BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
January 13, 1978
COLLAGENOLYTIC ACTIVITY
OF RABBIT V2-CARCINOMA GROWING AT MULTIPLE
C. Biswas,
W. P. Moran,
K. J. Bloch
and J.
33-38
SITES*
Gross
From the Developmental Biology Laboratory and the Clinical Immunology and Allergy Units Arthritis, of the Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received
November 18,
1977
SUMMARY: Rabbit V2-carcinoma was maintained by serial transfer of cell suspensions prepared from solid invasive tumors growing in the thigh muscles of rabbits. No collagenolytic enzyme, active or activatable, was found in extracts of 17 of 19 tumors removed from this site. In contrast, collagenolytic activity was detected in all extracts prepared from V -carcinoma growing in a subcutaneous site in all of seven rabbits. In five zabbits, tumor growth was initiated both subcutaneously and intramuscularly. Homogenates prepared from these tumors showed collagenolytic activity in both sites. No activity was extracted from tumors growing at two different intramuscular sites in the same animal. These results suggest that the site of tumor growth influences collagenolytic activity and the expression of this enzyme at an intramuscular site may be influenced by a second, subcutaneous, neoplasm. INTRODUCTION: has led which
us to study
(IM)
et al.
site
prior
We report on collagenolytic
in rabbits
in the ascites
but
which
not
also
here,
our
activity
experiments.
transferred
They found
collagenase,
rabbit
many years.
strain
of this
of the
neo-
to an intramuscular levels
detected
in the ascites influence
for
high
active
invasion
of V2-carcinoma
passage
(2)
of an invasive
MATERIALS AND METHODS: Reagents sulfate (Schwarz-Mann, Orangeburg,
a strain
and Itzhaki
had been maintained on the
in tumor
from a different
spontaneously
studies
bearing
form but
Steven
extracts.
enzymes
by intramuscular
homogenates
to use in their
in tumor
of collagenolytic
transferred
(1) prepared
collagenase,
V2-carcinoma
role question
whic.h was carried
collagenase table
this
has .been serially
McCroskery plasm
The potential
of active
trypsin-activain extracts
of IM
form. site
of tumor
growth
carcinoma.
and their sources included: Ultrapure trypsin and mersalyl acid NY); Tris,
ammonium (Sigma
X
This is publication No. 741 of the Robert W. Lovett Memorial Group for the This research is supported by NIH grants Study of Diseases Causing Deformities. (CA19158) and (AM03564) and a grant from the Massachusetts Chapter, Arthritis Foundation. OOOS-ZYlX/78/08OI-0033$01.00/O
33
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chemical Corp., St. Louis, MO); l-tosylamido-Z-phenylethylchloromethyl ketone (TPCK)-trypsin and soybean trypsin inhibitor (Worthington Biochemical Corporation, Freehold, NJ); 14C-labelled acetic anhydride (New England Nuclear Corp., Boston, MA). Maintenance
of V2-Carcinoma
in Rabbits
The V2-carcinoma (3) was obtained from Dr. J. Folkman, Children's Hospital, The tumor was maintained by serial intramuscular passage Boston, Massachusetts. in rabbits. For transplantation, a rabbit bearing the tumor for lo-12 days was killed by injection of sodium pentobarbital and the tumor dissected from the surrounding muscle. The central tumor mass was cut into small slices in lactated Ringer's solution and pressed through a stainless steel grid (60 mesh). The resulting cell suspension was injected into each thigh muscle of a 2 kg New Zealand white male rabbit. 2.5 ml of cell suspension representing approximately 1 gm of tumor, was injected at each site. For subcutaneous initiation of tumor growth the same volume of cell suspension was injected subcutaneously in the scapular region. Preparation
of Tumor Extracts
Extracts were prepared from tumor initiated at intramuscular (IM) or subcutaneous (SC) sites 2-4 weeks earlier. Rabbits were killed and tumors excised as described above. The central tumor mass was sliced and homogenized at 4°C in 0.05M Tris-HCl (pH 7.4) - 1.0 M NaCl-5r&l CaC12-0.02% sodium azide in a Brinkmann polytron (PT-350) homogenizer at high speed for two 30-second intervals. The homogenate was frozen in liquid N , thawed at room temperature and centrifuged at 29,000 x g for 30 minutes (1 5 . The resulting pellet was re-extracted with buffer containing 0.05M Tris-HCl (pH 7.4), 0.2M NaCl-1mM CaC12 and 0.02% sodium azide, followed by centrifugation. The second supernatant was combined with the first and the combined supernatants were adjusted to 20% saturation with (hXti)2S04. The precipitate was saved for assay and the supernatant adjusted to 5 % saturation with (NH,.,)2SO4. The second precipitate and the supernatant were collected after centrifugation at 29,000 x g for 30 minutes and the precipitate was dissolved in .05M Tris-HCl (pH 7.4), 0.2M NaCl-lmM CaC12-0.02% sodium azide. Solutions were dialyzed overnight against the same buffer at 4°C and all fractions were tested for collagenolytic activity. In all instances, enzyme was found only in fractions precipitated by (NH4)2S04 between 20 and 50% saturation. Assay
for
Collagenolytic
Activity
Collagenolytic using reconstituted collagen solution mixed with 100-200 hours. Substrate ded as controls in activity per gm of of enzyme capable
activity was measured by the collagen fibril lysis assay (5) I4 C-acetylated rat tail tendon collagen (6); 100 ~1 of 0.2% having a specific activity of approximately 12,700 cpm/mg was pl of the enzyme preparation and incubated at 37°C for 4 plus buffer alone and substrate plus 0.01% trypsin were inclueach assay. Results were expressed as units of collagenase tumor tissue; 1 unit of collagenase represents the quantity of lysing 1 I-lg of collagen/minute.
Trypsin
of Tumor
Treatment
Extracts
The trypsin-activatable form of collagenase was detected after treatment of the tumor extract with TPCK-trypsin (90pg/ml.) at 37°C for 5 minutes. Trapsin activity was inhibited by the addition of a fivefold excess of soybean Non-enzymathe treated extract was assayed as described above. trypsin inhibitor; tic activation of tumor extracts was attempted by incubation of the enzyme preparation with 0.021 M mersalyl at 37°C for one hour (Harris, E.D., personal conununication). 34
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
For identification of collagen degradation products, reaction mixtures (0.3 ml) containing 100 ug of acid soluble rat tail tendon collagen in 10 mu Tris0.4M NaCl (pH 7.4), 200 ~1 of tumor extract and L-arginine (5OmM) (added to inhibit fibril formation) were incubated at 35°C for 2 hours. The reaction products were subjected to polyacrylamide disc gel electrophoresis at pH 4.0 as described by Nagai et al. (7). RESULTS AND DISCUSSION: A.
Effect
of site
Extracts genolytic
were
tive
prepared
activity
activatable
of tumor from
was assayed
preparations,
tested
their
ability
These
procedures
extracts.
3 M NaSCN (8);
4 minutes
in the extraction
instance.
Treatment
lagenolytic
activity.
In order tumor,
we examined
the
site.
in each of seven
detected
in all
in extracts
may determine
In an attempt between tumors
were
of the tumor
or not to account
V2-carcinoma
at either
astasis
to regional
intramuscular
Thus,
at an intramuscular No obvious cellular
infiltrate
or subcutaneous
lymph nodes.
35
failed
or
in any col-
of the
initiated
established
at
singly
collagenolytic
acti-
activity
was also
of tumor
implantation
produced.
in collagenolytic
activity
and a subcutaneous difference was noted. sites
homogen-
to elicit
of tumors
the site is
against
environment
trypsin,
collagenase
IM tumor
was detected
Collagenolytic
the difference
histologically.
or in surrounding
growths
mersalyl.
were
0.1% Triton-100,
such tumors
with
I).
extractable for
with
in extracts from
systems
of the tumor
of the in vivo
activation (Table
with
initiated
examined
prepared
Several
homogenate
also
mersalyl
activity
were
initial
No enzyme activity with
In 17 inac-
in inactive
the pellet
influence
Following
treated
whether
(10).
extracts
of the
of
IM extracts
Extracts
activity
Trypsin-
I).
in other
by centrifugation
extraction
collagenolytic
animals.
was detected
obtained
the possible
(Table
complexes
and colla
trypsin.
in any of the fractions.
dialysis
buffer
of the
to explore
a subcutaneous
vity
(9),
with
two of 19 extracts
collagenolytic
the pellet
of 19 rabbits
activation
enzyme-inhibitor
included
activity
growths
and after
in only
to reveal
heating
at 60°C for
5 M urea
before
no enzyme was detected
known to dissociate
for
on collagenolytic
the intramuscular
enzyme was detected
procedures
ate
growth
regularly
site,
the
in the appearance Rabbits exhibited
bearing met-
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I COLLAGENOLYTIC ACTIVITY
OF TUMOR INITIATED
AT A SINGLE SITE:
INTRAMUSCULAR OR SUBCUTAfiJEOUS Site of tumor implantation
No. of Animals
Total:
IM tumora
Muscle
17
0
1
II
1,
2.18
1
,I
(1
1.82
19 Subcutaneous
7
SC turnorb
a = Tumor implanted
in muscle
b = Tumor implanted
subcutaneously
c = Mean value
B.
f standard
tumor
the effect
by initiating
from both lagenolytic prepared
from
basis
although
there
tumor
extracts
were
could
possibly
from the
on the
were
found
This
.14’
If
rabbits.
disc
collagenase
gel
of an SC tumor be explained
production
36
prepared
extracts activity
equal Both
electrophoresis
leads
on a weight IM and SC
(5).
to the appearance
by the production into
intramuscular
in
to produce
collagenases
was released by the
col-
with
Collagenolytic
of the animal
such a substance
were
was confirmed
among animals.
might
of an IM
to have trypsin-activatable
variation
the presence
of an intra-
Extracts
was approximately
that
elicit
i
activity
same animal
characteristic
former.
collagenolytic
observation
of five
products
in an IM tumor
activity
simultaneously.
shown by polyacrylamide
activity by the
on collagenolytic
sites
was considerable
Our observation
stimulator
II).
IM and SC tumors
extracts
the same reaction
extracts
(Table
the
IM and SC tumor
collagenase
at both
and both
activity
tumor
of a SC tumor
growth
tumors
2.07
deviation
Effect of a subcutaneous muscular tumor
We tested
it
pg collagen degraded/ min./gm tumor tissue
Tissue assayed for collagenase activity
of
the circulation V -carcin2
of
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II COLLAGENOLYTIC ACTIVITY
OF TUMORS INITIATED
AT TWO SEPARATE SITES:
INTRAMUSCULAR AND SUBCUTANEOUS Site of tumor initiationa
No. of Expts. 1
ug collagen degraded/ min./gm tumor tissue
IM and SC
XM
1.0
11
SC
1.0
II
IM
1.45
II
SC
1.50
II
IM
0.19
11
SC
0.15
,I
IM
1.7
11
SC
3.2
II
IM
6.47
,I
SC
6.09
2
3
4
5
a = Tumor implanted
Tissue assayed for collagenase activityb
at both
sites
simultaneously
b = Tumor implanted intramuscularly and subcutaneously assayed separately for enzyme activity.
oma.
It
is not
IM tumor
extract
an inert
mass or an inflammatory
results.
known whether requires
We are We cannot
tumor sized,
(in or,
the presence
currently
as yet
the absence
the appearance
testing
explain
our
alternatively,
it
might
to extract
Conceivably, exist
would
site
lead
in the or whether
to similar
possibilities.
inability
of SC tumor).
site
were
activity
at a subcutaneous
at this
these
same animal
of collagenolytic
of tumor
reaction
in the
collagenase
enzyme might
as an irreversible
from the not
IM
be synthe-
enzyme-inhibitor
complex.
ACKNOWLEDGEMENT: tions
and for
his
We thank interpretation
Dr.
R. B. Colvin
for
of the pathologic
37
reviewing findings.
the histologic
sec-
Vol. 80, No. 1, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 0.
McCroskery, P.A., Richards, .J.F. and Harris, E.D. (1975), Biochem. J. --152. 131-142. Steven, F.S. and Itzhaki, S. (1977), Biochim. Biophys. Acta 496:244-246. Kidd. J.A. and Rous, R. (1940), J. Exptl. Med. 71:813-837. -Gross, J. and Lapiere, C.M. (1962), Proc. Natl.cad. Sci. U.S.A. -48:10141022. Nagai, Y., Lapiere, C.M. and Gross, J. (1966), Biochemistry 2:3123-3130. Gisslow, M.T. and McBride, B.C. (1975), Anal. Biochem. 68:70-78. Nagai, Y., Gross, J. and Piez, K.A. (1964), Ann. N.Y. AGd. Sci. _121:494500. Abe, S. and Nagai, Y. (1972), Biochim. Biophys Acta 278:125-132. Weeks, J.G., Halme, J. and Woessner, J.F. (1976), Biaim. Biophys. Acta 445:205-214. Wirl, G. (1975), Hoppe-Seyler's 2. Physiol. Chem. __ 356:1289-1295.
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