Characteristic incorporation of ganglioside GM3, which induces monocytic differentiation in human myelogenous leukemia HL-60 cells

Vol.

161,

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2, 1989

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1989

782-789

CHARACTERISTIC INCORPORATION OF GANGLIOSIDE GM3, WHICH INDUCES MONOCYTIC DIFFERENTIATION IN HUMAN MYELOGENOUS LEUKEMIA HL-60 CELLS Mitsuru Division

Received

Nakamura,

Hidetoshi Oginol Kitagawa and Masaki

of Hemopoiesis, Institute Minami-kawachi, School,

Hisao Nojiri2, Saito3

of Hematology, Tochigi 329-04,

Seiichi

JICHI Japan

Medical

May 1, 1989

Using tritiated gangliosides (r3H]-GM3 and [3Hl-GM1), characteristic incorporation of exogenous GM3 to HL-60 cells was demonstrated in association with differentiation induction. E3H]-GM3 was bound 4 - 5 times more than r3Hl-GM1 was. Scatchard analysis revealed high and low affinity patterns of binding to the cells. The concentration of GM3 that caused growth inhibition and cell differentiation corresponded well to that which showed the bi-phasic binding pattern. It was strongly suggested that GM3, which induces monocytic differentiation, was characteristically bound and incorporated to the cells around the concentration which caused growth inhibition and cell differentiation. 0 1989 ncxlem1c Press,Inc.

Glycosphingolipids mammalian cells

(GSLs.1 are ubiquitous

and are believed

constituents

to be localized

of

predominantly

on

the outer leaflet of the plasma membrane (1). They have long been suggested to play an important role in cell-cell, cellmatrix

interactions

demonstrated

the

and in cell following

growth

(2).

We have recently on human myelogenous leukemia cells

' Present address is Division of Applied Enzymology, Exploratory Research Laboratories, Ban'yu Pharmaceutical Co., LTD., Shimomeguro 2-3-9, Meguro, Tokyo 153, Japan. 2 Present address is Biomembrane Suite 305, Seattle, WA 98119.

Institute,

3 To whom all

correspondence

Abbreviations: GgOse4Cer;

GMl, GM3.p 4 13NeuAc-LacCer; IV (NeuAc)2,113(NeuAc)2GgOse4Cer;

GQlb,

IV3NeuAc,I13(NeuAc)2GgOse4Cer; 0006-291x/89 Copyright All rights

should

be addressed. I13NeuAc-

GSLs, glycosphingolipids.

$1.50

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

201 Elliott

782

GTlb'

Ave.

W.,

Vol. 161, No. 2, 1989

HL-60 which

are bipotent

1) Ganglioside entiation

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about

GM3 increased

differentiation

remarkably

and neolacto-series

gangliosides

tically during myeloid differentiation can induce monocytic differentiation gangliosides

can induce

myeloid

cate that the gangliosides, during cell differentiation, ger

for

induction

been put

of

tritium

ganglioside

at

In the

increase

the

cell

the present

ceramide

moiety

differGM3

These indi-

characteristically

Attention differentiation

study,

characteris-

(8).

a crucial

role

as a trighas now with exoge-

we used GM3 labeled

to investigate

GM3 was bound and incorporated

MATERIALS

monocytic

2) Ganglioside 3) Neolacto-series

differentiation

might play differentiation.

(3,4).

increased

(5). (6.7).

which

on the mechanism of

nous gangliosides. with

cell

during

direction

to

how exogenous

HL-60 cells.

AND METHODS

Materials : Ganglioside GM3 was isolated from human normal liver and GM1 from normal human brain. Both were finally purified to homogeneity using high performance liquid chromatography with an Iatrobeads IRSP-8005 column (Iatron, Tokyo, Japan) (9). GM3 and GM1 were tritiated by catalytic reduction of the sphingosine double bond with [3H]-NaBH4 (DuPont-New England Nuclear, Boston, MA, USA) as described (10) and freed from radioactive impurities using si:Lica gel chromatography. The purity of these tritium labeled-gangliosides were at least 98 % on two-dimensional high performance thin-layer chromatography according to the method described elsewhere (11). Specific radioactivities of tritiated and GM1 were 248 Ci/mole and 118 Ci/mole, respectively. GM3 Radioactivity was determined by liquid scintillation counter. The amounts of gangliosides in the tritiated preparations were estimated on thin-layer chromatography using resorcinol-HCl spraying followed by heating at 105OC. All other chemicals were of the highest grade available. Cell Culture : Human myelogenous leukemia HL-60 cells (12) were maintained in GIT medium (Wake Pure Chemical, Osaka, Japan) at 37*C in humidified 5 % carbon dioxide. Before experiments, cells were carefully spun down and suspended in a serum-free synthetic medium according to the method described (13). The cells were preincubated for 1 hr at 37OC in humidified 5 % carbon dioxide. The synthetic medium was Dulbecco's modified Eagle's minimum v/v) supplemented with 5 essential medium/Ham's F12 medium (l:l, m/ml insulin, 5 H/ml transferrin, 1.2 g/l sodium bicarbonate and 30 nM selenium dioxide. Tritiated gangliosides or cold gangliosides were added to the cell culture medium as described preCell counts were made by erythrosine B dye excluviously (7,8). sion. Uptake kinetics of [3H]-gangliosides into HL-60 cells : lo6 cells/ml of HL-60 cells were incubated with 50 fl f3H]-GM3 and [3H]-GM1 under cell culture conditions for various lengths of After incubation, the cells were isolated by centrifugatime. tion at 4OC at 1,000 x g and washed three times with phosphatebuffered saline. The cell pellet was resuspended in 1 % Triton 783

Vol.

161,

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2, 1989

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AND

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X-100 and transferred into a vial. rinsed once with 1 % Triton X-100, cell suspension, and the radioactivity

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The centrifuge tube the wash was combined was counted.

was with

the

Binding of [3H]-gangliosides with HL-60 cells : HL-60 cells (lo8 cells ml) were incubated with various concentrations of r3H]-GM3 and [ < HI-GM1 in microcentrifuge tubes. The mixture was incubated for 15 min, and separated into the cell fraction and supernatant as described (14). After quick centrifugation for 1 min, the supernatant was carefully removed and put into a scintillation vial and the radioactivity was counted. The radioactivity of the cell pellet was counted as described above.

RESULTS Effect

of

entiation

exogenous of

HL-60

concentrations pressed effect

gangliosides cells

of

: HL-60

non-labeled

cells

inhibited expression

WY I cytochemistry, phagocytic activity, typical (7) in the GM3 concentration differentiation than 15 m.

growth partial Not only induction

was That

of

the

and cell

cultured Cell

not is,

differ-

with growth

while GM1 had inhibited with

various was

sup-

no lo-25

with 50 m. Judged by morpholof surface marker antigens and cell differentiation between 25 - 50 m.

suppression cells

were

manner, partially

observed no significant

was observed under 5 m of but significant inhibition growth

growth

gangliosides.

by GM3 in a dose-dependent (Fig.1). Cell growth was

IJM GM3 and completely

cell less

on cell

but

the GM3 concentration suppression of cell

of

Between 10 and 15 PM, of cell growth was noticed.

GM3.

also

was found

at

was observed Characteristic

typical

between

differentiation-

25 and 50 m GM3-

Incorporation of [3H]-Gangliosides into HL-60 Cells : [3H]labeled GM3 was added to the serum free culture medium of HL-60 cells at a concentration of 50 *. The association of the GM3 with the cells occurred quite rapidly within 30 to 60 min (Fig.2).

The association

then

was slowed

down considerably.

[3H]-GM1 the same pattern of incorporation kinetics r3Hl-GM3, however, was bound and incorporated into least

four times HL-60 cells [3H]-gangliosides

as much as C3H]-GMl. were incubated with various at 37OC and the incorporation

For

was observed. the cells at

concentrations of patterns shown in

Fig.3 were observed. At low concentrations, under 8 m for C3H]GM3 and 2 m for [3H]-GM1, incorporation increased linearly. At higher concentrations, 10 to 50 m, however, incorporation of [3H]-gangliosides into the cells was leveled off and the patterns were not linear. Especially 10 and 50 VM significantly

for [3H]-GM3, concentrations between suppressed growth of HL-60 cells. 784

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161,

No.

:2, 1989

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=0-J $80 I

h

+ 01 o

4”

Days

02

6

0

1

2

6

3

Time

( hr 1

Effect of various concentrations of exogenous Fig.1 growth of HL-60 cells. HL-60 cells were seeded at concentration of 2.0 x lo5 cells per ml and grown free medium in the presence or absence of exogenous as described in MATERIALS AND METHODS: 0, without 5 pt.! of GM3;8. 10 JIM of GM2;A. 15 m of GM2;V. A,. 50 /A-l of GM3;0, 50 /JM of GMl-

GM2 on cell an initial in the serumgangliosides ganglioside; 0, 25 /JM of GM3;

fzi&

Uptake kinetics of trfitiated gangliosides into HL-60 . 50 JJM [ HI-GM5.and [ HI-GM1 were exogenously added to serum-free culture media of HL-60 cells. After incubation for the period indicated, the cells were collected and radioactivities were counted as described in MATERIALS AND METHODS: 0, r3H]GM3; m, t3H]-GM1.

Scatchard HL-60

Analysis Cells

sponding

: The data

plotted

of

according

be

in

to

into

for

a high

affinity

type

which

that

affinity

caused

cell

a low

affinity not

tiation. are

The summarized

concentration growth

apparent Table

for

1.

cells

GMl.

The of

And

cell

growth

BRAI values The

apparent

785

the

At cells

appeared however,

revealed first low

10 25

20

50

also

calculated KD

phase

values

was that

to

m, to

It

m,

well

to

but

bi-

affinity. to

well

low

and

It

corresponded

corresponded

of

KD and

into was

were

(Fig.4). to

[3H]-GM3,

inhibition. pattern,

and

manner.

one of

corre-

concentrations,

pattern,

inhibition

(15)

bound

higher as

into

with

supernatants

unlimited

second

binding

in

an

well

the

together

method

At

as

and

binding only

cell

were in

[3H]-Gangliosides

taken

[3H]-gangliosides GM3

the

high

caused

Scatchard

status. of

patterns noted

of

were in

cells

pre-saturation

phasic was

the

the

incorporation

showed

Incorporation Fig.3

[3H]-gangliosides

incorporated the

the

radioactivities

concentrations, to

of data

that

which that

cell from between

gave

which differenFig.4 13H]-

Vol. 161, No. 2, 1989

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

k5 . 5i

.

:i:-

\z

L

+ 1: ‘+

k .

. . .

\

3 L

03

10

Coffentration

30( pM 1

40

50

04

mO

10 Bound

l\

.

.‘\

\

20 30 40 50 ( pmol / lo6 cells )

Fig.3 Incorporation of tritium-labeled gangliosides into HL-60 Radiolabeled cells as a function of ganglioside concentration. gangliosides were added to the culture media of HL-60 cells. At 15 min, reactions were stopped and radioactivities were fract'oned and counted as described in MATERIALS AND METHODS:O. [ 3 Ii]-GM3; m, [3Hl-GM1. Fia.4 Scatchard analysis of tritium-labeled ganglioside incorporation into HL-60 cells. Scatchard analysis was made using the data in Fig.3 and the corresponding radioactivity data in the supernatant fractions as described in MATERIALS AND METHODS:@, [3H]-GM3; n , [3H]-GM1.

GM3 and the cells C3H]-GM1 and the HL-60

cells,

r3H]-GM1.

were not so different cells. The apparent

however, This

WLE tritiated

were 2.5

difference

1,

Summary of gangliosides

Affinity =D %lx

Low Affinity

higher

well

than

to that

those between

apparent K. and BRAI values of associated with HL-60 cellsa

r3HI-GM3 High

from the ones between h values of [3H]-GM3 to

or 4 times

corresponded

Type

r3H]-GM1

7.9 37

2.5 11

41 a7

20 36

Type

a The values of l$, and A$V,I are presented in fl and pmol/106 cells, respectively. HL-60 cells were cultured with various concentrations of tritiated gangliosides and radioactivities were fractioned and counted as described in MATERIALS AND METHODS. Values presented in the table were calculated from Fig-l. 786

.

l\

from the

BIOCHEMICAL

Vol. 161, No. 2, 1989

amount of

incorporation

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[3H]-GM3

into

cells

and [3H]-GMl

(see

above).

DISCUSSION The mechanism of stood.

Only

cell

differentiation

down regulation

of c-myc

is

not

fully

proto-oncogene

underhad been

considered as one of essential events of hematopoietic cell differentiation

(16,171. For the mechanism by exogenous gangliosides

no investigation

In the

analyzed into

has been conducted.

how exogenous

HL-60 cells

GM3 was associated,

as a first

step

in

shown that GM3 was characteristically HL-60 cells around a concentration

the

of

using

derivatives

stood that with cells,

and radio-labeled

study,

we

bound or incorporated investigation.

We have

bound and incorporated into which caused growth inhibition

and cell differentiation. Intracellular metabolism spin-

present

GSLs has been studied (18,19).

elsewhere

It

is

under-

there are two fractions of labeled GSLs associated trypsin-sensitive and trypsin-resistant fractions.

We recently found, mainly in the trypsin-resistant fraction, specific metabolism of GM3 in HL-60 cells (unpublished data). Together

with

poration

of GM-, into

suggested tion

sides

specific

to play

of HL-60

In gliosides

this

the

metabolism, cells

an important

reported role

the

characteristic

in the

in the

present

monocytic

incorstudy

was

differentia-

cells.

addition to our investigations about on hematopoietic cells, functional

in several

cell

lines

the effects of ganroles of ganglio-

have been demonstrated.

exerted some influence '343. GM1 and GM3 derivatives liferation and on membrane receptor kinase activities Furthermore, a nerve growth factor-like function of

Gangliosides on cell

pro-

(20-22). tetrasialyl

on membrane receptor kinase activity GSL, GQlb, and its effect have been reported (23.24). Including the report from Kreutter all these reports were focussed on the effects of et al. (25), gangliosides on the kinase activities of membrane receptors to several growth factors and on several protein kinases. Recently, neoganglioprotein however, Schnaar et al. newly synthesized 1231]-HSA) and revealed ganglioside-specific binding pro(GTlb- [ This finding may tein in a rat brain synaptosomal fraction (26). support our results into HL-60 cells.

about characteristic And such a technique 787

incorporation of GM3 may help elucidate

Vol. 161, No. 2, 1969

whether

there

hematopoietic

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are membrane receptors cells

provide a new direction in which gangliosides

like

specific

HL-60 cells.

So our

in the investigation act as a trigger of

for

GM3 in

present

into initiation

study

would

the mechanism of cell dif-

ferentiation.

ACKNOWLEDGMENTS This research was partly supported by grants-in-aid from Ministry of Education, Science and Culture, JAPAN. We give thanks to Ms. Jinko Yamanoi for her technical assistance.

the

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