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