Plasminogen activator and MIF-like activities in Kirsten virus transformed mouse NIH culture fluids

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 1164-1170

August 14,1978

PLASMINCGENACTIVATORANDMIF-LIKEACTMTIFS IN KIRSTEN VIRUS TRANSFORMEDMOUSE NIH CULTURE FLUIDS* J. Feder, R.C. Kimes, W.R. Tolbert,

and C. Cleveland

Monsanto Company St. Louis,Missouri 63166 M.E. Hammond, N.S. Orenstein,

J. Goodwin, and H. F. Dvorak

Department of Pathology Massachusetts General Hospital Boston, Massachusetts 02114 Received s-y

June 1,1978

:

Kirsten virus transformed mouse NIH cells produce both a macrophage migration inhibition activity for guinea pig and mouse peritoneal exudate cells and a plasminogen activator. The migration inhibition factor activity exhibited thermal stability up to 80'~ while the plasminogen activator was inactivated after 15 minutes at 7o"c. Separation of these activities was achieved by absorption of the migration inhibition activity on agarose-fucosamine or high speed centrifugation. Introduction It bed been shown earlier formed cells cells

inhibit

the migration

(GPPE) --in vitro

and that

This inhibition

tar (4,5).

that

culture

fluids

of guinea pig peritoneal

these fluids

was similar

contain

macrophage migration

More recently

it has been observed that MIF-like

tially virus

purified

following

preparations

40 transformed

guinea pig serum (7). to Kirsten

virus

inhibition

incubation

exudate

plasminogen

activa-

(MIF) (6).

activity

of human urokinase activator

for GPPE or par-

from Simian

(SV3T3) with medium containing

These observations

transformed

factor

of plasminogen

mouse 3T3 cells

trans-

to that produced by lympho-

cyte-produced

could be generated

from virus

have here been extended

mouse NIH cells

(KNIH).

Both MIF-like

*This work was supported in part by USPHS grant CA-16881 and CA-20822. We thank Ms. Pat Giovinco for technical assistance. 0006-291X/78/0833-1164$01.00/0 Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

1164

Vol. 83, No. 3, 1978

activity

BIOCHEMICAL

as measured against

have been obtained

differences Materials

GPPE and plasminogen

in serum-free

and shown to be separate

AND BIOPHYSICAL

entities

culture

fluids

by affinity

RESEARCH COMMUNICATIONS

activator

activity

of these cells chromatography

and

in thermostability. and Methods

Cells and Cell Culture: The NIH Swiss mouse embryo cells, Kirsten virus transformed NM cells (KNIH) (8), and SV3T3 cells were kindly supplied by Dr. Judah Folkman, Children's Hospital Medical Center, Boston, Mass. Cells were grown in Dulbecco's modification of Eagle's minimal essentsal medium supplemented with 5-10% fetal calf serum either in 75 cm plastic T-flasks (Falcon Plastics, Los Angeles) or in suspension culture in spinners. Confluent monolayer cultures were washed with serum free medium or phosphate buffered saline (PBS) and replaced with same for incubation overnight. Suspeyion grown cells were similarly washed, resuspended at about 6 x 10 cells/ml, and allowed to incubate with agitation in the cold for 4 hours. After centrifugation, culture fluid supernatants from both monolayer and suspension cultures were harvested as starting material for these studies. In some cases these fluids were dialyzed and lyophilized and stored frozen before use. MIF' Assay: Crude or partially purified culture fluids were incubated overnight (16-18 hours) at 17'C in elastic tubes in Eagle's MEM (x4)‘-containing 15% freshiy thawed guinea pig serum (GPS). This medium was then assayed for MIF-like activity in chambers containing capillaries of pelleted GPPE cells (7,9). After incubation of chambers at 37OC in 5% CO atmosphere for 18-20 hours, the area of migration was traced2and measured by planimetry. Cell viability was assessed by trypan blue exclusion and migration inhibition tests were considered valid only if at least 95% of the GPPE cells remained viable. Replicate assays were run and only inhibition of macrophage migration greater than 20% was judged to be significant. Plasminogen Activator (PA) Assay: PA activity was determined by a micro-adaptation (10) of the fibrin dish assay of Unkeless et al. (10, 11). Plasminogen-free human fibrinogen was the generous gift of Dr. Yu-lee Hao, American Red Cross, Washington, D.C. Plasminogen was prepared by the method of Deutsch and Mertz (12). Urokinase (WinKinase, 35,000 CTA units/mg protein, Sterling-Winthrop Laboratories, New York) was made available through the generosity of Dr. William P. Blackmore and Dr. Joseph C. Fratantoni. Samples were assayed with and without added plasminogen to distinguish between PA and fibrinolytic activityl25The PA activity was expressed as t e init'al rate of release of I-fibrin in terms of CPM x ml2. - xml -i . At intervals aliquot of lo-25 1.11of reaction solution were removed and counted. A unit of PA activity was defined as the amount of enzyme which led to the solubilization of a microgram of fibrin per hour. Given the specific activity of the fibrin, which was determined by solubilizing random control wells with NaOH and calculating cpm/!~ gm fibrin, units of activity were calculated from the initial rates of cpm release by the equation: (cpm x min-'l x ml-l) x 6O&=@ct&y units (in reaction) = v initial Specific

Activity 1165

of Fibrin

(CPW' WP~)

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

Effect of KNIH cell culture fluid on the initial rate of hydrolysis (details in text).

Figure 1.

RESEARCH COMMUNICATIONS

concentration of fibrin

Results and Discussion Table 1 shows the effect culture

fluid

protein

Preincubation observed.

on the inhibition

Culture fluids of protein

from untransformed NIH cells exhibited

no MIF-like

migration

fluids

of GPPEcells.

Wigration

(1 mg protein/

1 ml).

activity.

This is

that fluids

from SV40

fluid

quantitate

the PA activity

the linear

portion

(53%) by the KNIH

Figure 1 demonstrates that which exhibits

tion dependence of the rate of 125I-fibrin of culture

at the same

of mouse peritoneal

inhibited

these cells also produced PA activity

centrations

inhibition

but not from untransformed 3T3 cultures

exudate cells was also significantly culture

of GPPEcell migration.

to the observations reported earlier

transformed 3T3 cultures inhibit

of KNIH

with guinea pig serum enhanced the migration

concentration similar

of varying concentrations

protein.

linear

hydrolysis

at dilute

Consequently all

were restricted

of the concentration

1166

concentracon-

attempts to

to rate data obtained over

rate curve.

The KNIH PA

0.125 0.25 0.50 1.00

2339 2413 2155 2011

Fluid

45 42 48 52

1650 1722 1564 1432

NIH Culture

13 3 23

2604 2909 2284

3 0 11 16

0

0

2984

Preincubated with GPS 12-14 hrs Migration Area % Inhibition

2403

2 10 9 7 3 38 38 5

0

No Preincubation Area % inhibition

CULTURES;RESuLTS OFATYPICALEXPERlMENT

No addition

0.1 0.2 0.3 0.4 0.5 0.6 0.8 1.0

2676 2453 2498 2545 2646 1701 1686 2592

Fluid

KNM Culture

Migration 2735

Protein Cone. (mghnl.)

INFLKDSFROMKNfHANDNIH

No addition

Addition

CGi"IPARISONOFMIF-ACY?MZY

TABLE I

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 2 THERMAL STABILITY OF MIF AiD PA ACTIVITIES OF KNIH AND SV3T3 CELL CULTURE FLUIDS Treatment

PA Activity V initial (CPM x minlx

KNIR Culture

% Activity Remaining

ml;1

% Migration

Inhibition

Fluid

untreated 7o"c, 15 min. SV3T3 Culture

100 0

861 0

49 46

Fluid

untreated

691

100

28

34

5

27

7o"c, 15 min.

activity,

MU? Activity

similar

to that produced by SV3T3 cells,

was inactivated

by diisopropylfluorophosphate. Since it was shown earlier incubation attempt

was made to demonstrate activity

from KNIR cultures PA activity

thermal

stability

or non-identity

of the

of crude fluids

activity

lost

PA activity

Partially

purified

exhibited

MB activity

strongly

resided

In an attempt 500 ml of KNIH cell

similar

to the

after

affinity

and MIF-like

molecules.

to physically culture

fluids

stability.(Table

suggested that the PA activity

in different

inhibition

Culture

obtained

thermal

fluid

separate

the MIF and PA activities,

which had been dialyzed 1168

the

considerable

under conditions

(see below) showed similar

inactivated

migration

even up to 80°C for 30 minutes.

chromatography These results

The MD'-like

(7) an

completely

on the GPPE cell

upon

activity

Incubation

at 70°C for 15 minutes

(Table 2).

KNIR fluids.

the identity

and PA activity.

but had no effect

from SV3T3 cells

activity

activators

with guinea pig serum generate MIF-like

KNIH-MD-like

activity

that plasminogen

against

3).

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 83, No. 3, 1978

TABLE 3 THE ADSORPTION OF MD-LIKE ACTIVITY BY AGARCSE- AMINO-CAPROYLFUCCSAMINE (AF)FROMK?JMCULTUREFIJJlDS Fraction

PA Activity Total

% of Total

%Migration Inhibition

9600

100

44

Supernatsnt frcm AF 9803 Supernatant,70°C, 15 min. 0

102 0

41 43

KNM CultureFluid Untreated Starting Material

Units

MIF Activity

Eluate from AF Eluate, 70°C, 15 min.

water

75 0

were exposed to 20 ml of agarose-E-amino

at 4'C for

18 hr with

trifugation,

slow mixing.

washed four times

1% Gf'ucose

for

starting

and the eluate.

Units

activity

remaining

activity

was adsorbed

in the supernatant. and eluted

results

demonstrate

that

and thus demonstrate might

be generated

present

a fairly

Finally, fugation

that

essentially

the two activities

thermal-stable

at 35,000 xg for 18 hours.

MIF activity

as proposed

the MIF

The MIF activity the capexhibited

material.

to any MIF-like

These

activity

earlier,

there

separated which is also

entity.

were separated

by ultracentri-

The PA activity

and settled

all

can be physically

Ml?-like

these two activities

fractions

significant

to the crude starting

in addition

for all

due to overloading

The eluted

from PA activity,

became insolubilized

with

In contrast

was probably

comparable

20 ml of

from agarose-fucosamine,

from the resin.

of the agarose-fucosamine. stability

by cen-

with

were calculated

in the eluate

in the supernatant

thermal

and eluted

supernatant

of PA activity

than 1% was obtained

acity

saline

was collected

Table 3 shows the PA and MIF-like

material,

and less

remaining

with

66 51

caproyl-fucosamine

The resin

in the cold overnight.

activities

fluids

0.8 0

out of solution

1169

of the culture under these

Vol. 83, No. 3, 1978

Ultracentrifugation

conditions. PA activity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

while the supernatant

yielded solution

a pellet

contained

PA was solubilized

by treatment

with 0.1% Triton

These observations

suggest that

the PA activity

with a membrane fraction

similar

PA in sarcoma virus-transformed

containing

the

the MCF activity.

Xl00 and 3M KCl. may be associated

to that reported

by Quigley

chick embryo fibroblasts

(13) for

and hamster

SV40 cells. Solubilized

KNIH PA activity

was shown in preliminary

ments to be adsorbed by lysine-sepharose vity

did not bind to this

contain

separable

thermal

stabilities.

resin.

PA and MI&like

beads, whereas the MD? acti-

In conclusion, activities

experi-

KNIH culture

which exhibit

fluids

different

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Hamn?ond, M.E., Roblin, R.O., Dvorak, A.M., Selvaggio, S.S., Black, P.H., and Dvorak, H.F. (1974). Science 185: 955-957. Poste, G. (1975). Cancer Res. 35: 2558-2566. B&.$zzi, P.E., Yoshida, T., W&T P.A. and Cohen, S. (1975). Am. J. Path. 80: 69-78. Ossowski, L., Unkeless, J.C., Tobia, A., Quigley, J.P., Rivkin, and Reich, E. (1973). J. Exp. Med. 137: 112-1l6. &:.'I N Black, P.H., and Roblin, R.O. (1974). Nature 250: 738$4i." Dvorak, A.M., Hammond, M.E., Dvorak, H.F., and Ksrnovsky, M.J. (1972). Lab. Invest. 27: 561-574. Roblin, R.O., Hammond, M.E, Berm@, N.D.,Dvorak, A.M., Dvorak, H.F., and Black, P.H. (1977). Proc. Natl. Acad. Sci USA 74: 1570-1574. Aaronson, S.A. and Weaver, C.A. (1971). J. Gen. Vgl. 13: 245-252. David, J.R. and David, R. (1971). In In Vitro Methods KCellMediated Isxnunity, Bloom, B.R. and Glade, P.R. (eds.), pp. 249261, Academic Press, New York. Dvorak, H.F., Orenstein, N.S., Rypysc, J., Colvin, R.B., and Dvorak, A.M. (1978). J. Imnunol. 120: 766-773. Unkeless, J.C., Tobia, A., Ossowski, L., Quigley, J.P., Rivkin, D.B., and Reich, E. (1973). J. Exp. Med. 137: 85-126. Deutsch, D.G. and Mertz, E.T. (1970). Scienzl70: 1095-1096. Quigley, J.P. (1976). J. Cell Biol. -71: 472-48c

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