Ganglioside GM1 sensitizes tumor cells to growth inhibitory glycopeptides

Vol. 107, No'. 2. 1982 July 30, 1982

BIOCHEMICAL

GANGLIOSIDE

AND BIOPHYSICAL

GM1 SENSITIZES

TO GROWTH INHIBITORY Robert

J. Kinders,

Kansas Retzeived

June 18,

David

RESEARCH COMMUNICATIONS Pages 663-669

TUMOR CELLS

GLYCOPEPTIDES

A. Rintoul

and Terry

Division of Biology University, Manhattan,

State

C. Johnson

KS 66506

1982

We 'have purified and characterized a glycopeptide from surfaces of brain cortex cells that inhibits cellular protein synthesis in normal but not transformed cells. Data are presented that correlate the ability of C3H fibrosarcoma 1316 cells to avoid the inhibitor's activity with a reduced cellular level of the ganglioside GMl. When 1316 cells were incubated with GM1 under conditions in which the ganglioside is incorporated into the cells, the cells became sensitive to the inhibitor. Similar experiments with ceramide resulted in no change in 1316 sensitivity. In addition, when 1316 cells were incubated with GM1 for 18 h, they apparently metabolized GM1 to simpler gangliosides and lost their sensitivity to the glycopeptide inhibitor.

INTRODUCTION -____ We have described

the isolation,

released

mild

which

are potent

These

glycopeptides

may be naturally

their

by cell-cell

synthesis

(1,2).

regulators

that

mediate

similar

to that

proposed

irhibit

protein

synthesis

level ard/or pr,otein

of elongation

synthesis which

action

(2).

The inhibitor

lines,

somewhat and relatively

is

in a reversible

less

in the

active

or completely

growth

chains, (4).

concentration

against

of cell

active

cells

contact (3).

growth

in a manner

These

macromolecules

manner,

requiring

range

associated

against

primary

cells

tissue

culture

against

mediation

can inhibit

of 2 x lo5 molecules

ineffective

at the

membrane

The glycopeptides

established

by

and protein

occurring

and non-lethal

peptide

is most

cortical

and Stoker

by 50% at a concentration

cell,

culture,

effects

modification

target

of brain

inhibitors

by Dulbecco

of nascent

an intra-cellular

the surfaces

and characterization

of glycopeptides, proteolysis,

from

purification

with

transformed

per hormone in cell or

0006-291X/82/140663-07$01.00/0 663

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in an-v form reserved.

BIOCHEMICAL

Vol. 107, No. 2, 1982 tumor-derived kidney

cell

cells

lines

(1,s).

(pyBHK) were

glycopeptides

in a saturable

inactivated

the

glycopeptides

1316 cells

escape

inhibition

the glycopeptide

inhibitors

remains

cell

MATERIALS

in their

AND BIOPHYSICAL Polyoma

able

to bind

virus

transformed

the biological

fashion

but escaped

(5).

We now report

by a different only

RESEARCH COMMUNICATIONS

occurs

activity inhibition that

mechanism. when the

baby hamster

intact

of the because

they

mouse fibrosarcoma Sensitivity

to

ganglioside

GM1

membranes.

AND METHODS

Isolation and Purification of Glycopeptides. The glycopeptides were prepared from cerebral cortex cells of 6 week-old Swiss albino outbred mice by mild proteolysis as described previously (2). The macromolecules released by proteolysis were purified by ethanol precipitation and chloroform/methanol extraction, followed by gel filtration on BioGel P-100 and the active material eluting at apparent MW 30,000 was concentrated by ultrafiltration before use. Some experiments were performed with material purified by affinity chromatography with agarose bound Ulex europaeus agglutinin I (UEA-I), eluted with 0.1 M fucose and dialyzed against HKM buffer (20 ti- Hepes; 120 fiJ KCl; 5 mMMgC12; pH 7.1) (2). Antisera. Antisera to the inhibitor were prepared from hyperimmunized Balb/c mice. Blood was obtained from tail bleedings, clotted, the sera were decomplemented, and then clarified by centrifugation at 10,000 x g for 1 hr. The clarified sera were diluted in an equal volume of borate saline buffer (BSB) (162 mM sodium borate, 133 mM sodium chloride pH 8) and salt fractionated with-(NH4)2SO4 as described by Heide and Schwick (6), dialysed, then fractionated on DEAE-Sephadex and Sephadex G-200 The recovered IgG fraction was diluted to a final concentration of (7). 6 mg/ml in BSB and characterized by quantitative precipitation assays with anti-mouse IgG, and double antibody radioimmunoassays (RIA) with UEA-I purified inhibitor that had been labelled with 1251 using the method,of Wood --et al. (8). Cell Culture. Fibrosarcoma were provided University. C02; 95% air

BHK-21 cells were grown as described previously (5). 1316 cells, induced by LJV irradiation of C3H mice (lo), by Dr. G. Wm. Fortner, Division of Biology, Kansas State Cells were routinely grown as monolayers at 37OC in a 5% atmosphere in humidified incubators.

Ganglioside Analysis. Cells which had been incubated for 2 hours at 2-4'c, with or without added GMl, were washed three times with phosphate-buffered saline (0.1 M sodium phosphate, 0.09 M NaCl), scraped with a rubber policeman, and centrifuged for 5 minutes at 1000 x g. Cell pellets were extracted by the method of Irwin and Irwin (9), and gangliosides were purified by silicic acid column chromatography (9). Gangliosides and appropriate standards were resuspended in CHC13/CH30H (2/l, v/v), and spotted on 250 cm silica gel G thin layer chromatography (TLC) plates. Chromatograms were developed at room temperature with CHC13/CH3OH/12 + MgC12/ 15 1 NH40H (60/35/7.5/3,v/v) (12), air-dried, sprayed with 50% overnight at 120'. H2SG4, and charred Protein Synthesis Assays. Cellular protein synthesis was measured in 100 nl DMEM containing 25 mM hepes plus 25 ~1 HKM buffer (controls) or 25 ~1 HKM containing specified concentrations of glycopeptides as described previously (1,5). 664

Vol. 107, No. 2, 1982

BIOCHEMICAL 80

AND BIOPHYSICAL

A

RESEARCH COMMUNICATIONS

B

1

1 0 PQ Glycopeptides

PQ

Glycopeptides

of Cellular Protein Synthesis by Brain Cell Fig. 1. Inhibition Surface Glycopeptides. 2 x 106 BHK-21 or fibroaarcoma 1316 cells were incubated with 0.25 to 1.0 pg Ulex europaeus affinity purified glycopeptides for 30 min then radiolabelled with 35S-methionine. Panel A, (0) BIN-21 cells, (0) 1316 cells; Panel B, (0) 1316 cells incubated with GM1 2 h at O°C prior to protein synthesis assay, (0) 1316 cells incubated with ceramide 2 h at O°C prior to assay, (& 1316 cells incubated with GM1 2 h at O'C then 18 h at 37'C prior to assay. All results are means and standard deviations of 2 experiments, each performed in triplicate. of Inhibitor --Inactivation exposed to exponentially clarified by centrifugation Inactivation assays were

by Conditioned Media. Medium that had been growing 1316 cells for 16 h was collected and at 2,000 g then frozen at -2O'C until use. performed as described previously (5).

--RE.SULTS Treatment resulted tfesis

of 1316 cells

in only

a limited

compared

b] 70% (Fig. medium

to BHK-21

1A).

from the

the inhibitory

Unlike 1316 cells

glycoproteins

role

with

tl-te insensitivity

in an ice

degree

of inhibition

cells medium did

protein

from

not

not

conditions

levels

which

of cellular synthesis cells

protein could

syn-

be inhibited

(5),

conditioned

the biological

activity

GM1 as a receptor (11,12)

to the

1316 cells

glycopeptides

of

shown).

activity

of 1316 cells

inhibitory

pyBHK-21

inactivate

(data

physiological

of 5 x lo6 bath,

where

of ganglioside

be due to insufficient

Mcsnolayers

the growth

glycopeptides

The possible

might

with

were

incubated

were

selected

66.5

a number

suggested

glycopeptide

of GM1 in

for

growth

the cell with

to us that inhibitor

surface 0.7

to allow

of

membrane.

UM GM1 for incorporation

2

h

of

Vol. 107, No. 2, 1982 the ganglioside endocytosis were

into

washed

scraping

with

Affinity

purified then

to allow

cells

were

a rubber

that

to bind

at 37'C

to the

subsequent

protein

inhibitory

TLC analysis

of the band migrating 0.1

preincubated

with

with

GM1 were

with

fibrosarcoma

5% fetal

gangliosides, low

it

levels

peptide.

change

in

them to a 37'C GM1 by TLC.

was detectable

in

2, lane

a short

with incubator

By 18 h after

the cells

by either

(Fig. that

there

material

was

migrating

with

supplemented

GM1 and other the cells GM1 could

had such produce

to the inhibitory

1316 cells

could

glyco-

metabolize

2 h at O°C and then

2, 8 or 18 h and measuring the addition TLC (Fig. 666

1B).

The intensity that

in medium

contains

UM GM1 for for

influence

C).

sensitivity

0.7

not

indicated

any,

to us that

that

of

When the 1316 cells if

incubation

the cell's

did

B).

or

that

inhibitors

(Fig.

are cultured

the possibility

1316 cells

of the

little,

surprising

cellular

no GMl,

indicated

manner

and serum usually

was somewhat

We tested

by incubating

remaining

serum,

of GM1 and that

such a marked

ferring

calf

1316 cells

received

in this

2, lane

The

into

ceramide

present.

tested, (Fig.

GMl.

of GM1 indicated

pg of the ganglioside

that

the sensitivity

while

cells

bath

by a 30 min

that

GM1 increased

the Rf value

at the Rf of GM1 was evident Since

the

1316 cells,

incorporated

The results

treated

into

lo6

conditions

followed

in the presence

of 1316 cells

GM1 had been incorporated

approximately

cells.

by

10 min in an ice

under

to 1316 cells

with

harvested

of 35 S-methionine

glycopeptides

synthesis

for

30 min,

The incorporation and compared

The cells

to 1.0 ng per

membrane

for

limiting

by centrifugation.

to the cells

of surface

1316 cells

while

HKM buffer,

in suspension

the cells

of the

(13,14),

and pelleted

2.5 !JM ceramide/106

1316 cells

cold

with

was measured

preincubation

not

ice

at 0.25

period.

received

with

glycopeptides,

incubated

radiolabelling

membrane

RESEARCH COMMUNICATIONS

of the gangliosides.

policeman,

degradation then

surface

times

the glycopeptides

restrict

AND BIOPHYSICAL

degradation

three

reacted

would

protein

the cell

or enzymatic

then

were

BIOCHEMICAL

of the 2, lane

GM1 trans-

the

ganglioside, E) or cell

none sensi-

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ABCDE SF-

GMl-

Fig. 2. Thin-layer Chromatographic Analysis of 1316 Cell Gangliosides. 3 x lo6 cells were incubated on ice for two hours in minimal Eagl.e's medium (without serum) containing 0.7 uP1 of ganglioside GMl. Control cells were incubated in medium without ganglioside. After rinsing the monolayers three times with phosphate-buffered saline, the cells were removed with a rubber policeman, washed twice with 5.0 ml phosphate-buffered saline, extracted, and chromatographed as described in Materials and Methods. Lanes A and D = ganglioside GM1 standard, Lane B = cells incubated with GMl, Lane C = cells incubated without GMl, Lane E = cells incubated with GM1 and subsequently incubated for 18 hours at.37'C in medium + 5% calf serum. SF = solvent front. Results shown are typical results from one of four experiments.

t.Lvity

t:o

the

af:companied products, with

by of

GMI

glycopeptide the

inhibitor

(Fig.

appearance

of

other

Rf

were

not

greater

that

(compare

lanes

C and

previous

data

suggested

E,

The

1B).

gangliosides, detected

Fig.

2).

that

the

loss

of

GM1

presumably in

1316

cells

was metabolic

not

treated

D.TSCUSSION -~Our rl?quire

a membrane

mediation

of

their

growth

inhibitory

667

inhibitory

glycopeptides

activity.

Both

BHK-21

BIOCHEMICAL

Vol. 107, No. 2, 1982 cells,

which

are

not,

are

bind

cells

apparently

which

is

cells

have

more

into

(11,

cells

demonstrate increases

that

the

their

not

are

allowed

The

fit

the

transformation reduced GM1

the

as level

growth

regulatory

experiments

acting

as

presence confers

in

the

suggests

by

(12).

Hakomori

and

cell

sensitivity

to

expression

the

inhibitory

test

as

fibrosarcoma

fibro-

Fig.

1

sensitivity if

the

at

37oC,

fibrosarcoma malignant

is

the

cells,

case, the

the removal

sensitivity

to

underlying on

whether

glycopeptides,

membrane, 1316

one

gangliosides

wilL

GM1

reduced

reflect

of

laboratory the

surface

may

this these

their

mouse

substantially

or

upon

in

been

increased

these

If

observed

has

gangliosides.

that

simplification

of

in

ceramide

with

ganglioside

our for

The

simpler

cells

neuroblastoma

membranes

incubated

membranes,

the

a receptor in

GMl,

to

cultured

presented

cell

with

cells,

glycopeptide, with

1316

(16).

incorporation

mouse data

BHK

BHK cells

transformed

The

GM1

transformed

phenomenon

glycopeptides.

the

regulation

surface

associated

Further

of

including

in

(5),

of

This

preincubated

no

proposed

their

growth

event

of

from

are

or

subsequent

(12),

GM1 the

1316

little

and

transformed

inhibitor

untransformed incubation

which

virally

virus

that

(15).

metabolize

pattern

do

fibroblasts

of

the

polyoma

(12).

types

to

that

contain

cells

human

cells

observation

actually

cell

presence

to

than

cells,

The

for

that

(11)

erythrocytes

when

sites

membrane

of

sensitivity

observed

cells

plasma

normal chick

binding

uptake

pyBHK

fashion.

surfaces

in

RESEARCH COMMUNICATIONS and

a saturable

demonstrated

the

and

glycopeptides,

observation

their

a variety 13),

(14),

on

results

using

blasts

the

previously

glycolipid

observed

of

possessed

GM1

been

the

in

with

more

has

to

inhibitor

gangliosides

the

is

the

consistent

It with

sensitive

AND BIOPHYSICAL

or

a structural

transformation

the

cell

GM1

is

surface. directly

whether

its

element,

indirectly

cells.

ACKNOWLEDGEMENTS This the

National

study

was Cancer

supported Institute,

by

grants

HHS

no.

and

668

the

CA 27648 Kansas

and

Agricultural

CA 31531

from

Experiment

Vol. 107, No. 2, 1982 Station.

BIOCHEMICAL

This

Agricultural

is

AND BIOPHYSICAL

contribution

Experiment

82-411-3,

Station,

RESEARCH COMMUNICATIONS

Division

Kansas

State

of Biology,

the Kansas

University.

REFERENCES

:I.

Kinders, 43-50.

R.,

Johnson,

Kinders, Biochem.

R.J., Milenkovic, J. 190, 605-614.

Dulbecco, R. and Stoker, 66!, 204-214. Kinders, R.J., 255, 6368-6372.

Hughes,

A.G., M.G.P.

J.V.

Kinders,

I).

He].de, K. and Schwick, Immunology (D.M. Weir,

H.G. ed.)

Fahey,

E.W.

J.L.

and Johnson,

and Terry,

Wood, F.T., 33!)-349.

Wu, M.M.

Irwin,

and Irwin,

C.C.

L.N.

Gitchley, D.R. (1979) (R.O. Hynes, ed.), pp.

12.

Hakomori,

13.

Moss, R.O.,

li.

Fi:shman, P.H., Bradley, R.EI., J. Lip. Res. 19, 77-81.

15.

Fishman, P-H., 16, 1871-1875.

16.

Callies, (1977)

17.

Gahmberg, 413-415.

(1981)

M.

Kiehn,

(1980)

(1970)

Proc.

Nat.

T.C.

(1980)

Exp.

Cell

ibid., J.

pp.

(1975) Anal.

(1977)

J.

Acad.

Sci.

Ann.

Res.

136,

P.H., Manganiello, Nat. Acad. Sci. Moss,

Biochem.

Immunol.

94,

335-339.

118,

1483-1487. Cells

733-764.

J. and Manganiello,

G., Radsak, 80, 425-432. and Hakomori,

669

69,

V.C., Vaughan, M. and Brady, U.S.A. 73, 1034-1937.

.I. and Manganiello,

D.,

31-41.

8.1-8.16.

Biochem.

50,

Chem.

of Experimental

Anal.

Rev. Biochem.

U.S.A.

.J. Biol.

in Surfaces ~ of Normal and Malipnant 63-101. Wiley and Sons, New York.

Schwarzmann, J. Biochem.

C.G.,

T.C.

(1979)

11.

R., Eur.

P. and Johnson,

and Gerhart,

G.W. and Kripke,

Moss,

Nordin,

(1978)

Fortner,

J.L., Fishman, (1976) Proc.

24,

(1978) in Handbook Vol. 1, pp. 7.1-7.11.

10.

S.-I.

Sci.

(1981)

T.C.

(1979)

M.

and Johnson,

ii .

‘3.

R.J.

Life

T. and Rachmeler,

V.C. K.,

Siegert, S.-I.

V.C.

(1977)

(1978)

Biochemistry

R. and Weigandt, (1974)

Nature

248,

H.