Platelet derived growth factor induces ornithine decarboxylase activity in nih 3T3 cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 127, No. 3, 1985 darch 29, 1985

Pages 843-848

P L A T E L E T DERIVED GROWTH FACTOR INDUCES ORNITHINE D E C A R B O X Y L A S E

ACTIVITY IN NIH 3T3 CELLS Elisa Vicenzi, Marina Bianchi, Mario Salmona, Maria Benedetta Donati, Andreina Poggi and Pietro Ghezzi Istituto di Ricerche Farmacologiche "Mario Negri"-via Eritrea, 62 20157 MILANO, Italy

Received February 7, 1985

Snmmary - Incubation with highly purified humah Platelet Derived Growth Factor induced ornithine decarboxylase activity in quiescent NIH 3T3 cells concomitantly with mitogenic stimulation. Pretreatment of cells with a specific ornithine decarboxylase inhibitor, DL-~difluoromethyl-ornithine significantly inhibited the effect of the mitogen on DNA synthesis. These experiments suggest that the mitogenic activity of Platelet Derived Growth Factor, similarly to that of other serum growth factors or tumor promoters, polyamine levels. © 1985 A c a d ~ i c Press, Inc.

is mediated through rise in

Increasedintracellular levels of polyamines have been found in association with cell proliferation and related to enhanced activity of ornithine deearboxylase synthesis

(ODC), the rate-limiting enzyme in polyamine

(1,2). Induction of ODC activity has been found after cell

stimulation by different growth factors such as epidermal growth factor, fibroblast derived growth factor, insulin, vasopressin, interleukin-I and T cell growth factor

(3,4). A rapid increase in ODC activity can also

be induced by treatment of cells with known tumor promoters, tetradecanoyl-phorbol-13-acetate marker of tumor promotion

like 12-0-

(TPA) suggesting that ODC might be a

(5). A specific inhibitor of ODC, the

ornithine analogue DL-~-difluoromethyl-ornithine

(DFMO) inhibits

cancer cell proliferation in vitro and shows a potential anticancer activity in vivo

(6,7).

ABBREVIATIONS: PDGF, Platelet Derived Growth Factor; ODC, ornithine decarboxylase; DFMO, DL-~-difluoromethyl ornithine; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; TdR, methyl-/3H/-thymidine; DME, Dulbecco modified Eagle's medium; PBS, phosphate buffered saline; CO2/14C/carbondioxide.

843

0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PDGF is a potent mitogenic

factor for cells of mesenchymal

released during blood clot formation platelets

and found in serum

of immediate

of activated

(8). Addition of PDGF induces a series

and early events not related to DNA synthesis.

are activation uptake,

from the ~ - g r a n u l e s

of a tyrosine

stimulation

oriqin,

specific kinase,

Among them

increase of aminoacid

of sodium ion flux and protein

synthesis

(see for

review, ref. 9). In this paper we report that highly purified human PDGF induced ODC activity

in NIH 3T3 cells. This effect appeared to be relevant to

the mitogenic

activity of PDGF since incubation of cells with a specific

ODC inhibitor,

inhibited also the increase

(TdR) incorporation

in

methyl-/3H?-thymidine

induced by PDGF.

MATERIAL AND METHODS Cells - NIH 3T3 cells were obtained through the courtesy of Dr. Renato Baserga

(Philadelphia,

Dulbecco modified Eagle's medium serum and 2 mM L-glutamine

Pa, USA). Cells were grown in (DME) in the presence

in humidified

of 10% calf

5% CO2/95% air incubator

at 37°C. ODC determination calf serum 10%

- NIH 3T3 cells

quiescent

(4-5 days).

phosphate

buffered saline

the test samples incubated

(5x104/ml)

were plated in DME plus

in 35 mm Petri dishes and left to proliferate Confluent monolayers (PBS); fresh DME

(serum, plasma,

for further 4 hours.

maximal effect on ODC activity,

(I ml/dish)

TPA, or PDGF)

This incubation

the incubation

was added with

and cells were time gave the

as tested in preliminary

using human whole blood derived serum as reference. determination

experiments,

At the end of

time, cells were rinsed with PBS and frozen. cells were disrupted by repeated

and scraped using a rubber policeman. sodium phosphate

buffer

For ODC

freezing and thawing

Cells were resuspended

(pH 7.2) containing

until

were rinsed twice with

0.i m M p y r i d o x a l

in 0.05 M phosphate

and I mM EDTA. ODC activity in the supernatant was determined by measuring the /-14C7carbon dioxide (C02) release from L-/-I-14C~

ornithine

as previously

described

(i0).

Proteins were determined in cell lysate after trichloroacetic acid precipitation by the method of Lowry et al (11) and ODC activity was expressed Mitogenic

as release of nmoles of CO2/h/mg of protein. activity - The assay was based on the incorporation

TdR by quiescent

cells.

of

NIH 3T3 cells were seeded in Costar 24 well

plates at the density of 104 cells/well

in DME containing

10% calf

serum. After 4-5 days medium was removed, cells were rinsed twice with PBS and fresh medium with the test samples and/or various concentrations of calf serum was added. Fifteen hours later, TdR (2.5 ~Ci/well) was added; medium was then removed and trichloroacetic acid precipitable radioactivity

collected

and counted

(12).

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VoI. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Other Material - Highly purified human PDGF, prepared according to Heldin et al (13) was kindly provided by Dr. Ake WastesOn (Linkoping, Sweden). Partially purified PDGF was prepared from outdated platelets by boiling at 100°C, chromatography on Sulfadex G 75 and ultrafiltration with PM i0 membranes, according to Deuel et al (14). Human whole blood derived serum was prepared as described previously (15). TPA was obtained from Sigma Chemicals (St. Louis, Mo). DFMO was from Merrel Dow (Cincinnati, Oh). All reagents for tissue culture were from Flow Laboratories (Laboratory Supplies, Milano, Italy). Methyl-/ 3H/-thymidine (specific activity 6.7 Ci/nmoles S.A.) and L-/ 1-14C/ ornithine

(specific activity 5.7 mCi/mmole S.A.)

from New England Nuclear (Boston, Ma) and Amersham Radiochemieal Center (Amersham, UK) respectively. were

RESULTS AND DISCUSSION Figure 1 shows the effect of PDGF on ODC activity and TdR incorporation in NIH 3T3 cells. Incubation of cells with highly purified PDGF caused a dose dependent stimulation of ODC, reaching the half maximal effect with 2.5 ng/ml and the plateau with 5 ng/ml of PDGF. A parallel effect on TdR incorporation was seen in the same cells, suggesting that the biochemical effect induced by PDGF was closely related to its mitogenic activity.

-30

?

1

30-

.a .J la.i

o

E

n

-20

ca z

o u

O

20

0

E c

e¢

-I0

10

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o z ta.i z

o o

8 IE

i

i

1

,o

,oo PDGF

(nglml)

FIGURE 1 - Dose response effect of PDGF on ODC induction and /3H/ thymidine incorporation in quiescent NIH 3T3 cells. Each number represents mean+S.E, of 3 data,ODC activity (o-----o); _/3H/thymidine_inc°rp°rati°n_ (o---o). ODC activity was measured after 4h and/3H/ thymidine incorporation after 17h of exposure to PDGF.

845

0

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

z o

100_

n~ ~ 8 0 . o -J ~. ..J w

g~ u

60

40-

z _z

x

o

IE

~

u20-

0 DFMO

(raM)

FIGURE 2 - Effect of DFMO tested at different molar concentrations on /3H/thymidine incorporation in NIH 3T3 cells in the presence (o--o) o r absence (o---o) of 5 pg/ml of partially purified PDGF. Each point represents mean + S.E. of 3 data.

PDGF appeared compared

to have a h i g h e r

to known ODC inducers,

was 29.5ei.9

nmoles C O 2 / h / m g

values being

of 10%

(v/v),

release

was m u c h

serum.

containing

The

of human

stimulatory

In order to investigate the mitogenic

activity

ODC inhibitor,

DFMO,

shows

(5 ~ g / m l ) .

Similar

highly purified

the e f f e c t

5 ng/ml of PDGF,

induced

The higher m i t o g e n i c

on the presence

of other

elevation

of ODC was r e l a t e d

incorporation

of TdR.

Figure

induced by p a r t i a l l y

was o b t a i n e d

by testing

purified

PDGF

highly p u r i f i e d

of 4.7 m M and 2.8 mM of DFMO for p a r t i a l l y PDGF respectively.

DFMO lacked any direct toxic

we have shown that human PDGF, factors,

stimulated

846

similarly

ODC activity

in

to

PDGF

at the

purified

effect on the cells.

serum and other growth

2

with DFMO caused a dose d e p e n d e n t

l).The half maximal inhibition of T d R w a s o b s e r v e d

In conclusion

to

we t e s t e d the effect of a specific

incubation

of T d R i n c o r p o r a t i o n

concentrations

However

added at the c o n c e n t r a t i o n

of protein.

whether

of PDGF,

effect

i00 ng/ml TPA,

from PDGF.

inhibition

(5 ng/ml)(Table

latter,

on P D G F - i n d u c e d

that simultaneous

PDGF

lower than the effect of human

C02/h/mg

different

as

w i t h 5 ng/ml

with

C02/h/mg.

serum m i g h t be d e p e n d e n t

factors

activity

Indeed ODC activity

incubation

approximately

of 46.6 • 4.8nmoles

activity

after

1.4 ± 0.2 n m o l e s

of PDGF on ODC activity whole blood d e r i v e d

such as TPA.

after 4 h incubation

and 10.4 ± 0.2 nmoles C O 2 / h / m g control

ODC stimulatory

and

VoI. 127, No. 3, 1985

TABLE

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effect of DFMO on TdR incorporation in NIH 3T3 cells after exposure to highly purified PDGF (5 ng/ml)

I -

DME

PDGF

ADDITIONS

CPMx10-3/well

DME DFMO I 2 5 I0

11.3 15.4 12.0 11.4 13.0

mM mM mM mM

~ • • i •

% of inhibition

CPMx10-3/well

2.2 1.4 1.0 1.0 1.2

121.4 117.0 79.3 41.9 29.3

• i • • •

11.7 10.7 19.2 5.9* 15.0"

3.7 34.7 65.4 75.6

* p< 0.01 at Dunnett t test (compared to the respective controls) Each number is mean + S.E. of 3 data per group. Percent of inhibition was calculated as 100 - CPM sample x 100. CPM DME D F M O w a s added simultaneously with PDGF and incubated with quiescent NIH 3T3 cells for 15 hours. TdR (2.5 ~Ci/well) was incubated for further 2 hours. Acid precipitable radioactivity was than counted as indicated in "Methods".

r e s t i n g N I H 3T3 cells,

at the same molar c o n c e n t r a t i o n

able to

-i0 stimulate mitosis

(0.6-1.7xi0

M). This e f f e c t was o b s e r v e d

four

h o u r s after exposure of the cells to PDGF and p r e c e d e d D N A synthesis.

This phenomenon

h a d not been d e s c r i b e d yet for PDGF and

m i g h t be c o n s i d e r e d a n o t h e r early

("pleyotropic")

event e l i c i t e d

by the peptide. ODC a c t i v i t y cell growth.

Indeed i n h i b i t i o n of ODC by a synthetic analogue

almost completely i n d u c e d by PDGF between

seemed to be c a u ~ a l l y r e l a t e d to i n d u c t i o n of

abolished

s t i m u l a t i o n of TdR i n c o r p o r a t i o n

(75% of i n h i b i t i o n

with

i0 m M DFMO).

is not

It has been s u g g e s t e d that the rise in p o l y a m i n e

f o l l o w i n g ODC a c t i v a t i o n m i g h t in turn stimulate aminoacid uptake proteins

The r e l a t i o n s h i p

s t i m u l a t i o n of ODC and i n d u c t i o n of the cell cycle

w e l l known.

(15), thus p r o m o t i n g

d u r i n g cell r e p l i c a t i o n

that e l e v a t i o n

of o r n i t h i n e

on t y r o s i n e - p h o s p h o r y l a t i o n Better understanding other early b i o c h e m i c a l

(DFMO)

increase

levels

in

the synthesis of c y t o p l a s m i c

(9). M o r e o v e r

decarboxylase

it has been h y p o t h e s i z e d

activity m i g h t be d e p e n d e n t

i n d u c e d by g r o w t h factors

of the links b e t w e e n

such as PDGF

stimulation

of ODC and

events due to PDGF m i g h t help to c l a r i f y

m e c h a n i s m of action of this mitogen.

847

(16).

the

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACKNOWLEDGEMENTS

The authors wish to thank dr. Ake Wasteson, Clinical Research Center, University Hospital, Linkoping, Sweden, for providing purified PDGF, dr. Renato Baserga, Department of Pathology, Temple University Medical School, Philadelphia, Pa, USA, for providing NIH 3T3 cells, dr. Giorgio Zanini and dr. Patrizia Bonfiglioli of Centro Trasfusionale, Ospedale Maggiore, Bologna, Italy for providing outdated platelets and dr. W.J. Hudak,Merrel Research Center, Cincinnati, Oh, USA for providing DFMO. The technical assistance of Giorgio Ferrari and help of dr. Emma Paola Sturani, Dipartimento di Fisiologia e Biochimica Generale, Sezione di Biochimica Comparata, Universit~ di Milano, Italy are gratefully acknowledged. Judith Baggot, Ivana Garimoldi, Vanna Pistotti and Vincenzo and Felice de Ceglie helped prepare this manuscript. This Taper was partially supported by the Italian National Research Council (Progetto Oncologia) and Italy-USA Project (CNR n. 83.01460.04). E.V. is the recipient of a fellowship of the Associazione Italiana per la Ricerca sul Cancro.

REFERENCES ]. Russel, D., and Snyder, S.H. (1968) Proc. Nat. Acad. Sci. USA 60, 1420-]427. 2. Pegg, A.E., and McCann, P.P. (]982) Am. J. Physiol. 243, C212-C221. 3. Sreevalsan, T., Rozengurt, E., Taylor-Papadimitriou, J., and Burchell, J. (]980) J. Cell. Physiol. 104, ]-9. 4. Fidelus, R.K., Laughter, A.H., Twomey, J.J., Taffet, S.M., and Haddox, M.K. (1984) Transplantation 37, 383-387. 5. O'Brien, T.G., Simsiman, R.C., and Boutwel], R.K. (1975) Cancer Res~ 35, 2426-2433. 6. Kingsnorth, A.N., Russell, W.E., McCann, P.P., Diekema, K.A., and Malt, R.A. (1983) Cancer Res. 43, 4035-4038. 7. Koch-Weser J., Schechter, P.J. , Bey, P., Danzin, C., Fozard, J.R., Jung, M.J., Mamont, P.S., Sei]er, N.,Prakash, N.J., and Sjoerdsma, A. (]981) In Polyamines in Biology and Medicine (Morris, D.R. and Marton L.J. eds.), Marcel Dekker, New York, pp. 437-453. 8. Stiles, C.D.(1983) Cell 33, 653-655. 9. Deuel, T.F., and Huang, J.S. (1984) J. C1in. Invest. 74, 669-676. 10. Maxwell, M.D., and Patterson, M.K. Jr. (1980) J. Tissue Culture Methods 6, 5-7. 11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. ]93, 265-275. 12. Poggi, A., Rucinski, B., James, P., Holt, J.C., and Niewiarowski, S. (1984) Exp. Cell Res. 150, 436-441. 13. Heldin, C.-H., Westermark, B., and Wasteson, A. (198]) Biochem. J. 193, 907-913. 14. Deuel, T.F., Huang, J.S., P r o f i t t , R.T., Baenziger, J.U., Chang, D., and Kennedy, B.B. (198]) J. Biol. Chem. 256, 8896-8899. 15. Auberger, P., Samson, M., Le Cam, G., and Le Cam, A. (1983) J. Cell. Physiol. 117, 204-210. 16. Hunter, T., and Cooper, J.A. (1984) In Oncogenes and Viral Genes (Vande Woude,G.F., Levine, A.J., Topp, W.C., and Watson, J.D. eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 61-68

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