BIOCHEMICAL

Vol. 76, No. 2, 1977

THE RELATIONSHIP

AND BIOPHYSICAL

BETWEEN SURFACE PROTEASE ACTIVITY

THE RATE OF CELL PROLIFERATION

Victor

AND

IN NORMAL AND TRANSFORMED CELLS

B. Hatcher, Martin S. Oberman, Michael S. Wertheim, Choo Y. Rhee, Grace Tsien and Peter G. Burk

Department Albert Montefiore

Received

RESEARCH COMMUNICATIONS

April

of Biochemistry and Medicine, Einstein College of Medicine, Hospital and Medical Center, Bronx, New York 10467

8,1977 SUMMARY

Surface protease activity and secreted protease activity has been determined on several cell lines utilizing 3H-acetyl casein as substrate. When normal rabbit aortic smooth muscle cells and fibroblasts stopped proliferating at high No decrease cell density a decrease in surface protease activity was observed. in surface protease activity or in the rate of cell proliferation was observed in either transformed melanoma or epidermal cells. The decrease of surface protease activity was related to a decrease in cell proliferation.

INTRODUCTION Surface

proteolytic

transformed is

cell

associated

for

with

certain

cellular

function

cells

the plasma activator

in the

growth

inhibitors

We have in several between

membrane acting

rate,

serine fraction

through

proteases

activator,

and transformed

Although

is not

protease

cells

to be responsible

(5-10).

and morphology

exogeneous

and

plasminogen

appears

cells

agglutinability

in normal

proteinase,

plasmin

surface

either

demonstrated

of normal

in transformed

endogeneous

utilizing

the

clearly

understood,

of cultured (11-16)

or specific

'(13,17,18,19).

demonstrated

normal

has been

A specific

changes

of the

can be produced

protease

(l-4).

morphological

alterations

Copyright All rights

lines

Plasminogen

(2,4).

enzyme activity

non plasminogen

and transformed

non plasminogen

activator

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

cell

activator lines

surface

(3).

protease

surface There activity

protease is

activity

a relationship and doubling

time.

602 ISSN

0006-291

X

BIOCHEMICAL

Vol. 76, No. 2, 1977

Cells

with

while

cells

activity. during surface aortic (HM-34)

long

doubling

with

times

have

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

small

rapid

doubling

times

The maximal

surface

protease

mitosis protease fibroblasts cells

in several activity

amounts

have

cell

was investigated muscle

and mouse epidermal

amounts

activity

synchronized

and smooth

large

(UT-l)

protease

lines.

which

just

In the present inhibited

and in transformed cells

were

activity

of surface

was observed

in growth

cells

MATERIALS

of surface

not

protease before

or

investigation

normal

rabbit

human melanoma growth

inhibited.

AND METHODS

Rabbit aortic fibroblasts and smooth muscle cells were obtained from rabbit abdominal aorta. The human melanoma cells (HM-34) are from lines grown from melanoma tissue and were obtained from Dr. J.C. Bystryn, New York University, N.Y.C. The transformed epidermal (UT-l) cells were obtained from Dr. S. Yuspa, NCI, Bethesda, Md. The transformed epidermal cell line (UT-l) was produced by treating pregnant Balb/c mice in utero with urethane. Epidermal cells from the newborn mice were treated in culture with tetradecylphorbal acetate, (Yuspa, unpublished results). Rabbit smooth muscle cells (passage 20-25) and fibroblasts (passage 12-15) are grown in medium 199 containing unmodified Earl's salts with 10% fetal calf serum according to the method described by Ross (20). Human melanoma cells and transformed mouse epidermal cells are grown in Dulbecco's modified Eagles media containing 10% newborn calf serum. In experiments in which protease activity was determined at 24 hour intervals the media on each plate was changed at 24 hour intervals otherwise the media was changed at 48 hour intervals. The cells are counted on duplicate plates employing a hemacytometer (Bright-line, Spencer, American Optical Corporation, Buffalo, N.Y.). After each experiment was completed the cells was examined by Eosin-Y exclusion. Surface protease was determined according to method described by Hatcher et al (3). The [3H]-labelled casein had a specific activity of 3,840 cpm per pmol assuming a molecular mass of 121,700 daltons. The cell cultures grown in Falcon petri dishes are washed 6X with Dulbecco's modified Eagles media without serum and incubated for 1.5 h at 37'C under 7% CO2 in room air. The media is removed and 300 ~1 [3H]-labelled casein containing 20 ug casein and 2 x lo6 cpm previously dialyzed against serum free media (600 ~1) containing EACA in that the final concentration of EACA in the reacting mixture was 10 mg/ml. The plates are then incubated for 180 min at 37'C under 7% CO2 in room air. Following termination of the reaction by the addition of unlabelled casein (100 ~1, 3% w/v in 1 M KCl) to an aliquot of the media (100 ul) followed by chilled 6% TCA (200 ~1) the mixture is allowed to remain overnight in an ice slurry. The samples are then centrifuged at 15,000 rpm for 30 min and 200 1.11 of supernatant is added to 10 ml of Riafluor (New England Nuclear) and then the radioactivity determined. Surface protease activities are reported only when no secreted activity was detected since the surface protease assay also measure secreted activity. Secreted protease activity was determined as previously described (3). Replicate plates containing serum free media (900 ~1) are incubated for 180 min at 37°C under 7% CO2 in room air. The media is removed, centrifuged at 15,000

603

Vol. 76, No. 2, 1977

BIOCHEMICAL

2

4

AND BIOPHYSICAL

6

6

IO

12

14

RESEARCH COMMUNICATIONS

16

TIME(Days)

Figure

1. smooth muscle Surface protease activity of: A. Rabbit aortic cells dispersed in trypsin (0.25% w/v)-EDTA (0.5 mM) (Grand Island Biological Co., Grand Island, N.Y.). B. Rabbit aortic smooth muscle cells dispersed with sterilized glass roller beads (3mm, Thomas Philadelphia, Pa). C. Rabbit aortic fibroblasts dispersed in trypsin (0.25% w/v)-EDTA (0.5 mM). Time 0 refers to Each point the day the cells were dispersed and subcultured. on the graph represents means values of triplicate counts of duplicate determinations on separate plates.

for 120 min at 37°C with [3H]cpm for 15 min and an aliquot (100 1.11 incubated labelled casein (40 ~1, 20 pg, 2 x 10 b cpm). The reaction mixture contained c-aminocaproic acid (EACA) at a concentration of lOmg/ml. The reaction is terminated with unlabelled casein (100 ~1, 3% w/v in 1 M KCl) and 6% TCA (200 ~1). The mixture is allowed to remain overnight in an ice slurry after which the samples are centrifuged at 15,000 rpm for 30 min and 200 1.11 of supernatant is added to 10 ml of Riafluor (New England Nuclear) and then the radioactivity measured. RESULTS AND DISCUSSION In Figure dispersed subculturing,

IA surface

in trypsin-EDTA secreted

protease

activity

of rabbit

and subcultured protease

activity

are

shown.

was detected.

604

smooth

muscle

cells

At 0 and 1 day after The amount

of combined

Vol. 76, No. 2, 1977

BIOCHEMICAL

0 Figure

2

AND BIOPHYSICAL

4 6 8 TIME( Days)

IO

RESEARCH COMMUNICATIONS

12

2. Surface protease activity of human melanoma (HI+34) cells dispersed with A. Trypsin (0.25% w/v)-EDTA (0.5 mM). B. Sterilized glass roller beads. C. EDTA (1 mM). Day 0 refers to the time the cells were dispersed and subcultured. Each point in the graph represents means values of triplicate counts of duplicate determinations on separate plates.

secreted

and surface

(results

not

creted

activity

activity. (3,5

and 7 days) protease number

observed cells

Surface

was detected

were

protease

activity

protease

activity

was observed

mechanically

was greater

activity 1000-3000

was observed

than

30,000

only

reported

casein

between

cells.

on days

9, 11 and 13.

with

sterilized

605

glass

for

A complete

surface protease muscle loss

cells

of

No increase

to insure of trypsin beads

cells.

when no se-

smooth

cpm/105

In order

cpm/105

and secreted

growing

was not due to the effect

dispersed

assay

surface

of rapidly

on days 9, 11 and 13.

activity

is

the 3H-aeetyl

differentiate

was between

proteolytic

activity

since

does not

The surface

surface cell

shown).

activity

protease

protease

that

in the

and EDTA, the

and subcultured

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

35

It 30 25

-3 -2 -1 0

Figure

2

I

3

4

TIME

(Doys)

5

I 7

6

6

3. Surface protease activity of mouse (balbfc) transformed epidermal cells (UT-l's) dispersed with A. Trypsin (0.25% w/v)-EDTA (0.5 mM). B. EDTA (1 mM). The cells were dispersed and subcultured on day 0. The points on the graph represent mean values of triplicate counts of duplicate determination on separate plates.

(Figure

1B).

protease

levels

14 when the tease

On day 3 and 10 when no secreted of 8000 cpm/105

cells

activity.

spersed

with

fibroblasts

Surface trypsin-EDTA secreted

subculturing.

protease

increase

protease

there activity

amounts

activity

are

in the number was also

loss

aortic

presented

of protease

of surface

cell

was a total of rabbit

and subcultured

large

was detected,

and 2000 cpm/105

multiplying,

At day 8 a loss

was no subsequent Surface

stopped

cells

protease

were

activity

found.

On day

of surface fibroblasts

in Figure

up to and including

protease

surface

prodi-

1C.

The

day 1 after

was found

and there

of fibroblasts. measured

606

on human melanoma

(BM-34)

BIOCHEMICAL

Vol. 76, No. 2, 1977

cells

and transformed

In all off

experiments

did

the

the plates.

melanoma not

epidermal

(m-34)

cells

secrete

(UT-l)

transformed

In Fig

protease

dispersed

rapid

proliferation

surface

in

of transformed

presented

activity.

was observed

the rapidly

growing

activity.

protease

glass

Similar

roller

in rapid

when the

growing

early

passage

WI-38

present

at the

protease

protease

in

activity (Figure

EDTA did

protease

3A)

not

activity

fibroblasts

was

dispersed

with

WI-38

activity.

the

of cultured

is not that

and that

in cell

the

cells.

detectable

is loss

Bosmann et al

cells

to our

of surface

is

activity to divide.

cell

protease

and was not

(21)

late

activity

protease

WI-38

observed

of rapidly

protease

similar

proliferation

was not

in non dividing

of this

passage

aortic

protease

in homogenates

the loss

activity

study

of surface

the neutral

of late

protease In our

to a decrease

(22)

cells

inability

neutral

cellular

activity

of surface

in rabbit

and mouse UT-1

protease

surface

for

protease

cells

a loss

proliferating

The loss

They suggest

cell

this

stopped

cells.

neutral

cells.

possible

rate

muscle

passage

be responsible

Noonan

with

in

of surface

dispersed

of surface

obtained

although

trypsin-EDTA

we have demonstrated

cells

human EM-34 that

dividing

terminal

also

investigation

and smooth

have observed

related

were

The HM-34

fluctuation

Surface

The UT-1 cells no loss

2C).

a large

with

was detected

density

no loss

dispersed

when the cells

beads.

activity

fibroblasts

cells

cell

Although

HM-34 cells.

Again

results

In the present protease

in EDTA (Fig

cells

secrete

of human

activity

at high

up and came

activity

protease

dispersed

inhibited.

piled

No secreted

activity/lo5

shown.

densities

3-11

2C).

3B) are

growth

2-7 and days

(Fig.

(UT-l)

are not

at days

continued

epidermal

RESEARCH COMMUNICATIONS

protease

the plates

and EDTA (Figure

observed.

at high

in EDTA came off

protease

was detected

which

2B surface

from day 0 to day 10 in HM-34 cells cells

cells

cells

2A and Fig are

AND BIOPHYSICAL

may It

is

surface activity

associated

was

with

a

event. has suggested cells

by altering

that

exogeneous the plasma

607

proteases membrane

may affect to increase

the the

growth

Vol. 76, No. 2, 1977

availability

of nutrients

hypothesis

(23)

of nutrients events

BIOCHEMICAL

that

to cells.

the

AND BIOPHYSICAL

This

initiation

is

of the

or growth

factors

across

may be mediated

through

cell

the

in agreement

cell cell

surface

RESEARCH COMMUNICATIONS

cycle

is

membrane.

proteases

with

Holley's

due to increase In the cell

in the plasma

these membrane.

ACKNOWLEDGEMENTS We thank

Drs.

P. Barland

The assistant

of A. Miller

investigation

was supported

IjHL163870, Fibrosis

American Foundation

Cancer

and D. Hamerman and B. Shapiro by grants Society

is

from

for

gratefully the Public

Institutional

and the Dermatology

help

Grant

and discussion. acknowledged. Health IN-28R,

This

Service The Cystic

Foundation.

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Tokes, Z.A., and Chambers, S.M., (1975) Biochim. Biophys. Acta 389, 325-338. Christman, J.K., Acs, G., Silagi, S., and Silverstein, S-C., (1975) In Proteases and Biological Control (Edit by Reich, E., Rifkin, D.B., and Shaw, E.) pp. 827-839 Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Hatcher, V.B., Wertheim, M.S., Rhee, C.Y., Tsien, G., and Burk, P.G., (1976) Biochim. Biophys. Acta 451, 499-510. Quigley, J.P., (1976) J. Cell Biol. 71, 472-486. Unkeless, J.S., Tobia, A., Ossowski, L., Quigley, J.P., Rifkin, D.B., and Reich, E., (1973) J. Exp. Med. 137, 85-111. Ossowski, L., Quigley, J.P., Kellerman, G.M., and Reich, E., (1973) J. Exp. Med. 138, 1056-1064. Ossowski, L., Unkeless, J.C., Tobia, A., Quigley, J.P., Rifkin, D.B., and Reich, E., (1973) J. Exp. Med. 137, 112-126. Unkeless, J., Dano, K., Kellerman, G.M., and Reich, E., (1974) J. Biol. Chem. 249, 4295-4305. Christman, J.K., and Acs, G., (1974) Biochim. Biophys. Acta 340, 339-347. Goldberg, A.R., (1974) Cell 2, 95-102. Burger, M-M., (1970) Nature 227, 170-171. Sefton, B.M., and Rubin, H., (1971) Proc. Nat. Acad. Sci. USA 68, 3154-3157. Goldberg, A.R., (1974) Ann. N.Y. Academy Sci. 234, 348-354. Chen, L.B., and Buchanna, J.M., (1975) Proc. Nat. Acad. Sci. USA 72, 131-135. Blumberg, P.M., and Robbins, P.W., (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 131-135. Teng, N.N.H., and Chen, L.B., (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 413-417. Schnebli, H.P., and Burger, M.M., (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 3825-3827. Goetz, I.E., Weinstein, C., and Roberts, E., (1972) Cancer Res. 32, 2469-2474. Weber, M.J., (1975) Cell 5, 253-261. Ross, R.J., (1971) Cell Biol. 50, 172-186. Bosmann, H.B., Gutheil, R.L. and Case, K.R., (1976) Nature 261, 499-501. (1976) Nature 259, 573-576. Noonan, K.F., Holley, R.W., (1972) Proc. Nat. Acad. Sci. (USA) 61, 2840-2841. 608

The relationship between surface protease activity and the rate of cell proliferation in normal and transformed cells.

BIOCHEMICAL Vol. 76, No. 2, 1977 THE RELATIONSHIP AND BIOPHYSICAL BETWEEN SURFACE PROTEASE ACTIVITY THE RATE OF CELL PROLIFERATION Victor AND...
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