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Activation of human breast carcinoma collagenase through plasminogen activator
Life Sciences, Vol. 26, pp. 1223-1231 Printed in the U.S.A.
Pergamon Press
ACTIVATION OF HUMAN BREAST CARCINOMA COLLAGENASE THROUGH PLASMINOGEN ACTIVATOR Meera ParanJpe,* Linda Engel, + Nathanlel Young, + and Lance A. Liotta* Laboratory of Pathophyslology,* Laboratory of Pathology, + and Cell Biology Section, ° National Cancer Institute, Natlonal Institutes of Health, Bethesda, Maryland 20205 (Received In final form February Ii, 1980) SUMMARY L a t e n t c o l l a g e n a s e a c t i v i t y was d e t e c t e d i n t h e media o f a w e l l - c h a r a c t e r i z e d l i n e o f human b r e a s t c a r c i n o m a c e l l s m a i n t a i n e d f o r o v e r two y e a r s in culture. The media a l s o c o n t a i n e d s u f f i c i e n t p l a s m i n o g e n a c t i v a t o r t o convert extrinsically added p l a s m i n o g e n t o p l a e m t n w h i c h i n t u r n a c t i v a t e d the collagenase. During c u l t u r e of t h e b r e a s t carcinoma i n s e r u m - f r e e medium, c o l l a g e n a s e a c t i v i t y was maximum on day 12 w h e r e a s p l a s m i n o g e n a c t i v a t o r a c t i v i t y changed l i t t l e with time. Using type I c o l l a g e n as a s u b s t r a t e , t h e a c t i v a t e d b r e a s t tumor c o l l m g e n a s e p r o d u c e d 3 / 4 - 1 / 4 f r a g m e n t s c o n s i s t e n t w i t h a mammalian c o l l a g e n a s e . These f i n d i n g s s u g g e s t a pathologic role of plasmlnogen activator in the activation of latent c o l l a g e n a s e d u r i n g tumor i n v a s i o n . A number o f i n v e s t i g a t o r s h a v e p o s t u l a t e d t h a t p r o t e a s e s may p l a y a r o l e i n tumor i n v a s i o n ( 1 - 5 ) . C o l l a g e n a s e I s one s u c h p r o t e a e e w h i c h i s a c t i v e a t n e u t r a l pH and s p e c i f i c a l l y c l e a v e s t r i p l e h e l i c a l c o l l a g e n i n t o two (3/4 - 1/4) fragments (6). Secretion of collagenase by ttmor cells m i g r a t i n g from t h e p r l m a r y m a s s p r o v i d e s an a t t r a c t i v e h y p o t h e s i s f o r t h e mechanism o f tumor i n v a s i o n of s u r r o u n d i n g h o s t c o n n e c t i v e t l s s u e - - s l n c e t h e l o c a l e n v i r o n m e n t would l i k e l y be a t n e u t r a l pH. C o n s e q u e n t l y , a number o f i n v e s t i g a t o r s h a v e r e p o r t e d s i g n i f i c a n t l e v e l s o f c o l l a g e n a s e a c t i v i t y i n a wlde v a r i e t y o f t u m o r s ( 7 - 1 4 ) . Abramson ( 1 3 ) h a s c o r r e l a t e d a g g r e s s i v e i n v t v o g r o w t h i n c a r c i n o m a s o f t h e head and n e c k w i t h c o l l a g e n a s e activity, and K u e t t n e r e t a l . (14) have p o s t u l a t e d t h a t t n h t b i t o r s of e o l l a g e n a s e may p r e v e n t t u m o r s from i n v a d i n g c a r t i l a g e . C o l l a g e n a s e i s p r o d u c e d I n b o t h l a t e n t and a c t i v e forms ( 6 ) . The l a t e n t form c a n be a c t i v a t e d w i t h b r i e f p r o t e a s e t r e a ~ e n t (15). S i n c e one o f t h e p r o f e s s e s c a p a b l e of a c t i v a t i n g c o l l a g e n a s e i s p l a s m i n ( 1 5 ) , t h e p o s s i b i l i t y a r o s e t h a t tumor c e l l s c o u l d a c t i v a t e c o l l a g e n a s e t h r o u g h p l a s m l n o g e n a c t i v a tor. P l a s m l n o g e n a c t i v a t o r s e c r e t e d by tumor c e l l s ( 4 , 5 ) c o u l d c o n v e r t p l a s m l n o g e n zymogen t o p l a s m i n which would i n t u r n a c t i v a t e l a t e n t tumor collagenase. T e s t i n g t h l s h y p o t h e s i s i n v i t r o was t h e s u b j e c t o f t h e present study. P r e v i o u s s t u d i e s on c o l l a g e n a s e from human c a r c i n o m a ( 7 , 1 3 , 1 4 ) h a v e s u f f e r e d from t h e drawback t h a t c o n t a m i n a t i n g i n f l a u m a t o r y c e l l s and f l b r o b l a s t s may h a v e b e e n t h e s o u r c e o f t h e c o l l a g e n a s e . T h e r e f o r e , we h a v e s t u d i e d c o l l a g e n a s e p r o d u c t i o n from c u l t u r e d human b r e a s t c a r c i n o m a c e l l s *To whom a l l
c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . 0024-3205/80/151223-09502.00/0
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
which have been well characterized to be mammary epithelial in origin, malignant in karyocype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasmlnogen. Finally, to verify that the tumor-secreted collegenase cleaved type I collagen at a slngle locus, enzyme degradation products were studied by gel electrophoreels.
Materials
A.
and Methods
Cell Cultures
Human breast carcinoma cell cultures Three human breast carcinoma cell llnes, ZR-75-1, ZR-75-30 and ZR-75-31A (ZR-75-31, attached subculture), were employed in these studies. ZR-75-I and ZR-75-30 cells were established from malignant ascltlc fluids, while the specimen from which ZR-75-31A cells were obtained was a malignant pleura1 effusion. Establishment and characterization of these cell lines to document their m a l i g n a n t human a n d mammary o r i g i n h a v e b e e n d e s c r i b e d i n d e t a i l p r e v i o u s l y (16,19). For each of the three donors, a reliable histopathological diagnosis (infiltrating duct carcinoma) has been recorded. Each cell line possesses a human karyotype distinct from HeLa and from other known cells by trypsin-Glemsa banding techniques. Enzyme phenotypes further serve to distinguish them from each o c h e r as well as from other cell lines. Each possesses epithelial morphology by light and electron microscopy. Cells in culture closely resemble those seen in biopsies or preparations of exfollated cells from their respective donors. In addition, they exhibit ultrascructurel features characteristic of breast carcinoma cells, such as desmosomes, tonoflbrils, end intracytoplasmlc vacuoles (17). Their organ specificity is strongly suggested by expression of differentiated function, including secretory activity as observed by ultrastructure, possession of sex steriod hormone receptors and responsiveness to estrogen, androgen or progesterone (16,19). The cell lines were all establlshed and have been maintained in RPMI 1640 medium supplemented with 25 mM HEPES buffer, I0% fetal calf serum, I00 units of penicillin and I00 ~g of streptomycin per all in serial passage for a period greater than 2 years. Cells were incubated at 370C in a humidified atmosphere of 95% air and 5% CO 2. ZR-75-1 cells studied in these experiments were between passages 63 and 71, while ZR-75-30 and ZR-75-31Acells were between passages 31 and 35. Control cultures H S 5 7 8 B s t , a human c e l l l i n e d e r i v e d f r o m n o r m a l t i s s u e p e r i p h e r a l co e breast tumor, possesses features characteristic of a non-malignant myoeplthellal origin as Judged by ultrastructural features (18). These cells were m a i n t a i n e d i n D u l b e c c o ' s m o d i f i e d MEM s u p p l e m e n t e d w i t h 10Z FCS, I n s u l l n , 1 mg/ml, end nonessential amino acids, and were studied at passages 14-15.
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Activation of Breast Carcinoma Collagenase
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HEK, normal (human embryonic k i d n e y ) e p i t h e l i a l c e l l s ( M i c r o b i o l o g i c a l A s s o c i a t e s , Bethesda, Maryland) were r e c e i v e d as c o n f l u e n t monolayers and were s t u d i e d a t the f i r s t s u b c u l t u r e . C u l t u r e s were e s t a b l i s h e d and m a i n t a i n e d i n Eagles ~ supplemented w i t h 8 p e r c e n t c a l f serum and 25 mH ~ P E S b u f f e r . B.
N a i n t e u a n c e o f C e l l L i n e s and H a r v e s t i n ~ of C u l t u r e F l u i d s f o r Assays of C o l l a s e n a s e and P l a s a i n o s e n A c t i v a t o r
B r e a s t carcinoma c e l l s were m a i n t a i n e d i n c u l t u r e media as s p e c i f i e d above. The c e l l s were d i v i d e d a t s p l i t r a t i o s ( 1 : 3 f o r ZR-75-31A and ZR75-30 c e l l s , 1:5 f o r ZR-75-1 c e l l s ) v a r i e d to a d j u s t f o r d i f f e r e n c e s i n growth r a t e 8o t h a t c u l t u r e s would r e a c h c o n f l u e n c y s i m u l t a n e o u s l y . When monolayers became a p p r o x i m a t e l y 90X c o n f l u e n t , c e l l s were e x t e n s i v e l y (4x) r i n s e d and growth medium was r e p l a c e d w i t h s e r u m - f r e e medium. C e l l s were then i n c u b a t e d and t h e a e d i u m w a 8 exchanged a t i n t e r v a l s of 48 h o u r s . ZR-75-31A c e l l s a p peared t o t o l e r a t e s e r u m - f r e e c o n d i t i o n s w i t h growth a r r e s t as Judged by c e l l number, b u t w i t h no a p p a r e n t l o s s of v i a b i l i t y f o r up to 15 d a y s . ZR-75-1 and ZR-75-30 c e l l s could n o t be maintaSned beyond 8 days u n d e r serumf r e e c o n d i t i o n s . HEK and HS578Bst c e l l s were m a i n t a i n e d i n complete medium w i t h o u t i n s u l i n p r i o r to r e p l a c e m e n t w i t h s e r u m - f r e e medium and f u r t h e r i n c u bation. At a p p r o p r i a t e i n t e r v a l s , t h e c u l t u r e f l u i d s from r e p l i c a t e f l a s k s were d e c a n t e d , c e n t r i f u g e d to remove c e l l u l a r d e b r i s , and p o o l e d . Sample8 were c o n c e n t r a t e d 5 n - f o l d w i t h an A~tcon IM-IO membrane (Amicon C o r p o r a t i o n , L e x i n g t o n , }4ass.) and a s s a y e d f o r c o l l a g e n a s e and plamainogen a c t i v a t o r activity. A c t i v a t i o n of l a t e n t c o l l a s e n a s e by t r y p s i n and p l a s a i n and a s s a y s f o r c o l l a ~enase a c t i v i t y Cuinea pig c o l l a g e n ( t y p e I ) , employed as a s u b s t r a t a f o r c o l l a g e n a s e a s s a y s , was p u r i f i e d by the method of Cross and L a p i r ( 2 0 ) , and l a b e l e d w i t h 3H-sodium b o r o h y d r i d e (New England N u c l e a r , Boston, M a s s . ) , u s i n g b r i e f r e d u c t i v e a l k y l a t i o n as d e s c r i b e d by Means and Feeny (22) and B a r r e t e t a l . ( 2 3 ) . This l a b e l i n g method d i d n o t s i g n i f i c a n t l y d e n a t u r e the c o l l a g e n as Judged by a) normal m i g r a t i o n p a t t e r n on g e l e l e c t r o p h o r e s i s , b) a b i l i t y to form f i b r i l s , and c) low s u s c e p t i b i l i t y to t r y p s i n (5Z). S p e c i f i c a c t i v i t y of the type I c o l l a g e n was 1 x 106 c p m / ~ . In collateral studies guinea p i g s k i n c o l l a g e n was b i o s y n t h e t i c a l l y l a b e l e d w i t h 14C and employed as a s u b s t r a t e f o r c o l l a g e n a s e a s s a y s u s i n g the method of Nagai e t e l . ( 2 4 ) . To d e t e r m i n e a c t i v a t i o n of l a t e n t c o l l a g e n a s e , 200 ~1 of t h e c o n c e n t r a ted s e r u m - f r e e s u p e r n a t a n t from each c e l l c u l t u r e was i n c u b a t e d w i t h 50 ~1 of O.OIZ t r y p s i n ( W o r t h i n g t o n B i o c h e m l c a l s ) f o r 5 mln a t 37"C, w l t h s u b s e q u e n t a d d i t i o n of f l v e - f o l d excess (50 ~ i , 0.05Z) of soybean t r y p s i n i n h i b i t o r (Sigma, S t . L o u i s , H o . ) . The t r y p s i n and soybean i n h i b i t o r was made up i n 0.05 M Trls-HC1, 0.2 M Nat1, 5 ~4 CaCI2, pH 7 . 6 . To d e t e r m i n e a c t i v a t i o n of l a t e n t c o l l a g e n a s e by p l a s m l n o g e n s t i m u l a t i o n , one unlC of human p l a s mlnogen ( W o r t h i n g t o n B i o c h e m l c a l s ) i n 50 vl was added t o the a l i q u o t (200 ~ i ) of c u l t u r e f l u l d and i n c u b a t e d f o r 30 mln a t 37°C f o l l o w e d by a d d i t i o n of soybean t r y p s i n i n h i b i t o r (50 ~ l , 0.05Z) t o i n a c t i v a t e the p l a s m l n . To a s s a y f o r c o l l a g e n a s e , 5 ~g of l a b e l e d c o l l a g e n d i s s o l v e d i n 50 u l b u f f e r ( 0 . 0 5 M Tris-HC1, 0.2 M NaC1, 5 aM CaC12, pH 7 . 6 ) was t h e n added to t h e t u b e s c o n t a i n l n $ 200 ~1 o f t r y p s i n or p l a s m i n a c t i v a t e d media sgmples. Adequate c o n t r o l s were i n c l u d e d : I ) Boiled media samples served as n e g a t i v e c o n t r o l s ; 2) B a c t e r l a l c o l l a g e n a s e (Advanced B i o f a c t o r s , Long I s l a n d , N.Y.) was added to e q u a l amounts of the 3 H - l a b e l e d c o l l a g e n to d e t e r m i n e t o t a l
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
collagenase labile radioactivlty in each reaction tube; 3) Fifty ~i of 0.01% trypsin was added to the labeled collagen to obtain counts released by nonspecific proteolytlc activity. These counts were less than 5% of the total activity and were subtracted from the counts obtained with the test samples to obtain the final counts. Collagenase activity in the assay was terminated by addition of equal volumes of i0% TCA, 0.5% tannic acid and 2 mM L-proline with subsequent incubation at 4°C for 30 mln. Precipitates were pelleted by centrifugation at 2,800 rpm for I0 mln. One hundred ~I allquots of the labeled collagen peptides from the supernatant were counted in a Packard scintillation counter after addition of i0 ml of Hydromlx.
Assays f o r plaemino~en a c t i v a t o r a c t i v i t y Allquots of serum-free supernatant from breast carcinoma cells were tested for extracellular plasmlnogen activator activity by adding them to 1311-labeled fibrln-coated plates. The reaction mixture consisted of 250 ~1 of 0.I M Trls-HCl buffer, pH 8.1, and human plasmlnogen (Worthington Biochemicals). Plates were incubated for 4 hours at 37°C and enzyme activity was assayed by measuring the labeled product of flbrinolysls (25).
Polyacrylamide gel e l e c t r o p h o r e s i s Polyacrylamide gel electrophoresis was performed at acid pH as described by Nagal et al. (26). Type I collagen (I mg) was gelled and digested by overnight incubation (27"C) with activated harvest flulds from ZR-75-31A human breast carcinoma cells. Samples containing the degradation products were denatured prior to electrophoresls. Results and Discussion Table 1 describes latent collagenase production by breast cancer cells and control cultures. Normal myoepithellal cells (HS578Bst) and normal eplthelial cells (HEK) did not elaborate collagenase as measured in these assays. Low levels of enzyme activity were measured for ZR-75-1 and ZR-75-30 cells, while substantially higher amounts of collagenase were obtained for ZR-75-31A cells. This difference is not due to differences in cell number since adjustment for cell density at confluency shows ZR-75-31A cells to produce more collagenase per cell than the other cell ~ines studied (data not shown). Collagenase from these cells was therefore used in all subsequent studies. Collagenase activity in ZR-75-31A cells was dependent on enzymatic activatlon. Figure i depicts collagenase activity in these cells before and after trypsin activation. Without activation less than 5% of the total collagen was degraded, whereas trypsin activation produced a ten-fold enhancement of activity, with peak activity on day 12.
Plasminogen a c t i v a t o r e l a b o r a t i o n remained r e l a t i v e l y constant with time and was recorded f o r each of the breast cancer c e l l l i n e s , ZR-75-1, ZR-75-30 and ZR-75-31A (Table 2). In experiments with serum-free ~rowth medium harvested from cell llneZR-75-31A, the label released from ~H-sodlum borohydrlde-labeled collagen was indicative of collagenase activity. Collagenase activity was also observed when 14C biosynthetically labeled collagen was used as a substrate (Table 3). However, addition of i unit of human plasmlnogen triggered a two- to three-fold increase in activity. These results are consistent with reports that this enzyme is commonly elaborated by neoplastic cells (5,28).
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Activation of Breast Carcinoma Collagenase
TABLE 1 C o l l a g e n a s e Activity* o f S e r u m - f r e e Media H a r v e s t e d from C u l t u r e d Human C e l l s
Enzyme source
Amount o f s u b s t r a t e ( t y p e 1 c o l l a g e n ) degraded (cpm, mean o f d u p l i c a t e s , range < IOZ o f mean)
128
Human m y o e p i t h e l i a l cells HS578Bst day 3
Human embryonic k i d n e y cells HEK day 3
96
Human breast carcinoma cells ZR-75-1 day 5 ZR-75-30 day 5 ZR-75-31A day 5
324 480 1464
T r y p s i n 0.011
162
Bacterial c o l l a g e n a s e 10 u n i t s
2100
* R e a c t i o n a t 37°C f o r 3 h o u r s . radioactivity.
I0 ~g s u b s t r a t e w i t h 2300 cpm
TABLE 2 Plasminogen A c t i v a t o r A c t i v i t y o f C e l l - f r e e S u p e r n a t a n t F l u i d H a r v e s t e d from Human B r e a s t Tumor C e l l s i n Continuous C u l t u r e Cell llne
ZR-75-1 ZR-75-30 ZR-75-31A
% of t o t a l
6 hr 17.6 15.3 19.8
radlo-actlvlty
12 h r 20.8 20.5 24.2
released
24 hr 26.0 29.0 30.9
1227
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Activation of Breast Carcinoma Collagenase
le00
~mmC o h g m
Activity aftor Trypsin Ac0vstion
Vol. 26, No. 15, 1980
m
Cohgenue Activity before Trypsin Activation AGttvity of I.aUmt Colbgenm <1%
.~Not Done o
io
-
O.-.___.O~
O" ~
_
I-I
5Days 6~7" (Cumulative) -,
II 88.9
" 0 " ~
,
, 0 . ..
---0
~ O T~ Z o O~
II
10 Et 11"* 12 8" 13 DAY
~0o~
m0D 600o_~
14 8" 15
Cm ~Z
Bm
FIG. 1 Bar graphs show collagenase activity of serum-free culture fluids from ZR-75-31A b r e a s t tumor c e l l s a f t e r b r i e f t r y p s i n a c t i v a t i o n . Solid llne 40- - 0) d e n o t e s plasmlnoEen a c t i v a t o r a c t i v i t y o f t h e same h a r v e s t f l u i d at the respective tlse points. Harvest fluids from non-transformed myoeplthellal cells 4HS578Bst) did not show any significant activity at any time point.
The data show that human breast tumor cell llnes have the capacity to elaborate collagenase desplCe prolonaed conclnuous culcure. The collagenase conCalned in culture fluids exists as a lacent form or as a latent zymogen or e n z y m e - l n h l b l t o r complex which c o u l d be a c c i v a t e d by C r y p s l n or p l a s m l n . D e t e c t a b l e c o l l a g e n a s e a c C l v l C y was a b s e n t i n harvesC f l u l d s o f normal (HS578BsC) myoeplthellal cells and normal (HEK) eplchellal cells. Collagenage produced by the breast carcinoma cell llne ZR-75-31A cleaved type I collagen into the characteristic fragments of m = ~ e l l a n collagenases (Fig. 2). Metastatic tumors may elaborate a collagenase which preferentially degrades type IV baeement membrane collagen 427). The substrata specificity of breast tumor collaaanase(s ) will be the subject of a subsequent report.
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Activation of Breast Carcinoma Collasenase
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TABLE 3
A c t i v a t i o n o f L a t e n t Human B r e a s t Tumor C o l l a g e n a s e Throush P l a s n / n
Source o f enzyme
cpm o f l a b e l e d p e p t i d e r e l e a s e d *
ZR-75-31A Breast tumor cell eupernatant
880
ZR-75-31A Breast tumor supernatant and p l a s m i u o g e n
1900
P l a s m i n o g e n and b u f f e r Bacterial
no d e t e c t a b l e
aetlvity
2415
collagenase
*Guinea p i g s k i n c o l l a g e n t y p e 1 was l a b e l e d b i o s y n t h e t i c a l l y and used ms a s u b s t r a t e as i n t h e a e t h o d o f N a s a l e t e l . (24). Seven day s e r u a - f r e e c u l t u r e f l u l d h a r v e s t e d f r o u c u l t u r e s o f c e l l l l n e Z R - 7 5 - 3 1 A w e s used a s a s o u r c e o f t h e enzyme. C o n t r o l c o u n t s o b t a i n e d by i n c u b a t i o n w l t h 0.01Z t r y p s i n were s u b t r a c t e d t o g i v e t h e f l u e 1 c o u n t s shown h a r e . Mean o f f o u r e x p e r i a e n t s w i t h r a n s e l e s s t h a n 15.1 o f u e a u . D ! e e s t i o ~ were a t pR 7 . 6 , 370C f o r 3 h o u r s .
FIG. 2 Collagenase degradation products; lathrytlc rat skin collasen Incubated a t 27°C f o r 20 h o u r s w l t h o u t (A) or w l t h (B) b r e a s t t u m o r - a c t l v a t e d c o l l a g e n • Be •
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
The data further suggest a similarity between collagenase produced by breast tumor cells in culture and rheumatoid synovial collagenase as demons t r a f e d by Its sensitivity Co activation by trypsin or plasmin (15). Malignant cells of animal and human orlgin have been shown Co elaborate increased amounts of plasmlnogen acCivaCor (28). The present results suggest thaC a pachologlc role of tumor-produced plasmlnogen activator is to enhance the acclvlCy of tumor-produced latent collagenase , thereby promoclng meCasCasls. Acknowled~nents We t h a n k Dr. S h t g e t o Abe f o r h e l p i n g w i t h g e l e l e c t r o p h o r e s i s t e c h n i q u e s , and Karen Kniska f o r her e x c e l l e n t t e c h n i c a l a s s i s t a n c e . References 1. 2. 3. 4. 5. 6. 7.
A. FISCHER, (Whilhelm Roux) Arch. EnCw. Mech. Ar~. 104 201-261 (1925). B. SYLVEN, Eur. J. Cancer 4 463-474 (1968). A. R. POOLE, Cancer Ree. 302252-2259 (1970). L. OSSOWSKI, J. P. QUIGLEY, G. M. KELLERMAN, and E. REICH, J. Exp. Med. 138 1056-1064 (1973). J. C. UN~LESS, A. TOBIA, L. OSSOWSKI, J. P. QUIGLEY, D. B. RIFKIN, and E. REICH, J. Exp. Had. 137 85-111 (1973). J. GROSS, The Harvey Lecture Series 68, pp. 351-432, Academlc Press, New York (1974). M. R. DRESDEN, S. A. HEILMAN, and J. D. SCHMIDT, Cancer Res. 32 993-996
(1972). 8.
19. 20. 21. 22. 23.
M. K. DABBONS, D. SOBHY, A. N. ROBERTS, and S. B. BRINKEY, Molec. Cell. B£ochem. 16 37 (1977). Y. YAMANI~-HI, M. K. DABBONS and K. HASHIMOTO, Cancer Res. 32 2551-2560 (1972). Y. YAMANISHI~--E. MAEYENS, M. K. DABBONS, H. OHYAMA, and K. HASHIMOTO, Cancer Res. 33 2507-2512 (1973). K. HASHIMOTO, Y. YAMANISHI, E. MAEYENS, M. K. DABEONS, and T. KANZAKI, Cancer Ree. 33 2790-2801 (1973). K. R. LABEOSSE, AND I. E. LIENER, Molec. Cell. Biochem. 19 181-189 (1978). M. ABRAMSON, R. W. SCHOLLING, C. C. HUANG, and R. G. SALOME, Ann. 0Col. Rhlnol. LarT,6ol. 8 4 158 (1975). K. E. KUETTNER, L. SOBLE, R. L. CROXEN, B. MAECQYNSKA, J. HITI, and E. HARPER, Science 196 643-654 (1977). Z. WERB, C.'~2-----------'~I, C. A. VATER, AND E. D. HARRIS, New End1. J. )fed, 296 1017-1023 (1977). L . W . ENCIKL, N. A. YOUNG, T. S. TRALKA, M. E. LIPPMAN, S. J. O'BRIEN, and M. J. JOYCE, Cancer Res. 38 3352-3364 (1978). G. C. BUEEHIMG, and A. J. HACKETT, J. Natl. Cancer Inst. 53 621-629 (1974). A.J. HACKETT, H. S. SMITH, E. L. SPRINGER, R. B. OWENS, W. A. NELSON-REES, J. L. RIGGS, and M. B. GARDNER, J. Natl. Cancer Inst. 58 1795-1803 (1977). L . W . ENGEL, and N. A. YOUNG, Cancer Res. 38 4327-4339 (---1978). J. L. GROSS, and C. M. LAPIER, Proc. Natl. Acad. Sol. U.S. 48 1014 (1962). P. A. JONES, W. E. LANG, and W. F. BENEDICT, Cell 6 245-252 (-'-1975). G. E. MEANS, and R. E. FEENY, Biochemistry 7 2192-2201 (1968). J. C. BARRET, B. D. CRAWFOED, and P,O.P. TSTO, Cancer Res. 37 1182-1185
24. 25.
Y. NAGAI, C. M. LAPIERE, end J. GROSS, B~ochemistr~ ~ 3123-3131 (1966). M. PARANJPE, S. EATON, and C. W. BOONE, J. Cell Physiol. 96 63-72 (1978).
9. 10.
11. 12. 13. 14. 15. 16. 17. 18.
(1977).
Vol. 26, No. 15, 1980
26.
27. 28.
Activation of Breast Carcinoma Collagenase
Y. N&CAI, 2. CROSS, and K. A. PIEZ, Ann. N.Y. Acad. S c i . 121 495-500 (1964). L. A. LIOTTA, S. ABE, P° ~ H R O N ROBEY, and G. R. MARTIN, Proc. Natl. Acsd. Scl. 76 2268-2272 (1979). D. B. RII~IN, and R. POLLACK, J. Cell Biol. 73 47-55 (1977).
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Pergamon Press
ACTIVATION OF HUMAN BREAST CARCINOMA COLLAGENASE THROUGH PLASMINOGEN ACTIVATOR Meera ParanJpe,* Linda Engel, + Nathanlel Young, + and Lance A. Liotta* Laboratory of Pathophyslology,* Laboratory of Pathology, + and Cell Biology Section, ° National Cancer Institute, Natlonal Institutes of Health, Bethesda, Maryland 20205 (Received In final form February Ii, 1980) SUMMARY L a t e n t c o l l a g e n a s e a c t i v i t y was d e t e c t e d i n t h e media o f a w e l l - c h a r a c t e r i z e d l i n e o f human b r e a s t c a r c i n o m a c e l l s m a i n t a i n e d f o r o v e r two y e a r s in culture. The media a l s o c o n t a i n e d s u f f i c i e n t p l a s m i n o g e n a c t i v a t o r t o convert extrinsically added p l a s m i n o g e n t o p l a e m t n w h i c h i n t u r n a c t i v a t e d the collagenase. During c u l t u r e of t h e b r e a s t carcinoma i n s e r u m - f r e e medium, c o l l a g e n a s e a c t i v i t y was maximum on day 12 w h e r e a s p l a s m i n o g e n a c t i v a t o r a c t i v i t y changed l i t t l e with time. Using type I c o l l a g e n as a s u b s t r a t e , t h e a c t i v a t e d b r e a s t tumor c o l l m g e n a s e p r o d u c e d 3 / 4 - 1 / 4 f r a g m e n t s c o n s i s t e n t w i t h a mammalian c o l l a g e n a s e . These f i n d i n g s s u g g e s t a pathologic role of plasmlnogen activator in the activation of latent c o l l a g e n a s e d u r i n g tumor i n v a s i o n . A number o f i n v e s t i g a t o r s h a v e p o s t u l a t e d t h a t p r o t e a s e s may p l a y a r o l e i n tumor i n v a s i o n ( 1 - 5 ) . C o l l a g e n a s e I s one s u c h p r o t e a e e w h i c h i s a c t i v e a t n e u t r a l pH and s p e c i f i c a l l y c l e a v e s t r i p l e h e l i c a l c o l l a g e n i n t o two (3/4 - 1/4) fragments (6). Secretion of collagenase by ttmor cells m i g r a t i n g from t h e p r l m a r y m a s s p r o v i d e s an a t t r a c t i v e h y p o t h e s i s f o r t h e mechanism o f tumor i n v a s i o n of s u r r o u n d i n g h o s t c o n n e c t i v e t l s s u e - - s l n c e t h e l o c a l e n v i r o n m e n t would l i k e l y be a t n e u t r a l pH. C o n s e q u e n t l y , a number o f i n v e s t i g a t o r s h a v e r e p o r t e d s i g n i f i c a n t l e v e l s o f c o l l a g e n a s e a c t i v i t y i n a wlde v a r i e t y o f t u m o r s ( 7 - 1 4 ) . Abramson ( 1 3 ) h a s c o r r e l a t e d a g g r e s s i v e i n v t v o g r o w t h i n c a r c i n o m a s o f t h e head and n e c k w i t h c o l l a g e n a s e activity, and K u e t t n e r e t a l . (14) have p o s t u l a t e d t h a t t n h t b i t o r s of e o l l a g e n a s e may p r e v e n t t u m o r s from i n v a d i n g c a r t i l a g e . C o l l a g e n a s e i s p r o d u c e d I n b o t h l a t e n t and a c t i v e forms ( 6 ) . The l a t e n t form c a n be a c t i v a t e d w i t h b r i e f p r o t e a s e t r e a ~ e n t (15). S i n c e one o f t h e p r o f e s s e s c a p a b l e of a c t i v a t i n g c o l l a g e n a s e i s p l a s m i n ( 1 5 ) , t h e p o s s i b i l i t y a r o s e t h a t tumor c e l l s c o u l d a c t i v a t e c o l l a g e n a s e t h r o u g h p l a s m l n o g e n a c t i v a tor. P l a s m l n o g e n a c t i v a t o r s e c r e t e d by tumor c e l l s ( 4 , 5 ) c o u l d c o n v e r t p l a s m l n o g e n zymogen t o p l a s m i n which would i n t u r n a c t i v a t e l a t e n t tumor collagenase. T e s t i n g t h l s h y p o t h e s i s i n v i t r o was t h e s u b j e c t o f t h e present study. P r e v i o u s s t u d i e s on c o l l a g e n a s e from human c a r c i n o m a ( 7 , 1 3 , 1 4 ) h a v e s u f f e r e d from t h e drawback t h a t c o n t a m i n a t i n g i n f l a u m a t o r y c e l l s and f l b r o b l a s t s may h a v e b e e n t h e s o u r c e o f t h e c o l l a g e n a s e . T h e r e f o r e , we h a v e s t u d i e d c o l l a g e n a s e p r o d u c t i o n from c u l t u r e d human b r e a s t c a r c i n o m a c e l l s *To whom a l l
c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . 0024-3205/80/151223-09502.00/0
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
which have been well characterized to be mammary epithelial in origin, malignant in karyocype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasmlnogen. Finally, to verify that the tumor-secreted collegenase cleaved type I collagen at a slngle locus, enzyme degradation products were studied by gel electrophoreels.
Materials
A.
and Methods
Cell Cultures
Human breast carcinoma cell cultures Three human breast carcinoma cell llnes, ZR-75-1, ZR-75-30 and ZR-75-31A (ZR-75-31, attached subculture), were employed in these studies. ZR-75-I and ZR-75-30 cells were established from malignant ascltlc fluids, while the specimen from which ZR-75-31A cells were obtained was a malignant pleura1 effusion. Establishment and characterization of these cell lines to document their m a l i g n a n t human a n d mammary o r i g i n h a v e b e e n d e s c r i b e d i n d e t a i l p r e v i o u s l y (16,19). For each of the three donors, a reliable histopathological diagnosis (infiltrating duct carcinoma) has been recorded. Each cell line possesses a human karyotype distinct from HeLa and from other known cells by trypsin-Glemsa banding techniques. Enzyme phenotypes further serve to distinguish them from each o c h e r as well as from other cell lines. Each possesses epithelial morphology by light and electron microscopy. Cells in culture closely resemble those seen in biopsies or preparations of exfollated cells from their respective donors. In addition, they exhibit ultrascructurel features characteristic of breast carcinoma cells, such as desmosomes, tonoflbrils, end intracytoplasmlc vacuoles (17). Their organ specificity is strongly suggested by expression of differentiated function, including secretory activity as observed by ultrastructure, possession of sex steriod hormone receptors and responsiveness to estrogen, androgen or progesterone (16,19). The cell lines were all establlshed and have been maintained in RPMI 1640 medium supplemented with 25 mM HEPES buffer, I0% fetal calf serum, I00 units of penicillin and I00 ~g of streptomycin per all in serial passage for a period greater than 2 years. Cells were incubated at 370C in a humidified atmosphere of 95% air and 5% CO 2. ZR-75-1 cells studied in these experiments were between passages 63 and 71, while ZR-75-30 and ZR-75-31Acells were between passages 31 and 35. Control cultures H S 5 7 8 B s t , a human c e l l l i n e d e r i v e d f r o m n o r m a l t i s s u e p e r i p h e r a l co e breast tumor, possesses features characteristic of a non-malignant myoeplthellal origin as Judged by ultrastructural features (18). These cells were m a i n t a i n e d i n D u l b e c c o ' s m o d i f i e d MEM s u p p l e m e n t e d w i t h 10Z FCS, I n s u l l n , 1 mg/ml, end nonessential amino acids, and were studied at passages 14-15.
Vol. 26, No. 15, 1980
Activation of Breast Carcinoma Collagenase
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HEK, normal (human embryonic k i d n e y ) e p i t h e l i a l c e l l s ( M i c r o b i o l o g i c a l A s s o c i a t e s , Bethesda, Maryland) were r e c e i v e d as c o n f l u e n t monolayers and were s t u d i e d a t the f i r s t s u b c u l t u r e . C u l t u r e s were e s t a b l i s h e d and m a i n t a i n e d i n Eagles ~ supplemented w i t h 8 p e r c e n t c a l f serum and 25 mH ~ P E S b u f f e r . B.
N a i n t e u a n c e o f C e l l L i n e s and H a r v e s t i n ~ of C u l t u r e F l u i d s f o r Assays of C o l l a s e n a s e and P l a s a i n o s e n A c t i v a t o r
B r e a s t carcinoma c e l l s were m a i n t a i n e d i n c u l t u r e media as s p e c i f i e d above. The c e l l s were d i v i d e d a t s p l i t r a t i o s ( 1 : 3 f o r ZR-75-31A and ZR75-30 c e l l s , 1:5 f o r ZR-75-1 c e l l s ) v a r i e d to a d j u s t f o r d i f f e r e n c e s i n growth r a t e 8o t h a t c u l t u r e s would r e a c h c o n f l u e n c y s i m u l t a n e o u s l y . When monolayers became a p p r o x i m a t e l y 90X c o n f l u e n t , c e l l s were e x t e n s i v e l y (4x) r i n s e d and growth medium was r e p l a c e d w i t h s e r u m - f r e e medium. C e l l s were then i n c u b a t e d and t h e a e d i u m w a 8 exchanged a t i n t e r v a l s of 48 h o u r s . ZR-75-31A c e l l s a p peared t o t o l e r a t e s e r u m - f r e e c o n d i t i o n s w i t h growth a r r e s t as Judged by c e l l number, b u t w i t h no a p p a r e n t l o s s of v i a b i l i t y f o r up to 15 d a y s . ZR-75-1 and ZR-75-30 c e l l s could n o t be maintaSned beyond 8 days u n d e r serumf r e e c o n d i t i o n s . HEK and HS578Bst c e l l s were m a i n t a i n e d i n complete medium w i t h o u t i n s u l i n p r i o r to r e p l a c e m e n t w i t h s e r u m - f r e e medium and f u r t h e r i n c u bation. At a p p r o p r i a t e i n t e r v a l s , t h e c u l t u r e f l u i d s from r e p l i c a t e f l a s k s were d e c a n t e d , c e n t r i f u g e d to remove c e l l u l a r d e b r i s , and p o o l e d . Sample8 were c o n c e n t r a t e d 5 n - f o l d w i t h an A~tcon IM-IO membrane (Amicon C o r p o r a t i o n , L e x i n g t o n , }4ass.) and a s s a y e d f o r c o l l a g e n a s e and plamainogen a c t i v a t o r activity. A c t i v a t i o n of l a t e n t c o l l a s e n a s e by t r y p s i n and p l a s a i n and a s s a y s f o r c o l l a ~enase a c t i v i t y Cuinea pig c o l l a g e n ( t y p e I ) , employed as a s u b s t r a t a f o r c o l l a g e n a s e a s s a y s , was p u r i f i e d by the method of Cross and L a p i r ( 2 0 ) , and l a b e l e d w i t h 3H-sodium b o r o h y d r i d e (New England N u c l e a r , Boston, M a s s . ) , u s i n g b r i e f r e d u c t i v e a l k y l a t i o n as d e s c r i b e d by Means and Feeny (22) and B a r r e t e t a l . ( 2 3 ) . This l a b e l i n g method d i d n o t s i g n i f i c a n t l y d e n a t u r e the c o l l a g e n as Judged by a) normal m i g r a t i o n p a t t e r n on g e l e l e c t r o p h o r e s i s , b) a b i l i t y to form f i b r i l s , and c) low s u s c e p t i b i l i t y to t r y p s i n (5Z). S p e c i f i c a c t i v i t y of the type I c o l l a g e n was 1 x 106 c p m / ~ . In collateral studies guinea p i g s k i n c o l l a g e n was b i o s y n t h e t i c a l l y l a b e l e d w i t h 14C and employed as a s u b s t r a t e f o r c o l l a g e n a s e a s s a y s u s i n g the method of Nagai e t e l . ( 2 4 ) . To d e t e r m i n e a c t i v a t i o n of l a t e n t c o l l a g e n a s e , 200 ~1 of t h e c o n c e n t r a ted s e r u m - f r e e s u p e r n a t a n t from each c e l l c u l t u r e was i n c u b a t e d w i t h 50 ~1 of O.OIZ t r y p s i n ( W o r t h i n g t o n B i o c h e m l c a l s ) f o r 5 mln a t 37"C, w l t h s u b s e q u e n t a d d i t i o n of f l v e - f o l d excess (50 ~ i , 0.05Z) of soybean t r y p s i n i n h i b i t o r (Sigma, S t . L o u i s , H o . ) . The t r y p s i n and soybean i n h i b i t o r was made up i n 0.05 M Trls-HC1, 0.2 M Nat1, 5 ~4 CaCI2, pH 7 . 6 . To d e t e r m i n e a c t i v a t i o n of l a t e n t c o l l a g e n a s e by p l a s m l n o g e n s t i m u l a t i o n , one unlC of human p l a s mlnogen ( W o r t h i n g t o n B i o c h e m l c a l s ) i n 50 vl was added t o the a l i q u o t (200 ~ i ) of c u l t u r e f l u l d and i n c u b a t e d f o r 30 mln a t 37°C f o l l o w e d by a d d i t i o n of soybean t r y p s i n i n h i b i t o r (50 ~ l , 0.05Z) t o i n a c t i v a t e the p l a s m l n . To a s s a y f o r c o l l a g e n a s e , 5 ~g of l a b e l e d c o l l a g e n d i s s o l v e d i n 50 u l b u f f e r ( 0 . 0 5 M Tris-HC1, 0.2 M NaC1, 5 aM CaC12, pH 7 . 6 ) was t h e n added to t h e t u b e s c o n t a i n l n $ 200 ~1 o f t r y p s i n or p l a s m i n a c t i v a t e d media sgmples. Adequate c o n t r o l s were i n c l u d e d : I ) Boiled media samples served as n e g a t i v e c o n t r o l s ; 2) B a c t e r l a l c o l l a g e n a s e (Advanced B i o f a c t o r s , Long I s l a n d , N.Y.) was added to e q u a l amounts of the 3 H - l a b e l e d c o l l a g e n to d e t e r m i n e t o t a l
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
collagenase labile radioactivlty in each reaction tube; 3) Fifty ~i of 0.01% trypsin was added to the labeled collagen to obtain counts released by nonspecific proteolytlc activity. These counts were less than 5% of the total activity and were subtracted from the counts obtained with the test samples to obtain the final counts. Collagenase activity in the assay was terminated by addition of equal volumes of i0% TCA, 0.5% tannic acid and 2 mM L-proline with subsequent incubation at 4°C for 30 mln. Precipitates were pelleted by centrifugation at 2,800 rpm for I0 mln. One hundred ~I allquots of the labeled collagen peptides from the supernatant were counted in a Packard scintillation counter after addition of i0 ml of Hydromlx.
Assays f o r plaemino~en a c t i v a t o r a c t i v i t y Allquots of serum-free supernatant from breast carcinoma cells were tested for extracellular plasmlnogen activator activity by adding them to 1311-labeled fibrln-coated plates. The reaction mixture consisted of 250 ~1 of 0.I M Trls-HCl buffer, pH 8.1, and human plasmlnogen (Worthington Biochemicals). Plates were incubated for 4 hours at 37°C and enzyme activity was assayed by measuring the labeled product of flbrinolysls (25).
Polyacrylamide gel e l e c t r o p h o r e s i s Polyacrylamide gel electrophoresis was performed at acid pH as described by Nagal et al. (26). Type I collagen (I mg) was gelled and digested by overnight incubation (27"C) with activated harvest flulds from ZR-75-31A human breast carcinoma cells. Samples containing the degradation products were denatured prior to electrophoresls. Results and Discussion Table 1 describes latent collagenase production by breast cancer cells and control cultures. Normal myoepithellal cells (HS578Bst) and normal eplthelial cells (HEK) did not elaborate collagenase as measured in these assays. Low levels of enzyme activity were measured for ZR-75-1 and ZR-75-30 cells, while substantially higher amounts of collagenase were obtained for ZR-75-31A cells. This difference is not due to differences in cell number since adjustment for cell density at confluency shows ZR-75-31A cells to produce more collagenase per cell than the other cell ~ines studied (data not shown). Collagenase from these cells was therefore used in all subsequent studies. Collagenase activity in ZR-75-31A cells was dependent on enzymatic activatlon. Figure i depicts collagenase activity in these cells before and after trypsin activation. Without activation less than 5% of the total collagen was degraded, whereas trypsin activation produced a ten-fold enhancement of activity, with peak activity on day 12.
Plasminogen a c t i v a t o r e l a b o r a t i o n remained r e l a t i v e l y constant with time and was recorded f o r each of the breast cancer c e l l l i n e s , ZR-75-1, ZR-75-30 and ZR-75-31A (Table 2). In experiments with serum-free ~rowth medium harvested from cell llneZR-75-31A, the label released from ~H-sodlum borohydrlde-labeled collagen was indicative of collagenase activity. Collagenase activity was also observed when 14C biosynthetically labeled collagen was used as a substrate (Table 3). However, addition of i unit of human plasmlnogen triggered a two- to three-fold increase in activity. These results are consistent with reports that this enzyme is commonly elaborated by neoplastic cells (5,28).
Vol. 26, No. 15, 1980
Activation of Breast Carcinoma Collagenase
TABLE 1 C o l l a g e n a s e Activity* o f S e r u m - f r e e Media H a r v e s t e d from C u l t u r e d Human C e l l s
Enzyme source
Amount o f s u b s t r a t e ( t y p e 1 c o l l a g e n ) degraded (cpm, mean o f d u p l i c a t e s , range < IOZ o f mean)
128
Human m y o e p i t h e l i a l cells HS578Bst day 3
Human embryonic k i d n e y cells HEK day 3
96
Human breast carcinoma cells ZR-75-1 day 5 ZR-75-30 day 5 ZR-75-31A day 5
324 480 1464
T r y p s i n 0.011
162
Bacterial c o l l a g e n a s e 10 u n i t s
2100
* R e a c t i o n a t 37°C f o r 3 h o u r s . radioactivity.
I0 ~g s u b s t r a t e w i t h 2300 cpm
TABLE 2 Plasminogen A c t i v a t o r A c t i v i t y o f C e l l - f r e e S u p e r n a t a n t F l u i d H a r v e s t e d from Human B r e a s t Tumor C e l l s i n Continuous C u l t u r e Cell llne
ZR-75-1 ZR-75-30 ZR-75-31A
% of t o t a l
6 hr 17.6 15.3 19.8
radlo-actlvlty
12 h r 20.8 20.5 24.2
released
24 hr 26.0 29.0 30.9
1227
1228
Activation of Breast Carcinoma Collagenase
le00
~mmC o h g m
Activity aftor Trypsin Ac0vstion
Vol. 26, No. 15, 1980
m
Cohgenue Activity before Trypsin Activation AGttvity of I.aUmt Colbgenm <1%
.~Not Done o
io
-
O.-.___.O~
O" ~
_
I-I
5Days 6~7" (Cumulative) -,
II 88.9
" 0 " ~
,
, 0 . ..
---0
~ O T~ Z o O~
II
10 Et 11"* 12 8" 13 DAY
~0o~
m0D 600o_~
14 8" 15
Cm ~Z
Bm
FIG. 1 Bar graphs show collagenase activity of serum-free culture fluids from ZR-75-31A b r e a s t tumor c e l l s a f t e r b r i e f t r y p s i n a c t i v a t i o n . Solid llne 40- - 0) d e n o t e s plasmlnoEen a c t i v a t o r a c t i v i t y o f t h e same h a r v e s t f l u i d at the respective tlse points. Harvest fluids from non-transformed myoeplthellal cells 4HS578Bst) did not show any significant activity at any time point.
The data show that human breast tumor cell llnes have the capacity to elaborate collagenase desplCe prolonaed conclnuous culcure. The collagenase conCalned in culture fluids exists as a lacent form or as a latent zymogen or e n z y m e - l n h l b l t o r complex which c o u l d be a c c i v a t e d by C r y p s l n or p l a s m l n . D e t e c t a b l e c o l l a g e n a s e a c C l v l C y was a b s e n t i n harvesC f l u l d s o f normal (HS578BsC) myoeplthellal cells and normal (HEK) eplchellal cells. Collagenage produced by the breast carcinoma cell llne ZR-75-31A cleaved type I collagen into the characteristic fragments of m = ~ e l l a n collagenases (Fig. 2). Metastatic tumors may elaborate a collagenase which preferentially degrades type IV baeement membrane collagen 427). The substrata specificity of breast tumor collaaanase(s ) will be the subject of a subsequent report.
Vol. 26, No. 15, 1980
Activation of Breast Carcinoma Collasenase
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TABLE 3
A c t i v a t i o n o f L a t e n t Human B r e a s t Tumor C o l l a g e n a s e Throush P l a s n / n
Source o f enzyme
cpm o f l a b e l e d p e p t i d e r e l e a s e d *
ZR-75-31A Breast tumor cell eupernatant
880
ZR-75-31A Breast tumor supernatant and p l a s m i u o g e n
1900
P l a s m i n o g e n and b u f f e r Bacterial
no d e t e c t a b l e
aetlvity
2415
collagenase
*Guinea p i g s k i n c o l l a g e n t y p e 1 was l a b e l e d b i o s y n t h e t i c a l l y and used ms a s u b s t r a t e as i n t h e a e t h o d o f N a s a l e t e l . (24). Seven day s e r u a - f r e e c u l t u r e f l u l d h a r v e s t e d f r o u c u l t u r e s o f c e l l l l n e Z R - 7 5 - 3 1 A w e s used a s a s o u r c e o f t h e enzyme. C o n t r o l c o u n t s o b t a i n e d by i n c u b a t i o n w l t h 0.01Z t r y p s i n were s u b t r a c t e d t o g i v e t h e f l u e 1 c o u n t s shown h a r e . Mean o f f o u r e x p e r i a e n t s w i t h r a n s e l e s s t h a n 15.1 o f u e a u . D ! e e s t i o ~ were a t pR 7 . 6 , 370C f o r 3 h o u r s .
FIG. 2 Collagenase degradation products; lathrytlc rat skin collasen Incubated a t 27°C f o r 20 h o u r s w l t h o u t (A) or w l t h (B) b r e a s t t u m o r - a c t l v a t e d c o l l a g e n • Be •
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Activation of Breast Carcinoma Collagenase
Vol. 26, No. 15, 1980
The data further suggest a similarity between collagenase produced by breast tumor cells in culture and rheumatoid synovial collagenase as demons t r a f e d by Its sensitivity Co activation by trypsin or plasmin (15). Malignant cells of animal and human orlgin have been shown Co elaborate increased amounts of plasmlnogen acCivaCor (28). The present results suggest thaC a pachologlc role of tumor-produced plasmlnogen activator is to enhance the acclvlCy of tumor-produced latent collagenase , thereby promoclng meCasCasls. Acknowled~nents We t h a n k Dr. S h t g e t o Abe f o r h e l p i n g w i t h g e l e l e c t r o p h o r e s i s t e c h n i q u e s , and Karen Kniska f o r her e x c e l l e n t t e c h n i c a l a s s i s t a n c e . References 1. 2. 3. 4. 5. 6. 7.
A. FISCHER, (Whilhelm Roux) Arch. EnCw. Mech. Ar~. 104 201-261 (1925). B. SYLVEN, Eur. J. Cancer 4 463-474 (1968). A. R. POOLE, Cancer Ree. 302252-2259 (1970). L. OSSOWSKI, J. P. QUIGLEY, G. M. KELLERMAN, and E. REICH, J. Exp. Med. 138 1056-1064 (1973). J. C. UN~LESS, A. TOBIA, L. OSSOWSKI, J. P. QUIGLEY, D. B. RIFKIN, and E. REICH, J. Exp. Had. 137 85-111 (1973). J. GROSS, The Harvey Lecture Series 68, pp. 351-432, Academlc Press, New York (1974). M. R. DRESDEN, S. A. HEILMAN, and J. D. SCHMIDT, Cancer Res. 32 993-996
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M. K. DABBONS, D. SOBHY, A. N. ROBERTS, and S. B. BRINKEY, Molec. Cell. B£ochem. 16 37 (1977). Y. YAMANI~-HI, M. K. DABBONS and K. HASHIMOTO, Cancer Res. 32 2551-2560 (1972). Y. YAMANISHI~--E. MAEYENS, M. K. DABBONS, H. OHYAMA, and K. HASHIMOTO, Cancer Res. 33 2507-2512 (1973). K. HASHIMOTO, Y. YAMANISHI, E. MAEYENS, M. K. DABEONS, and T. KANZAKI, Cancer Ree. 33 2790-2801 (1973). K. R. LABEOSSE, AND I. E. LIENER, Molec. Cell. Biochem. 19 181-189 (1978). M. ABRAMSON, R. W. SCHOLLING, C. C. HUANG, and R. G. SALOME, Ann. 0Col. Rhlnol. LarT,6ol. 8 4 158 (1975). K. E. KUETTNER, L. SOBLE, R. L. CROXEN, B. MAECQYNSKA, J. HITI, and E. HARPER, Science 196 643-654 (1977). Z. WERB, C.'~2-----------'~I, C. A. VATER, AND E. D. HARRIS, New End1. J. )fed, 296 1017-1023 (1977). L . W . ENCIKL, N. A. YOUNG, T. S. TRALKA, M. E. LIPPMAN, S. J. O'BRIEN, and M. J. JOYCE, Cancer Res. 38 3352-3364 (1978). G. C. BUEEHIMG, and A. J. HACKETT, J. Natl. Cancer Inst. 53 621-629 (1974). A.J. HACKETT, H. S. SMITH, E. L. SPRINGER, R. B. OWENS, W. A. NELSON-REES, J. L. RIGGS, and M. B. GARDNER, J. Natl. Cancer Inst. 58 1795-1803 (1977). L . W . ENGEL, and N. A. YOUNG, Cancer Res. 38 4327-4339 (---1978). J. L. GROSS, and C. M. LAPIER, Proc. Natl. Acad. Sol. U.S. 48 1014 (1962). P. A. JONES, W. E. LANG, and W. F. BENEDICT, Cell 6 245-252 (-'-1975). G. E. MEANS, and R. E. FEENY, Biochemistry 7 2192-2201 (1968). J. C. BARRET, B. D. CRAWFOED, and P,O.P. TSTO, Cancer Res. 37 1182-1185
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Y. NAGAI, C. M. LAPIERE, end J. GROSS, B~ochemistr~ ~ 3123-3131 (1966). M. PARANJPE, S. EATON, and C. W. BOONE, J. Cell Physiol. 96 63-72 (1978).
9. 10.
11. 12. 13. 14. 15. 16. 17. 18.
(1977).
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27. 28.
Activation of Breast Carcinoma Collagenase
Y. N&CAI, 2. CROSS, and K. A. PIEZ, Ann. N.Y. Acad. S c i . 121 495-500 (1964). L. A. LIOTTA, S. ABE, P° ~ H R O N ROBEY, and G. R. MARTIN, Proc. Natl. Acsd. Scl. 76 2268-2272 (1979). D. B. RII~IN, and R. POLLACK, J. Cell Biol. 73 47-55 (1977).
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