Prostaglandin production by mouse fibrosarcoma cells in culture: Inhibition by indomethacin and aspirin

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PROSTAGLANDIN PRODUCTION BY MOUSEFIBROSARCOMA CELLSIN CULTURE: INHIBITION BY INDOMETHACIN ANDASPIRIN* Lawrence Levine Graduate Patricia

Denartment of Biochemistry. Waltham. Massachusetts

M. Hinkle,

Edward

F. Voelkel,

Brandeis 02154

University,

and Armen H. Tashjian,

Department of Pharmacology. Harvard School of Dental and Harvard Medical School, Boston, Massachusetts

Received

April

Jr.

Medicine 02115

18, 1972

Summary -- Five clonal strains of mouse tumor cells (HSDM ) synthesize and secrete large quantities (0.70-2.0 vg/mg cell protein/24 hrj of prostaof control cells did not synthesize significant glandin E . Five lines amounts o ? prostaglandins. HSDM cells produce prostaglandin E2 during both the logarithmic and stationary p ?iases of the cell growth cycle. Prostaglandin production was inhibited by aspirin-lik drugs; for example, 50s inhibiWe conclude tion was obtained with as little as 3 X 10 -5 M indomethacin. that the HSDM cell system will serve as a useful model system to study prostaglandin syn Ehesis and secretion. Growth systems cial

of functional

in which

cell

to study

it

possible

cells.

prostaglandin

the measurement of these here

a potent

Goldhaber,

1966;

Publication University.

bone Voelkel,

No.

a transplantable

resorption-stimulating Tashjian,

82.5 from

the Graduate

model

has made C2. fatty

oxygenated of large (HSDM1)

quantities

grown

Department

(Goldhaber, 1972).

acids of

in culture.

mouse fibrosarcoma factor

of spespecific

of prostaglandins

and Goldhaber,

888

Copyright O1972,by AcademicPress,Inc.

and relatively

cyclic,

of cells

useful

and secretion

the production line

from

may provide

of sensitive

synthesis

We describe

was derived

to produce

deis

the

for

culture

of the biosynthesis

by a fibroblast-like

The HSDMl line

*

control

procedures

to study

by cultured

in tissue

The availability

products.

radioimmunoassay

cells

known 1960;

The chemical

of Biochemistry,

and

Bran-

Vol. 47, No. 4,1972

biological

properties

led Voelkel

described

(1972)

--

Establishment

(Voelkel

13 months calf

cells

by single

dishes

serum

to suggest

(complete cell

it

might

and the

1972),

Five

clonal

in microcloning

(50 X 15 mm, Falcon)

containing

were

15% horse

strains

were the

grown

Medium

has been

serially

with

propagated

serum and 2.5%

derived

from HSDML

clonal

strains

in plastic

tissue

3 ml of complete

of 5% CO2 and 95s air.

in culture

have been

dishes;

Cells

HSDMlC5.

initially

be a prostaglandin. of cells

cells

supplemented

FlO).

plating

atmosphere

that

material

of the HSDMl line

--et al.,

HSDMICL through

humidified

resorption-stimulating

in Ham's F10 medium

fetal

designated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the bone

--et al.

Methods

for

BIOCHEMICAL

FlO medium

was changed

were culture

at 37'

every

in a

3 or 4

days. Medium stored

for

radioimmunoassay

frozen.

scraped

For

in 2 ml buffer

2 min at l-2'

acetic

Model

at 40'

DFlOl

by the method

Radioimmunoassay

ether

of Lowry

immediately

PGBl

(Levine

et al.,

PGE to PGF, 0.01 tions

*

(0.01

M Tris,

Abbreviations: series, respectively.

1971)

All

0.14 M NaCl,

Where

assay

assays

were

and treated

24 hr.

for treat-

To assay

of the medium was adjusted with

equal

volumes

to dryness

for

assay.

out

according

indicated,

(pH 11.5-12.0,

were

dilution

mg of NaBH4 was added

and

of

under

Protein

was

(1951).

(1971).

at a final

within

evaporated

was carried

before

*

cells

The material

3 times

in buffer

of prostaglandins

PGE or FGA to PGB.

were

--et al.

treated to convert

extracted

dissolved

and Van Vunakis

alkali

oscillator.

the pH value

layers

Gutierrez-Cernosek, with

pH 7.6)

at 4' and assayed

from plates

washed

M NaCl,

sonic

medium,

and the residue

material, 0.14

HCl,

and the medium

The combined

ether.

determined

M Tris

culture

acid

was removed

of intracellular

was stored

of cell

to 3.5 with

nitrogen

(0.01

oscillation

extracts

diethyl

assay

in a Raytheon

ed by sonic ether

the

of prostaglandins

carried

to 1 ml of tritiated

pH 7.5)

PGE, PGA, and PGB for

889

or to appropriate

prostaglandins

samples 100'

out with

of 1:130,000.

to Levine,

For

for

were 5-K

min)

antiserum

to

reduction

prostaglandin serum-free

of solu-

tissue

of the E, A, and B

Vol. 47, No. 4, 1972

culture with

fluids

and allowed

2 M citric

trality

with Results

ability

BIOCHEMICAL

acid.

After

the

and Discussion

to inhibit

are

30 min.

bubbling

RESEARCH COMMUNICATIONS

The solutions stopped,

were

then

acidified

the pH was adjusted

to neu-

1 N NaOH. --

the binding

1 and as previously antiserum

to stand

AND BIOPHYSICAL

specific

described for

Medium

from HSDMl cells

[5~]~~~,

of

by Levine PGB; i.e.,

to anti-PGBl --et al.

the&

I Nonogromr

bind

IO

(lgl),

was tested serum. the

its

As shown in Fig

antibodies

PGB more effectively

loo

for

in this than

PGA

1000

Pro8laplondin

of [%)PGB anti-PGB binding. A. PGA (O), PGB2 (0), Fig. 1. Inhibition PGB2 was obtaiked by alk&line treatment of ?GE and PGE2 (X). g&g (zzl culture medium before (A) and after (A) alkaline treatment. plete FlO) was a 3-day collection from HSDMl cells containing 0.41 rng cell protein. Complete F10 medium alone did not inhibit the binding of [ H]PGBl to anti-PGB at the levels used in the assay. C. HSDMl serum-free culture mediumlbefore (m) and after (0) alkaline treatment.

890

Vol. 47, No. 4, 1972

BIOCHEMICAL

Whereas

and "GE.

AND BIOPHYSICAL

50%of the

270 "g of PGB2 inhibit

5 ng of PGA2 and 1RO ng of PGE2 are required IB shows that anti-F'GBl

small

binding

by treatment tatively

alkali.

extracted

into

material tive

secrete

that

the

ether

appear

from

complete

inhibition

capacities

If

amount

relatively

(see

is

['H]PGB~ enhanced

table).

large

and after

Fig.

can be quantiThus,

quantities

of PGB and PGA, as judged

of the medium before

binding,

the

material

HSDMl medium

FlO medium

to be a mixture

inhibit

by the medium

active

acidified

anti-PGBl inhibition.

medium

produced

serologically

['H]PGBl

equivalent

culture

inhibition

This

into

for

of HSDMl cell

and that

with

cells

HSDM,

amounts

RESEARCH COMMUNICATIONS

of

by the rela-

treatment

with

alkali. a small

ditions

of these

PGBl.

(Under

plete

experiments,

the PGA-like

to PGB before line

treatment

tion

than

treatment

to be a mixture

the prostaglandin

present

in the

rated

could

found

in the To test

complete

complete whether

medium

lacking

table,

HSDMl cells

glandin active

Under

produced medium

was partly

we have found

suggested

that

alka-

in

inhibi-

increase

1, A and B).

medium from

Thus,

HSDMl cells that

com-

isomerized

observation

a smaller

COI

to

from

F10 medium

the

the app'ears

PGE, is not

stable

is converted

to PGAl and PGB . Therefore, 1 by HSDMl cells were PGE, dehydrases

synthesized

convert

isomerized

it

to PGA; the PGA that

to PGB giving

the mixture

was gene-

of PGA and PGB

medium.

HSDMl cells

serum.

in serum-free material

culture

the

PGA (Fig.

However,

medium would

then be partly

produced

in the complete

some of it

being

explain

of authentic

of PGB and FGA.

FlO medium;

recovered

of HSDMl cells

would

medium

material

quantitati~vely

of PC%1 in complete

that

of HSDMl culture

F10 medium under

30%of the FGAl is converted

FGBl is

in the medium

- findings

active

in complete

approximately

instabil.ity

material

assay

serologically

if

This

alkaline

in complete

incubated

the same conditions,

FlO medium.)

that

of PGAl is

produce

these smaller than

in HSDMl medium

PGE, they

conditions but

highly

significant serum.

serum behaved

891

incubated

PGE was stable.

in medium with lacking

were

for

3 days in

As shown in the amounts

of prosta-

The serologically on treatment

with

alkali

Vol. 47, No. 4, 1972

like

BIOCHEMICAL

PGE and not

about

300-fold

only

like

PGA. For

15-fold

(Fig.

If

PGE were

ed by the then

activities

reduction

anti-PGF.

(converted

to PGF) as either

--et al.,

material

NaBH4 treatment.

PGEl

Control

with

did

not

not

react

NaBHl, did

not

of known

product,

On the basis

out

with

serologically rived

anti-PGFa

serum-free

and anti-

medium

1971).

these

inhibited

anti-PGFla

bind-

treated

with

PGA treated

As expected,

PGE2> on treatment but

28’

In our the

before

with

medium

[3~l~~~ln

the

PGF2S cross

laboratory,

yield

with

reacts

after

reduc-

of serologically

active

we are

tentatilvely All

and calculation curve)

subsequent

assays

the prostawere

of prostaglandin

was based

in base-treated

designating

content

on the assumption

HSDMl medium

carried

and cells

(by

that

the

was de-

from PGE. In order

culture

material

containing

to HSDMl cells),

and PGF

NaBH4,

the PGE

effectively

anti-PGF.

as PGE2.

samples,

with

and

was 50%.

findings,

a PGB2 calibration active

addition

with

anti-PGF&,

by HSDMl cells

on alkaline-treated

comparison

with

medium

or anti-PGFIQI.

of PGE2 with

of these

produced

(before

--et al.,

to identify

very

than

with

of anti-PGFla

Serum-free

the

as indicat

by analysis

the HSDMl serum-free

of PGFa

(Levine

be measured

react

(Corey,

treatment,

alkaline

possible

more effectjvely

serologically

quantities

when assayed

glandin

enhanced

medium

specificities

however,

to a mixture

anti-PGF&

and after

make it

with

anti-PGFZa

react

serum-free

NaBH4 and assayed

react

medium

with

NaBH4, is converted

tion

borohydride

relative

NaBH4 treatment,

serum-free

c 0.5% with

sodium

(PGE, PGA, and PGB do not After

did

with in the

or PGE2.

with

[3~l~~~au anti-PGFpCI binding

NaBH4,

the

was treated

The anti-PGFs

ing.

have been

before

1971) would

(Levine

anti-PGFs.)

was enhanced

would

of PGE to PGF could

In addition,

anti-PGF&

PGFlo.

activity

PGA, it

by HSDMl cells

serologic

NaBH4 catalyzed

PGE-like

itNere

to PGF by treatment

produced

relative

specific

if

serological

LA).

PGE can be converted

1971).

its

example,

UC); whereas

(Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

medium

to test from

whether several

the other

production

of PGE2 is

unique

cell

was assayed

for

types

892

to HSDMl cells, the presence

of

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROSTAGLANDIN PRODUCTION BY CELLS IN CULTURE

Cell

Culture

type

Complete

HSDMl

medium

FlO ether

HSDMl FlOl 11

HSDMl HSDMl HSDMlCl HSDMlC2

soluble

no seLLi I, 11 , ether

Complete 11

FL0 11

7,

II

HSDMlC?

Prostaglandin production* (kg PGEdmg cell protein/ 24 hr) 1.12

s

1.16 0.25

w

soluble

0.22

2.04 1.28 2.00

HSDMICl+

0.80

HSDM,C5

0.72

Mouse

fibroblasts

Mouse neuroblastoma Rat glial

< 0.03

(Cl-1D)

< 0.01

(41A3)

-=L 0.01

(Cb)

< 0.01

Human HeLa Mouse

adrenal

-c 0.01

(ATlo)

*

Medium samples were glandin after alkaline ** Medium was extracted

x-day collections treatment. with

As shown in the

prostaglandins. produced wise,

significant

quantities

none of these

prostaglandins. quantities clones

cell

Five of PGE2 (see

was sufficiently

cell

could

ried

out with It

be measured a single

was of interest

were

assayed

for

prosta-

as described in the text. Cells were washed in complete F10 medium. serum serum, and for 3 days F10 medium without

"i HSDMl cells were grown twice with medium lacking was added to the dishes.

ether

that

types clonal table). large in this clone,

and assayed,

table,

none

of material contained lines

that measurable

derived

from

The quantity so that

of the other

the

with

amount

of cells anti-PGBl.

intracellular

levels

the HSDMl cells

produced

of PGE2 produced

Subsequent

system.

reacted

types

by several

of PGE2 produced experiments

whether

893

large

quantities

Likeof large of the

by a single

have been

HSDMIC1.

to determine

tested

of PGE are

car-

Vol. 47, No. 4, 1972

stored

BIOCHEMICAL

in HSDMLCL cells.

intracellular

Cells

material

300 ng/mg

amount

secreted

into

growth

does not

affect

of medium

were

2, the rate were

the

collected

protein;

culture

the

disrupted

this

medium

rate

quantity

in about

times

remained or were

is

3-l/2

the

equivalent

subculture.

of cell

cells.

Samples

As shown in Fig.

the

stationary

to the

The stage

by HSDMICl

after

and the

of intracellular

hr.

approximately in

oscillation

The amount

of PGE2 production

at various

logarithmically

RESEARCH COMMUNICATIONS

by sonic

by immunoassay.

cell

of PGE2 production

growing

were

examined

PGE2 was at most

AND BIOPHYSICAL

same whether

the

cells

phase. i 2 . .E

6.0 2 4.0 g 20 e

0

4 Days

6 After

12

16

Subculture

Fig. 2. Production of PGE2 by HSDMICL cells. HSDMICL cells were innoculated At zero time in replicate dishes 12 hr before the start of the experiment. fresh medium was added to each dish. Every 2 to 4 days the medium was collected and changed. Duplicate dishes were washed twice with saline and frozen Medium was treated with alkali and for the determination of cell protein. assayed for PGE2, as described in the text.

Since thesis

it

has been

and release

Moncada,

reported

and Vane,

1971;

Smith

(acetylsalicylic

production

by HSDMLCl cells.

of PGE2 production, level

of 1 rig/ml

dose

level

of 10 wg/ml rate

in

and

several

sodium

Indomethacin

(6 X 10e5 M) (Fig. of PGE2 synthesis

aspirin

894

1971;

examined

the Ferreira,

level

on PGE2 inhibitor

of PGE2 synthesis

caused

a 50s decrease

salicylate

syn-

the effects

to be a potent

Sodium

at a dose

inhibit

and indomethacin

in the rate

3).

(Vane,

we have

was found

whereas

drugs

systems

salicylate,

a 50s reduction

(3 X 10s9 M);

related

1971),

and Willis,

acid),

causing

dose

in the

aspirin

of prostaglandins

of aspirin

decrease

that

caused

of 50 pg/ml.

at a at a <20$

In cells

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 10-4 10-s IO-L IQ’

100 IO’

102

pa/ml

of PGE2 production by Fig. 3. Inhibition dium containing either no additions or the methacin or aspirin was added to duplicate later the medium was collected and frozen with saline and frozen for the determination treated with alkali and assayed for PGE2, untreated dishes produced 1.94 Fg PGE2/mg dishes treated with aspirin and indomethacin this control value.

treated

with

levels

up to 50 rig/ml

of PGE2 did

not

the drugs

do not act

PGE2 into

the

Data Ferreira

obtained

the

than

potency Ferreira

is

which

to study

other

established

prostaglandins whether tion

with

HSDMICl cells

support

by inhibiting

Therefore,

the

greater

than

that

HSDMl cells

the biosynthesis cell

lines

table).

arachidonic

acid

of arachidonic

acid,

is

the observations

secretion

of

of Vane

(1971),

cells.

observed

in other

that

should

and secretion did

not appear

It

was interesting

LOOO-fold This

more potent

difference

systems

a useful

in

(Vane,

and unique

of prostaglandins. to produce in this

substrate

895

drugs

1971;

(1971).

provide

up to 167 pg/ml,

is

by HSDMlCl

and Willis

a limiting

aspirin-like

Indomethacin

PGE2 synthesis

(1971), and Smith

(see

intracellular

cultures.

(1971) that

and Willis

of prostaglandins.

considerably

We conclude

in untreated

merely

in inhibiting

--et al.

aspirin,

medium.

synthesis

aspirin

those

cells

(1971), and Smith

--et al.

inhibit

from

on HSDMlCl

culture

or 50 i.lg/ml

indomethacin

differ

indomethacin and aspirin. Fresh meindicated concentrations of indodishes of HSDMlCl cells. Three days for assay. Dishes were washed twice of cell protein. Medium was as described in the text. Cells from cell protein/24 hr. Results for are expressed as percentages of

measurable context

in PGE2 synthesis.

to cultures

system

A variety

in of

quantities

of

to examine The addi-

of HSDM~C~ cells

for

3

Vol. 47, No. 4, 1972

days

led

the

supply

BIOCHEMICAL

to no change of arachidonic

does not

enter

material

causes glandins

produced

are

of the partially ent with

for

possible.

This

American

Cancer

ican

Kalamazoo, purchased

is

no direct

the medium.

Either

arachidonic

acid

or exogenous

cells

is

resorption

evidence the

(Voelkel

bone

that

the

same substance --et al.,

resorption,

serologically as that

1972).

and the

HSDMl bone-resorption

-expert

The authors

act.

which

However,

chemical

stimulating

Society

prosta-

properties

factor

No. PRP-21)

Michigan

is

are

an American

Cancer

and P. M. H. is thank

Dr.

from New England

Nuclear

cells

consist-

studies

grants

from

of Health

(AM-

Society

Professor

a postdoctoral

fellow

Boston,

and

discovery

made these

Institutes

The tritiated

Corp.,

Barowsky

whose

by research

John Pike

the prostaglandins.

N. J.

Goldhaber,

in part

and the National

The authors for

Paul

to Misses

HSDMl tumor

was supported

(lC-1OK) L. L.

Society.

and to Dr.

transplantable

investigation

(Award

are grateful

assistance

of the

and DE-02849).

Cancer

limiting

into

of a prostaglandin.

and establishment

chemistry

not

to stimulate

Acknowledgments

11011

is

by HSDMlCl

purified

those

D. J. Jensen

there

of bone

known

of PGE2 secreted

cells.

time

stimulation

amount

acid

HSDMlCl

At the present ive

in the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the Upjohn

the

of Bioof the AmerCompany,

prostaglandins

were

Massachusetts.

REFERENCES In Prostaglar ldins, Ramwell, P. W. and Shaw, J. E., editors. Ann. Corey, E. J. N. Y. Acad. Sci. l&, 24 (1971). Ferreira, S. H., Moncada, S., and Vane, J. R. Nature New Biology a, 237 (1971). Goldhaber; P. Proc. Amer. Assoc. Cancer Res. 2, 113 (1960). to NO. 3) Gg, 490 (1966). ---------, -* J. Dental Res. (Supplt. Levine, L., Gutierrez-Cernosek, R. M., and Van Vunakis, H. J. Biol. Chem. s,

6782 (1971).

Lowry, 0. K., Rosebrough, N. J., Farr, A. L., and Randall, R. J, J. Biol. Chem. 193, 265 (1951). Smith, J. B. and Willis, A. L. Nature New Biology 3, 235 (l-971). Vane, J. R. Nature New Biology 231, 232 (1971). Voelkel, E. F., Tashjian, A. H., Jr., and Goldhaber, P. In Calcium, Parathyroid Hormones and the Calcitonins, Proc. 4th Parathyroid Conference, Talmage, R. V. and Munson, P. L., editors. Amsterdam, Excerpta Medica Foundation, ICS No. 243, 1972.

896