Vol. 68, No. 1, 1976

ON THE

BIOCHEMICAL

THE EFFECT FIBRINOLYTIC

W.E. Division

Received

Laug’,

AND BIOPHYSICAL

OF CYCLIC ACTIVITY

P.A.

AMP AND PROSTAGLANDI NS OF MOUSE NEUROBLASTOMA

Jones*,

C.A.

of Hematology-Oncology, 4650 Sunset Boulevard,

October

17,

RESEARCH COMMUNICATIONS

Nye

and

W.F.

CELLS

Benedict3

Childrens Hospital Los Angeles, Ca

of 90027

Los

Angeles

1975

SUMMARY. The Cl300 mouse neuroblastoma cell line was found to produce plasminogen activator which is secreted into the growth medium. The intraand extracellular activities of this enzyme were markedly increased (up to 14 fold) by treatment with cyclic AMP agents. Prostaglandins El and E2 and butyric acid were the most efficient inducers followed by propionic acid and dibutyryl cyclic AMP. Theophylline was found to be ineffective. The highest enzyme activities were found in cells exposed simultaneously to prostaglandin and dibutyryl cyclic AMP. I NTRODUCTI

ON.

Many

activator

which

appears

This

activity

is

species

including

induced

to

agents

in

cells

to

found

in

human

be

vitro

(6),

in

activity,

since

activator

can

of

the

glucocorticoids

have

induced can

in

vitro

with

the derived

neuroblastoma

(5).

Since

and

their

enzymatic

change to

mouse

study

Cl300 that

the

these

that

in

(7).

Also,

inhibit

the

Address: Address:

3To

the

reprint

whom

FCS,

Wigler

114 Copyrighf A It rights

0 I976 by Academic Press, of reprodzcction in any form

Inc. reserved.

PBS,

cells

can

these

agents

on

We were

of

et.

al.

CH3000,

AMP the

fibrinolytic plasminogen (8)

have of

Berne,

shown

tumor

passage 62 cells were IO% (v/v) were seeded The Cal.

63, Tygerberg

be

particularly

influence

activity

Box

and

cyclic

secretion

fibrinolytic

fetal calf serum; Adenosine 3’:5’-cyclic El and E2.

tissues

by

of

might

(l-4).

functions

cells.

University Childrens Hospital, Dept. Medical Biochemistry, South Africa. requests should be addressed.

Abbreviat’ons used: dbcAMP, N 6 , 02-Dibutyryl and PGE2, prostaglandins

phenotype several

METHODS : The uncloned Cl300 line was kindly provided in The Finkels tein, Harbor General Hospital, Torrance, Cal. Minimum Essential Medium (GIBCO) supplemented with Eagle’s and streptomycin (100 units/ml). Cells (5x105) penici 1 lin 100 mm tissue culture dishes obtained from Falcon, Oxnard, 1 Present *Present

a plasminogen

neuroblastoma

effects

agents

found

strongly

from

neuroblastoma

recently vivo

produce transformed

cells

possibility we

be

associated

we decided

activity

grown

transformed

“differentiate”

fibrinolytic interested

malignant

El

cells. by J.Z. grown in FCS and into growth

Switzerland. 7505,

phosphate buffered monophosphoric acid;

that

saline; PGE,

Vol.

68,

No.

BIOCHEMICAL

1, 1976

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

medium

was changed to test medium 24 hr later. Test medium always contained (v/v) FCS since FCS does not interfere with the fibrinolysis assay but, on the contrary, appears to stimulate protease secretion (7). Control cultures were fed with the growth medium supplemented with calcium and magnesium free PBS (GIBCO) or 96% ethanol respectively at the same concentration as used as solvents for the cyclic AMP-agents. dbcAMP (Sigma), theophylline (Calbiochem), butyric acid and propionic acid were dissolved in PBS and added to the growth medium. PGEL and PGE2 (personal gifts from Dr. J. Pike, Upjohn Co.,Kalamazoo, Mich.), were dissolved in 96% ethanol. Growth medium supplemented with the solvents and compounds respectively was changed after 48 hr incubation at 37’. Extracellular fibrinolytic activity was determined in aliquots of the superAt the same time photomicrographs were made of dishes natant taken after 96 hr. seeded with lo5 cells and the percentage of “differentiated” ccl Is determined on a minimum sample of 100 cells (9). After 96 hr of exposure to the compounds the medium was removed and the cell cultures washed once with PBS. The cells were then either trypsinized and counted in a Coulter counter or scraped off the dish with a rubber policeman when they were to be lysed for fibrinolysis assays. Gel lular protein levels were determined by scraping the cells directly into cold 5% perchloric acid and determining the protein by the method of Lowry et. al. (10). Intracellular plasminogen activator was determined in cell lysates prepared with 0.5% Triton X-100 as previously described (7). Lysates( lo-20 ul) were incubated with 2.5 ml of freshly prepared 0. IM Tris-HCI pH 8.1 containing 1% acid treated pooled human serum as plasminogen source onp25l]fibrin plates. The appearance of radioactivity in the supernatant was used as a measure of fibrinolytic activity with one unit of activity defined as previously (7). The plasminogen activator secreted by the cells was assayed in IO-20 ul aliquots of growth medium incubated on p251]fibrin plates exactly as outlined above.

10%

RESULTS.

Table

growth

of

Cl300

extensions

the

cells.

was

not

particularly

All

simply

“neurite” these

two

that

the

when

compared

exposed The lysates fibrinolytic added

level

compounds

caused

to

effect of

activity

ccl

plasminogen

and

these is

(i.e.

growth

cultures

shown the

and on

the

as

a

lysates acid

treated

not

both

and

human

again due

115

to

dose medium

in was result

dbcAMP

and

1 also

shows

in

not

cells

inhibition Triton

X-100

1.

totally

of

increased

growth

Figure

in-

level

greatest

of

a

severly

agents

activity

serum,

not

and

ex-

induce

highest

these

with

the

not

Table

was

of growth

did

inhibition.

fibrinolytic function

of

PGE,

with

cells with

did

which

simply

of

cells

acid)

to

increase

was

of

The

treated

This

number

extensions.

growth

1s.

the

and

effects

dbcAMP

exposed

cells

morphology

number

(butyric

of

the

inhibitory

Also,

of

PGEl agents

of

the

considerable

untreated

cells

The

compound

in

on

exposure.

percentage

seen

agents increased

“neurites.”

levels

dbcAMP

these

compounds hr

to

protein

of

Cl300

96

a higher was

both

the

inhibitory of

outgrowth

cellular

to

most

caused

growth,

of

of

related

the

high

t

effect

after

since

chemicals,

on

shows

(“neurites”)

tensions

hibi

I

The dependent

shown).

Ex-

Vol.

68, No.

1, 1976

BIOCHEMICAL

AND

BIOPHYSICAL

Table Effects

of

Cyclic

AMP Agents

on

Treatment

Neuroblastoma

Control PGE,

(~xIO-~M)

dbcAMP

(IO-‘M)

PGEl + dbcAMP (3~10-5M)(lO-~M) Butyric Propionic Theophylline

Cell

%

Acid Acid

( 10m3M) (10m3M) (10v3M)

96

Morphology

and

Protein/ Cell. % Control

Cells

hr

COMMUNICATIONS

I

“Differentiated” 24

RESEARCH

Cell Dish.

Growth

Number/ % Control

hr

21

4

27

23

137

70

44

29

121

89

72

63

175

29

22

21

110

25

16

13

118

101

27

17

106

87

Cl300 cells were treated with the indicated doses of compounds 24 hr after seeding and the percentage of cells with long extensions (“differentiated” cells) counted at 24 and 96 hr after exposure, The cell number and protein content per cell were also determined 96 hr after exposure and compared to control cultures. t 96 hr were about 13 x lo6 cells/dish Control values with a protein content of 97 ug/lO % cells.

I

I

400 -I g 300!? 8 g 200 loo-

i

/ffj IO-7

10-s

IO-5

IO-4

IO-3

IM1 Effect of cyclic AMP agents on the intracellular fibrinolytic Figure I. activity of Cl300 cells. Cultures of Cl300 cells were exposed to the agents 24 hr after seeding and the cells harvested after 96 hr treatment. Cell centrifuged and plasminogen activator depellets were lysed in Triton X-100 termined in the supernatant using t251]fibrin plates incubated with 1% acidThe protein content of the lysates was determined and the treated human serum. specific activity of the enzyme compared to that of untreated controls. The agents used were ; PGEl ( A ); PGE2 ( A ); butyric acid ( ); propionic acid ( l ); dbcAMP ( n ); theophylline ( 0 ). Control values ranged from units/mg protein and the points represent the mean values obtained 5.9 - 14.7 from duplicate dishes in 2-4 separate experiments.

0

116

BIOCHEMICAL

Vol. 68, No. 1, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fiqure 2. Effect of cyclic AMP agents on the secretion of plasminogen activator by Cl300 cells. Cultures of Cl300 cells were treated as in Figure 1 and plasmino en activator determined in the culture fluids after 96 hr treat9 ment using 1*5l]fibrin plates incubated with 1% acid-treated human serum. The cell number present after treatment was also determined and the amount of enzyme secreted was normalized for cell number and then compared to untreated controls. The agents used were; PGEl ( A ); butyric acid ( 0 ); propionic acid ( l ); dbcAMP ( m ); theophylline ( Q ). Control values ranged from g-29 units/l06 cells and the points represent the mean values obtained from duplicate cultures in 2-3 separate experiments.

periments with

with

lysates

increasing

protein

Therefore

although

they

not

were

aliquots PGEl

and

inhibitors

measured

pionic

its

were

this

enzyme.

tory

properties

of

can

be due

creases the

enzyme

over

the x

minogen

Since

also

capable

there these to

10m5M)

and

activator

range

acids

Cl300

used.

dbcAMP (not

and

ug

of of

(10V3M)

all

1)

it

and found

that a 650%

shown).

117

30

assays

were

conducted

20-50

ug/assay).

present

specific

seems

the

in in

the

unlikely

Both of

that

plas-

and pro-

the

activity

growth

that

shows

lysates,

on

Butyric

increases

linear

ug/ml).

in

activity

used.

1 also that

was

be

large

Figure

caused

(i.e.

differences

(Table

assay

ug/assay

concentrations inducing

the

might

the

substantial

was

that

(range

increased

lysates It

75

protein

cytotoxicity. in

range

75

were

to

fibrinolysis

PGE2 the

showed

up

assay

than

over

activity dose

this

relative

activator

cells

of

by less

close

acids

untreated

concentration

containing

mi nogen

(3

of

inhibi-

these dbcAMP

theophylline

was

ineffective

a combined

treatment

with

increase

in

intracellular

of

inraised

PGEl plas-

Vol. 68, No. 1, 1976

Figure

2 shows

plasminogen those

the

observed

into

for

than

PGE,

(3 x 10m5M)

extracel

lular

the

propionic

DISCUSSION.

and

The

dbcAMP

agents

which

enzymes

(4

is

unlikely

(II), amongst to

be

40

fold)

that

the

were

in

the

range

ante

of

the

morphological

to The

ferase Also, of

activity

butyric

shape

(13)

and

acid

differentiation

increases butyric of

the

the

active treated

the acid role

in

cultured

endogenous

of

CAMP

in

cells.

in

the

treated

effec-

dose

of of

2 could

also

and

that

of

activity

these of

agents.

It

inhibitors

of

concentrations we

confirmed

indicative

of

increased

as

these

fibrinolytic

with

Although be

such

sialytrans-

increase

protein

to

including

enzymes

levels at

fatty of

a lesser

of

more

which the

appear-

differentiation,

fibrinolytic

activity

are

stage.

HeLa

acids

interest. cells

It

and

induces

alkaline extent

level

CAMP Thus increase

cells

introduces

many

we

at

of

cannot fibrinolytic

dbcAMP.

118

has

been

acid cells

Cl300

induce

large

that

in

sialyltrans-

(16). problems this

are

(15).

stage activity

increases

reported

increases

propionic

of

can

phosphatase

erythroleukemic

CAMP

derivatives

intermediate with

to

cells

in

decreasing

membrane-bound

and

to

level

cells

several

range

considered

also

of

the

of

irreversible

(6)

assay.

short-chain is

of

conducted the

similar

Figure

neuroblastoma

show

and

this

that

activity

were

thus was

in

in

many

clearly

to

“differentiation”

observation

alters

due

of

at

induce

a wide

Cl300

changes

establish

fibrinolytic

is

linearity

between

difficult

butyrate

of

is

in

assays

increase shown

activities

results

increase

since

correlations

our

secretion

A combined

1400%

Cl300

There

the

agents.

used

the

dbcAMP

acetyltransferase

increased the

have

and

a

are

that

increases

the

choline

and

secretion.

COMMUNICATIONS

influence results

enzyme

cultured

others.

markedly

fibrinolysis,

in

in

(6)

acetylcholinesterase (12)

of

we

The except

caused

RESEARCH

to

enzyme

The

addition

agents

medium.

shown).

alterations

ferases

growth

( 10m3M)

(not

BIOPHYSICAL

these

inducing

functions

morphological

also

in

after

“differentiated”

can

the

acid

24 hr

of

intracellular

enzyme

detected

AND

abilities

activator

tive

be

BIOCHEMICAL

activity

(14).

potent

inducers

Butyric

acid

Clearly, in be

sodium

the the

of

interpretation

certain in

use

also

Cl300

of cells

the

BIOCHEMICAL

Vol. 68, No. 1, 1976

The treated

large with

secretion ently ing

increases these

of

cell

agents factor

investigating whether

No.

recipient

of

types

This NOl-CP-55641 a Career

is

activity

of

interest

be

induced

biochemical

cell

ACKNOWLEDGEMENTS.

fibrinolytic

can

the

other

Contract

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in in

nature

respond

to

investigation from

Development

the

was National Award

view vivo of

these

from

seen of

when

our

(7). the

Cl300

cells

observation We are

induction

that

therefore as

are

well

the curr-

as

determin-

agents.

supported

by

Cancer

Institute.

the

N.I

Public

Health W.B.

Service is

the

.H.

REFERENCES

1. 2. 3. 4.

2:

2 9. 10. 11. 12. 13. 14. 15. 16.

Unkeless, J.C., Tobia, A., Ossowski, L., Quigley, J.P., Rifkin, D.B., and Reich, E., (1973) J. Exptl. Med., m 85-111. Ossowski, L., Unkeless, J.C., Tobia, A., Quigley, J.P., Rifkin, D.B., and Reich, E., (1973) J. Exptl. Med., u 112-126. Laug, W.E., Jones, P.A., and Benedict, W.F., (1975) J. Natl. Cancer Inst., 173-179. 5!I! Pollack, R., Risser, R., Conlon, S., and Rifkin, D., (1974) Proc. Natl. Acad. Sci. U.S., 2 4792-4796. Wachsman, J.T., and Biedler, J.L., (1974) Exptl. Cell. Res -9 -86 264-268. Prasad, K.N., and Kumar, S., (1974) Control of Proliferation in Animal Cells, pp. 581-594. Cold Spring Harbor Laboratory, New York. Jones, P.A., Laug, W.E., and Benedict, W.F., (1975) Cell 6 245-252. -Wigler, M., Ford, J.P., and Weinstein, I.B., (1975) Proteases and Biological Control, in press, Cold Spring Harbor Laboratory, New York. Schubert, D., and Jacob, F., (1970) Proc. Natl. Acad. Sci. U.S., G 247-254. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., (1951) J. Biol. Chem. 191 265-275. Prasad, K.N., Vernadakis, A., (197.2) Exptl. Ceil. Res. a 27-32. Moskal, J.R., Gardner, D.A., and Basu, S., (1974) Bochem. Biophys. Res. Commun., & 701-708. Fishman, P.H., Simmons, J.L., Brady, R.O., and Freese, E., (1974) Biophys. Res. Commun.,jj Biochem. 292-299. Griffin, M.J., Price, G.H., and Bazzel, K.L., (1974) Arch. Biochem. Biophys., 164 619-623. Leder, A., and Leder, P., (1975) m 5 319-322. Glazer, R. I., and Schneider, F.H., (1975) J. Biol. Chem., w 2745-2749.

119