Vol.

179,

No.

September

2, 1991

16,

Adhesion

BIOCHEMICAL

AND

of

COMMUNICATIONS Pages

Human

Cancer

Katsuyuki Akihiro

Takada*, Tsuyuoka*,

*Department

Cells

of

July

to

Vascular

Antigen,

Endothelium

Sialyl

Lewis

713-71

g

Mediated

A1

Ohmori", Naofumi Takahashi*, Kiyotaka Yago*, Koichi Zeni&a*, Akira Hasegawa+ and Reiji Kannagi

Laboratory Medicine,

+Department of School of Agriculture, Received

RESEARCH

1991

by a Carbohydrate Akiko

BIOPHYSICAL

Medicine, -Kyoto,

Kyoto University, 606, Japan

Applied Gifu

Bio-organic University,

School

of

Chemistry, 510-11, Japan

29, 1991

Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-l) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-l-mediated binding of human cancer cells to activated endothelial cells. B 1991 Academic Press, Inc. SUMMARY:

Many have

been

carbohydrate

antigens

introduced

as

Recently,

the

associated

carbohydrate

ligand the

for surface

Cytokines factor cells ELAM-1

the of

sialyl

as

and thought

the

be

To whom correspondence

should

been

shown

endothelial

of

antibodies

ELAM-1

and

recruitment

expressed cells tumor

part by grants-in-aid of Education, Science

at (3-6).

necrosis endothelial

of of

cancer-

be a specific

on human

significance

(1,2).

these to

human (1Ll)beta

the

of

molecule

phyisiological to

one

adhesion

expression

'This work was supported in Research from the Ministry Japan (03557114 and 03258218). 2

cell

has

interleukin-1

induce

monoclonal antigens

antigen,

cytokine-activated

(TNF)alfa

is

x

antigens, the

by

cancer-associated

Lewis

ELAM-1,

such

(7-IO),

defined

expressed

leukocytes

to

for Scientific and Culture,

be addressed. 0006-291X/91 $1.50 713

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

179, No. 2, 1991

stimulated On the

vascular

other

cancer

cells

hematogeneous Here

chemical

suggested

metastasis

molecule

on activated

the be

the

process

sialyl

cells

Lewis

inflammation.

in

x antigen

on

process

of

the

(11,12). cancer-associated

a antigen, to

on cancer as a ligand

endothelial

of

Lewis

important

related

serves

RESEARCH COMMUNICATIONS

involved

cancer

expressed

also

the

an another

closely

mainly

of to

of

that

structure

(14-16),

in

expression

antigen,

and is

system

hand, is

AND BIOPHYSICAL

endothelium

we report

carbohydrate

(IS),

BIOCHEMICAL

that cells for

which of

has

a

sialyl

Lewis

x

the

digestive

of the

cell-adhesion

cells.

MATERIALS

AND METHODS

The cultured human colon cancer cells, Antibodies: and human leukemia cells, HL-60, are maintained in our laboratory in suspension culture, with RPM11640 medium The monoclonal antisupplemented with 10% fetal calf serum. Lewis a antibodies used for flow cytometric analysis and blocking experiments were N19-9 (murine IgGl, obtained from Centcor, Malvern, PA, USA)( 13), 2D3 and lH4 (murine IgM and IgG3, respectively. Established in our laboratory). The anti-Lewis x antibodies were FH-6, SH3 (both murine IgM, kindly supplied by Dr. Sen-itiroh Hakomori, Seattle, WA) (4,17) and 4F9 (murine IgM, produced in our laboratory). Monoclonal anti-ELAM-I and antiICAM-I antibodies (BBA2 and BBAI, both murine IgGl) were obtained from British Biotechnology Ltd., Abington, Oxon, UK. Monolayer cell adhesion assay using HUVECs: HUVECs (2-6 passages after isolation, obtained from Kurabou Co. Ltd., Osaka, Japan) were stimulated with Ing/ml of rILlb for 4 hrs in 24-well To this, ~010 201 (o.5X?o6/o.5ml/well) or ~~-60 plates(7-IO). (l.OXlo6/o.5ml/well) cells were added and incubated for 20 min at room temperature with rotation (100 rpm). The number of attached cells was counted directly under a microscope. The rILlB was obtained from the Central Research Laboratory of Otsuka Pharmaceutical Co., Tokushima Japan, and the recombinant basic fibroblast growth factor used for the in vitro culture of HUVECs was from the Central Research Laboratory of Takeda Pharmaceutical co., Juso, Japan. Preparation of liposomes containing pure glycolipids: Pure sialyl Lewis x glycolipid was chemically synthesized in our laboratory (18). The sialyl Lewis a glycolipid was purified from cultured human colon cancer SW11 16 cells as described (13). The liposome contained the indicated amount of the respective 20 pg of cholesterol and 40 pg of phosphatidylglycolipid, choline. The control liposome contained the same amount of cholesterol and phosphatidylcholine, but no glycolipid.

Cells c010201,

and

The human colon Lewis

a and sialyl

RESULTS

AND

cancer

cells,

Lewis

x antigens 714

DISCUSSION

Co10 201, (Fig.

I),

express as is

both the

sialyl

case with

Vol.

179,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

lo4

Fluorescence

RESEARCH

a-ELAM-l-

Intensity

a-ICAM-

-

-

+

-

-

-

+

Fiq. 1. Flow cytometric analysis of sialyl Lewis a antigens in Co10 201 and HL-60 cells ELAM-1 dependent adhesion of these cells HUVECs (right panel). HUVECswere pretreated

COMMUNICATIONS

Lewis x and sialyl (left panel) and the to rILlB-activated

with anti-ELAM-1 or anti-ICAMantibody (40 pg/ml) for 30 min at room temperature prior to the incubation with Co10 201 or HL-60 cells.

many

other

contrast, ly

cancer cultured

used for

ELAM-1

cells

cell

adhesion

When applied

recombinant

ILIB-activated

significant

adhesion

only the

the

i.e.,

to

attachment

HUVECs with

to

adhesion

kinds

been of

inhibited of

2).

HUVECs was much less

inhibited

these

the

cells

affected

were

treated 715

the

and of

the

conditions

were

pretreated

was with

x antibodies: of

Co10

by

three

the

(4,51,

these

adhesion

with

HL-60

1).

authors

adhesion

of

a

HUVECs was seen

(Fig.

Lewis

instead,

that

pretreatment

HL-60 cells

significantly

antibodies; when the

several

using

such as rILlB,

antibody

anti-sialyl

However,

to

by

to HUVECs under

when the

monoclonal

cells

Lewis

exhibited

to

by cytokines

by

x and

assays

cells

201

inhibited

common-

x and no sialyl

similar

201

anti-ELAM-1

cells

Co10

In

Lewis

cell-adhesion

very

Co10

reported

HL-60

FH6 and 4F9 (Fig.

with

of

activated

a monoclonal

significantly three

HUVECs,

are

sialyl

Lewis

monolayer

was completely

has

attachment

sialyl

organs.

which

involving

HUVECs,

when HUVECs were

As

only

digestive

HL-60 cells,

assays

express

a antigen.

from

human leukemia

(3-5,12),

cells;

derived

the

201

SH3, cells

to

pretreatment was

kinds

clearly of

anti-

Vol.

179, No. 2, 1991

BIOCHEMICAL

Anti-

-

AND BIOPHYSICAL

SH3 FH6 4F9

-

a-Sialyl

MY

‘-

NC)-9 2D3 1 H4

a-SL3lyl Lea

LG

Fiq.

2.

Lewis I-IL-60

The effect a antibodies (lower panel)

RESEARCH COMMUNICATIONS

of monoclonal anti-sialyl on the adhesion of Co10 cells to rIL18-activated

Lewis x and sialyl 201 (upper panel) and HUVECs. Co10 201 or

HL-60 cells were pretreated with monoclonal anti-sialyl Lewis a or anti-sialyl Lewis x antibody (20 pg/ml) for 30 min at room temperature prior to the incubation with HUVECs.

sialyl

Lewis

a antibodies:

was in clear to

HUVECs was not

Lewis

at

treatment.

antigen,

role

to

in

not

the

the

inhibited

but

inhibited

These the

HL-60 cells,

all

a antibodies,

antibody a

contrast

sialyl

adhesion

2D3 and lH4

N19-9,

only

process

with

that

x antigen,

to

HUVECs in

the

which

anti-sialyl Lewis

the

plays

This

of

by anti-sialyl

suggest

Lewis

2).

the attachment

by treatment

findings

(Fig.

sialyl

x

Lewis

an essential

case

of

Co10

201

cells. This liposome

was futher

confirmed

suspension

containing

preparation

having

and tested Co10

201

for

the

the

cells

a pure

following

adhesion

was

when HUVECs were pretreated

with

Co10

completely

liposome

antigen,

not

by treatment

with

indicating

that

again

31,

expressed adhesion

on of

Co10

these

201

cells

suspension

cells to

cells.

201

inhibited

the

(Fig.

Lewis

a

a glycolipid

structure,

HUVECs with but

sialyl

with

is

HUVECs. 716

by

containing

a control

the

sialyl

intimately

The adhesion pretreating sialyl

liposome Lewis involved

of the

Lewis

a

suspension a antigen in

the

Vol.

179,

No.

2, 1991

BIOCHEMICAL

rTT.lB ___.r Limo-

seine

-

+

-

-

AND

BIOPHYSICAL

COMMUNICATIONS

+++

+++

, 0 5 20 Sialil LeX Liposome

(l&7)

RESEARCH

,o 5 20, Sialyl Lea Liposome

Fiq. 3. Effect of pretreatment suspension containing pure sialyl glycolipid on the adhesion of Co10 (lower panel) cells to rILlb-activated

of HUVECs with liposome Lewis x or sialyl Lewis a 201 (upper panel) and HL-60 HUVECs. HUVECs were

with liposome suspensions, which contained the respective pure glycolipid of the indicated amounts, for 30 min at room temprature prior to the incubation with Co10 201 or HL-60 cells. pretreated

Interestingly, also

significantly

sialyl

Lewis

not

cells,

Co10

suspension the

as

cells

(Fig.

well

the

that 3).

of

pure

the

with

same extent liposomes

These

findings

HUVECs for closely same.

antibody

could

HUVECs, the ELAM-1,

or

containing

sialyl

from clearly

sialyl

related

cells

the

the

of

HUVECs

Lewis

do

HL-60 with

a

x ylycolipid

Cer,

that Lewis with

the

the

even

if

they

that

inhibit

the

adhesion

site

on HUVECs is

to

a monoclonal

ELAM-1

717

at

least

of

are

3).

sites

x antigens

finding

closely

(Fig.

binding

Lewis

other,

pretreated

a ylycolipid

carbohydrate

a and sialyl each

HUVECs were

Lewis

carbohydrate-binding is

HL-60

adhesion

sialyl

as was seen when the

and compete Judging

HUVECs with

structure,

indicate

the

HUVECs was

Conversely,

as the

NeuAc~2+3Gal81+4GlcNAc~l+3Ga181+4Glc~l+l FuccxlA3 to

to the

fact

pretreatment

containing

following

the

a antigen

by

HL-60

preincubatiny

by

cells,

201

of

despite

Lewis

inhibited

was

having

inhibited

sialyl

of

liposome

adhesion

a liposomes,

express

adhesion

the

on

overlap not

the

anti-ELAM-1 both most

cells probably

antiyenically.

to

Vol.

BIOCHEMICAL

179, No. 2, 1991

It

should

isomer

be noted of

structure

the

that

the

sialyl

not

may

react

discriminate

key enzymes

for

the

biological

studies

synthesizes

the

both

synthesis

of

sialyl

always

discriminate

antigens

mediated

adhesion

sialyl

(20

Kannagi, does not course

not

from

clear

a antigen

and

a significant

are

known

presence

of

an enzyme

which

that

even

enzymes

do not

The sialyl

the

between

cancer

cells

cancers

of

of the

sialyl

Lewis

in the

pancreas the

surface

role

the

in

the

originating

is

lung,

is

expressed

x antigen

these

of

may well

on cancer organs

be involved

2.

frequently

on

extent,

on

other

hand,

much more intensely and

is

probably of

metastasis

R.

J. Natl. -~

Cancer

AInst

A. N. & Scheinberg,

D. A,

Semin. ---

Oncol.

(1986). 718

the

originating

cancer

organs.

(1983).

Houghton, 179

S. & Kannagi,

in

metastasis.

cells

(15-77,22),

hematogeneous

the

a antigen

a lesser On the

(18,21).

during

Lewis

known to be expressed

digestive

process

sialyl

and

probably

adhesion

to

A.,

antigen

and hematogeneous

organs

and other

in

leukocyte

and,

human

most

Konomoto,

the

cells

ovary,

digestive

Lewis

T.,

on

Since

REFERENCES 1. Hakomori, 251

two

a antigen-

study.

detected

the

cancer

Lewis

present

Yoneda,

in

invasion

a antigen

sialyl

of

x antigen the

K.,

sialyl

the

not

However,

of vascular Lewis

the

observations),

inflammation. at

is

Ohmori,

play

process

cells

of

unpublished

of

involved

The

and molecular

difference

functions

R.,

expressed

than

are

Lewis

leukocytes

the

x antigen

and

(19).

The physiological

the

structural

them,

antigens.

enzymological

the

an

such as ELAM-1

between

Lewis

indicating

the

is

carbohydrate

carbohydrate

and recent

antigens,

its

differences

have disclosed

both

in

molecule

with

as 3-fucosyltransferases,

a antigen

x antigen

structural

well

RESEARCH COMMUNICATIONS

Lewis

A lectin-like

the

equally

sialyl

Lewis

as shown above.

may

AND BIOPHYSICAL

71,

13,

231165-

Vol.

179,

3. 4.

No.

Lowe, J. Berhend, Phillips, Singhal, 1130-1132

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719