BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CYCLICAMP PHOSPHODIESTERASE INHIBITORS DEPRESS PRODUCTION OF PLASMINOGEN ACTIVATORBY CHINESEHAMSTEROVARYCELLS1 David M. Mott, The Institute Received

Phyllis

H. Fabiech and SamSorof

for Cancer Research, Fox Chase Cancer Center, Philadelphia, April

PA 19111

20,1976

Summary> Oncogenic transformation in a limited number of cell systems has been shownby others to be associated with an increased production of extracellular proteolytic activators that convert the plasma proenzyme, plaeminogen, to the active protease, plasmin. In the present study, two cyclic AMP phoephodiesterase inhibitors (theophylline, papaverine) markedly depressed the production of intracellular and extracellular plaeminogen activator by Chinese hamster ovary cells of the CHO-Kl line in serum-free medium. Proetaglandin El had a moderately similar effect on the production of only extracellular plasminogen activator. The ability to control experimentally the level of production of plasminogen activator should be of value in elucidating the possible biological role of the proteolytic action of plasmin on the surface of CHOcells, and the cell surface alterations which accompany oncogenic transformation.

Proteolytic

alteration

basis of certain of cells verting

(1).

of cell surfaces has been suggested

phenotypes associated with the oncogenically Cells liberate

plaeminogen activators

transformed cells in culture lytic

(fibrinolytic)

Haiever , a variety

exhibit

these properties

Neoplaetically intracellular

cyclic

duce proliferation

liberate

activity,

(4-7).

transformed state

which are capable of con-

to lyse labeled fibrin

Abstract

(2-4).

high levels of this extracellular

Certain proteo-

compared to low levels from nontransformed cells of other nontransformed and transformed cells do not

(6-9).

and virally

transformed cells usually have lower levels of

AMP than have nontransformed cells in resting

cultures

generally

while cessation of cell proliferation 1

Copyright All rights

be at the

the plasma proenzyme, plaaminogen, to the active protease, plaemin,

which has-been measured by its ability

tion,

to

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

1150

reduce cyclic

raises it

in part in (1975) Fed. Proc. a(3),

(10).

(11,12).

533.

Agents that inAMP concentra-

Vol. 70, No. 4,1976

Guided conditions would

bring

BIOCHEMICAL

by these which about

two bodies

reportedly the

raise

depression

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of evidence, the level

we undertook of cyclic

of the production

to determine

AMP in

whether

transformed

of fibrinolytic

cells

activity.

Materials and Methods: Cells and medium. The Chinese hamster ovary cell line, CHO-Kl, was studied because these cells exhibit high levels of production of fibrinolytic activity (8), and several of their phenotypes of transformation can be reversed by addition of dibutyryl cyclic AMP (13,14). The cells were grown in Ham's F-12 medium with L-glutamine (GIBCO), 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 pg/ml). Cells were propagated (37", 5% CO2) to near confluency in 25 and 75 cm2 plastic tissue culture flasks (Falcon), 1:250) in Dulbecco phosphate buffered disaggregated with 0.25% trypsin (Difco, saline, and plated in plastic culture dishes for experiments. Extracellular fibrinolytic activity. Extracellular fibrinolytic activity was measured in serum-free medium by the method of Unkeless et al. (4) as outlined in our previous report (8). Cells were seeded in 25 and 75 cm2 tissue culture flasks, or 60 mm dishes, in medium containing 10% fetal bovine serum. After 1 day the density was approximately 3.0 x 104 cells/cm2. The monolayers were then washed 3 times with serum-free medium, and each 5 cm2 of growth surface was overlayed with 1 ml of serum-free medium containing or lacking drug. After incubation for 18 hr in 5% CO2 at 37", extracellular fluids were centrifuged to yield cell-free supernatants ('harvest medium"). For the sterile purified fibrinogen below, and 61 pg of fibrin-coated 35 mm The 1251 in 1 ml of

enzymatic digestion of the 125 I-fibrin films (U)], 2 ml of the serum-free harvest medium, purified human plasminogen (16) were added to and incubated for 18 hr at 37' in 5% dishes, the clear supematant fluids was then counted.

[prepared from ailuS& as the ICO atmosphere. 2

The concentration of plasminogen activator in the centrifuged serum-free culture medium was normalized on the basis of activator liberated by lo5 cells per 5 cm2 per ml (100% ltharvest medium"), The harvest medium was diluted with fresh serum-free medium, so that the fibrinolytic activities were proportional to the concentration of activator, i.e. % harvest medium. The fibrinolytic activity was expressed as % of total counts of solubilized 1251-fibrin. The activities were corrected for the radioactivity released by plasminogen alone (no harvest medium), and by harvest medium alone (no plasminogen). Both corrections included activities due to small amounts of plasminogen bound to the purified fibrinogen. As in our previous studies (8,9), the sum of these background values rarely exceeded 2% of the original total radioactivity of the fibrin film. Intracellular fibrinolytic activity. Levels of intracellular plasminogen activator were determined on cell lysates by a method based on that of Unkeless 2.8 x 105 cells were plated in-60 mm dishes and maintained in medium et al. (4). After the 18 hr incubation with or containing 10% fetal bovine serum for 24 hr. without drug in serum-free medium, the cell density was close to 3 x 104 cells/cm2. The medium and pooled cells of 2 to 4 replicate cultures were analyzed for extraThe cells were washed cellular (above) and intracellular activators, respectively. twice with a solution of 25 ti- Tris-Cl, pH 7.4, 137 ti-NaCl, 5 nrM_KCl, 0.7 tiNa2HP04 (TD buffer), suspended by rubber policeman, and centrifuged in 1.5 ml of TD buffer per plate. The cells were homogenized by 25 to 30 strokes in sterile 1 ml glass Dounce-type homogenizers in 0.3 ml of 0.1 g Tris-Cl buffer, pH 8.0, containing 500 pg/ml Triton X-100. The sterile cell lysates were diluted with serum-free medium and an appropriate volume of the Tris-Triton buffer to give different concentrations of intracellular activator at a constant level of Triton (125 ug/ml in early experiments, 75 pg/ml thereafter). The content of intracellular activator was then assayed as for external activator.

1151

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Results:

We investigated

following

four reagents would depress production of plasminogen activator:

serum-free medium, cyclic

whether the exposure of CHOcells to any of the

AMPphosphodiesterase inhibitors,

and

AMP. As indicated below, the combined use of phosphodiesterase

dibutyryl

cyclic

inhibitor

and the serum-free state succeeded in doing so:

Extracellular

plasminogen activator.

of plasminogen activator (8).

prostaglandins,

CHOcells liberate

considerable amounts

into serum-free mediumduring 18 hr of incubation

Increasing concentrations

of plasminogen activator

in 2 ml of serum-free

medium (% harvest medium), together with 61 ug of plasminogen, brought about the proportional

digestion of up to 70% of the total

125I-fibrin

of coated dishes

during an 18 hr digestion period. The 2 cyclic consistently activator tive

AMP phosphodiesterase inhibitors,

theophylline

and markedly depressed the level of extracellular

line or 130 ug papaverine reduced the ability

phylline activator

plasminogen

released by CHOcells in serum-free medium. Table 1 shows representa-

results of 3 experiments in which the presence of either

activator

and papaverine,

of CR0cells

in serum-free mediumduring 18 hr incubation. reduced the level of fibrinolytic

activity

depressed the level of fibrinolysis

to accumulate the The presence of theo-

resulting

to 6-21% of that with the control CHOcells.

1.0 mM_ theophyl-

from extracellular

Papaverine correspondingly

to 6-12X.

Prostaglandin El, which has been reported to stimulate the adenyl cyclase system (12), also caused a drop in the level into serum-free extracellular with the cyclic

fluid.

of plasminogen activator

liberated

However, the drop was not as extensive

as

AMP phosphodiesterase inhibitors.

18 hrs lowered the level

Prostaglandin E1 at 45 @ for fibrinolytic activity to 42-54X of that

of extracellular

of control CR0 cells. Dibutyryl

cyclic

AMP at 0.1 mMand 1.0 mMdid not significantly

level of production of extracellular dibutyryl

cyclic

extracellular

AMP contributed

fibrinolytic

activity

activator.

little

Further,

alter

the

the presence of

to the depression of the level of the

by 1.0 mMtheophylline.

1152

Neither equimolar

Vol. 70, No. 4,1976

BIOCHEMICAL

Table 1.

plasminogen activator

Extracellular

AND BIOPHYSICAL

Activity,

RESEARCH COMMUNICATIONS

produced by CHOcells % 125I-Fibrin

Solubilized

Drug

20% harvest Medium

None

33

67

2

5

2

5

Ethanol, 27 mM_

43

74

Prostaglandin El, 45 L&J and ethanol, 27 mM

18

49

Theophylline,

1.0 mM_

Papaverine, 130 pM

40% harvest Medium

CHOcells were plated on 60 mm'plastic tissue culture plates with 2.8 x lo5 cells in Ham's F-12 mediumcontaining 10% fetal bovine serum. 24 hr later, the cell monolayers were washed 3 times with mediumlacking serum, and were overlayed with serum-free mediumcontaining or lacking drug. After 18 hr, the cell-free mediumwas diluted as indicated with serum-free medium (no drug) on the basis of cell density (ca 3 x lo4 cell 1/cm2) at that time, so that "100% harvest medium" was from lO~cells/ml/5 cm of dish surface. Two ml of each diluted mediumwas incubated for 18 hr at 37" with 61 pg of purified human plasminogen in 35 mmplastic tissue culture dishes coated with purified 1251fibrin. The % fibrinolysis was computed on the basis of the radioactivity of soluble 1251-fibrinopeptides in the medium, and the total radioactivity originally present in the dishes (174,000 - 188,000 cpm). 5'-adenylic cyclic

acid nor twice equimolar sodium butyrate,

AMP, had a significant

effect

on the liberation

both relative

to dibutyryl

of activator

by the CHO

cells. Experiments involving at the above concentrations,

direct

addition

of either

to the serum-free medium from control

showed that the action of the drugs was neither process of activation

after

Further,

by the high, rather

intentional

chemically

different

cell

CHOcells

on the extracellular

enzymatic

per -se is indicated

than low, proteolytic

activity

death; by the lowering of fibrinolytic agents, namely papaverine,

El; and by normal cellular

diges-

that the depression of fibrinolytic

was not brought about by cell toxicity

following:

or papaverine,

of plasminogen to plasmin, nor on the proteolytic

tion by the plasmin per se. activity

theophylline

theophylline

morphology under phase contrast 1153

by the

of the medium activity

by three

and prostaglandin microscopy.

Vol. 70, No. 4,1976

Table 2.

6lOCHEMlCAL

Intracellular

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

plasminogen activator

produced by CHOcells 125I-Fibrin

Cells x 104

Drug None

Theophylline,

1.0 I@

Papaverine, 130 ukJ

Ethanol, 27 "IM

Solubilized %

2.0 10.0 20.0

4 29 47

1.8 9.1 18.2

2 2 3

(3)=

1.5 7.5 15.0

2 4 7

(9)

2.0 10.0 20.0

1 19 36

1.9 9.3

19

Prostaglandin El, 45 pg, and ethanol, 27 mM_

2 35

18.6

(38)

CHOcells of Table 1 were washed twice, pooled, and homogenized in 0.1 M Tris-Cl buffer, pH 8.0, containing 500 ug/ml Triton X-100. The cell lysates were diluted with both serum-free mediumand the Tris-Triton buffer to yield different levels of fibrinolysis brought about by intracellular activator, corresponding to quantities derived from different numbers of cells listed in the table, all assayed in 75 ug/ml Triton. The diluted cell lysates were analyzed for plasminogen activator exactly as summarized under Table 1. a % 125I-fibrin

Intracellular presence of either

solubilized,

normalized to 2.0 x lo5 cells The incubation

plasminogen activator. 1.0 mM_ theophylline

the level of fibrinolytic 19-26%, respectively, on the fibrinolytic

activity

or 130 ~3 papaverine for 18 hr reduced

due to intracellular

of that with control activities

activator

activity

cells in 45 @ prostaglandin Discussion:

The cyclic

activator

CHOcells.

These values are based

(Table 2).

No significant

was observed with similar

effect

incubation

on

of the CHO

El.

AMP phosphodiesterase inhibitors,

papaverine, markedly depress the production of intracellular plasminogen activator

to 6-22% and

associated with the highest number of cells for

each drug, normalized to 2.0 x lo5 cells intracellular

of Cl-IOcells in the

theophylline

and

and extracellular

by CHOcells in serum-free medium. Prostaglandin El evokes 1154

BIOCHEMICAL

Vol. 70, No. 4,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a similar but moderate response on the production of extracellular The presence of dibutyryl

cyclic

reported to bring about the reversal in CHOcells (13,14).

activator

only.

AMP in serum-containing mediumhas been of certain

phenotypes of cell transformation

Our CL-IOcells undergo such morphologic reversion in serum-

containing medium, but do not do so in serum-free medium. On the other hand, the depression of production of at least extracellular the cyclic

AMPphosphodiesterase inhibitors

serum (data not shown). Therefore, plasminogen activator

does not occur in the presence of

the observed depression of production of

namely, the serum-free condition and the presence of

AMP phosphodiesterase inhibitors.

It is as yet unknown if

mediated by a change in the level of intracellular It is noteworthy that four types of cells apparently

in response to regulatory

cyclic

that alter

AME'(13,14), of cyclic

their

phenotypes

Chinese hamster of dibutyryl

production of plasminogen activator

AMP phosphodiesterase inhibitors

cyclic

in the presence

in serum-free medium(this

report).

Secondly, mouseL cells

that grow in monolayer cultures as round, densely

packed, piled up cells,

grow in the presence of the glucocorticoid,

sone (10-7 M), as flat,

polygonal,

and less densely packed cells.

dexamethaThis

transition

is accompanied by a depression of production of plasminogen activa-

tor (17).

Thirdly,

three rat hepatoma cell lines also suppress production of

plasminogen activator

in response to dexamethasone (17).

neuroblastoma cells,

which can be induced to "differentiate"

enzymatic levels by cyclic increase their other regulatory activator

is

nucleotide.

Firstly,

which in serum are responsive to the level

depress their

this effect

molecules, react to those signals by changing

the levels of production of plasminogen activator. ovary CHOcells,

by

in Cl-IOcells appears to be brought about by two factors

that may act synergistically, cyclic

plasminogen activator

AM? agents in vitro,

Cl300 mouse

and to change

under such conditions markedly

production of plasminogen activator molecules to control

Fourthly,

(18).

The ability

of these and

the level of production of plasminogen

by cells should be of value in experiments designed to elucidate

1155

the

BIOCHEMICAL

Vol. 70, No. 4, 1976

possible biological cells,

role of the proteolytic

and in specific

differentiation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

actions of plasmin on the surfaces of

cell systems the cell surface alterations

which accompany

and oncogenic transformation.

Acknowledgements. Supported in part by grants CA-05945, CA-06927, and RR-05539 from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania. References 1.

Burger, M. M. (1971) in Current Topics in Cellular Regulation (Horecker, B. L. and Stadtman, E. R., eds.), 3, 135-193, Academic Press, NewYork. 2. Astrup, T. (1966) Fed. Proc, 5, 42-51. 3. Bernik, B. and Kwaan, H. C. (1969) J. Clin. Invest, 48, 1740-1753. 4. Unkeless, J. C., Tobia, A., Ossowski, L., Quigley, J. P,, Rifkin, D. B. and Reich, E. (1973) J. Exp. Med, 137, 85-111. 5. Ossowski, L., Unkeless, J. C., Tobia, A., Quigley, J. P., Rifkin, D. B. and Reich, E. (1973) J. Exp. Med, 137, 112-126. 6. Roblin, R., Chou, I. and Black, P. H. (1975) Adv. Cancer Res, 22, 203-260. S. C. and Acs, G. (1976) in Proteinases 7. Christman, J. K., Silverstein, of MammalianCells (Barret, A., ea.), Associated ScientiEc Publishers, Amsterdam, in press. 8. Mott, D. M., Fabisch, P. H., Sani, B. P. and Sorof, S. (1974) Biochem. Biophys. Res. Commun,6l, 621-627. 9. Chibber, B. A., Niles, R. M., Prehn, L. and Sorof, S. (1975) Biochem. Biophys. Res. Commun,65, 806-812. 10. Pastan, I., Anderson, W. B., Carchman, R. A., Willingham, M. C., Russell, T. R. and Johnson, G. S. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga,T., eds.), 563-570, Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork. 11. Sheppard, J. R. and Bannai, S., ibid, 571-579. 12. Pastan, I. H., Johnson, G. S. and Anderson', W. B. (1974) Ann. Rev. Biochem, 66, 491-522. 13. Hsie, A. W. and Puck, T. T. (1971) Proc. Natl. Acad. Sci. USA68, 358-361. 14. Puck, T. T., Waldren, C. A. and Hsie, A. W. (1972) Proc. Natl. Acad. Sci. USA69, 1943-1947. 15. Laki., K. (1951) Arch. Biochem. Biophys, 32, 317-324. 16. Deutsch, D. G. and Mertz, E. T. (1970) Science, 170, 1095-1096. 17. Wigler, M., Ford, J. P. and Weinstein, I. B. (1975) in Cold Spring Harbor Symposiumon Proteases and Biological Control (ReichTE., Rifkin, D. B. and Shaw, E., eds.), 849-856, Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork. 18. Laug, W. E., Jones, P. A., Nye, C. A. and Benedict, W. F. (1976) Biochem. Biophys. Res. Commun,68, 114-119.

1156

Cyclic AMP phosphodiesterase inhibitors depress production of plasminogen activator by Chinese hamster ovary cells.

BIOCHEMICAL Vol. 70, No. 4, 1976 AND BIOPHYSICAL RESEARCH COMMUNICATIONS CYCLICAMP PHOSPHODIESTERASE INHIBITORS DEPRESS PRODUCTION OF PLASMINOGEN A...
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