparagloboside; Ü,the component correspondingto «2—»6 sialosylparagloboside; •,GDU. B, GM3 synthase activity levels ofK562 cells after treatment with TPA and hemin. Paniculate fractions of K562cells after treatment with 6 nM TPA for 3 days and 50 /IM hemin for 6 days wereprepared and analyzed for CM, synthase activity assay as described in "Materialsand Methods." Column, mean of two determinations. SD < 15 %.

The K.562 cell line was derived from a patient with chronicmyeloid leukemia in acute blastic crisis (13). It was reportedthat K562 cells had glycophorin as a marker of erythroid cells(14) and synthesized embryonic hemoglobin in response tochemical inducers, sodium butyrate, and hemin (15, 16), indicating that the cells had potential to differentiate into anerythroid lineage. It was also reported that K562 cells hadantigens recognized by 80 H5 and anti-Myl as markers ofmyeloid cells (18, 34, 35). On the other hand, when induced byTPA, K562 cells expressed Gp lili 'I Ila and a moderate number

of induced cells became positive for PPO (18, 19), indicatingthat the cells had potential to differentiate into a megakaryocy-tic lineage. Furthermore, K562 cells could be differentiated intoerythrocytic, granulocytic, and monocytic lineages spontaneously in long-term culture (17). K562 cells, however, did nothave lymphocyte markers (36). On the basis of these studies, itwas considered that K562 cells were derived from pluripotenthemopoietic stem cells and that the cells could potentiallydifferentiate into erythrocytic, monocytic, granulocytic, andmegakaryocytic lineages. In our experiments, we investigatedtwo differentiation lineages, megakaryocytoid and erythroidlineages, which were induced by chemical inducers, TPA andhemin, respectively, because monocytic and granulocytic differentiation in long-term culture seemed spontaneous (17) andwere difficult to subject to repetitive analyses.

Suzuki et al. (37) have reported that GM,, GM2, and GD,awere the major components of gangliosides in K562 cells.Confirming these molecular structures by immunostaining, wefound that the cells had minor components in addition, «2—»3 and «2—»6 sialosylparaglobosides. However, the structuresof these two acidic glycolipids were yet to be characterized moreclearly. After induction of differentiation by TPA and hemin,the two minor gangliosides decreased, whereas the major GM2and GDia increased. However, GM., behaved in a totally different manner in both lineages. While GM., decreased in erythrocytic differentiation induced by hemin, GM., increased in megakaryocytic differentiation induced by TPA, indicating thatthe megakaryocytic, but not the erythrocytic, lineage was "GM3-rich." It was previously reported that human platelets had GM3

as a major component of gangliosides (38). While sialosylparagloboside was the major component, GM3 was only 25.7% ofthe total lipid-bound sialic acid in human erythrocytes (39).

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Our present results indicate that about 40% of acidic glyco-sphingolipids consist of GM, in nontreated K562 cells. Thissuggests that the GM., content in human hematopoietic cellsdecreased along with erythrocytic differentiation. A major component of gangliosides in human mature monocytes was GM.,(40), and GM., was increased along with monocytic differentiation of human leukemia HL-60 cells (10). On the other hand,sialosylparaglobosides and other neolacto-series gangliosideswere major components in human mature granulocytes (41),and the neolacto-series gangliosides were increased along withgranulocytic differentiation of human leukemia HL-60 cells asreported earlier (9). Consequently, megakaryocytic and monocytic lineages can be considered as GMj-rich lineages in thedifferentiation of human hematopoietic cells, among erythrocytic, monocytic, granulocytic, and megakaryocytic cells.

Using K562 cells, we examined whether or not several gangliosides could inhibit cell growth. Among them, only GM3could inhibit growth of the cells, while other gangliosides, GM,,GM2, GDla, a bovine brain ganglioside mixture, and free NeuAchad no effect. According to the morphological studies on thebasis of Wright-Giemsa staining, electron microscopy, andcytochemistry, the growth inhibition by GM., was thought tobe the result of terminal differentiation of K562 cells. Moreover,membrane glycoprotein (Gp Ilia) expression (26) was alsoutilized to evaluate cell differentiation. As a result of findingsfrom PPO staining, Gp Ilia expression, and the morphologicalstudies, it was considered that about 40% of GM.,-treated K562cells were differentiated into a megakaryocytic lineage. On theother hand, about 10% of GM.,-treated K562 cells showedmacrophage-like morphology and expressed «-naphthyl butyr-ate esterase activity. These findings indicate that GM., couldinduce differentiation into a monocytoid lineage, as well as amegakaryocytoid lineage, in a small population of K562 cells.We have already reported that GM., could induce differentiationinto a monocytic lineage in HL-60 and U-937 cells (11). InK562 cells, however, it was shown that exogenous GM., couldinduce differentiation not only into monocytic but also intomegakaryocytic lineage. From the studies using HL-60 cells, itwas hypothesized that GM., induced differentiation of the cellsinto a GM.,-rich lineage and neolacto-series gangliosides induced differentiation of the cells into a "neolacto-series gan-glioside-rich" lineage (11, 12). The present studies support the

assumption that some molecular species of gangliosides mayhave critical roles in determining the direction of differentiationof leukemic cells; exogenous GM, induces the differentiationof K562 cells into GM,-rich lineages. The difference in directions of differentiation between GM.,-treated K562 cells andHL-60 cells might be explained by the fact that the differentiation direction was also dependent on their potential and stagesof differentiation.

There have been several reports that gangliosides had physiological effects in cell growth and differentiation. GangliosideGQib was reported to have nerve growth factor-like activitiesthrough the effect upon protein kinase (5, 42). It was reportedthat GM, and GM,, which were decreased in transformed Swiss3T3 cells, inhibited cell growth (7,43). These findings indicatedthat cell surface gangliosides play a role as a regulator or aninhibitor of cell growth via deactivating growth factor receptorkinases. However, in hematopoietic cells the mechanism of celldifferentiation by gangliosides was not yet clear. We analyzedGM, metabolism in HL-60 cells and suggested that GM, incorporation into the cells was mediated by a receptor-likemechanism (44). Furthermore, it was reported that there was a

ganglioside-specific binding protein in rat brain (45). Likewise,a GM.,-specific binding protein might exist also in hematopoietic cells, but it remains to be elucidated more clearly. Onthe other hand, the GM., increase in monocytic differentiationof HL-60 cells induced by TPA was the result of an activationof LacCer:CMP-NeuAc «2—»3sialyltransferase as previouslyreported (46). Our present results also indicate that the GM.,increase in megakaryocytic differentiation of K562 cells is basedon an activation of the same sialyltransferase, while the GM.,decrease in erythrocytic differentiation is the result of a lowlevel of sialyltransferase activity. Together with the observationsreported earlier (46), GM., levels and synthetic activity levelsand the direction of differentiation induced by TPA, which isrelated to protein kinase C (47), correspond well with eachother in leukemic cell differentiation. In order to understandmore clearly these relationship and the mechanism of celldifferentiation by gangliosides, it is of interest to determine thesialyltransferase activity in GM,-treated K562 or HL-60 cellsand to elucidate whether protein kinase C directly activatesGM., synthase activity. Although these remain to be elucidated,GM., synthase and its product, GM,, might play important andcritical roles in megakaryocytic differentiation of K562 cells aswell as in monocytic differentiation of HL-60 cells.

ACKNOWLEDGMENTS

We give special thanks to Dr. Y. Enomoto. Department of Pathology,School of Medicine, Keio University, for the ultrastructural analysis,and to Dr. M. Ohta in our laboratory' and Dr. J-Q. Zhou now in Peking,

China, for their assistance with cytochemical studies. We also thankDrs. K. Motoyoshi, S. Kitagawa, M. Ohta, and Y. Furukawa in ourlaboratory for their valuable discussions.

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1991;51:1940-1945. Cancer Res   Mitsuru Nakamura, Keita Kirito, Jinko Yamanoi, et al.   Human Leukemia Cell Line K562 Cells

Can Induce Megakaryocytoid Differentiation of3Ganglioside GM

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