[CANCER RESEARCH 32, 2551-2560,
November 1972]
Effect of Collagenolytic Activity in Basal Cell Epithelioma of the
Skin on Reconstituted Collagen and Physical Properties and
Kinetics of the Crude Enzyme1
Yuji Yamanishi, Mustafa K. Dabbous, and Ken Hashimoto2
Memphis Veterans Administration Hospital and Division of Dermatology, Department of Medicine, The University of Tennessee College of
Medicine, Memphis, Tennessee 38104 ¡Y. Y., K. ff.J, and Department of Biochemistry, College of Medical Sciences, The University of Tennessee,
Memphis, Tennessee 38i 03 [M. K. D.]
SUMMARY
Human basal cell epithelioma was shown to contain
collagenolytic
enzymes. With the use of reconstituted
radioactive collagen substrate, the crude enzyme (tumor
homogenate) was shown to release radioactivity 4 to 70 times
greater than the release from normal control skin. Disc
electrophoresis of the same incubation mixture demonstrated
degradation products of collagen such as aA and j3A. Kinetic
studies with the pooled specimens revealed a linear increase of
collagenolytic
activity with respect to crude enzyme
concentration or the length of incubation time. The optimal
pH ranged between 7 and 8.5. Normal human serum, cysteine,
and ethylenediaminetetraacetate
significantly inhibited the
enzyme activity, whereas a trypsin inhibitor (soybean) did not.
Viscometric studies demonstrated that basal cell epithelioma
crude enzyme decreased the specific viscosity of acid-soluble
calf skin tropocollagen. Polarimetrie studies indicated that, at
temperatures
below the denaturation
temperature
of
tropocollagen, the ordered structure of the triple helical
macromolecule was not affected during this treatment. The
denaturation temperature of tropocollagen was decreased by
5° as a result of incubation with basal cell epithelioma
homogenates.
INTRODUCTION
In BCE3 of the skin, it has been observed with the light
microscope that the stroma immediately surrounding the
parenchyma shows rarefaction of the connective tissue or
empty spaces. This clear zone, commonly referred to as a
retraction band, was thought to represent artifactual shrinkage
during preparative procedures of the stroma, which underwent
mucinous degeneration (17). The retraction band often
exhibits metachromasia with basic dyes (7,18), indicating the
presence of acid mucopolysaccharides. Since degradation of
'This is Paper 1 of 2 papers of a series, entitled "Collagenolytic
Activity in Basal Cell Epithelioma of the Skin."
2This research was supported by a Part I-Designated Research Grant,
Medical Investigatorship from the Veterans Administration, and by
USPHS Grants 243301-1534R10 and IN85F.
3The abbreviation used is: BCE, basal cell epithelioma of the skin.
Received November 11, 1971; accepted July 24, 1972.
collagen often releases metachromatic substances, and since
fragmented collagen fibrils and sometimes absence of the basal
lamina could be demonstrated in this region (11), Hashimoto
and Lever (11) postulated that collagenolytic enzymes are
responsible for the production of the retraction band.
The present study was undertaken to test this hypothesis,
with 4 methods: (a) by the incubation of proline-14C- or
glycine-14C-labeled rat or guinea pig skin collagen (either
acid- or salt-extracted) with tumor homogenate and counting
of released radioactivity; (¿>)by the disc electrophoresis of
collagen-tumor mixtures and demonstration of the appearance
of new bands representing degraded molecules of collagen ;(c)
by viscometric studies demonstrating a decrease in the specific
viscosity of substrate collagen, due to BCE crude enzyme; and
(d) by denaturation temperature determination showing a
significant decrease of denaturation temperature midpoint of
substrate collagen through the action of BCE crude enzyme.
MATERIALS AND METHODS
Fifty-one specimens of BCE and 7 of squamous cell
carcinoma were obtained either by curettage or excision under
1% procaine anesthesia. All BCE's occurred on the face, ear,
neck, back, or other sun-exposed parts of the upper
extremities. All squamous cell carcinomas occurred in the skin,
tongue, or the vermilion border of the lip. All tumors were
diagnosed histologically except for a few, clinically very
typical BCE's. For the control studies, skin from the lower
extremities and face was similarly obtained.
Preparation of Tumor Homogenate. After removal of s.c. fat
and normal skin from excised specimens and elimination of
blood clots and necrotized debris, the specimens were minced
and homogenized, with a ground-glass homogenizer, in 2 to 4
ml of 0.05 M Tris-HCl buffer, pH 7.6, containing 0.001 M
CaClj. Since tonofibrils were very poorly developed and the
stroma in most tumors consisted of half-degenerated collagen
(11), it took only 2 to 3 min to homogenize these tumors.
Homogenization was performed in an ice bath. Quantitative
determination of protein was done on each homogenate by
UV absorption by the method of Warburg and Christian (24).
Preparation of Substrate. Uniformly labeled proline-14 C
(Schwarz/Mann, Orangeburg, N. Y.) was injected either in a
single dose of 25 /uCi i.p. into the Holtzman strain of young
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2551
Y. Yamanishi, M. K. Dabbous, and K. Hashimoto
rats weighing about 100 g or into randomly bred albino guinea
pigs (200 g) in 2 doses of 25 juCi each. In the other experiment
uniformly labeled glycine-14C (Schwarz/Mann) was injected
i.p. into the same type of guinea pig in 2 doses of 50/iCi each.
The rats were killed by decapitation 24 hr after the injection.
Guinea pigs were sacrificed 48 hr after the injection. The pelt
was removed and s.c. tissue was scraped off mechanically.
Acid-soluble rat collagen was prepared by the method of
Gallop and Seifter (9), and acid-soluble guinea pig collagen was
prepared by the method of Rubin et al. (22). Neutral
salt-soluble collagen was extracted by the method of Gross
(10). The final products were lyophilized and stored in a
desiccator at —¿20°.
Preparation of Incubation Mixture. For production of
reconstituted collagen fibers, the method of Nagai et al. (20)
was followed. A highly viscous solution was thus prepared.
Aliquots of 0.5 ml of this solution were pipetted into plastic
centrifuge tubes and allowed to gel in a 37°water bath for at
least 12 hr. Prior to admixture with the tumor homogenate,
the gelatinized substrate was disrupted with a steel needle
to ensure a good contact with the homogenate (20). An equal
amount (0.5 ml) of the tumor homogenate (enzyme solution)
was added to each tube. All tubes were incubated at 37°for
18 hr with constant agitation. Normal skin homogenate was
similarly admixed with the substrate and incubated. After
incubation, tubes were centrifuged at 59,000 X g at room
temperature for 30 min to sediment undissolved collagen. A
0.5-ml aliquot of the supernatant was added to 10 ml of
Insta-Gel (Packard Instrument Co., Downers Grove, 111.),and
the radioactivity
was counted in a liquid scintillation
spectrometer.
Kinetic Studies. Since a large quantity of the crude enzyme
was required for these studies, 12 specimens from different
patients were pooled and homogenized in 6 ml of 0.05 M
Tris-HCl buffer, pH 7.6, containing 0.001 M CaCl2. Aliquots
of 0.1 ml were used for the studies of (a) enzyme
concentration and collagenolytic activity and (b) incubation
time and collagenolytic
activity.
For the study of
pH-dependent collagenolytic activities, aliquots of 0.1 ml of
the same crude enzyme solution was incubated at pH's 5 and
5.6 in acetate buffer; at pH's 6.0 and 6.6 in Tris-maleate
buffer; at pH's 7.0, 7.6, and 8.3 in Tris-HCl buffer; and at pH
9.0 in glycylglycine buffer. In the other experiment, 15
tumors were pooled and homogenized in the same way.
Aliquots of this homogenate were used in the same kinetic
studies with the use of salt-soluble, instead of acid-soluble,
reconstituted
guinea pig collagen that was labeled with
proline-14C.
Caseinolytic Activity. Caseinolytic activity of the tumor
homogenate was measured, with the methods of Kunitz (14)
and Nagai et al. (20).
Inhibition Studies. For these studies, 9 specimens from
different patients were pooled and homogenized in 5 ml of
Tris-HCl buffer, pH 7.6, containing 0.001 M CaCl2. Aliquots
of 0.1 ml were added to various reaction mixtures containing
0.5 ml of acid-extracted rat collagen that had been labeled
with proline-14C, 0.3 ml of the same buffer, and one of the
following inhibitors to make a total of 1.0 ml of each reaction
mixture: (a) normal human whole serum at final dilutions of
2552
10, 50,250, 500, and 1000; (b) EDTA at a final concentration
of 0.01 M and (c) 50 and 100 jug of soybean trypsin inhibitor
(Sigma Chemical Co., St. Louis, Mo.).
In another experiment with 6 tumors from different
patients, proline-14C-labeled, salt-soluble collagen was used as
substrate. In this experiment, in addition to normal human
whole serum and EDTA, the inhibitory effect of cysteine was
studied at concentrations of 0.01 and 0.001 M.
Disc Electrophoresis.
Aliquots of several incubation
mixtures were analyzed by disc electrophoresis through
acrylamide gels with a Canalco apparatus. In addition,
Fractions 2A and 2B of acid soluble tropocollagen were
prepared from calf skin by the method of Rubin et al. (22)
and used as substrate. The purity of these fractions was
checked by amino acid analysis. The data will be provided in
another paper (12). The reaction mixture was first incubated
for 18 hr at 27°,the temperature below that of tropocollagen
denaturation. EDTA (0.01 M) was added to the reaction
mixture to prevent further action of the enzymes, and then
the reaction mixture was denatured at 45°for 10 min at pH
4.8. A 0.1-ml of the mixture was run at pH 4.0 at room
temperature. The method described by Nagai et al. (19) was
used except that 0.05 M potassium acetate buffer, pH 4.5, was
used in the lower buffer tray. No sample gel was used; instead
the sample was applied in 5% sucrose directly on the
separating gel. The current was applied for a period of 3.5 hr.
Viscometric Method. Viscosity was measured in Ostwald
viscometers with a flow time for water of 75 to 90 sec. The
temperature of the water bath was maintained at 27 ±0.1°.
The reaction mixture consisted of 4 to 5 mg of Fractions 2A
or 2B of acid-soluble tropocollagen in 3 ml of 0.05 M Tris-HCl
buffer, pH 7.6, containing 0.04 M CaCl2 and 1.0 ml of tumor
homogenate (homogenates were always well shaken before use
and 1.0-ml aliquot rapidly transferred). After various periods
of incubation the samples were centrifuged at 10,000 X g for 5
min at 4°,and the viscosity of each supernatant was measured
at 27°.Prior to viscosity measurements each supernatant was
allowed to stand at 27°for 5 min with gentle agitation.
Optical Rotation and Melting Curves. A Zeiss polarimeter
with a hydrogen light source (365 nm) was used to measure
optical rotations. The concentration of tropocollagen used in
these experiments was calculated from the optical rotation
measurement obtained on denatured samples with -460° as
the specific optical rotation of denatured gelatin. Melting
curves were constructed by measuring the optical rotation of
samples as the temperature was increased from 27°to 42°;
before each measurement the samples were allowed to
equilibrate at that temperature for 30 min. Samples, at pH 3.8,
were kept in water-jacketed, 1-dm polarimeter tubes during
measurement.
RESULTS
Collagenolytic
Activities
on
Acid-extracted,
Proline-14C-
labeled Rat Collagen
Release of Radioactivity.
Collagenolytic
activities on
reconstituted
fibers (collagen gel) were expressed by the
radioactivity found in the solubilized supernatant of the
CANCER RESEARCH VOL. 32
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Collagenolytic Activities in BCE
reaction mixture. In Table 1, these activities were tabulated in
terms of cpm, percentage of lysis of collagen gel, and
percentage of lysis of collagen gel per mg protein. The highest
activity (22.9% lysis per mg protein) was found in the
specimen obtained from the ear (Specimen 1). The lowest
activity (2.0% lysis per mg protein) was recorded in the
specimen taken from the cheek. The highest activity found in
the normal skin control was 0.4% lysis per mg protein and was
therefore less than the lowest value of the cheek specimen.
The statistical treatment of the values of percentage lysis per
mg protein of BCE's and those of the normal skin showed that
the difference was significant at p < 0.005. Compared with the
collagenolytic activity of squamous cell carcinoma on the same
substrate (Table 2), the activity of BCE's was significantly
higher, although
squamous
cell carcinoma
showed a
significantly higher activity than the normal skin control (p <
0.05) (Table 3).
Caseinolytic activities (trypsin-like activity) of BCE were
expressed in fig equivalence per mg crude enzyme protein.
These activities did not correlate with their collagenolytic
activities (Table 1).
Kinetic Studies. Collagenolytic activity increased linearly
with the increase of crude enzyme concentration (Chart 1) and
with the time of incubation (Chart 2). The optimum pH for
the enzyme activity was between 7 and 8.5 (Chart 3). Below
or above these values, activity decreased rapidly and
significantly (Chart 3).
Enzyme Inhibitors. Human serum at a dilution of 1:10
inhibited the collagenolytic activity of the reaction mixture to
90.4% of the control and at a dilution of 1:1000 to 54.3%
(Table 4). Almost complete inhibition of collagenolytic
activity was observed with a final concentration of 0.01 M
EDTA (Table 4). Soybean trypsin inhibitor at a concentration
of 100 jUg/ml did not significantly inhibit the collagenolytic
activity of BCE's (Table 4). Since collagenolytic activity of
trypsin on the same substrate was 79.7% inhibited by 100 ng
of the same soybean trypsin inhibitor (Table 4), the lack of
significant inhibition on collagenolytic activity of BCE's might
indicate that trypsin was not responsible for the collagenolytic
activity of the tumor.
Table 1
BCE collagenolytic and caseinolytic activities on
acid-extracted, praline- ' *C-labeled rat skin collagen
lysis/mg
gel
solubilized
protein
activity*9.32.44.25.13.73.71.71.43.63.8
protein22.915.413.112.59.08.36.35.63.83.02.72.62.52.0Caseinoly
lysed
(%)20.630.816.927.521.523.222.832.333.121.88.826.08.86.5%
(above
blank)88013107201175920990970138014109303751105375275Collagen
Location
ml)EarScalpNeckNeckNoseTempleBackTempleNeckNoseBackNeckNoseCheek0.92.01.32.22.42.83.65.88.77.23.39.83.53.2cpm"
(mg/0.5
Specimen1234567891011121314Tissue
a Total radioactivity per incubation mixture was 4265 cpm.
b Expressed as Mgtrypsin equivalence/mg crude enzyme protein.
Table 2
Squamous cell carcinoma collagenolytic activities
on acid-extracted, proline-1 " C-labeled rat skin collagen
lysis/mgprotein2.40.90.70.60.50.50.3
protein(mg/0.5
gellysed
ml)9.63.87.413.37.414.09.0cpm°solubilized(above
blank)985150225360170290110Collagen
(%)23.13.55.38.44.06.82.5%
Specimen1234567LocationTongueEarWristFaceLipBack
handCheekTissue
of
a Total radioactivity per incubation mixture was 4265 cpm.
NOVEMBER 1972
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2553
Y. Yamanishi, M. K. Dabbous, and K. Hashimoto
Table 3
Normal skin collagenolytic activities on acid-extracted,
proline-1 "C-labeled rat skin collagen
protein(mg/0.5
gellysed
lysis/mgprotein0.40.30.20
blank)115140950Collagen
Specimen1234LocationCheekTempleLower
ml)7.612.810.52.7cpm"solubilized(above
(%)2.73.32.20%
legThighTissue
' Total radioactivity per incubation mixture was 4265 cpm.
aoo
3
bl
tn
150
I 50
100
t
100
o
50
50
0 V
i
0 0.2
HOURS
0.6
I
mg CRUDE ENZYME
1.4
1.8
PROTEIN
Chart 2. Degradation of collagen by BCE crude collagenase as a
function of time is measured by release of radioactivity from
14C-labeled reconstituted collagen (see text for details).
Chart 1. Degradation of acid-extracted, proline-14C-labeled collagen
by BCE collagenase as a function of crude enzyme concentration is
measured by release of radioactivity from l4 C-labeled reconstituted
d
LÜ
300
collagen after 18 hr incubation (see text for details).
Collagenolytic Activities on Salt-extracted,
Pro line-'4 C-labeled
Guinea Pig Collagen
Release of Radioactivity. The highest collagenolytic activity
(21.8% lysis per mg protein) was found in a tumor excised
from the neck (Table 5), and the lowest activity (3.0%
lysis/mg protein) was found in a specimen taken from the neck
of another individual (Table 5). The highest activity of the
normal control skin with the same substrate was 0.7% lysis per
mg protein; the value was less than one-fourth of the least
active tumor. The difference of activities percentage of lysis
per mg protein) between tumors and the normal skin controls
was significant (p < 0.02).
Kinetic Studies. The linear increase of collagenolytic
activity with the increase of crude enzyme concentration
(Chart 4) and with the time of incubation (Chart 5) was
similar to that observed for the pooled crude enzyme on
acid-soluble rat collagen substrate (Charts 1 and 2). The
2554
<=>200
V
o
<
u
100
N
ÃœJ
5.0
70
SO
9.0
pH
Chart 3. Degradation of collagen by BCE crude collagenase as a
function of pH dependence is measured by release of radioactivity from
14C-labeled reconstituted collagen after 18 hr incubation (see text for
details).
CANCER RESEARCH VOL. 32
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Collagenolytic Activities in BCE
optimum pH for the enzyme action was between 7 and 8.5,
and the crude enzyme activity dropped rapidly and
significantly below and above this pH range (Chart 6).
Enzyme Inhibitors.
Inhibition
of crude enzyme was
observed with normal human whole serum at a dilution of
1:1000 (66.1%) (Chart 7), with 0.01 M EDTA (97.4%), and
0.01 M cysteine (74.6%) (Table 7). The mode of inhibition of
each inhibitor tested on this substrate was very similar to that
observed in the experiments with acid-extracted collagen
substrate labeled with proline-14 C (Table 4). Soybean trypsin
600r
Table 4
Effects of collagenase and trypsin inhibitors on acid-extracted,
proline-14 C-labeled rat skin collagen
UJ
4OO
(above
blank)530558514020024015430485255650%
inhibition90.484.273.261.954.397.418.98.079.7
BCE
completeWith
(1:10)With
serum
(1:50)With
serum
(1:250)With
serum
(1:500)With
serum
(1:1000)With
serum
M)With
EDTA (0.01
inhibitor(50
soybean trypsin
reactionmixture)With
Mgin 1 ml
Q_
O
>
H
>
200
inhibitor(lOUMgin
soybean trypsin
reactionmixture)Trypsin
1 ml
mlreaction
alone (50 Mgin 1
mixture)With
inhibitor(
soybean trypsin
reactionmixture)cpm"
100 Mgin 1 ml
100/jg 200pg
CRUDE
a Total radioactivity per incubation mixture was 4265 cpm.
0 Trypsin, 50 tig, lysed 6% of the substrate collagen.
400 ug
ENZYME
800,i'g
PROTEIN
Chart 4. As,the crude enzyme concentration increases, degradation
of salt-extracted, proline-'4 C-labeled collagen progresses linearly (cf.
Chart 1).
Table 5
BCE collagenolytic activities on salt-extracted, proline-14 C-labeled
guinea pig skin collagen and caseinolytic activities
gellysed
lysis/mgprotein21.88.97.56.33.73.23.0Caseinolyticactivity67.53
protein(mg/0.5
blank)73030013016018580100Collagen
(%)17.47.13.03.84.41.92.4%
ml)0.80.80.40.61.20.60.8cpm"solubilized(above
Specimen1234567LocationNeckNoseNeckNoseEarTempleNeckTissue
a Total radioactivity per incubation mixture was 4205 cpm.
b Expressed as fig,trypsin equivalence/mg crude enzyme protein.
Table 6
Normal skin collagenolytic activities on salt-extracted, proline-' 'C-labeled
guinea pig skin collagen and caseinolytic activities
gellysed
lysis/mgprotein0.70.60.40.3Caseinolyticactivity64.35.42.23.0
protein(mg/0.5
(%)1.51.01.61.4%
ml)2.21.84.54.7cpm°solubilized(above
blank)65406560Collagen
Specimen1234LocationCheekCheekCheekTempleTissue
Total radioactivity per incubation mixture was 4205 cpm.
b Expressed as Mgtrypsin equivalence/mg crude enzyme protein.
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2555
Y. Yamanishi, M. K. Dabbous, and K. Hashimoto
Collagenolytìc
Activities
on
Glycine-'4 C-IabeledGuinea Pig Collagen
600
Release of Radioactivity. Three BCE's tested on this
substrate released significantly higher radioactivities (Table 8)
than those of the normal skin incubated with the same
substrate (Table 9); namely, the collagenolytic activities of
these tumors were about 30 times higher than those of the
normal skin controls.
Kinetic studies and enzyme inhibitor studies were not
repeated on this substrate.
O
lu
CO
LU
ce
Salt-extracted,
40O
CL
O
Disc Electrophoresis
o
<
The control tropocollagen yielded a characteristic pattern
(Fig. 1). The fast-moving a chains (a2 and a1) were
200
LU
100
LU
-300
75I
23456
O
UJ
Vi
HOURS
Chart 5. As incubation time increases; degradation of salt-extracted,
proline-1 *C-labeled collagen progresses linearly (cf. Chart 2).
Z
O
-200
50-
m
X
-100
„¿25-1
600r
l/'500
'1000
'250
fc»
a.
o
l/_
J/
'50'10
UJ
cc
SERUM
400
CL
o
Table 7
Effects of collagenase and trypsin inhibitors on salt-extracted,
proline-1 "C-labeledguinea pigskin collagen
UJ 2OO
M
blank)BCE
LU
5.0
6.0
70
8.0
9.0
100
pH
6.
The
highest
degradation
of
salt-extracted,
Chart
proline-' 'C-labeled collagen occurs between pH's 7 and 8.5 (cf. Chart
3).
inhibitor (100 ¿ig)
did not inhibit the collagenolytic activity of
BCE crude enzyme (Table 7), whereas the collagenolytic
activity of trypsin on the same substrate was 81.9% inhibited
by soybean trypsin inhibitor (100 ßg)
(Table 7).
2556
DILUTION
Chart 7. Inhibition of BCE collagenolytic activity by serially diluted
normal human whole serum is measured by reduction of release of
radioactivity from ' 4C-labeled, salt-extracted collagen after 18 hr
incubation (cf. Chart 4). »,% inhibition; »,cpm release.
cpm"
(above
inhibition92.785.577.670.566.1
completeWith
(1:10)With
serum
(1:50)With
serum
(1:250)With
serum
(1:500)With
serum
(1:1000)With
serum
M)With
EDTA (0.01
trypsininhibitor
soybean
(100|ig)With
M)With
cysteine (0.01
M)Trypsin
cysteine (0.001
Mg)With
alone (50
trypsininhibitor
soybean
(100 Mg)3802555851101301038095375325b60%
a Total radioactivity per incubation mixture was 4205 cpm.
b Trypsin, 50 Mg,lysed 7.5% of the substrate collagen.
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Collagenolytic Activities in BCE
Table 8
BCE collagenolytic activities on salt-extracted, glycine-' *C-labeled
guinea pig skin collagen and caseinolytic activities
protein
(mg/0.5
ml)1.6
Specimen12
Scalp
NoseTissue
3LocationScalp
1.6
1.3cpm"
solubilized
(above
blank)1300
gel
lysed
(%)20.8
1295
900Collagen
20.7
14.5%
lysis/ mg
protein13.0
activity610.6
9.4
12.9
11.2Caseinolytic 7.1
" Total radioactivity per incubation mixture was 6265 cpm.
b Expressed as tig trypsin equivalence/mg crude enzyme protein.
Table 9
Normal skin collagenolytic activities on salt-extracted, glycine-1 ^-labeled
guinea pig skin collagen and caseinolytic activities
Specimen1
LegTissue
2LocationFace
protein
(mg/0.5
ml)3.2
1.5cpm"
solubilized
(aboveblank)80
gel
lysed
(%)1.2
35Collagen
0.5%
lysis/mg
protein0.3
activity62.5
0.4Caseinolytic 7.7
" Total radioactivity per incubation mixture was 6265 cpm.
b Expressed as Mgtrypsin equivalence/mg crude enzyme protein.
sequentially followed by the relatively slow-moving j3 chains
(ßi2 and ßii), the slow-moving 7-chains, and the higher
molecular components. The pattern produced by the reaction
products was different in that there appeared discrete sets of
new bands. Simultaneously, the density of all the bands seen
in the control (a2, Qt1', ßit, ßn, and 7) became diminished
A
(Fig. 1), indicating that the new bands were produced by the
degradation of tropocollagen molecules. Compared with the
bands seen in the control (Fig. 1), a fast-moving dense band
appeared in front of a2, representing a set of aA. The band
corresponding to that designated as a2 in the control
disappeared and the band corresponding to a1 became faint
B
ex
(Fig. 1). In the ßchain region, a new set of bands moving
faster than (312 appeared in front of 0i2 (Fig. 1). This new
band region seemed to represent j3A. A thin band of low density
appeared, lagging behind ßt\ ; the identity of this band was
obscure.
In
this
experiment,
small-molecular-weight
components which, if present, appear between aA and the
buffer front, were not present in any significant amount.
Fig. 1. Polyacrylamide disc electrophoretic patterns of control
tropocollagen after denaturation (A), and BCE-treated denatured
tropocollagen after a 24-hr incubation at 27°(B). Bands are labeled as
follows: a-, ßand 7- for single-, double- and triple-chain components,
respectively; aA and 0A, the modified a- and (3-components, respec
Viscosity Changes
tively.
The viscosity of acid-soluble tropocollagen was reduced as a
result of incubation with BCE crude enzyme. At 27°, a
specific viscosity also varied between specimens; including the
data obtained from specimens other than BCE I, II, and III, it
ranged between 37 and 60% of the original value after 18 to
24 hr incubation.
temperature well below that of collagen denaturation, the
specific viscosity of dilute tropocollagen solution at pH 7.6
was reduced by about 50 (BCE III), 40 (BCE II), and 30%
(BCE I). No decrease of viscosity was detected at pH 3.8 to
4.0. When the progress in specific viscosity of tropocollagen as
a function of time (the time at which samples were removed
from the incubation bath), an initial lag period was observed
(Chart 8). Its duration varied from one specimen to another
and was followed by a continuous loss of viscosity until a
limiting value was reached (Chart 8). The maximum loss of
Denaturation Temperature Change
At 27°no detectable change of negative optical rotation of
tropocollagen solutions was observed. This indicated that at
this temperature
the BCE crude enzyme did not cause
denaturation
of the tropocollagen macromolecule to any
significant extent. When the temperature of the reaction
NOVEMBER 1972
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2557
Y. Yamanishi, M. K. Dabbous, and K. Hashimoto
enzymes cannot be eliminated. By this reasoning, the
comparatively low collagenolytic activity of BCE reported
(23) may not necessarily mean absolutely low collagenase
activity.
On the other hand, the results obtained with more sensitive
methods used in the present investigation suggest that BCE
indeed contains collagenolytic enzyme or enzymes. In this
study different substrates and isotopes were used. The
advantage of salt-extracted collagen over acid-soluble collagen
was the rapid growing animals. Its specific activity was
therefore much higher than that extracted with acid, namely,
4205 versus 535 cpm/mg in proline-14C-labeled collagen and
6265 versus 870 cpm in glycine-14C-incorporated
collagen.
Glycine-14C was used because it is uniformly distributed in
the collagen molecule, whereas proline-14 C is more specific for
IO
09
08
t—
^
>^
07
*
06
05
458
TIME
10
(hr)
Chart 8. Viscometric determination of the collagenolytic activity of
BCE homogenates (1 ml/4 ml reaction mixture) at 27 ±0.1°.The
collagen concentration was 0.1% in 0.05 M Tris-0.04 M CaCl2 (pH 7.6).
The fractional change in specific viscosity (n spt¡r\spta) at each
measurement is plotted as a function of time. Curves I, II, and ///,
different BCE preparations described under "Materials and Methods."
TEMPERATURE
Chart 9. Melting curves of normal acid-soluble tropocollagen control
(•)and BCE-treated tropocollagen (•,»).Curves I and //, BCE
preparations described under "Materials and Methods." Denaturation
was carried out in 0.05% acetic acid (pH 3.8), and the temperature was
raised in discrete steps to obtain the melting curves.
mixture was raised in steps, a decrease of negative optical
rotation became apparent. The denaturation
temperature
midpoint, i.e., melting temperature (Tm), of BCE-treated
tropocollagen dropped to 35°,which compares to 40°for the
acid-soluble native tropocollagen (Chart 9).
DISCUSSION
In earlier studies, collagenolytic activity of various tumors
was assayed in a tissue culture system (23). Compared to other
tumors, the collagenolytic activity of BCE was not impressive
in such a study (23). In the tissue culture method, it was
difficult to demonstrate coUagenase activity alone, since
activities of trypsin-like enzymes and other proteolytic
2558
collagen. In spite of the use of various substrates and labels,
the results obtained were similar: (a) collagenolytic activities
of BCE's were always significantly higher than those of the
normal skin controls, (b) crude enzyme activities increased
linearly with respect to time and enzyme concentration, (c)
crude enzymes showed a pH optimum of 7 to 8.5, and (d) the
pattern of crude enzyme inhibition by the same inhibitors was
similar. Since accurate determination of the molecular weight
and a precise dilution of reconstituted collagen gel were
difficult, K^ determination
could not be done. We are
therefore unable to tell whether single or multiple enzymes are
involved.
Like BCE collagenolytic enzyme, collagenases of human
origin such as the skin, granulocytes, and rheumatoid
synovium have a neutral pH optimum, and are inhibited by
0.001 to 0.01 M EDTA (4, 5, 15,16) and 0.01 M cysteine (4,
15). In BCE, lymphocytes and plasma cells are seen in the
stroma but granulocytes are a distinct minority. Thus, it is
unlikely that the collagenolytic activities demonstrated in the
tumor homogenates included activity of granulocyte origin. As
demonstrated in this study, BCE collagenase is inhibited more
than 90% by normal human whole serum at a dilution of 1:10,
whereas human granulocyte collagenase is not inhibited at this
dilution (15). It is therefore presumed that the main source of
BCE collagenase is the tumor cells per se.
Disc electrophoresis of BCE homogenate and highly purified
calf skin tropocollagen mixture demonstrated new bands such
as j3A and aA, which represent cleaved molecules of
tropocollagen (1). Similar degradation of tropocollagen by
various mammalian collagenases has been reported previously
(1, 4, 13, 15). The possibility that proteolytic enzymes other
than collagenase were mainly responsible for the observed
degradation of tropocollagen should be ruled out. Available
information (2) indicates that at temperatures below the
denaturation
temperature
of
tropocollagen-proteolytic
enzymes other than collagenase do not change the main
structural features of tropocollagen. The action of proteolytic
enzymes
is restricted
to extrahelical
regions, called
telopeptides, where most of the inter- and intramolecular
cross-linkages are located (2); by the action of proteolytic
enzymes, more a chains are produced at the expense of j3and
7 chains and their aggregates (2). The action of BCE
homogenate, on the other hand, brought about scission of the
tropocollagen molecules beyond the telopeptide regions, and
CANCER RESEARCH VOL. 32
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Collagenolytic Activities in BCE
the quantity (density) of a chain bands in electrophoresis was
reduced. In addition, new discrete bands corresponding to aA
and |3A appeared in front of a and ß,indicating that the
enzymatic attack was on a susceptible region at a point
three-fourths of the length of the molecule from the A end (5,
6). This conclusion is supported by our electron microscopic
studies (12). In the electrophoretic pattern produced in the
present experiments, however, the band representing small
molecular fragments (aB or one-quarter length pieces) was not
found. Two possibilities exist to explain their absence. Under
the experimental conditions used (3.5 hr electrophoresis time)
their mobility exceeded the gel length, or they became
susceptible to proteolysis after scission and were digested
completely. Both possibilities seem to be valid because
electrophoresis under different conditions demonstrated the
presence of «B components
and electron microscopic
examination of ATP-reconstituted crystallites showed some
erosions from both ends of one-quarter fragments (12).
Viscometric study showed that at 27°the specific viscosity
was reduced by 30 to 50% with no significant decrease in
negative optical rotation. This suggests that collagenolytic
enzyme(s) in BCE homogenates produces a limited cleavage of
tropocollagen without detectable denaturation of the triple
helical structure of the molecule. The BCE homogenate used
in our experiments is only a crude preparation and may
contain components that affect the enzymatic activity. This
fact, together with the possibility that BCE collagenolytic
enzyme has physicochemical characteristics different from
those enzymes derived from other tissues, may explain some
observed differences between BCE enzyme and other tissue
collagenases. For example, there was a lag period observed
prior to the decrease in specific viscosity of tropocollagen
solutions, although the subsequent decrease progressed as a
function of time. In addition, the loss in specific viscosity
reached a limiting value (30 to 50% of the original).
The introduction
of cross-linkages in the ordered
tropocollagen helical structure was shown to increase the
denaturation temperature (20, 21). The observed decrease in
the melting temperature of BCE-treated collagen suggests that
the cleavage of tropocollagen by BCE enzyme decreased the
amount of stabilizing structures (such as cross-linkages in the
B-end region) in the molecule. Dresden and Gross (3) reported
that Tm of protease-treated tropocollagen is also 35°.Similar
Tm values were obtained when collagen was treated with
collagenases from other tissues (8, 13).
Some variation of collagenolytic activity from tumor to
tumor as observed in this study may be due to several factors
such as (a) type of the tumor (solid, cystic, adenoid, keratotic,
etc.); (b) stages of growth (young and active or old and
necrotic); (c) ratio of tumor parenchyma to stroma and,
among others, the amount of serum (serum inhibitor) that
contaminated the specimen at the time of operation and the
amount of keratin (cysteine) of the covering epidermis and/or
that contained in the tumor itself.
The significance of collagenase in BCE is obvious in relation
to the histogenesis and spreading of the tumor. BCE does not
metastasize but is locally very invasive, often encroaching into
cartilage and bone. Such a local destructive property is
apparently related to its collagenolytic enzymes. The presence
NOVEMBER 1972
of collagenolytic activity in squamous cell carcinoma, although
much less in activity than those found in BCE, raises a
question of whether or not all invasive tumors of epidermal or
mucous membrane
origin have quantitatively
elevated
collagenase or collagenases that are qualitatively different from
that of the normal skin. Further studies are in progress in our
laboratory to determine the identity or nonidentity of BCE
collagenase with the normal skin collagenase (4) and to see
whether invasiveness of various skin tumors correlate with
their level of collagenase activities.
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CANCER
RESEARCH
VOL. 32
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Effect of Collagenolytic Activity in Basal Cell Epithelioma of the
Skin on Reconstituted Collagen and Physical Properties and
Kinetics of the Crude Enzyme
Yuji Yamanishi, Mustafa K. Dabbous and Ken Hashimoto
Cancer Res 1972;32:2551-2560.
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