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.
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