Znt. J . Cancer: 45, 1137-1 142 (1990) 0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union internationale Contre le Cancer
EXTRACTION OF TYPE-IV COLLAGENASE/GELATINASE FROM PLASMA MEMBRANES OF HUMAN CANCER CELLS Stanley ZUCKER's273*6, Ute M. MOLL^, Rita M. LYSIK',Elizabeth I. DIMASSIMO',William G.STETLER-STEVENSON', Lance A. LIOTTA5 and Janine w. SCHWEDES' Departments of 'Research and 2Medicine, VeteransAdministration Medical Center, Northport, NY I 1768; Departments of 3Medicine and 4Pathology,Health Science Center, State University of New York, at Stony Brook, NY 11 794; and sLaboratory of Pathology, National Cancer Institute, Bethesda, MD, USA. Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octylglucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62kDa. lmmunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenasel gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagendgelatinase of 68kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.
Studies dealing with mechanisms of collagen degradation during cancer metastasis have focused primarily on cellsecreted collagenolytic enzymes (Liotta et al., 1980; Salo et al., 1983; Gabrisa et al., 1987; Nakajima et al., 1987; Spinucci et al., 1988; Bonfil et al., 1989). Cancer cells have also been shown to have collagen- and gelatin-degrading metalloproteinases localized to their plasma membranes (Zucker et al., 1985a,b, 1987b). We have proposed that the surface membrane localization of these proteinases is of crucial importance in the process of cancer invasion and metastasis. In the current study we have used an organic solvent and a non-ionic detergent to extract metalloproteinases from human pancreatic cancer plasma membranes. These membrane enzymes have been partially purified by chromatography and have been characterized by gelatin zymography and immunoblotting procedures. Type-IV collagenase/gelatinase was also identified in the plasma membranes of human melanoma cells. MATERIAL AND METHODS
Chemicals were purchased from Sigma, St. Louis, MO and Boehringer Manheim, Indianapolis, IN. The human RWP-1 human pancreatic cancer cell line was kindly provided by Dr. D.L. Dexter. Partial purijkation and characterization of metalloproteinases from tumor-cell membranes Tumor heterotransplantation of RWP- 1 human pancreatic cancer cells in HO NIH Swiss nude mice was performed as described by Zucker et al. (1985b). Tumor-cell suspensions were prepared and post-nuclear cell membranes were isolated using nitrogen cavitation followed by differential centrifugation. Plasma membrane fractions were isolated by sucrose density gradient centrifugation (Zucker et al., 1985~).Extrinsic membrane proteins were removed by 2~ KC1 treatment (Zucker et al., 1987b). The RWP-1 crude membranes were then extracted with 2.5% N-butanol (4:l volume ratio of butanol to membrane pellet) for 60 min at 4°C. After centrifuga-
tion at 50,000 g for 20 min, the lower protein and upper lipid phases of the butanol extraction were separated (Penefsky and Tzagoloff, 1971). The protein-rich phase was concentrated by 040% (NH,),SO, precipitation. The 50,000 g pellet remaining following butanol extraction was then extracted with 1% n-octyl glucoside in buffer for 2 hr at 4°C (Zucker et al., 1987~).The extracted proteins were dialyzed against 25 nm cacodylate/5 nm CaCl,/O.OS% Brij 35, PH 7.2, and applied to a Mono Q HR 5/5 strong anion exchange column. After application of the sample, a 42-ml gradient of 0-1 M NaCl was used to elute the bound proteins (Zucker et al., 1987~).Fractions rich in gelatinolytic and collagenolytic activity were combined, dialyzed against HEPES buffer, PH 7.2, containing 0.5 M NaCl, concentrated by ultrafiltration through an Amicon YM-10 membrane, and applied to a Superose gel permeation column operated at a flow rate of 0.5 mYmin. One-ml fractions were collected and assayed for enzymatic activity. Human A2058 melanoma cells were cultivated in DulbecCO'S modified Eagle's medium with 10% fetal bovine serum (Hoyhtya et al., 1988). Plasma membranes were prepared from cultivated cells following cell disruption in 0.02 M borate, 0.2 m EDTA, PH 10.2 as described by Thom et al. (1977). Plasma membranes were layered over a 35% (w/v) sucrose solution and isolated by centrifugation at 24,000 g for 1 hr. Serum-free conditioned medium was harvested from RWP- 1 cells (lo7 per tissue culture flask) as described by Spinucci et al. (1988). Assays for metalloproteinases The degradation of soluble native collagen at 27°C and heatdenatured collagen (gelatin) at 37°C was measured using rat skin type-I collagen labelled in vitro with [3H] formaldehyde (Zucker et al., 1987~).Degradation of type-IV collagen was measured as described by Salo et al. (1983). Enzyme activity was generally assayed after activation with trypsin. After several months of storage of the tumor fractions at 4"C, collagenolytic activity was in the active state and did not require prior activation with trypsin. The degradation of [3H]carboxymethylated (CM)transfemn was employed as a screening assay for proteoglycan-degrading activity (Okada et al., 1986). Na-p-tosyl L-lysine chloromethylketone (250 pglml) was added to the above proteinase assays to inhibit cysteine proteinases and trypsin-like serine proteinases, thereby making these assays more specific for metalloproteinases. Inhibition of gelatinolytic activity was determined by preincubating enzyme in assay buffer with various inhibitors for 1 hr at 37°C (Zucker et al., 1987~). The PH dependence of enzymatic activity was studied using
6To whom reprint requests should be sent, at the Department of Research, Veterans Administration Medical Center, Northport, NY 11768, USA. Received: December 29, 1989 and in revised form February 6, 1990.
1138
ZUCKER ET AL.
sodium acetate (PH 4-6), sodium cacodylate (PH 5-8), and Tris/HCl (PH 7-9) buffers (Zucker et al., 1 9 8 7 ~ ) . Gelatinase zymography was performed in SDS gels using 11% and 7.5% polyacrylamide cast in the presence of gelatin (0.15 mg/ml) by a modification of the method of Heussen and Dowdle (1980). Samples were not boiled prior to electrophoresis. After electrophoresis the gels were washed 3 times in 50 rtm Tris-HC1, 5 rm CaCl,, 2.5% Triton X-100 (v/v), PH 7.6 and incubated in the above buffer containing 1% Triton X-100 and 1 rm aminophenylmercuric acetate for 40 hr at 37°C. Zones of enzymatic activity were characterized by absence of Coomassie blue staining. Protein standards (BioRad, Richmond, CA) were run concurrently and approximate molecular weights of sample proteins were determined by plotting the relative mobilities of known proteins. Affinity-purified rabbit polyclonal antibodies to human typeIV collagenase were prepared which recognized a specific peptide sequence (MGPLLVATFWPELPEK) of type-IV collagenase which is not homologous to stromelysin or type-I collagenase (Hoyhtya et al., 1988). Immunoblotting for type-IV collagenase was performed using polyclonal antibodies prepared against the type-IV collagenase synthetic peptide. After blocking the non-specific binding sites with 1% bovine serum albumin, immune antibodies were incubated with nitrocellulose strips for 2 hr at room temperature. Horseradish-peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibodies were incubated for 1 hr at room temperature. The peroxidase reaction was visualized using 4-chloro- 1-naphthol and Hz02 substrates. Protein determinations were made by the method of Bohlen et al. (1973). RESULTS
-M r l k l
3.
2
1
4
5
6
200-116-
92
~
66 -
45
1
-
2
3
4
5
6
Mr Ikl
92
70 62
Pancreatic cancer cells 52 Plasma membranes were isolated from RWP-1 cancer cells following nitrogen cavitation and sucrose density gradient centrifugation and solubilized in 1% n-octyl glucoside. Gelatin zymograms of detergent-solubilized RWP- 1 plasma membrane fractions demonstrated that the crude membrane fraction and membrane bands 2 and 3 were most highly enriched in gelatb inolytic activity with zones of lysis detecting 4 proteins with gelatinolytic activity noted at 120, 92, 7 0 , and 62kDa (Fig. FIGURE1 - (a) Zymogram showing the digestion of gelatin by l a ) . Pancreatic cancer plasma membrane band 3 has been RWP-1 pancreatic cancer subcellular fractions extracted with N-octyl shown to be composed primarily of linear arrays of microvil- glucoside. Gel slabs containing 11% polyacrylamide, 0.1% SDS, and lous cell-surface membrane with a glycocalyceal coat (Zucker 0.15% co-polymerized gelatin were cast. Samples were electrophoet al., 19876). Membrane band 1 consists of large, round, resed as follows: lane 1, molecular weight standards: lane 2, cancer smooth, single profiles consistent with plasma membranes. cytosol; lane 3, crude membranes; lane 4, membrane band 1; lane 5, membrane band 2; lane 6, membrane band 3 . After the electrophoresis Band 2 is made up of components of bands 1 and 3. gels were washed in a Triton X-100containing buffer, incubated High salt treatment with 2 M KCl, which extracts extrinsic the for 24 hr, stained with Coomassie brilliant blue and destained. Moplasma membrane proteins, resulted in minimal solubilization lecular weight standard proteins were run simultaneously and are listed of metalloproteinase activity (Table I and Fig. 16). Treatment in the left-hand margin: myosin (200 m a ) , P-galactosidase (116.25 of the membranes with n-butanol resulted in partial extraction m a ) phosphorylase B (92.5 m a ) , bovine serum albumin (66.2 kDa), of metalloproteinases. The non-ionic detergent, n-octyl gluco- and ovalbumin (45 m a ) . (b) Extraction of gelatinolytic activity from side, was highly effective in solubilizing additional membrane the plasma membranes of RWP-I cancer cells. Pancreatic cancer cells metalloproteinase activity. Gelatin zymography demonstrated were disrupted by nitrogen cavitation and crude plasma membranes the presence of major gelatinase species at approximately 92, were isolated. The membrane pellet was washed in buffered 150 n w (PH7.4) by centrifugation. After washing, aliquots were treated 70 and 62kDa, with the predominance of proteinase activity NaCl with 2 M KCl at 4°C for 20 min, then centrifuged. The supernate and noted in the n-octyl glucoside extract which was concentrated pellet were separated and the pellet was washed in buffered NaCl. The by 040% (NH4)$04 precipitation. The 92-kDa band was pellet was then treated with 2.5%n-butanol and centrifuged to sepamost prominent in the membrane detergent extract. These 3 rate a solubilized upper lipid and a lower protein phase. The remaining gelatinolytic bands in the zymogram were inhibited by EDTA pellet was then treated with 1% n-octyl glucoside and centrifuged. (data not shown). The broad gelatin-degrading species of Each of the supernates was dialyzed against buffer to remove salts and 52kDa and over 15OkDa were relatively insensitive to EDTA detergents. Gelatin zymography was then performed. The contents of inactivation and therefore do not meet the criteria for metallo- the lanes are as follows: 1, whole-cell homogenate (79 pg protein applied); 2, KCl extract (39 pg); 3, butanol extract, upper lipid phase proteinases. (4 kg); 4, butanol extract, lower protein phase (9 pg); 5, n-octylgluActivation of freshly prepared membrane-extracted gelati- coside extract, 0 4 0 % (NH4),S0, precipitated proteins (10 kg); 6, nase and type-IV collagenase with trypsin was essential to n-octylglucoside extract, -100% (NH4)2S04 precipitated proteins demonstrate enzymatic activity in the soluble assays. After 1 (42 pg). Molecular weight markers are noted.
1139
CANCER PLASMA MEMBRANE GELATINASE
Tumor extracts
KC1 n-Butanol n-Octyl glucoside
Degradation of ’H-protein substrates during 2-hr incubation (bg per mg protein) Gelatin Type-IV collagen CM-transfenin
0.5 17.0 7.4
0.3 15.4 17.9
0.2 0.6 2.2
‘Pancreatic cancer cell organelles were isolated by nitrogen cavitation and differential centrifugation. The crude plasma membrane preparation was washed with buffer X 3 and then treated with 100 ml of ZM KCI at 4°C for 20 min. The pellet and supernatant were separated by centrifugation. The pellet was treated with 100 ml of 2.5% n-butanol at 4°C for 60 min. The extracted phase was separated by centrifugation. The remaining pellet was then treated with 50 ml of 1 % n-octyl glucoside for 2 hr and the supernatant and pellet were separated by centrifugation. All of the supernatants were then dialysed against buffer and assayed for protein content, gelatinolytic, type-IV collagenolytic, and carboxymethylated (CM)-transfemn degrading activity.
was 195 pg of gelatin degraded per mg protein per 2-hr incubation. Insufficient protein (400 and 15kDa metalloproteinases which are localized to the plasma mem(Fig. 3a). The specific activity of the major proteinase peak branes of human cancer cells. We have shown that mild or-
0.8
1.o
-
-
1
LI
I
0.6-
Y A
Y
0.5
0 OD (y
0
M z
0.4-
0
0
20
C 0
40, Y
I
0
Y
10
0
0
Elution Volume (ml) FIGURE 2 - Anion exchange chromatography of detergent-extractedpancreatic cancer plasma membrane proteins. Following extraction of RWP-1 human pancreatic cancer cells with n-octyl glucoside, the solubilized proteins were dialyzed against 25 m~ sodium cacodylate, 5 m~ CaCl,, 0.05%Brij, PH 7.2, and applied to a Mono Q HR5/5 strong anion exchange column (Pharmacia, Uppsala, Sweden). Proteins were eluted using a 0-1 M NaCl gradient as shown. Absorbance at 280 nm, [3H]gelatin, [3H]collagen, and [3H]CM transfemn degradationwere measured. The enclosed arrow at the top of the Figure indicates the fractions that were pooled and applied to the gel permeation column in Figure 3a.
1140
ZUCKER ET AL.
a
.09
I
.06 CI
2
1
-I
b
1v\-
Ve I Vo
0 00
Mr Ikl 200-
II
(Y
40
1
3
92.03
66 -
45 0 0
5
10
15
25
30
20-
Elution Volume (ml)
FIGURE3 - (a)Gel permeation chromatography of RWP- 1 pancreatic cancer metalloproteinases. Pancreatic cancer gelatinase active fractions from an anion exchange column were pooled, dialyzed against HEPES buffer containing 0.5 M NaCl, concentrated, and applied to a Superose 12 HR 10/30 column. A flow rate of 0.5 mVmin was maintained and 1-ml fractions were collected. The protein standards used for calculation of molecular weights (see insert) were blue dextran (Vo), apofemtin (443kDa), (3-amylase (200kDa), alcohol dehydrogenase (150kDa), bovine serum albumin (66kDa), ovalbumin (45kDa), and carbonic anhydrase (29kDa). Four peaks of activity against the different substrates were noted. (b) Immunoblot identification of type-IV collagenase isolated from detergent-extracted human pancreatic cancer plasma membranes. The gelatinase-active peak II noted in Figure 3a was treated with SDS-PAGE (7.5%) followed by electro-transfer to a nitrocellulose filter. After blocking of non-specific binding sites with 1% bovine serum albumin, immune antibodies were incubated with the strips for 2 hr at room temperature. Horseradish peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibodies were incubated with the nitrocellulose strips for 1 hr at room temperature. The peroxidase reaction was visualized with the use of 4-chloro-1-naphthol and H,O, substrate. Lane 1 contains type-IVcollagenase/gelatinase which was purified from the cytosol of human small-cell lung cancer cells (Zucker et al., 1989). Lane 2 contains the gelatinolytic peak I1 which was isolated from pancreatic cancer membranes by anion exchange and gel permeation chromatography. Both enzyme preparations had been stored for more than 1 month and contained active as well as latent forms of type-IV collagenase. Lane 3 contains molecular weight standards.
ganic solvent treatment of tumor membranes with n-butanol was able to extract a portion of the gelatinase and type-IV collagenase activities. Nakajima et al. (1989) have demonstrated that n-butanol is also capable of extracting type-IV collagenase/gelatinase from viable rat breast adenocarcinoma cells, suggesting that the 64-kDa proteinase is localized on the cell membrane outer surface. In our study the non-ionic detergent, n-octyl glucoside, was most effective in extracting gelatinasekype-IV collagenase activity from the pancreatic tumor membranes as shown in Figure 1. These data support the concept that metalloproteinases are tightly bound to the plasma membranes of cancer cells. On gelatin zymography of well-characterized pancreatic cancer plasma membrane components, 4 distinct molecular weight species with metalloproteinase activity were identified. Better resolution of the gelatin-degrading species of 92, 70, and 62kDa was achieved on zymograms containing sequentially solubilized proteinases, The extrinsic membrane proteins extracted with 2 M KCl had minimal 92-kDa gelatinolytic activity. Anion exchange chromatography was used to further sepa-
rate the metalloproteinases present in detergent-solubilized pancreatic cancer plasma membranes. The apparent higher molecular weights of 2 of the protein peaks identified on gel permeation chromatography as compared to the gelatin zymograms may be due to the presence of protein aggregates or the continued association of membrane lipids with the proteinases. A discrepancy in apparent molecular weight of tumor gelatinase on gel permeation chromatography as compared to SDSPAGE has been noted previously (Zucker et al., 1989). A similar degree of gelatinase enrichment could be achieved by using gelatin Sepharose chromatography (chromatogram not shown) followed by gel permeation chromatography (1 10 pg gelatin degradecUmg protein), but this procedure provided no advantage in purification. Pancreatic cancer membrane gelatinase was identified as a 70-kDa type-IV collagenase using affinity-purified polyclonal antibodies which specifically recognize the following amino acid sequence of type-IV collagenase; MGPLLVATFWPELPEK. This sequence is derived from a portion of the type-IV collagenase molecule which is non-homologous with interstitial collagenase and stromelysin.
1141
CANCER PLASMA MEMBRANE GELATINASE
1
D
34
6
YrlkDa] -220 -110 - 72 - 45
- 29 FIGURE4 - Zymogram showing the digestion of gelatin by human A2058 melanoma subcellular fractions (lanes 1 4 ) and immunoblot identification of type-IV collagenase (lanes 5 and 6). Plasma membranes from human melanoma cells were prepared by hypotonic lysis
and isolated by centrifugation in sucrose. Gelatin zymography was performed following application of 10 p.g of protein to each lane. Lane 1 contains whole-cell homogenate; lane 2 contains melanomaconditioned media; lane 3 contains plasma membranes; lanes 4 and 6 contain molecular weight markers. Lane 5 is an immunoblot identification of type-IV collagenase from melanoma plasma membranes. The immunoblotting procedure is the same as that described in Figure 36. A 68-kDa type-IV collagenase/gelatinasc was similarly identified from plasma membranes of human melanoma cells. On immunoblotting, pancreatic cancer and melanoma plasmamembrane type-IV collagenase appeared similar to the 75-kDa type-IV collagenase/gelatinase that we have previously purified from the cytosol of human small-cell lung cancer cells (Zucker et al., 1989) suggesting that this proteinasc is a common component of highly invasive and metastatic human cancers. The small differences in molecular weight determination between human type-IV collagcnasc isolated from pancreatic cancer cells versus lung cancer cells may be related to differences in glycosylation or in the activation state of each enzyme. The 92-kDa gelatinase that we isolated from pancreatic cancer membranes did not react with antibodies to 68- to 75-kDa type-IV collagenase, which suggests that this proteinase is derived from a different gene product. Collier et al. (1988) have shown that oncogene-transformed human bronchial epithelial cells secrete a 72-kDa gelatin degrading type-IV collagenase which is identical to the gelatinase secreted by normal fibroblasts. Mono-specific antibodies to the 66- to 72-kDa enzyme did not recognize a 92-kDa gelatinase secreted by transformed fibroblasts or polymorphonuclear leukocytes. Wilhelm et al. (1989) purified a 92-kDa type-IV collagenase/gelatinase from transformed human lung fibroblasts which was not detectable in the parental cell line. Ballin et al. (1988) and Yamagata et al. (1989) proposed that the secretion of a 92- to 95-kDa glycoprotein with gelatinolytic and type-IV collagenolytic activity by tumor cell lines correlates more closely with increased metastatic potential than does the secretion of the 60- to 70-kDa gelatinase. In the current study we noted that human pancreatic cancer cells release relatively small amounts of type-IV collagenasel gelatinase into conditioned media. In contrast, melanoma cells secrete high levels of type-IV collagenase/gelatinase relative to
enzyme concentrations in the plasma membranes. Other human cancer cell lines (HT-1080 fibrosarcoma, U-937 monocytic leukemia, and HL-60 promyelocytic leukemia) also release considerably higher levels of gelatinase than RWP- 1 cells (data not shown), which suggests that the intracellular localization and secretion of type-IV collagenaselgelatinase varies between different cancer cell lines. Detailed protein turnover studies will be required to quantitate the relative amounts of secreted versus membrane-bound enzymes in cancer cells. We proposed that the critical issue in tumor degradation of extracellular matrix during metastasis may not be the species of type-IV collagenase/gelatinase produced, but rather the surface membrane localization of the enzyme. It is tempting to speculate that the localization of proteinases on the plasma membrane places these enzymes in an optimal location for invasion of surrounding extracellular matrix. In contrast, Eisenbach et al. (1985) demonstrated that the plasma membranes of Lewis lung carcinoma displayed little type-IV collagenase activity as compared to the cytosol and, in fact, may contain a collagenase inhibitor. Differences in proteinase production by various tumor cell lines represent a well-described phenomenon (Zucker, 1988). In a previous cell culture study, we demonstrated that malignant melanoma cells shed collagenase and gelatinase into conditioned media as a component of intact membrane vesicles rather than as soluble enzymes (Zucker et ul., 1987~).This raises the possibility that the expression of metalloproteinases on tumor cell membranes may be a transient phenomenon that precedes the shedding of portions of the plasma membrane as membrane vesicles. It is a matter of speculation, however, whether the phenomenon of membrane shedding occurs in vivo as well. Sas ef ul. (1986) reported that clearing and release of basement membrane fibronectin, laminin, and type-IV collagen occurred only beneath or along the path of the tumor cell and beneath cellular processes, suggesting that it IS due to cellsurface proteinases, rather than to secreted enzymes. Experiments by Chen and Chen (1987) have indicated that extracellular matrix fibronectin degradation by Rous sarcoma virustransformed fibroblasts is highly localized and appears to occur at cell substrate contact sites within the matrix. An integral membrane metalloproteinase of 15OkDa was extracted by detergents from tumor plasma membranes and was absent in normal cells. Our demonstration of the enrichment of metalloproteinases in the plasma membranes of cancer cells differs from descriptions of rapid secretion of collagenase from normal mesenchyma1 cells. Valle and Bauer (1979) and Nagase et al. (1983) noted the appearance of collagenase in the culture media of skin and synovial fibroblasts within 20 min after the initial appearance of radiolabelled enzyme in mesenchymal cells. These observations in mesenchymal cells led to the general conclusion that intracellular transport of collagenase after synthesis on polyribosomes follows a pathway common to other secretory proteins, and that negligible intracellular storage occurs. The mechanism whereby malignant cells, but not their normal counterparts, localize metalloproteinases in their plasma membranes remains an area of considerable interest. ACKNOWLEDGEMENTS
The authors thank Mr. D. Moutsiakis for his technical assistance. This research was supported by Merit Review Research Funds from the Veterans Administration Medical Center and the Schermerhorn Cancer Foundation.
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SALO,T., LIOTTA,L.A. and TRYGVASON, L., Purification and characterization of a murine basement membrane collagen-degrading enzyme secreted by metastatic tumor cells. J . bid. Chem., 258, 3058-3063 (1983). SAS,D.F., MCCARTHY, J.B. and FURCHT,L.T., Clearing and release of basement membrane proteins from substrates by metastatic tumor cell variants. Cancer Res., 46, 3082-3089 (1986). SPINUCCI, C., ZUCKER,S., WIEMAN,J.M., LYSIK,R.M., IMHOF,B., RAMAMURTHY, N., LIOTTA,L.A. and NAGASE,H., Purification of a gelatin degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts. J . nat. Cancer Inst., 80, 1416-1421 (1988). THOM,D., POWELL,A.J., LLOYD,C.W. and REES,D.A., Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem. J. 168, 187-194 (1977). VALLE,K-I. and BAUER,E.A., Biosynthesis of collagenase by human skin fibroblasts in monolayer culture. J. biol. Chem., 254, 10115-10122 (1979). WILHELM, S.M., COLLIER,I.E., MARMER, B.L., EISEN,A.Z., GRANT, G.A. and GOLDBERG, G.I., SV40-transformedhuman lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J . bid. Chem., 261, 17213-17221 (1989). S., TANAKA, R., ITO, Y. and SHIMIZU,S., Gelatinases of YAMAGATA, murine metastatic tumor cells. Biochem. biophys. Res. Comm., 158, 228234 (1989). ZUCKER,S., A critical appraisal of the role of proteolytic enzymes in cancer invasion: Emphasis on tumor surface proteinases. Cancer Invest., 6, 219-231 (1988). S., LYSIK,R.M., RAMAMURTHY, M.S., GOLUB,L., WIEMAN, J. ZUCKER, and WILKIE,D.P., Diversity of melanoma membrane metalloproteinases: Inhibition of collagenolytic and cytolytic activity by minocycline. J . nut. Cancer Inst., 75, 517-525 (1985~). ZUCKER,S., LYSIK,R.M., WIEMAN,J., WILKIE, D.P. and LANE,B., Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinasesin collagenolysisand cytolysis. Cancer Res., 45, 6168-6178 (19856). ZUCKER, S., TURPEENNIEMI-HUJANEN, T., RAMAMURTHY, N., WIEMAN, J., LYSIK,R., GOREVIC,P., LIOTTA,L.A., SIMON,S.R. and GOLUB, L.M., Purification and characterization of a connective tissue degrading metalloproteinase from the cytosol of metastatic melanoma cells. Biochem. J., 245, 429-437 (1987a). ZUCKER, S., WIEMAN, J., LYSIK,R.M., IMHOF,B., NAGASE, H., RAMAMURTHY, N., LIOTTA, L.A. and GOLUB,L.M., Gelatin degrading type IV collagenase isolated from human small cell lung cancer cells. Invas. Metast., 9, 167-181 (1989). ZUCKER, S., WIEMAN, J.M., LYSIK,R.M., WILKIE,D., RAMAMURTHY, N.S., GOLUB,L.M. and LANE,B., Enrichment of collagen and gelatin degrading activities in the plasma membranes of human cancer cells. Cancer Res., 47, 1608-1614 (19876). S., WIEMAN,J.M., LYSIK,R.M., WILKIE,D., RAMAMURTHY, ZUCKER, N. and LANE,B., Metastatic mouse melanoma cells release collagengelatin degrading metalloproteinases as components of shed membrane vesicles. Biochim. biophys. Acta, 924, 225-237 (1987~).
Publication of the International Union Against Cancer Publication de I'Union internationale Contre le Cancer
EXTRACTION OF TYPE-IV COLLAGENASE/GELATINASE FROM PLASMA MEMBRANES OF HUMAN CANCER CELLS Stanley ZUCKER's273*6, Ute M. MOLL^, Rita M. LYSIK',Elizabeth I. DIMASSIMO',William G.STETLER-STEVENSON', Lance A. LIOTTA5 and Janine w. SCHWEDES' Departments of 'Research and 2Medicine, VeteransAdministration Medical Center, Northport, NY I 1768; Departments of 3Medicine and 4Pathology,Health Science Center, State University of New York, at Stony Brook, NY 11 794; and sLaboratory of Pathology, National Cancer Institute, Bethesda, MD, USA. Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octylglucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62kDa. lmmunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenasel gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagendgelatinase of 68kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.
Studies dealing with mechanisms of collagen degradation during cancer metastasis have focused primarily on cellsecreted collagenolytic enzymes (Liotta et al., 1980; Salo et al., 1983; Gabrisa et al., 1987; Nakajima et al., 1987; Spinucci et al., 1988; Bonfil et al., 1989). Cancer cells have also been shown to have collagen- and gelatin-degrading metalloproteinases localized to their plasma membranes (Zucker et al., 1985a,b, 1987b). We have proposed that the surface membrane localization of these proteinases is of crucial importance in the process of cancer invasion and metastasis. In the current study we have used an organic solvent and a non-ionic detergent to extract metalloproteinases from human pancreatic cancer plasma membranes. These membrane enzymes have been partially purified by chromatography and have been characterized by gelatin zymography and immunoblotting procedures. Type-IV collagenase/gelatinase was also identified in the plasma membranes of human melanoma cells. MATERIAL AND METHODS
Chemicals were purchased from Sigma, St. Louis, MO and Boehringer Manheim, Indianapolis, IN. The human RWP-1 human pancreatic cancer cell line was kindly provided by Dr. D.L. Dexter. Partial purijkation and characterization of metalloproteinases from tumor-cell membranes Tumor heterotransplantation of RWP- 1 human pancreatic cancer cells in HO NIH Swiss nude mice was performed as described by Zucker et al. (1985b). Tumor-cell suspensions were prepared and post-nuclear cell membranes were isolated using nitrogen cavitation followed by differential centrifugation. Plasma membrane fractions were isolated by sucrose density gradient centrifugation (Zucker et al., 1985~).Extrinsic membrane proteins were removed by 2~ KC1 treatment (Zucker et al., 1987b). The RWP-1 crude membranes were then extracted with 2.5% N-butanol (4:l volume ratio of butanol to membrane pellet) for 60 min at 4°C. After centrifuga-
tion at 50,000 g for 20 min, the lower protein and upper lipid phases of the butanol extraction were separated (Penefsky and Tzagoloff, 1971). The protein-rich phase was concentrated by 040% (NH,),SO, precipitation. The 50,000 g pellet remaining following butanol extraction was then extracted with 1% n-octyl glucoside in buffer for 2 hr at 4°C (Zucker et al., 1987~).The extracted proteins were dialyzed against 25 nm cacodylate/5 nm CaCl,/O.OS% Brij 35, PH 7.2, and applied to a Mono Q HR 5/5 strong anion exchange column. After application of the sample, a 42-ml gradient of 0-1 M NaCl was used to elute the bound proteins (Zucker et al., 1987~).Fractions rich in gelatinolytic and collagenolytic activity were combined, dialyzed against HEPES buffer, PH 7.2, containing 0.5 M NaCl, concentrated by ultrafiltration through an Amicon YM-10 membrane, and applied to a Superose gel permeation column operated at a flow rate of 0.5 mYmin. One-ml fractions were collected and assayed for enzymatic activity. Human A2058 melanoma cells were cultivated in DulbecCO'S modified Eagle's medium with 10% fetal bovine serum (Hoyhtya et al., 1988). Plasma membranes were prepared from cultivated cells following cell disruption in 0.02 M borate, 0.2 m EDTA, PH 10.2 as described by Thom et al. (1977). Plasma membranes were layered over a 35% (w/v) sucrose solution and isolated by centrifugation at 24,000 g for 1 hr. Serum-free conditioned medium was harvested from RWP- 1 cells (lo7 per tissue culture flask) as described by Spinucci et al. (1988). Assays for metalloproteinases The degradation of soluble native collagen at 27°C and heatdenatured collagen (gelatin) at 37°C was measured using rat skin type-I collagen labelled in vitro with [3H] formaldehyde (Zucker et al., 1987~).Degradation of type-IV collagen was measured as described by Salo et al. (1983). Enzyme activity was generally assayed after activation with trypsin. After several months of storage of the tumor fractions at 4"C, collagenolytic activity was in the active state and did not require prior activation with trypsin. The degradation of [3H]carboxymethylated (CM)transfemn was employed as a screening assay for proteoglycan-degrading activity (Okada et al., 1986). Na-p-tosyl L-lysine chloromethylketone (250 pglml) was added to the above proteinase assays to inhibit cysteine proteinases and trypsin-like serine proteinases, thereby making these assays more specific for metalloproteinases. Inhibition of gelatinolytic activity was determined by preincubating enzyme in assay buffer with various inhibitors for 1 hr at 37°C (Zucker et al., 1987~). The PH dependence of enzymatic activity was studied using
6To whom reprint requests should be sent, at the Department of Research, Veterans Administration Medical Center, Northport, NY 11768, USA. Received: December 29, 1989 and in revised form February 6, 1990.
1138
ZUCKER ET AL.
sodium acetate (PH 4-6), sodium cacodylate (PH 5-8), and Tris/HCl (PH 7-9) buffers (Zucker et al., 1 9 8 7 ~ ) . Gelatinase zymography was performed in SDS gels using 11% and 7.5% polyacrylamide cast in the presence of gelatin (0.15 mg/ml) by a modification of the method of Heussen and Dowdle (1980). Samples were not boiled prior to electrophoresis. After electrophoresis the gels were washed 3 times in 50 rtm Tris-HC1, 5 rm CaCl,, 2.5% Triton X-100 (v/v), PH 7.6 and incubated in the above buffer containing 1% Triton X-100 and 1 rm aminophenylmercuric acetate for 40 hr at 37°C. Zones of enzymatic activity were characterized by absence of Coomassie blue staining. Protein standards (BioRad, Richmond, CA) were run concurrently and approximate molecular weights of sample proteins were determined by plotting the relative mobilities of known proteins. Affinity-purified rabbit polyclonal antibodies to human typeIV collagenase were prepared which recognized a specific peptide sequence (MGPLLVATFWPELPEK) of type-IV collagenase which is not homologous to stromelysin or type-I collagenase (Hoyhtya et al., 1988). Immunoblotting for type-IV collagenase was performed using polyclonal antibodies prepared against the type-IV collagenase synthetic peptide. After blocking the non-specific binding sites with 1% bovine serum albumin, immune antibodies were incubated with nitrocellulose strips for 2 hr at room temperature. Horseradish-peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibodies were incubated for 1 hr at room temperature. The peroxidase reaction was visualized using 4-chloro- 1-naphthol and Hz02 substrates. Protein determinations were made by the method of Bohlen et al. (1973). RESULTS
-M r l k l
3.
2
1
4
5
6
200-116-
92
~
66 -
45
1
-
2
3
4
5
6
Mr Ikl
92
70 62
Pancreatic cancer cells 52 Plasma membranes were isolated from RWP-1 cancer cells following nitrogen cavitation and sucrose density gradient centrifugation and solubilized in 1% n-octyl glucoside. Gelatin zymograms of detergent-solubilized RWP- 1 plasma membrane fractions demonstrated that the crude membrane fraction and membrane bands 2 and 3 were most highly enriched in gelatb inolytic activity with zones of lysis detecting 4 proteins with gelatinolytic activity noted at 120, 92, 7 0 , and 62kDa (Fig. FIGURE1 - (a) Zymogram showing the digestion of gelatin by l a ) . Pancreatic cancer plasma membrane band 3 has been RWP-1 pancreatic cancer subcellular fractions extracted with N-octyl shown to be composed primarily of linear arrays of microvil- glucoside. Gel slabs containing 11% polyacrylamide, 0.1% SDS, and lous cell-surface membrane with a glycocalyceal coat (Zucker 0.15% co-polymerized gelatin were cast. Samples were electrophoet al., 19876). Membrane band 1 consists of large, round, resed as follows: lane 1, molecular weight standards: lane 2, cancer smooth, single profiles consistent with plasma membranes. cytosol; lane 3, crude membranes; lane 4, membrane band 1; lane 5, membrane band 2; lane 6, membrane band 3 . After the electrophoresis Band 2 is made up of components of bands 1 and 3. gels were washed in a Triton X-100containing buffer, incubated High salt treatment with 2 M KCl, which extracts extrinsic the for 24 hr, stained with Coomassie brilliant blue and destained. Moplasma membrane proteins, resulted in minimal solubilization lecular weight standard proteins were run simultaneously and are listed of metalloproteinase activity (Table I and Fig. 16). Treatment in the left-hand margin: myosin (200 m a ) , P-galactosidase (116.25 of the membranes with n-butanol resulted in partial extraction m a ) phosphorylase B (92.5 m a ) , bovine serum albumin (66.2 kDa), of metalloproteinases. The non-ionic detergent, n-octyl gluco- and ovalbumin (45 m a ) . (b) Extraction of gelatinolytic activity from side, was highly effective in solubilizing additional membrane the plasma membranes of RWP-I cancer cells. Pancreatic cancer cells metalloproteinase activity. Gelatin zymography demonstrated were disrupted by nitrogen cavitation and crude plasma membranes the presence of major gelatinase species at approximately 92, were isolated. The membrane pellet was washed in buffered 150 n w (PH7.4) by centrifugation. After washing, aliquots were treated 70 and 62kDa, with the predominance of proteinase activity NaCl with 2 M KCl at 4°C for 20 min, then centrifuged. The supernate and noted in the n-octyl glucoside extract which was concentrated pellet were separated and the pellet was washed in buffered NaCl. The by 040% (NH4)$04 precipitation. The 92-kDa band was pellet was then treated with 2.5%n-butanol and centrifuged to sepamost prominent in the membrane detergent extract. These 3 rate a solubilized upper lipid and a lower protein phase. The remaining gelatinolytic bands in the zymogram were inhibited by EDTA pellet was then treated with 1% n-octyl glucoside and centrifuged. (data not shown). The broad gelatin-degrading species of Each of the supernates was dialyzed against buffer to remove salts and 52kDa and over 15OkDa were relatively insensitive to EDTA detergents. Gelatin zymography was then performed. The contents of inactivation and therefore do not meet the criteria for metallo- the lanes are as follows: 1, whole-cell homogenate (79 pg protein applied); 2, KCl extract (39 pg); 3, butanol extract, upper lipid phase proteinases. (4 kg); 4, butanol extract, lower protein phase (9 pg); 5, n-octylgluActivation of freshly prepared membrane-extracted gelati- coside extract, 0 4 0 % (NH4),S0, precipitated proteins (10 kg); 6, nase and type-IV collagenase with trypsin was essential to n-octylglucoside extract, -100% (NH4)2S04 precipitated proteins demonstrate enzymatic activity in the soluble assays. After 1 (42 pg). Molecular weight markers are noted.
1139
CANCER PLASMA MEMBRANE GELATINASE
Tumor extracts
KC1 n-Butanol n-Octyl glucoside
Degradation of ’H-protein substrates during 2-hr incubation (bg per mg protein) Gelatin Type-IV collagen CM-transfenin
0.5 17.0 7.4
0.3 15.4 17.9
0.2 0.6 2.2
‘Pancreatic cancer cell organelles were isolated by nitrogen cavitation and differential centrifugation. The crude plasma membrane preparation was washed with buffer X 3 and then treated with 100 ml of ZM KCI at 4°C for 20 min. The pellet and supernatant were separated by centrifugation. The pellet was treated with 100 ml of 2.5% n-butanol at 4°C for 60 min. The extracted phase was separated by centrifugation. The remaining pellet was then treated with 50 ml of 1 % n-octyl glucoside for 2 hr and the supernatant and pellet were separated by centrifugation. All of the supernatants were then dialysed against buffer and assayed for protein content, gelatinolytic, type-IV collagenolytic, and carboxymethylated (CM)-transfemn degrading activity.
was 195 pg of gelatin degraded per mg protein per 2-hr incubation. Insufficient protein (400 and 15kDa metalloproteinases which are localized to the plasma mem(Fig. 3a). The specific activity of the major proteinase peak branes of human cancer cells. We have shown that mild or-
0.8
1.o
-
-
1
LI
I
0.6-
Y A
Y
0.5
0 OD (y
0
M z
0.4-
0
0
20
C 0
40, Y
I
0
Y
10
0
0
Elution Volume (ml) FIGURE 2 - Anion exchange chromatography of detergent-extractedpancreatic cancer plasma membrane proteins. Following extraction of RWP-1 human pancreatic cancer cells with n-octyl glucoside, the solubilized proteins were dialyzed against 25 m~ sodium cacodylate, 5 m~ CaCl,, 0.05%Brij, PH 7.2, and applied to a Mono Q HR5/5 strong anion exchange column (Pharmacia, Uppsala, Sweden). Proteins were eluted using a 0-1 M NaCl gradient as shown. Absorbance at 280 nm, [3H]gelatin, [3H]collagen, and [3H]CM transfemn degradationwere measured. The enclosed arrow at the top of the Figure indicates the fractions that were pooled and applied to the gel permeation column in Figure 3a.
1140
ZUCKER ET AL.
a
.09
I
.06 CI
2
1
-I
b
1v\-
Ve I Vo
0 00
Mr Ikl 200-
II
(Y
40
1
3
92.03
66 -
45 0 0
5
10
15
25
30
20-
Elution Volume (ml)
FIGURE3 - (a)Gel permeation chromatography of RWP- 1 pancreatic cancer metalloproteinases. Pancreatic cancer gelatinase active fractions from an anion exchange column were pooled, dialyzed against HEPES buffer containing 0.5 M NaCl, concentrated, and applied to a Superose 12 HR 10/30 column. A flow rate of 0.5 mVmin was maintained and 1-ml fractions were collected. The protein standards used for calculation of molecular weights (see insert) were blue dextran (Vo), apofemtin (443kDa), (3-amylase (200kDa), alcohol dehydrogenase (150kDa), bovine serum albumin (66kDa), ovalbumin (45kDa), and carbonic anhydrase (29kDa). Four peaks of activity against the different substrates were noted. (b) Immunoblot identification of type-IV collagenase isolated from detergent-extracted human pancreatic cancer plasma membranes. The gelatinase-active peak II noted in Figure 3a was treated with SDS-PAGE (7.5%) followed by electro-transfer to a nitrocellulose filter. After blocking of non-specific binding sites with 1% bovine serum albumin, immune antibodies were incubated with the strips for 2 hr at room temperature. Horseradish peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibodies were incubated with the nitrocellulose strips for 1 hr at room temperature. The peroxidase reaction was visualized with the use of 4-chloro-1-naphthol and H,O, substrate. Lane 1 contains type-IVcollagenase/gelatinase which was purified from the cytosol of human small-cell lung cancer cells (Zucker et al., 1989). Lane 2 contains the gelatinolytic peak I1 which was isolated from pancreatic cancer membranes by anion exchange and gel permeation chromatography. Both enzyme preparations had been stored for more than 1 month and contained active as well as latent forms of type-IV collagenase. Lane 3 contains molecular weight standards.
ganic solvent treatment of tumor membranes with n-butanol was able to extract a portion of the gelatinase and type-IV collagenase activities. Nakajima et al. (1989) have demonstrated that n-butanol is also capable of extracting type-IV collagenase/gelatinase from viable rat breast adenocarcinoma cells, suggesting that the 64-kDa proteinase is localized on the cell membrane outer surface. In our study the non-ionic detergent, n-octyl glucoside, was most effective in extracting gelatinasekype-IV collagenase activity from the pancreatic tumor membranes as shown in Figure 1. These data support the concept that metalloproteinases are tightly bound to the plasma membranes of cancer cells. On gelatin zymography of well-characterized pancreatic cancer plasma membrane components, 4 distinct molecular weight species with metalloproteinase activity were identified. Better resolution of the gelatin-degrading species of 92, 70, and 62kDa was achieved on zymograms containing sequentially solubilized proteinases, The extrinsic membrane proteins extracted with 2 M KCl had minimal 92-kDa gelatinolytic activity. Anion exchange chromatography was used to further sepa-
rate the metalloproteinases present in detergent-solubilized pancreatic cancer plasma membranes. The apparent higher molecular weights of 2 of the protein peaks identified on gel permeation chromatography as compared to the gelatin zymograms may be due to the presence of protein aggregates or the continued association of membrane lipids with the proteinases. A discrepancy in apparent molecular weight of tumor gelatinase on gel permeation chromatography as compared to SDSPAGE has been noted previously (Zucker et al., 1989). A similar degree of gelatinase enrichment could be achieved by using gelatin Sepharose chromatography (chromatogram not shown) followed by gel permeation chromatography (1 10 pg gelatin degradecUmg protein), but this procedure provided no advantage in purification. Pancreatic cancer membrane gelatinase was identified as a 70-kDa type-IV collagenase using affinity-purified polyclonal antibodies which specifically recognize the following amino acid sequence of type-IV collagenase; MGPLLVATFWPELPEK. This sequence is derived from a portion of the type-IV collagenase molecule which is non-homologous with interstitial collagenase and stromelysin.
1141
CANCER PLASMA MEMBRANE GELATINASE
1
D
34
6
YrlkDa] -220 -110 - 72 - 45
- 29 FIGURE4 - Zymogram showing the digestion of gelatin by human A2058 melanoma subcellular fractions (lanes 1 4 ) and immunoblot identification of type-IV collagenase (lanes 5 and 6). Plasma membranes from human melanoma cells were prepared by hypotonic lysis
and isolated by centrifugation in sucrose. Gelatin zymography was performed following application of 10 p.g of protein to each lane. Lane 1 contains whole-cell homogenate; lane 2 contains melanomaconditioned media; lane 3 contains plasma membranes; lanes 4 and 6 contain molecular weight markers. Lane 5 is an immunoblot identification of type-IV collagenase from melanoma plasma membranes. The immunoblotting procedure is the same as that described in Figure 36. A 68-kDa type-IV collagenase/gelatinasc was similarly identified from plasma membranes of human melanoma cells. On immunoblotting, pancreatic cancer and melanoma plasmamembrane type-IV collagenase appeared similar to the 75-kDa type-IV collagenase/gelatinase that we have previously purified from the cytosol of human small-cell lung cancer cells (Zucker et al., 1989) suggesting that this proteinasc is a common component of highly invasive and metastatic human cancers. The small differences in molecular weight determination between human type-IV collagcnasc isolated from pancreatic cancer cells versus lung cancer cells may be related to differences in glycosylation or in the activation state of each enzyme. The 92-kDa gelatinase that we isolated from pancreatic cancer membranes did not react with antibodies to 68- to 75-kDa type-IV collagenase, which suggests that this proteinase is derived from a different gene product. Collier et al. (1988) have shown that oncogene-transformed human bronchial epithelial cells secrete a 72-kDa gelatin degrading type-IV collagenase which is identical to the gelatinase secreted by normal fibroblasts. Mono-specific antibodies to the 66- to 72-kDa enzyme did not recognize a 92-kDa gelatinase secreted by transformed fibroblasts or polymorphonuclear leukocytes. Wilhelm et al. (1989) purified a 92-kDa type-IV collagenase/gelatinase from transformed human lung fibroblasts which was not detectable in the parental cell line. Ballin et al. (1988) and Yamagata et al. (1989) proposed that the secretion of a 92- to 95-kDa glycoprotein with gelatinolytic and type-IV collagenolytic activity by tumor cell lines correlates more closely with increased metastatic potential than does the secretion of the 60- to 70-kDa gelatinase. In the current study we noted that human pancreatic cancer cells release relatively small amounts of type-IV collagenasel gelatinase into conditioned media. In contrast, melanoma cells secrete high levels of type-IV collagenase/gelatinase relative to
enzyme concentrations in the plasma membranes. Other human cancer cell lines (HT-1080 fibrosarcoma, U-937 monocytic leukemia, and HL-60 promyelocytic leukemia) also release considerably higher levels of gelatinase than RWP- 1 cells (data not shown), which suggests that the intracellular localization and secretion of type-IV collagenaselgelatinase varies between different cancer cell lines. Detailed protein turnover studies will be required to quantitate the relative amounts of secreted versus membrane-bound enzymes in cancer cells. We proposed that the critical issue in tumor degradation of extracellular matrix during metastasis may not be the species of type-IV collagenase/gelatinase produced, but rather the surface membrane localization of the enzyme. It is tempting to speculate that the localization of proteinases on the plasma membrane places these enzymes in an optimal location for invasion of surrounding extracellular matrix. In contrast, Eisenbach et al. (1985) demonstrated that the plasma membranes of Lewis lung carcinoma displayed little type-IV collagenase activity as compared to the cytosol and, in fact, may contain a collagenase inhibitor. Differences in proteinase production by various tumor cell lines represent a well-described phenomenon (Zucker, 1988). In a previous cell culture study, we demonstrated that malignant melanoma cells shed collagenase and gelatinase into conditioned media as a component of intact membrane vesicles rather than as soluble enzymes (Zucker et ul., 1987~).This raises the possibility that the expression of metalloproteinases on tumor cell membranes may be a transient phenomenon that precedes the shedding of portions of the plasma membrane as membrane vesicles. It is a matter of speculation, however, whether the phenomenon of membrane shedding occurs in vivo as well. Sas ef ul. (1986) reported that clearing and release of basement membrane fibronectin, laminin, and type-IV collagen occurred only beneath or along the path of the tumor cell and beneath cellular processes, suggesting that it IS due to cellsurface proteinases, rather than to secreted enzymes. Experiments by Chen and Chen (1987) have indicated that extracellular matrix fibronectin degradation by Rous sarcoma virustransformed fibroblasts is highly localized and appears to occur at cell substrate contact sites within the matrix. An integral membrane metalloproteinase of 15OkDa was extracted by detergents from tumor plasma membranes and was absent in normal cells. Our demonstration of the enrichment of metalloproteinases in the plasma membranes of cancer cells differs from descriptions of rapid secretion of collagenase from normal mesenchyma1 cells. Valle and Bauer (1979) and Nagase et al. (1983) noted the appearance of collagenase in the culture media of skin and synovial fibroblasts within 20 min after the initial appearance of radiolabelled enzyme in mesenchymal cells. These observations in mesenchymal cells led to the general conclusion that intracellular transport of collagenase after synthesis on polyribosomes follows a pathway common to other secretory proteins, and that negligible intracellular storage occurs. The mechanism whereby malignant cells, but not their normal counterparts, localize metalloproteinases in their plasma membranes remains an area of considerable interest. ACKNOWLEDGEMENTS
The authors thank Mr. D. Moutsiakis for his technical assistance. This research was supported by Merit Review Research Funds from the Veterans Administration Medical Center and the Schermerhorn Cancer Foundation.
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