Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF CHLOROQUINE ON CULTURED FIBROBLASTS: RELEASE OF LYSOSOMAL HYDROLASES AND INHIBITION

Ulrich

N. Wiesmann,

Stephano

Pediatric

Received

August

25,

DiDonato*

OF THEIR UPTAKE

and Norbert

Department, University Berne, Switzerland

N. Herschkowitz

of Berne

1975

SUMMARY: Incubation of normal human fibroblasts with l-5 uM chloroquine at physiological pH for 8 hr produces granular cytoplasmic inclusions, release of lysosomal enzymes into the medium and decrease of intracellular lysosomal enzyme activities. The effects are dose dependent and reversible. The uptake of arylsulfatase A into fibroblasts genetically deficient in arylsulfatase A (grown from skin biopsies of patients with metachromatic leukodystrophy) is completely inhibited by pretreating the cells with 5 pM chloroquine. Arylsulfatase A, which has been taken up as exogenous enzyme from the medium into the cells, is partially released into the culture medium upon incubation with chloroquine. The data suggest that chloroquine competes with the binding of lysosomal enzymes to the cell membrane and to the membranes of pinocytotic vacuoles and causes release of previously internalized exogenous enzyme.

INTRODUCTION Chloroquine istics

is

of which

digestive tured

a well-known

have been

vacuoles cells

function enzymes a change

(3).

recently

of malaria It

the

In cultured

pH of the

The cells

35 SO4-labeled

mucopolysaccharides

patients

Mucolipidosis

lysosomal the

with

modification

Fellow

Copyright All rights

a rare

This

(5,6).

and to reenter

of the

Institute

that

from the of Neurology

o 19 75 by Academic Press, Irzc. of reproduction in my fmw reservrd.

chloroquine

lysosomes

and/or

thus

inhibiting

loss

of these is believed

normally

"C. Besta", 1338

produces

stop

degrading cultured

from the

from

by multiple cells

into

to be due to a genetic

enables

extracellular

rapidly

characterized

enzymes

of cul-

lysosomal

fibroblasts

disorder

in the

lysosomal

and they

resembling

genetic

lysosomes

inhibits

1 uM chloroquine

due to loss

of an enzyme marker

the lysosomes

*

fluid

that

character-

accumulates

as in the

become granulated (4)

II,

enzyme deficiencies

extracellular

as well

human fibroblasts

of morphology.

Chloroquine

(1).

(2)

speculated

the lysosomotropic

drug,

reviewed

parasites

has been

by increasing (1).

antimalarial

the enzymes

to stay

within

fluid

(7,8).

Our working

Milan,

Italy.

Vol. 66, No. 4,1975

hypothesis

BIOCHEMICAL

was that

enzyme retention

chloroquine

mechanism

mimicking

Mucolipidosis

MATERIALS

AND METHODS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

would

interfere

to produce

with

the normal

a morphological

function

and biochemical

of the picture

II.

Fibroblasts were grown from skin biopsies of patients with metachromatic leukodystrophy (MLD) and from normal control patients undergoing minor surgical interventions. The cultures were grown in Eagle MEM (GIBCO) pH 7.4 as described previously (9). For the experiments fibroblasts were subcultured and replicate cultures were started in 250 ml Falcon plastic flasks with 50,000 cells per flask and grown to confluence. Chloroquine was purchased from Sigma. Arylsulfatase A was partially purified from human urine as described previously (92. The specific activity of the preparation used was 20-40 nmol/min/mg protein. C-dextran (80,000 MW) was obtained from Amersham (England). Arylsulfatase A was determined according to Baum --et al. (lo), S-glucuronidase was determined using 4-methylumbelliferyl-B-glucuronide as a substrate (11). The cells were removed from the culture flasks with buffered 0.2% trypsin in Hank's solution pH 7.4. After two washings the cells were sonicated in distilled water with a Branson ultrasonicator at 4' C for two times 30 sec. Protein determinations were performed in an aliquot of the homogenate by the Lowry method (12). Lactic acid dehydrogenase and malic acid dehydrogenase were determined using a Boehringer (Boehringer GMBH, Germany) test kit. RESULTS Effect

of chloroquine In normal

fibroblasts

lysosomal

enzymes

exposure,

decreased

Recovery

on intracellular

enzyme activity.

pretreated

S-glucuronidase with

with

24 hr after

concentrations

of chloroquine

(Table

was noted

to 1 and 2 pM chloroquine.

The recovery

cells

An inhibition

at the

concentration

In media cultures,

used

collected

elevated

extracellular

found

as compared

(Fig.

1).

The increased

enzymes,

reduction lactic

medium were

not

Arylsulfatase

to the

acid

of the

control

activities

complete

after

exposure

I). exposure of the

by chloroquine

had been washed

of arylsulfatase in proportion

in the media

accounted

and malic

hr following

enzyme activities

cultures,

in the cells.

dehydrogenase

48-72

the

was excluded.

the chloroquine activities

of activities

within

was less

experiments

24 hr after

were

of the

in our

of the

A, measured

activities

to 5 PM chloroquine.

the activity

and arylsulfatase

increasing

of the two enzyme

chloroquine,

from

the

A and B-glucuronidase to the dose applied for

The activities acid

out

more than

two-thirds

of two non-lysosomal

dehydrogenase

in the

culture

elevated. A activity

in the

culture

1339

medium was followed

for

72 hr after

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I EFFECT OF CHLOROQUINE ON THE INTRACELLULAR ACTIVITY

OF

ARYLSULFATASE A AND &GLUCURONIDASE Chloroquine

Arylsulfatase

A*

B-glucuronidase* *

nmolfminjmg

protein.

concentration

(@i)

-0

I

-2

I

11.2

10.5

8.2

6.8

2.5

2.2

1.9

1.5

Mean values

of two cultures

Normal fibroblasts were grown to confluence in 250 ml Falcon culture flasks; duplicate cultures were exposed to chloroquine for 8 hr. Then the cultures were washed twice with 10 ml of medium and they were incubated for 24 hr with 7 ml of fresh medium prior to harvest.

Fig. 1. Effect of chloroquine on the subsequent release of arylsulfatase A and B-glucuronidase from normal fibroblasts. Replicate cultures were exposed Fresh medium was then added and the cells were into chloroquine for 8 hr. Then the media were dialyzed and arylsulfatase A and cubated for 24 hr. B-glucuronidase were measured in an aliquot. Arylsulfatase lines represent

Vertical

exposure return

to chloroquine to control

Seventy-two was still

values

hr after significantly

A, striated bars; $-glucuronidase, range of values of three cultures.

by changing

the media

was found

72 hr after

exposure higher

at 24 hr invervals. exposure

to 5 PM chloroquine than

control

1340

black

values.

the

bars.

A complete

to 2 uM chloroquine. activity

of arylsulfatase

A

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0!-+++44 CHLOROWINE

CONCENTRATION

$A~I

Fig. 2. Effect of chloroquine on the subsequent uptake of exogenous arylsulfatase A into MLD fibroblasts. Replicate cultures of MLD fibroblasts were exposed to chloroquine for 8 hr. After through washing three times with medium, the cells were incubated for 24 hr with partially purified arylsulfatase A preparation (40 nmol/min/ml medium). Control cultures which had not been exposed to chloroquine were also incubated with arylsulfatase A preparation. Intracellular arylsulfatase A activity was determined in the homogenate of the trypsinized and washed cells and corrected for the small amount of endogenous activity.

TABLE II EFFECT OF CHLOROQUINE ON MLD CELLS CONTAINING EXOGENOUS ARYLSULFATASE A Arylsulfatase (nmol/min/mg cell Chloroquine

(PM) -

Intracellular activity

A protein)

Recovered in medium

Percent released

0

14.0

0.3

2

5

8.2

3.5

30

Replicate cultures of MLD fibroblasts were incubated with 40 nmol/min/ml arylsulfatase A for 24 hr. The cultures were washed with medium twice and exposed to 5 pM chloroquine for 13 hr. Arylsulfatase A was determined in the cells exposed to chloroquine and in control culture. The media were dialyzed overnight against three liters of 0.05 M Na acetate buffer, pH 5.6 and arylsulfatase A was measured in an aliquot.

1341

Vol. 66, No. 4, 1975

Effect

BIOCHEMICAL

of chloroquine Partially

on the pinocytosis

purified

taken

up into

5 a,

completely

MLD fibroblasts blocked

the

was found

to be reversible

Effect

Exposure

could

be almost

the other for

conditions

on MLD cells

arylsulfatase

hand,

13 hr retained

enzyme

A into

The uptake

the

prelabeled

with

the intracellular

cells.

the

2.

The inhibition The inhibition 14 C-dextran

in the

loss

of 30% of previously

The missing

incubation

C-dextran

(13).

enzyme.

from MLD fibroblasts. from

Chloroquine,

of macromolecular

exogenous

14

(9).

by chloroquine

13 hr resulted

recovered

is efficiently

as shown in Fig.

containing

A activity

the cells

in that

was unaffected

for

quantitatively

human urine

of arylsulfatase

4-5 days.

to 5 uM chloroquine

internalized

deficient

dose-dependent

within

experimental

of chloroquine

are

uptake

of enzyme was clearly

identical

A from normal

which

A and 14 C-dextran.

of arylsulfatase

arylsulfatase

of uptake

under

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

medium

and exposed

activity

(Table

11).

On

to 5 uM chloroquine

radioactivity.

DISCUSSION Dark granular of 8 hr incubation the

with

concentration

cells

inclusions

acid

damaged.

Loss of lysosomal

reciprocal

medium,

relation

The inhibition fibroblasts 80,000

rather

enzymes were

not

enzymes than

and the

or cellular

elevated

extracellular

lactic

as would

(1)

especially unaffected.

acid

be expected

Dextran

1342

for

enzymes of the

used during

varied

with

changes,

the

noted

during

dehydrogenase if

cells

were into

the intra-

tested.

The

same enzyme were

the preincubation,

may be assumed.

of arylsulfatase because

end

fibroblasts

accounts

increase

of chloroquine

of pinocytosis

were

chloroquine-pretreated

of the two observations

MW was completely

debris

of the two lysosomal

concentrations

was unexpected,

of the granulation

in the medium,

from

at the

of the morphological

enzyme inhibition

of the activity

reduction at all

so a direct

no dead cells

dehydrogenase

reduction

intracellular

But in spite

Non-lysosomal

and malic

by phase microscopy

The intensity

chloroquine.

to be healthy;

the experiments.

cellular

be observed

of chloroquine.

appeared

the culture

could

A into pinocytosis

enzyme-deficient 14 C-dextran of

was used as a measure

MLD of

of pinocytosis

Vol. 66, No. 4,1975

because it

is

its

BIOCHEMICAL

molecular

assumed not Studies

of lysosomal

weight

to bind

enzymes

enhanced

is

alteration

of the

of lysosomal If uptake

the membrane

and/or

enzymes

The release

a possible

fibroblasts.

requires

binding

out in our

is bound

of the

avidly

the pinocytosis enzymes,

presumably

Lack of recognition

due to

II

in the

patients

fluids

results

(6). at the

by the assumption carried

and because

that

cell

membrane

chloroquine

laboratory to the

with cell

(14),

competes

with

14 C-chloroquine

surface

of normal

(15). vacuoles,

effected

recognition

of Mucolipidosis

enzymes

enzymes

have shown that

extracellular

chloroquine

In lysosomal

lysosomal

the

of lysosomal

(13).

and by us (8)

enzymes

Experiments that

membrane

of the

into

to that

by specific

may be explained

sites.

fibroblasts

from

lysosomal

of lysosomal

have demonstrated

(7)

membrane

enzymes

observations

the binding

to the cell --et al.

to the cell

our

comparable

by Hickman

by binding

loss

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

into

chloroquine interfere

the

mechanism

the

arylsulfatase

suggests for

with

displace localization

bound

lysosomal

enzyme

of newly-synthetized

lysosomes.

of exogenous

by chloroquine

could

lysosomal

that

A from MLD fibroblasts

enzyme displacement

into

from secondary

the medium lysosomes

enzyme loss.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

De Duve, Ch., De Barsy, Th., Poole, B., Trouet, A., Tulkens, P., Van Hoof, F., Biochemical Pharmacology g, 2495 (1974). Polet, H. and Barr, C. F., J. Pharmacol. Exp. Ther. l&, 380 (1968). Wibo, M., Poole, B., J. Cell Biol. 2, 430 (1974). Lie, S. L. and Schofield, B., Biochem. Pharmacology 2, 3109 (1973). Wiessmann, U. N., Vassella, F. and Herschkowitz, N., Acta Paediat. Stand. 63, 9 (1973). Neufeld, E. F., Lim, T. W. and Shapiro, L. J., Ann. Rev. Biochem. 44, 357 (1975). Hickman, S., Shapiro, L. J. and Neufeld, E. F., Biochem. Biophys. Res. Commun. 57, 55 (1974). Wiesmann, U. N. and Herschkowitz, N., Ped. Res. 8, 865 (1974). Wiesmann, U. N., Rossi, E. E. and Herschkowitz, N., Acta Paediat. Stand. 61, 296 (1972). Baum, H., Dodgeson, K. S. and Spencer, B., Clin. Chem. Acta 4, 453 (1959). Wollen, J. W. and Walker, P. G., Proc. Assoc. Clin. Biochem. 2, 14 (1961). Lowry, 0. H., Rosebrough, N. .I., Farr, A. L. and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Wiesmann, U. N., Enzyme Therapy in Lysosomal Storage Diseases, Tager, J. M., Hooghwinkel, G. J. M. and Daems, W. Th. (Eds.),North Holland Publ., Amsterdam, 1974. Hickman, S. and Neufeld, E. F., Biochem. Biophys. Res. Commun. 49, 992 (1972). DiDonato, S., Wiesmann, U. N. and Herschkowitz, N., in preparation. 1343

is