CIinica Chimica Acta, 203 (1991) 225-234 0 1991 Elsevier Science Publishers B.V. All rights
225 reserved
C009-8981/91/503.50
CCA 05138
Assay for type III collagenolytic activity in lung cancer tissue Masafumi Kawamura ‘, Ryoichi Kato ‘, Koji Kikuchi *, Koichi Kobayashi Tsuneo Ishihara I, Tetsuichi Shibata 2 and Seiichi Inayama ’
‘,
’ Department of Surgery and ’ Pharmaceutical Institute, School of Medicine, Keio Unictwity, Tokyo 160 (Japan) (Received
31 May 1991; revision
Key words: Type III collagen:
received
Collagenolytic
30 August
enzyme;
1991: accepted
Collagenase:
3 September
1991)
Fibril assay; Lung cancer
Summary We developed a method for measuring the activity of type III collagenolytic enzyme in lung cancer tissue, using as substrate, type III collagen purified from human placenta. In this method [3H]propionate is used for labeling type III collagen, with bacterial collagenase used for making the standard curve. It, therefore, becomes possible to compare type III collagenolytic activity with those of other collagen subtypes (types I and IV). As this method is a fibril assay it is not susceptible to trypsin or other proteases. The average type III collagenolytic enzyme activity was higher in squamous cell carcinoma than in adenocarcinoma, while that of lung cancer tissue exceeded that of normal lung tissue. The activity of type III collagenase increased with the progression from one disease stage to the next.
Introduction The main protein constituent of mammalian connective tissue is collagen, of which there are reportedly more than 10 subtypes. In human lung tissue, for example, the parenchyma is constructed of types I and III, while the basement membrane of the alveoli and capillaries is mainly constructed of type IV (11.
Correspondence to: Masafumi Kawamura, sity, 35 Shinanomachi, Shinjuku-ku, Tokyo
Department 160, Japan.
of Surgery,
School
of Medicine,
Keio Univer-
226
Type I collagen is not easily degraded by common proteases except for its specific metalloprotease called collagenase. It has been determined recently that collagenase is not necessarily specific for other collagen subtypes [2,3]. For example, type III collagen is cleaved by some serin proteases and/or SH-proteases as well as by collagenase [4-61. On the other hand, collagen contains a characteristically larger amount of hydroxyproline (Hyp) than do other proteins, such as elastin. We previously measured the amount of Hyp in the urine of patients with lung cancer by the KISO method [7] and showed that the amount excreted was significantly higher than normal. This observation suggests that collagen metabolism is accelerated in lung cancer patients. To evaluate the collagen subtypes involved in the acceleration of collagen degradation in the lung tissue of patients with lung cancer, we established a method for measuring the activity of the enzyme that cleaved type III collagen (type III collagenolytic protease). We then determined the activity of this enzyme in human lung cancers. The fibril assay for type III collagenolytic enzyme activity using i3H]type III collagen as the substrate as described below was first used as a highly convenient method for the assay of type I collagenase activity. Bacterial collagenase (EC 3.4.24.31, which can cleave all subtypes of collagen, is used as the standard in assays for determining collagenolytic activities in vertebrates. On the other hand, type III collagen is labeled with [N-propionate-2,33H]succinimidyl propionate, as are other labeled subtypes (types I and IV) of collagen. Thus, the measured activity of type III collagenolytic enzymes should be compared with that determined by using collagenolytic enzymes against other collagen subtypes. Methods Preparation of 3H-type III collagen Purification of type III collagen. Pure type III collagen was prepared from human placenta according to the method of Glanville et al. [8]. Human placental tissues (550 g), stored in a deep freeze just after delivery, were used. The umbilical cord, chorion and amnion membrane were removed. After the blood was washed off, the chorionic plate with its attached villi was minced, washed in 0.4 M sodium acetate solution, and digested with 1 g pepsin (Boehringer Mannheim) to provide a collagen solution. This solution was purified chromatographically through the column of DEAEcellulose (DE-52, Whatmann). After dialysis the collagens in the 0.04 M acetate buffer (pH 4.8 2 M urea) were made to adhere to CM-cellulose (CM-52 Whatmann). Type III was separated from type IV collagen through the CM-cellulose columns (CM-52, Whatmann) with a linear gradient of NaCl concentration of O-O.2 M. After dialysis against 0.05 M acetic acid followed by lyophilization, a white cottonlike substance (280 mg) was obtained as a pure collagen.
227
Amino acid analysis. The purified coUagen (1 mg) was hydrolyzed in 6 N HCI under N, at 110°C for 24 h and dried to provide a hydrolysate. Analysis of the collagen hydrolysate was performed with a Beckmann System 6300 Amino Acid Analyzer (Beckmann, Fullerton, CA). Unlike in other published data [9], this collagen fiber was identified as type III collagen, because it included cysteine and less hydroxylysine than type IV (Table I). Polyacrylamide gel electrophoresis. This purified collagen, standard type I, and type II collagen in acetic acid buffer were electrophoresed by using a 4.5% polyactylamide disc gel containing sodium dodecyl sulfate (SDS) and stained with Coomassie brilliant blue. Standard human type III collagen was not commercially available. The difference between standard type I and the purified type III collagen is seen on the B-band (Fig. 1). Labeling of collagen. Type III collagen (7.5 mg) was dried in a vacuum and labeled with 10 mCi of [ N-propionate-2,3-JH]succinimidyl propionate (56 Ci/mmol) after removal of the solvent (hexan and ethyl acetate) under a stream of
TABLE
I
Analysis a of amino acids in extracted Extracted
type 111 collagen
collagen
a,Wl)
b
a,(IV)
h
a#V)
HYP
124
125
122
110
ASP
47
42
45
49
THR
16
13
19
30
SER
37
39
38
30
GLU
76
71
78
65
PRO
104
107
85
73
GLY
335
350
334
324
ALA
98
96
30
4-l
I
2
0
2
16
14
33
27
CYS/Z VAL MET
7
8
15
14
ILE
12
13
32
38 56
LEU
24
22
52
TYR
2
3
5
7
PHE
9
8
27
36
HYL
11
5
50
36
LYS
24
30
6
7
HIS
6
6
6
6
50
46
22
42
999
999
ARG
999 ’ Analysis performed h Data extracted
with Beckmann
from Methods
System 6300 Amino
in Enzymology
191.
acid analyzer.
b
228
A
Fig.
1. Sample
of collagen
analyzed
A
using SDS-PAGE:
A,
purified
type
III;
B, standard
type
I;
C, standard type II.
dry N, gas at 50 o C in a 20 ml vial. Five milliliters of dried l+dioxane were added to the vial. Following vigorous shaking for 12 h at 15°C and centrifugation (lo4 x g, 15 min), the remaining fibrous precipitate was dissolved in 0.5 mol/l acetic acid and was then dialyzed repeatedly against 5 mmol/l acetic acid. The characteristics of [3H]labeled type III collagen are: (i) concentration, 0.5 mg/ml; (ii) radioactivity, 42.6 pCi/ml; and (iii) specific activity, 89.6 pCi/mg collagen. Assay for determining type III collagenolytic enzyme activity in lung cancer tissue
There are several steps in assaying enzyme activity in lung cancer tissue using [3H]labeled type III collagen as described below. Preincubation (collagen fibril formation). In acetic acid solution the natural collagen fiber tends to be a single chain which is susceptible to protease. The single chain was regenerated to a triple chain (the fibril) during preincubation in pH 7.6 Tris-HCI buffer (50 mmol/l Tris, 5 mmol/l CaCl,, 0.2 mol/l NaCI) for 90 min at 37°C. The fibril was separated out as a precipitate by centrifugation at 15 000 X g for 10 min from the single chain or degenerated collagen in solution. Optimal conditions for enzyme activity. Incubation temperature: To obtain the most appropriate temperature for degradation, the incubation temperature was selected from two conditions: 37°C and 21°C. The reaction occurred at 37°C but not at 21°C. Thus the former was chosen as the temperature for this reaction (Fig. 2). Incubation time: The optimal incubation time was determined from five .tests at 37°C.
229
I. O13
6
16(h)Resction
9
time
Fig. 2. Mixtures of substrate and tumor tissue were incubated for 16 h at 37°C and 21°C. respectively. No digestion was observed at 21°C.
The gap between measured activities of the homogenates of the two individual cancers was greater after incubation for 6 h than for 16 h, but there was a large difference after 6 h. By contrast, the difference became smaller after 16 h of incubation (Fig. 31. According to these experiments, the optimal conditions for measuring the enzyme activity were as follows: (1) an aliquot of acetic acid solution of 3H-type III collagen (0.02 pCi/50 ~1) was incubated in pH 7.6 Tris-HCI buffer (50 mmol/l Tris, 5 mmol/l CaCl,, 0.2 mol/l NaCI) for 90 min at 37°C. (2) About 10 mg wet weight of tumor tissue was homogenized in the Tris buffer (pH 7.6, 2 ml) and diluted lo-25 times to a suitable concentration for this assay; this homogenate was added to the incubation tube with regenerated fibril collagen. (3) The tube was capped and the contents were mixed gently and incubated at 37°C for 16 h. (4) The reaction tube was placed on ice for 10 min just after incubation, and reaction tube was centrifuged at 15 000 x R for 10 min. (5) Fifty microliters or 100 ~1 of supernatant including the cleaved collagen was placed in the scintillation vial
DPM
. 5wo
0
Tumor
1
Tumor
2
k--
I 16(h) Reaction time ‘13 6 9 Fig. 3. Period of incubation and collagenase activities of hvo lung tumors. After incubation for 16 h at 37”C, digestion reached a plateau.
230
with scintillator (4 ml), and the amount of degenerated mined by means of a liquid scintillation counter.
L3H]collagen was deter-
Standard curve using bacterial collagenase. Purified collagenase from Clostridium histolyticum (EC 3.4.24.3 - bacterial collagenase type II@, Sigma, St. Louis,
MO) was dissolved in Tris-HCl buffer to provide the standard solution concentrated at 1 g/l, which was diluted to various concentrations (10°-10-5 g/l) to create the standard curve. The constructed standard curve had a good lineation (r = 0.9942). An activity unit of collagenolytic
enzyme per g of wet tissue was defined as the weight of bacterial collagenase (EC 3.4.24.3) having the same collagenolytic activity (mg bacterial collagenase/g wet tissue weight). Materials Twenty-two primary lung cancer patients (aged 40-75 yr; four females and 18 males) were examined. All had undergone operations. Neither chemotherapy nor radiotherapy was performed before the operation. The activity of type III collagenolytic enzyme was determined in the primary lung cancer tissue from 22 patients and in the normal lung tissue obtained from the same resected lobe of 4 patients without another diffuse pulmonary disease and sufficiently distant from cancer tissue. The tissue was frozen at - 80°C immediately after the operation and was assayed by the method described above. Diagnoses included 12 cases of squamous cell carcinoma and 10 of adenocarcinoma. Concerning the disease stage, 6 were of postsurgical stage I, 9 of stage III A, and 7 of stage IV due to pulmonary metastasis.
mg bac~collagenase/g~tissu~
I
0369 0293
i 150 I
Fig. 4. Type III collagenolytic enzyme activity in lung tissue from 22 patients with cancer (12 squamous cell carcinoma and 10 adenocarcinoma) and in normal lung tissue from 4 patients without another diffuse pulmonary disease.
231
Results
The average type III collagenolytic enzyme activity values in squamous cell carcinoma, adenocarcinoma, and normal lung tissue were 91.8, 24.0, and 5.5 mg of bacterial collagenase/g of wet tissue weight, respectively. The differences between the collagenolytic enzyme activity in the carcinomas vs. normal lung tissue, and between the squamous cell carcinoma vs. adenocarcinoma were statistically significant (Fig. 4). The average type III collagenolytic enzyme activity values of stage I, III A, and IV were 21.5, 68.7, and 84.7 mg of bacterial collagenase/g of wet tissue weight, respectively. Thus, type III collagenolytic enzyme activity increased according to disease progression (Fig. 5). Discussion
The ratio of the collagen subtypes in the lung parenchyma has not been previously reported. As types II and IV belonging to group 2 collagen are distributed in the cartilage and basement membrane, respectively, their content in the lung parenchyma was thought to be negligible. In contrast, types I and III contribute so much to the lung parenchyma that the metabolism of collagen in the lung is considered to depend mainly on the activity of the proteases that degrade type I and III collagens. These proteases also seem to be related to the biological behavior of lung cancer; for example, tendency toward local invasion, tumor necrosis or metastasis. Type I collagen is not easily digested by the common proteases except for the specific metalloprotease, type I collagenase [2,31. On the other hand, it has been
mg bac,collagenase/g.tissue l369
!
0293
150.
8
I oo-
50 l SC! 0 Ad
l
I
01
0
. -
a
I
S'RP",' I StagemA n-9 (2s
Fig. 5. Type
III collagenolytic enzyme postsurgical
(Z
8
StageIY n=l (zz:
activity and disease stage of 22 patients stage I, III A, and IV lung cancer.
with
lung
cancer:
232
found that type III collagen is cleaved not only by type III collagenase but also by some other proteases such as elastase [lO,ll]. Therefore, it is believed important to measure the activities not only of the protease that cleaves type I collagen (type I collagenase), but also of those that cleave type III (type III collagenase and other proteases). A method for measuring type III collagenolytic enzyme activity has been reported in a few.publications. Christner et al. [12] measured the activity of type III collagenolytic enzyme in bronchoalveolar lavage fluid using iZI-type III collagen as the substrate. This method is cumbersome, especially when many samples are involved. in contrast, one can assay more than 1000 samples at one time with our method. Birkedal-Hansen [13] also reported a method using the collagen fibril and indicated that it was preferable to the solution assay for measuring the activity of type III collagenolytic enzyme because the latter was susceptible to trypsin and/or other proteases. However, the fibril assay used was frequently affected by contamination. In contrast, our method is simple to perform and is thus useful in evaluating large numbers of samples at one time. In this assay, type III collagen is labeled with [3Hlpropionate instead of E3H]acetic anhydride. Although the latter is frequently used to label type I collagen, it’is susceptible to the acetyl-transferase contained in tissue since it can be cleaved at the acetamide bond. The former is commonly used to label the other collagen subtypes (types I and IV>. Thus it becomes possible to compare the coIlagenolytic activities of the different subtypes. The standard curve was constructed by using the purified enzyme from Clostridium histolyticum (EC 3.4.24.3 - bacterial collagenase type II@‘, Sigma) which can cleave collagen at Xj-Gly in the repetition of Gly-Pro-Xj-Gly-Pro-Xj, regardless of collagen subtype. This enzyme is useful in this assay for comparing type III cohagenolytic activity with that of other subtypes (types I and IV). The enzyme activity of the samples was represented as the weight of bacterial collagenase to make it possible to standardize the enzyme activity in cleaving not only type III, but also type I and IV collagen. These results demonstrate that the average type III collagenolytic‘ enzyme activity in pulmonary carcinoma is significantly higher than that of normal lung tissue. This suggests that acceIeration of collagen metabolism in patients with pulmonary carcinomas is related to the high average type III ~llagenolytic enzyme activity in the pulmonary tumor tissue. A significant difference in enzyme activity was found between the squamous cell carcinomas and adenocarcinomas. In general, these malignancies differ in respect to their biological behavior in that squamous cell carcinomas are apt to invade locally, while adenocarcinomas are likely to metastasize distantly. The difference in type III collagenolytic enzyme activity between these carcinomas might be related to their tendency toward local invasion. References 1 Miller U, Gay S. The Colfagens: An overview and update. Methods Enzymol 1987;144:3-41. 2 Harris ED, Vater CA, Jr. Vertebrate collagenases. Methods Enzymoi 1982;82:423-439.
233 3 Goldberg GI, Wilhelm SM, KronBerger A, Batter EA, Grant GA, Eizen AZ. Human fibroblast collagenase. J Biol Chem 1986;261:6600-6605. 4 Mainardi CL, Hasty DL, Seyer JM, Kang AH. Specific cleavage of human type III collagen by polymorphonuclear leukocyte elastase. J Biol Chem 1980;255:12006-12010. 5 Macartney HW, Tschesche H. Latent and active human polymorphonuclear collagenases - isolation, purification and characterization. Eur J Biochem 1983;130:71-78. 6 Hotwitz AL, Hance AJ, Crystal RG. Granulocyte collagenase: selective digestion of type I relative to type III collagen. Proc Natl Acad Sci USA 1977;74:897-901. 7 Inayama S, Shibata T, Ohtsuki J, Saito S. A new microanalytical method for determination of hydroxyproline in connective tissues. Keio J Med 1978;27:43-46. 8 Glanville RW, Rauter A, Fietzek PP. Isolation and characterization of a native placental basement membrane collagen and its component a chains. Eur J Biochem 1979;95:383-389. 9 Miller EJ, Gay S. Collagen: An overview. Methods Enzymol 1982;182:20. 10 Janoff A. Elastase in tissue injury. Annu Rev Med 1985;36:207-216. 11 Weissmann G, Smolen JE, Korchak HM. Release of inflammatory mediators from stimulated neutrophils. N Engl J Med 1980;303:27-34. 12 Christner P, Fein A, Goldberg S, Lippmann M, Abrams W, Weinbaum G. Collagenase in the lower respiratory tract of patients with adult respiratory distress syndrome. Am Rev Respir Dis 1985;131:690-695. 13 Birkedal-Hansen H. Catabolism and turnover of collagens: collagenases. Methods Enzymol 1987;144:140-171.
225 reserved
C009-8981/91/503.50
CCA 05138
Assay for type III collagenolytic activity in lung cancer tissue Masafumi Kawamura ‘, Ryoichi Kato ‘, Koji Kikuchi *, Koichi Kobayashi Tsuneo Ishihara I, Tetsuichi Shibata 2 and Seiichi Inayama ’
‘,
’ Department of Surgery and ’ Pharmaceutical Institute, School of Medicine, Keio Unictwity, Tokyo 160 (Japan) (Received
31 May 1991; revision
Key words: Type III collagen:
received
Collagenolytic
30 August
enzyme;
1991: accepted
Collagenase:
3 September
1991)
Fibril assay; Lung cancer
Summary We developed a method for measuring the activity of type III collagenolytic enzyme in lung cancer tissue, using as substrate, type III collagen purified from human placenta. In this method [3H]propionate is used for labeling type III collagen, with bacterial collagenase used for making the standard curve. It, therefore, becomes possible to compare type III collagenolytic activity with those of other collagen subtypes (types I and IV). As this method is a fibril assay it is not susceptible to trypsin or other proteases. The average type III collagenolytic enzyme activity was higher in squamous cell carcinoma than in adenocarcinoma, while that of lung cancer tissue exceeded that of normal lung tissue. The activity of type III collagenase increased with the progression from one disease stage to the next.
Introduction The main protein constituent of mammalian connective tissue is collagen, of which there are reportedly more than 10 subtypes. In human lung tissue, for example, the parenchyma is constructed of types I and III, while the basement membrane of the alveoli and capillaries is mainly constructed of type IV (11.
Correspondence to: Masafumi Kawamura, sity, 35 Shinanomachi, Shinjuku-ku, Tokyo
Department 160, Japan.
of Surgery,
School
of Medicine,
Keio Univer-
226
Type I collagen is not easily degraded by common proteases except for its specific metalloprotease called collagenase. It has been determined recently that collagenase is not necessarily specific for other collagen subtypes [2,3]. For example, type III collagen is cleaved by some serin proteases and/or SH-proteases as well as by collagenase [4-61. On the other hand, collagen contains a characteristically larger amount of hydroxyproline (Hyp) than do other proteins, such as elastin. We previously measured the amount of Hyp in the urine of patients with lung cancer by the KISO method [7] and showed that the amount excreted was significantly higher than normal. This observation suggests that collagen metabolism is accelerated in lung cancer patients. To evaluate the collagen subtypes involved in the acceleration of collagen degradation in the lung tissue of patients with lung cancer, we established a method for measuring the activity of the enzyme that cleaved type III collagen (type III collagenolytic protease). We then determined the activity of this enzyme in human lung cancers. The fibril assay for type III collagenolytic enzyme activity using i3H]type III collagen as the substrate as described below was first used as a highly convenient method for the assay of type I collagenase activity. Bacterial collagenase (EC 3.4.24.31, which can cleave all subtypes of collagen, is used as the standard in assays for determining collagenolytic activities in vertebrates. On the other hand, type III collagen is labeled with [N-propionate-2,33H]succinimidyl propionate, as are other labeled subtypes (types I and IV) of collagen. Thus, the measured activity of type III collagenolytic enzymes should be compared with that determined by using collagenolytic enzymes against other collagen subtypes. Methods Preparation of 3H-type III collagen Purification of type III collagen. Pure type III collagen was prepared from human placenta according to the method of Glanville et al. [8]. Human placental tissues (550 g), stored in a deep freeze just after delivery, were used. The umbilical cord, chorion and amnion membrane were removed. After the blood was washed off, the chorionic plate with its attached villi was minced, washed in 0.4 M sodium acetate solution, and digested with 1 g pepsin (Boehringer Mannheim) to provide a collagen solution. This solution was purified chromatographically through the column of DEAEcellulose (DE-52, Whatmann). After dialysis the collagens in the 0.04 M acetate buffer (pH 4.8 2 M urea) were made to adhere to CM-cellulose (CM-52 Whatmann). Type III was separated from type IV collagen through the CM-cellulose columns (CM-52, Whatmann) with a linear gradient of NaCl concentration of O-O.2 M. After dialysis against 0.05 M acetic acid followed by lyophilization, a white cottonlike substance (280 mg) was obtained as a pure collagen.
227
Amino acid analysis. The purified coUagen (1 mg) was hydrolyzed in 6 N HCI under N, at 110°C for 24 h and dried to provide a hydrolysate. Analysis of the collagen hydrolysate was performed with a Beckmann System 6300 Amino Acid Analyzer (Beckmann, Fullerton, CA). Unlike in other published data [9], this collagen fiber was identified as type III collagen, because it included cysteine and less hydroxylysine than type IV (Table I). Polyacrylamide gel electrophoresis. This purified collagen, standard type I, and type II collagen in acetic acid buffer were electrophoresed by using a 4.5% polyactylamide disc gel containing sodium dodecyl sulfate (SDS) and stained with Coomassie brilliant blue. Standard human type III collagen was not commercially available. The difference between standard type I and the purified type III collagen is seen on the B-band (Fig. 1). Labeling of collagen. Type III collagen (7.5 mg) was dried in a vacuum and labeled with 10 mCi of [ N-propionate-2,3-JH]succinimidyl propionate (56 Ci/mmol) after removal of the solvent (hexan and ethyl acetate) under a stream of
TABLE
I
Analysis a of amino acids in extracted Extracted
type 111 collagen
collagen
a,Wl)
b
a,(IV)
h
a#V)
HYP
124
125
122
110
ASP
47
42
45
49
THR
16
13
19
30
SER
37
39
38
30
GLU
76
71
78
65
PRO
104
107
85
73
GLY
335
350
334
324
ALA
98
96
30
4-l
I
2
0
2
16
14
33
27
CYS/Z VAL MET
7
8
15
14
ILE
12
13
32
38 56
LEU
24
22
52
TYR
2
3
5
7
PHE
9
8
27
36
HYL
11
5
50
36
LYS
24
30
6
7
HIS
6
6
6
6
50
46
22
42
999
999
ARG
999 ’ Analysis performed h Data extracted
with Beckmann
from Methods
System 6300 Amino
in Enzymology
191.
acid analyzer.
b
228
A
Fig.
1. Sample
of collagen
analyzed
A
using SDS-PAGE:
A,
purified
type
III;
B, standard
type
I;
C, standard type II.
dry N, gas at 50 o C in a 20 ml vial. Five milliliters of dried l+dioxane were added to the vial. Following vigorous shaking for 12 h at 15°C and centrifugation (lo4 x g, 15 min), the remaining fibrous precipitate was dissolved in 0.5 mol/l acetic acid and was then dialyzed repeatedly against 5 mmol/l acetic acid. The characteristics of [3H]labeled type III collagen are: (i) concentration, 0.5 mg/ml; (ii) radioactivity, 42.6 pCi/ml; and (iii) specific activity, 89.6 pCi/mg collagen. Assay for determining type III collagenolytic enzyme activity in lung cancer tissue
There are several steps in assaying enzyme activity in lung cancer tissue using [3H]labeled type III collagen as described below. Preincubation (collagen fibril formation). In acetic acid solution the natural collagen fiber tends to be a single chain which is susceptible to protease. The single chain was regenerated to a triple chain (the fibril) during preincubation in pH 7.6 Tris-HCI buffer (50 mmol/l Tris, 5 mmol/l CaCl,, 0.2 mol/l NaCI) for 90 min at 37°C. The fibril was separated out as a precipitate by centrifugation at 15 000 X g for 10 min from the single chain or degenerated collagen in solution. Optimal conditions for enzyme activity. Incubation temperature: To obtain the most appropriate temperature for degradation, the incubation temperature was selected from two conditions: 37°C and 21°C. The reaction occurred at 37°C but not at 21°C. Thus the former was chosen as the temperature for this reaction (Fig. 2). Incubation time: The optimal incubation time was determined from five .tests at 37°C.
229
I. O13
6
16(h)Resction
9
time
Fig. 2. Mixtures of substrate and tumor tissue were incubated for 16 h at 37°C and 21°C. respectively. No digestion was observed at 21°C.
The gap between measured activities of the homogenates of the two individual cancers was greater after incubation for 6 h than for 16 h, but there was a large difference after 6 h. By contrast, the difference became smaller after 16 h of incubation (Fig. 31. According to these experiments, the optimal conditions for measuring the enzyme activity were as follows: (1) an aliquot of acetic acid solution of 3H-type III collagen (0.02 pCi/50 ~1) was incubated in pH 7.6 Tris-HCI buffer (50 mmol/l Tris, 5 mmol/l CaCl,, 0.2 mol/l NaCI) for 90 min at 37°C. (2) About 10 mg wet weight of tumor tissue was homogenized in the Tris buffer (pH 7.6, 2 ml) and diluted lo-25 times to a suitable concentration for this assay; this homogenate was added to the incubation tube with regenerated fibril collagen. (3) The tube was capped and the contents were mixed gently and incubated at 37°C for 16 h. (4) The reaction tube was placed on ice for 10 min just after incubation, and reaction tube was centrifuged at 15 000 x R for 10 min. (5) Fifty microliters or 100 ~1 of supernatant including the cleaved collagen was placed in the scintillation vial
DPM
. 5wo
0
Tumor
1
Tumor
2
k--
I 16(h) Reaction time ‘13 6 9 Fig. 3. Period of incubation and collagenase activities of hvo lung tumors. After incubation for 16 h at 37”C, digestion reached a plateau.
230
with scintillator (4 ml), and the amount of degenerated mined by means of a liquid scintillation counter.
L3H]collagen was deter-
Standard curve using bacterial collagenase. Purified collagenase from Clostridium histolyticum (EC 3.4.24.3 - bacterial collagenase type II@, Sigma, St. Louis,
MO) was dissolved in Tris-HCl buffer to provide the standard solution concentrated at 1 g/l, which was diluted to various concentrations (10°-10-5 g/l) to create the standard curve. The constructed standard curve had a good lineation (r = 0.9942). An activity unit of collagenolytic
enzyme per g of wet tissue was defined as the weight of bacterial collagenase (EC 3.4.24.3) having the same collagenolytic activity (mg bacterial collagenase/g wet tissue weight). Materials Twenty-two primary lung cancer patients (aged 40-75 yr; four females and 18 males) were examined. All had undergone operations. Neither chemotherapy nor radiotherapy was performed before the operation. The activity of type III collagenolytic enzyme was determined in the primary lung cancer tissue from 22 patients and in the normal lung tissue obtained from the same resected lobe of 4 patients without another diffuse pulmonary disease and sufficiently distant from cancer tissue. The tissue was frozen at - 80°C immediately after the operation and was assayed by the method described above. Diagnoses included 12 cases of squamous cell carcinoma and 10 of adenocarcinoma. Concerning the disease stage, 6 were of postsurgical stage I, 9 of stage III A, and 7 of stage IV due to pulmonary metastasis.
mg bac~collagenase/g~tissu~
I
0369 0293
i 150 I
Fig. 4. Type III collagenolytic enzyme activity in lung tissue from 22 patients with cancer (12 squamous cell carcinoma and 10 adenocarcinoma) and in normal lung tissue from 4 patients without another diffuse pulmonary disease.
231
Results
The average type III collagenolytic enzyme activity values in squamous cell carcinoma, adenocarcinoma, and normal lung tissue were 91.8, 24.0, and 5.5 mg of bacterial collagenase/g of wet tissue weight, respectively. The differences between the collagenolytic enzyme activity in the carcinomas vs. normal lung tissue, and between the squamous cell carcinoma vs. adenocarcinoma were statistically significant (Fig. 4). The average type III collagenolytic enzyme activity values of stage I, III A, and IV were 21.5, 68.7, and 84.7 mg of bacterial collagenase/g of wet tissue weight, respectively. Thus, type III collagenolytic enzyme activity increased according to disease progression (Fig. 5). Discussion
The ratio of the collagen subtypes in the lung parenchyma has not been previously reported. As types II and IV belonging to group 2 collagen are distributed in the cartilage and basement membrane, respectively, their content in the lung parenchyma was thought to be negligible. In contrast, types I and III contribute so much to the lung parenchyma that the metabolism of collagen in the lung is considered to depend mainly on the activity of the proteases that degrade type I and III collagens. These proteases also seem to be related to the biological behavior of lung cancer; for example, tendency toward local invasion, tumor necrosis or metastasis. Type I collagen is not easily digested by the common proteases except for the specific metalloprotease, type I collagenase [2,31. On the other hand, it has been
mg bac,collagenase/g.tissue l369
!
0293
150.
8
I oo-
50 l SC! 0 Ad
l
I
01
0
. -
a
I
S'RP",' I StagemA n-9 (2s
Fig. 5. Type
III collagenolytic enzyme postsurgical
(Z
8
StageIY n=l (zz:
activity and disease stage of 22 patients stage I, III A, and IV lung cancer.
with
lung
cancer:
232
found that type III collagen is cleaved not only by type III collagenase but also by some other proteases such as elastase [lO,ll]. Therefore, it is believed important to measure the activities not only of the protease that cleaves type I collagen (type I collagenase), but also of those that cleave type III (type III collagenase and other proteases). A method for measuring type III collagenolytic enzyme activity has been reported in a few.publications. Christner et al. [12] measured the activity of type III collagenolytic enzyme in bronchoalveolar lavage fluid using iZI-type III collagen as the substrate. This method is cumbersome, especially when many samples are involved. in contrast, one can assay more than 1000 samples at one time with our method. Birkedal-Hansen [13] also reported a method using the collagen fibril and indicated that it was preferable to the solution assay for measuring the activity of type III collagenolytic enzyme because the latter was susceptible to trypsin and/or other proteases. However, the fibril assay used was frequently affected by contamination. In contrast, our method is simple to perform and is thus useful in evaluating large numbers of samples at one time. In this assay, type III collagen is labeled with [3Hlpropionate instead of E3H]acetic anhydride. Although the latter is frequently used to label type I collagen, it’is susceptible to the acetyl-transferase contained in tissue since it can be cleaved at the acetamide bond. The former is commonly used to label the other collagen subtypes (types I and IV>. Thus it becomes possible to compare the coIlagenolytic activities of the different subtypes. The standard curve was constructed by using the purified enzyme from Clostridium histolyticum (EC 3.4.24.3 - bacterial collagenase type II@‘, Sigma) which can cleave collagen at Xj-Gly in the repetition of Gly-Pro-Xj-Gly-Pro-Xj, regardless of collagen subtype. This enzyme is useful in this assay for comparing type III cohagenolytic activity with that of other subtypes (types I and IV). The enzyme activity of the samples was represented as the weight of bacterial collagenase to make it possible to standardize the enzyme activity in cleaving not only type III, but also type I and IV collagen. These results demonstrate that the average type III collagenolytic‘ enzyme activity in pulmonary carcinoma is significantly higher than that of normal lung tissue. This suggests that acceIeration of collagen metabolism in patients with pulmonary carcinomas is related to the high average type III ~llagenolytic enzyme activity in the pulmonary tumor tissue. A significant difference in enzyme activity was found between the squamous cell carcinomas and adenocarcinomas. In general, these malignancies differ in respect to their biological behavior in that squamous cell carcinomas are apt to invade locally, while adenocarcinomas are likely to metastasize distantly. The difference in type III collagenolytic enzyme activity between these carcinomas might be related to their tendency toward local invasion. References 1 Miller U, Gay S. The Colfagens: An overview and update. Methods Enzymol 1987;144:3-41. 2 Harris ED, Vater CA, Jr. Vertebrate collagenases. Methods Enzymoi 1982;82:423-439.
233 3 Goldberg GI, Wilhelm SM, KronBerger A, Batter EA, Grant GA, Eizen AZ. Human fibroblast collagenase. J Biol Chem 1986;261:6600-6605. 4 Mainardi CL, Hasty DL, Seyer JM, Kang AH. Specific cleavage of human type III collagen by polymorphonuclear leukocyte elastase. J Biol Chem 1980;255:12006-12010. 5 Macartney HW, Tschesche H. Latent and active human polymorphonuclear collagenases - isolation, purification and characterization. Eur J Biochem 1983;130:71-78. 6 Hotwitz AL, Hance AJ, Crystal RG. Granulocyte collagenase: selective digestion of type I relative to type III collagen. Proc Natl Acad Sci USA 1977;74:897-901. 7 Inayama S, Shibata T, Ohtsuki J, Saito S. A new microanalytical method for determination of hydroxyproline in connective tissues. Keio J Med 1978;27:43-46. 8 Glanville RW, Rauter A, Fietzek PP. Isolation and characterization of a native placental basement membrane collagen and its component a chains. Eur J Biochem 1979;95:383-389. 9 Miller EJ, Gay S. Collagen: An overview. Methods Enzymol 1982;182:20. 10 Janoff A. Elastase in tissue injury. Annu Rev Med 1985;36:207-216. 11 Weissmann G, Smolen JE, Korchak HM. Release of inflammatory mediators from stimulated neutrophils. N Engl J Med 1980;303:27-34. 12 Christner P, Fein A, Goldberg S, Lippmann M, Abrams W, Weinbaum G. Collagenase in the lower respiratory tract of patients with adult respiratory distress syndrome. Am Rev Respir Dis 1985;131:690-695. 13 Birkedal-Hansen H. Catabolism and turnover of collagens: collagenases. Methods Enzymol 1987;144:140-171.
