©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Plasma Membrane-associated Component of Ovarian Adenocarcinoma Cells Enhances the Catalytic Efficiency of Matrix Metalloproteinase-2 (*)

(Received for publication, November 21, 1994)

Timothy N. Young Salvatore V. Pizzo M. Sharon Stack (1)(§)

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity. The purpose of this study was to determine if association of MMP-2 with the plasma membrane also modulates the catalytic efficiency of the active enzyme. Using density gradient centrifugation, we isolated the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA 432, previously described either to express MMP-2 or to express no gelatinolytic metalloproteinases, respectively. While DOV 13 cells contained plasma membrane-associated MMP-2 and OVCA 432 did not, both cell types were able to bind exogenous MMP-2. Furthermore, plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [^14C]gelatin I substrate by both MMP-2bullettissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory activity was dose-dependent, soluble in Triton X-100, and abolished by trypsin treatment of the membranes, but was stable to heat treatment. Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis. These data suggest that, in addition to promoting zymogen activation, cell surface binding of MMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) nevertheless may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis.


INTRODUCTION

Proteolytic degradation of the extracellular matrix is a postulated mechanism by which tumor cells initiate tissue invasion and metastasis(1) . Overexpression of plasminogen activators (PAs) (^1)has long been associated with malignancy and, more recently, elevated secretion of matrix metalloproteinases (MMPs) by tumor cells also has been demonstrated (2, 3, 4) . The combined actions of the broad specificity serine proteinase plasmin (formed upon conversion of the zymogen plasminogen by PAs) and MMPs on the glycoprotein, proteoglycan, and collagenous components of basement membranes enable tumor cells to penetrate these tissue barriers. Furthermore, increasing evidence demonstrates that cell surface localization of proteinases is a common cellular strategy for regulating pericellular proteolysis, as exemplified by urinary-type PA (u-PA), the activity of which is localized predominantly on the cell surface via binding to a specific receptor, u-PA receptor(5, 6, 7, 8, 9) .

Although recent evidence suggests that MMPs may also be associated with the cell surface, the mechanism of these interactions as well as the potential relevance to tumor metastasis remains to be elucidated. Previous studies employing subcellular fractionation and electron microscopic immunolocalization have demonstrated MMP-2 and MMP-9-like type IV collagenases as well as interstitial collagenase (MMP-1) associated with the plasma membranes of human lung and pancreatic cancer cells(10, 11, 12) . Furthermore, a class of specific, saturable cell surface binding sites for MMP-2 (K = 2 nM) has been described on breast cancer cells(13) , although no membrane protein that functions as a metalloproteinase receptor has been identified. In addition, a plasma membrane-associated component that activates the zymogen of MMP-2 (proMMP-2) has been described(14, 15, 16, 17) . This activator is detergent-soluble and trypsin- and heat-sensitive, and its function requires the carboxyl-terminal domain of MMP-2(14, 15, 16, 17) . A newly discovered ``membrane-type'' MMP (MT-MMP), an MMP family member with a transmembrane domain, catalyzes the activation of proMMP-2 when overexpressed in HT1080 cells (18) and may be related to the previously described membrane activator. These data indicate that a distinct cell surface binding molecule as well as a membrane-tethered activator may be present on the cell surface. Taken together, these findings suggest that cell surface localization may function as a mechanism by which the activity of MMPs, particularly MMP-2, is regulated.

The current study is designed to assess the effect of membrane association on the catalytic activity of active MMP-2. To address this question, we have prepared the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA 432, which either express MMP-2 or express no gelatinolytic metalloproteinases, respectively (19) . These membrane fractions were utilized to determine the effects of plasma membrane association on the rate of [^14C]gelatin I hydrolysis by active MMP-2. Our data demonstrate that the catalytic activity of MMP-2 is enhanced by plasma membrane association, suggesting a mechanism by which the activity of MMPs may be regulated.


EXPERIMENTAL PROCEDURES

Materials

Percoll and density marker beads were the products of Pharmacia, Uppsala, Sweden. Human TIMP-2-free MMP-2 was the generous gift of Dr. Hideaki Nagase, University of Kansas Medical Center, Kansas City, KS. Bovine collagen type I, p-aminophenylmercuric acetate (APMA), and 1,10-phenanthroline (o-phenanthroline), 3,4-dichloroisocoumarin (DCI), trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), and soybean trypsin inhibitor (SBTI) were purchased from Sigma. Gelatin type I was prepared by denaturation of collagen type I at 60 °C for 20 min. Collagen type I was radiolabeled with [^14C]acetic anhydride according to the method of Cawston and Barrett(20) . [^14C]Acetic anhydride was supplied by DuPont NEN. All other chemicals were of reagent grade.

Purification of MMP-2bulletTIMP-2 Complex

Human proMMP-2bulletTIMP-2 complex was purified from DOV 13 ovarian adenocarcinoma cell serum-free conditioned medium. The first purification step employed gelatin-agarose chromatography (21) followed by dialysis against 50 mM HEPES, 10 mM CaCl(2), 0.05% Brij 35, pH 7.2. The second step employed DEAE-Sephacel chromatography, in which detergent exchange was carried out during washing with 50 mM HEPES, 10 mM CaCl(2), 0.02% CHAPS, pH 7.2. Following a 1.0 M NaCl elution and concentration by ultrafiltration, the protein was desalted into 50 mM Tris-HCl, 0.02% CHAPS, pH 7.5, using a NAP-5 column (Pharmacia).

Cell Culture and Subcellular Fractionation

The human ovarian adenocarcinoma cell lines DOV 13 and OVCA 432 were the gift of Dr. Robert C. Bast, Jr., M.D. Anderson Cancer Center, Houston, TX, and were maintained in culture as described previously(22) . DOV 13 and OVCA 432 cells were fractionated essentially as described by Zucker et al.(10) , except that a Percoll gradient rather than sucrose was used. Briefly, confluent adherent cells (1 times 10^8 cells total) were incubated in serum-free medium for 18 h and harvested with phosphate-buffered saline containing 2 mM EDTA. After the cells were swelled hypotonically and subjected to nitrogen cavitation at 300 p.s.i. of N(2) for 20 min on ice, 0.05 mM DCI, 0.01 mM E-64, and 20 µg/ml SBTI were added, the nuclear fraction was removed, and the membrane pellet was obtained as described previously(10) . Following resuspension of the crude membrane fraction using a Potter-Elvehjem homogenizer, the membranes were applied to a 20% (v/v) Percoll solution in 20 mM Tris-HCl, 0.25 M sucrose, pH 7.4 (Tris-sucrose) (12 ml total). A parallel gradient was prepared including density marker beads. The membrane fractions were formed by centrifugation of the Percoll solution at 50,000 times g for 30 min at 4 °C. A total of six approximately 1-ml fractions were collected and rendered free of Percoll by repeated centrifugation at 50,000 times g for 1 h until membranes formed an adherent pellet. The 5`-nucleotidase activity of membrane fractions was determined as described previously(23) . Membrane extracts were prepared by incubating aliquots of membrane fractions with 1.0% Triton X-100 at 4 °C for 16 h, followed by clarification by centrifugation (50,000 times g for 30 min). Protein determinations on membrane extracts employed BCA assays (Pierce). For trypsin treatments, aliquots of membranes were incubated with 20 µg/ml trypsin (12,000 units/mg) at 37 °C for 2 h, inhibited with 0.05 mM DCI and 100 µg/ml SBTI, washed thrice with Tris-sucrose, and resuspended with Tris-sucrose to the original volume. For some experiments, membrane extracts of the trypsin-treated membranes were prepared with 1.0% Triton X-100 as above.

Zymography

The gelatinase activities present in membrane preparations were determined using SDS-PAGE zymography as described previously(24) . SDS-PAGE gels were prepared with 9% acrylamide and 0.1% gelatin, and samples were electrophoresed without reduction. Following removal of SDS, the gels were incubated in 20 mM glycine, 10 mM CaCl(2), 1 µM ZnCl(2), pH 8.3 for 24 h at 37 °C and stained.

Gelatin Degradation Assays

The degradation of [^14C]gelatin type I was measured essentially as described by Harris and Krane(25) . ProMMP-2bulletTIMP-2 complex (100 ng) or TIMP-2-free proMMP-2 (20 ng) was activated with 1 mM APMA at 37 °C for 1 h, and incubated in the presence or absence of membrane fractions for 1 h at 37 °C in 20 mM Tris-HCl, 5 mM CaCl(2), 0.25 M sucrose, pH 7.4 (Tris-Ca-sucrose), and 0.5 mM APMA. Reactions were initiated by addition of various quantities of [^14C]gelatin type I (1 mg/ml in 50 mM Tris-HCl, 0.2 M NaCl, pH 7.6) and proceeded at 37 °C for 2-4 h. The reactions were stopped with 15% trichloroacetic acid and soluble radioactivity determined as described previously(25) .


RESULTS AND DISCUSSION

Identification of Membrane-associated MMP-2 in Ovarian Cancer Cells

To determine whether ovarian carcinoma cells contain plasma membrane-associated MMP activity, DOV 13 and OVCA 432 cells were lysed and fractionated using a combination of differential and density gradient centrifugation. Gelatin substrate zymography of the membrane fractions (six fractions total) demonstrated that DOV 13 cells have an o-phenanthroline-inhibitable gelatinase comigrating with MMP-2 that fractionates with density fractions 2-4 (density = 1.042-1.048 g/ml) (Fig. 1, A, lane 1, and B). When the plasma membrane containing fractions were identified by measuring 5`-nucleotidase activity(10) , DOV 13 fractions 2-4 also were found to contain high levels of the plasma membrane marker (Fig. 2A), suggesting that the membrane-associated MMP-2 activity is bound to the plasma membrane. Plasma membrane-associated MMP-2 was dissociated from its membrane binding sites by washing with 4 M guanidine-HCl or 0.1 M glycine, pH 2.5, but not by 2 M NaCl (data not shown), suggesting the binding interaction is not ionic but may depend on the tertiary structure of MMP-2 and/or its binding molecule. Our data demonstrating membrane-associated MMP-2 are consistent with previous investigations(11, 14) . In contrast to DOV 13 cells, OVCA 432 cells lacked membrane-associated MMP activity (Fig. 1, A, lane 3, and C). A faint gelatinolytic band with higher molecular weight than MMP-2 detected in the crude membrane fraction of OVCA 432 (Fig. 1A, lane3) was not o-phenanthroline-inhibitable (data not shown).


Figure 1: MMP-2 binding to ovarian carcinoma cell membranes. DOV 13 cells (panelA, lanes 1 and 2, and panelB) and OVCA 432 cells (panel A, lanes 3 and 4, and panelC) were fractionated as described under ``Experimental Procedures,'' and aliquots of the membrane preparations were analyzed using gelatin zymography. PanelA, aliquots of the crude plasma membrane fraction of both cell types were incubated with 0.16 µM proMMP-2bulletTIMP-2 complex (lanes2 and 4) or with buffer only (lanes1 and 3) in Tris-Ca-sucrose for 1 h at 37 °C. Subsequently, the membrane samples were centrifuged at 50,000 times g for 30 min, the supernatant was discarded, and the membranes were resuspended and washed twice. Following resuspension to the original volume, 10-µl aliquots of the MMP-2-treated and untreated membranes were electrophoresed. Panel B, the Percoll gradient fractions 1-6 (lanes 1-6) of DOV 13 cells were subjected to gelatin zymography (2 µg of membrane protein/lane). Panel C, the Percoll gradient fractions 1-6 (lanes1-6) of OVCA 432 cells were subjected to gelatin zymography (2 µg of membrane protein/lane). The migration positions of proMMP-2 (72 kDa) and proMMP-9 (92 kDa) standards are indicated. The gelatinase activities observed were inhibited by o-phenanthroline in parallel experiments (data not shown), with the exception of the faint gelatinase activity migrating above proMMP-2 in crude OVCA 432 membranes (panel A, lane 3).




Figure 2: A plasma membrane-associated component enhances gelatinolysis by MMP-2. The rate of cleavage of [^14C]gelatin type I (714 nM) by MMP-2bulletTIMP-2 (5.38 nM) was determined in the absence (designated No on the x axis) and presence of density gradient membrane fractions (designated 1-6) from DOV 13 (panel A) and OVCA 432 (panel B) cells or their Triton X-100 extracts (1 µg of membrane protein added) as described under ``Experimental Procedures.'' As controls, the membrane fractions were incubated with substrate in the absence of exogenous MMP-2 TIMP-2. Data represent the means of five to seven experiments, and error bars signify standard deviation. The symbols are as follows: opencircles, membrane fraction + enzyme; filled circles, membrane extract + enzyme; open squares, membrane fraction alone; filled squares, membrane extract alone. The 5`-nucleotidase activity (µM phosphate/µg protein/h) of the membrane fractions are indicated. The fraction density values were: DOV 13, fraction 1 = 1.038 g/ml; fraction 2 = 1.042: fraction 3 = 1.045; fraction 4 = 1.048; fraction 5 = 1.050; fraction 6 = 1.052; OVCA 432, fraction 1 = 1.038 g/ml; fraction 2 = 1.043; fraction 3 = 1.044; fraction 4 = 1.047; fraction 5 = 1.049; fraction 6 = 1.052.



To determine the ability of membrane preparations to bind exogenous MMP-2, purified proMMP-2bulletTIMP-2 complex was incubated with the membranes and the unbound ligand was removed by washing. Increased MMP-2 activity was observed in association with both the DOV13 and OVCA 432 membranes incubated with proMMP-2bulletTIMP-2 (Fig. 1A), indicating that these cells contain a membrane-associated MMP-2 binding activity that may be related to the MMP-2 receptor detected previously on breast cancer cells(13) . In addition, these data demonstrate that cells such as OVCA 432, which do not express any gelatinolytic MMPs, nonetheless have the capacity to utilize exogenous MMP-2, suggesting that cell surface MMP-2 binding may be an important determinant of tumor cell invasion and metastasis.

Identification of a Cell Membrane Activity That Enhances Gelatinolysis by MMP-2

Since both DOV 13 and OVCA 432 membranes bind MMP-2, the effect of membrane fractions on the rate of degradation of [^14C]gelatin type I by both MMP-2bulletTIMP-2 complex and TIMP-2-free MMP-2 was analyzed. Preincubation of APMA-activated MMP-2bulletTIMP-2 complex with either membrane fractions or Triton X-100 membrane extracts resulted in significant enhancement of gelatinase activity (Fig. 2, A and B, fractions 2-4). The DOV 13 membranes stimulated MMP-2 gelatinase activity a maximum of 2.5-fold or 5.8-fold with added membranes or detergent extracts, respectively. OVCA 432 membranes or detergent extracts enhanced activity 3.2-fold or 8.2-fold, respectively. Additionally, the activity of TIMP-2-free MMP-2 was stimulated 5.9-fold by DOV 13 plasma membrane detergent extract (data not shown), suggesting that the stimulatory effect is not dependent on the presence of TIMP-2. Furthermore, the increased activity was not due to gelatinase activity associated with the membrane fractions themselves, since the fractions alone did not contain detectable activity under the assay conditions (Fig. 2, open and filled squares). It is noted, however, that o-phenanthroline-inhibitable gelatinase activity was detectable in DOV 13 fractions 2-4 observed to contained membrane-associated MMP-2 (Fig. 1B) following more extended incubation (24 h) with substrate (data not shown), consistent with earlier data demonstrating active gelatinase associated with tumor cell plasma membranes(10) . The gelatinase activity of the endogenous membrane-associated MMP-2 in the DOV 13 fractions 2-4 was approximately 0.15 pmol cleaved/h or 1-2% of the activity of membrane-stimulated purified MMP-2. The stimulatory activity corresponded well with both the 5`-nucleotidase plasma membrane marker and the membrane-bound MMP-2 activity in DOV 13 membranes (Fig. 1B) and partially corresponded with the 5`-nucleotidase marker in OVCA 432 cells (Fig. 2). The observation that the stimulatory activity in OVCA 432 cells is slightly offset from the plasma membrane marker may indicate the existence of distinct subpopulations of plasma membranes that bear the stimulatory factor.

To characterize further the plasma membrane-associated stimulatory activity, dose-response experiments were performed. Using DOV 13 plasma membranes and detergent extracts, a dose-dependent increase in MMP-2 gelatinase activity was observed in the presence of increasing amounts of membrane material (Fig. 3). Maximal stimulation required the addition of 1000 or 250 ng of protein for the membranes and detergent extracts, respectively. Pretreatment of the DOV 13 plasma membrane samples with trypsin abolished the stimulatory activity (Fig. 3, inset, column3), whereas heat treatment (100 °C, 10 min) had no effect (Fig. 3, inset, column4). In contrast, the previously described plasma membrane-associated proMMP-2 activator was observed to be both trypsin- and heat-sensitive(14, 16) . These data suggest that the stimulatory activity is a thermostable membrane protein distinct from the activator protein. The mechanism for the increase in stimulatory activity observed in the detergent extracts relative to the membrane fractions is not clear. This positive effect of solubilization does not appear to be related to the presence of inside-out vesicles in the membrane preparation, since solubilization of trypsin-treated membranes (as shown in Fig. 3, inset) did not partially restore the stimulatory activity abolished by trypsinization (data not shown), as would be predicted if a portion of the binding protein molecules were protected inside vesicles.


Figure 3: Characterization of the MMP-2 stimulatory activity. The dose dependence of the plasma membrane-associated MMP-2 stimulatory activity from DOV 13 cells (density gradient fraction 4) was determined by measuring the rate of cleavage of [^14C]gelatin type I (214 nM) by MMP-2bulletTIMP-2 (5.38 nM) in the presence of increasing amounts of plasma membranes (open circles) or plasma membrane detergent extracts (closed circles). Base-line gelatin cleavage in the absence of added membrane material is indicated by a solid horizontal line. Data represent the mean of five experiments. Inset, the effect of trypsin and heat treatment on the MMP-2 stimulatory activity. The rate of cleavage of [^14C]gelatin type I (714 nM) by MMP-2bulletTIMP-2 (5.38 nM) was determined in the absence of modulator (column1), in the presence of 1 µg of DOV 13 cell plasma membrane fraction 4 (column 2), in the presence of an equivalent volume of the same membranes trypsin-treated as described under ``Experimental Procedures'' (column3), and in the presence of 1 µg of the same fraction heated to 100 °C for 10 min (column4). Data represent the mean of three experiments.



The effects of DOV 13 and OVCA 432 membranes on the kinetics of gelatin hydrolysis by MMP-2 were determined. The reactions obeyed Michaelis-Menten kinetics in the presence and absence of membranes (Fig. 4). When the kinetic data was analyzed using a nonlinear regression fit to the Michaelis-Menten equation, the stimulatory mechanism was similar for membrane extracts from both cell types, with the observed stimulation resulting predominantly from an increase in k (3-3.5-fold) (Table 1). The small changes in K(m) do not appear to be significant. These data suggest that association with a plasma membrane protein causes a molecular alteration in MMP-2, such as a conformational shift, that promotes catalysis but does not alter the binding affinity for substrate. Overall, the catalytic efficiency (k/K(m)) of MMP-2 increased 4.6- and 3.8-fold when incubated with DOV 13 and OVCA 432 plasma membranes, respectively. To the authors' knowledge, this report is the first description of modulation of MMP activity by a protein that functions as a nonessential activator.


Figure 4: Kinetic analysis of MMP-2 catalysis in the absence and presence of membrane stimulation. Michaelis-Menten plot of the initial rates of [^14C]gelatin type I cleavage of MMP-2bulletTIMP-2 (5.38 nM) determined in the presence of varying amounts of [^14C]gelatin type I with no modulator (open circles) or with added DOV 13 (filled circles) or OVCA 432 (open triangles) membrane detergent extract (fraction 4; 100 ng of membrane protein). Data represent the mean of six experiments. Kinetic constants were derived from the data as described in Table 1.





To summarize, we have observed that endogenous MMP-2 is found in association with the plasma membranes of ovarian adenocarcinoma cells. These membranes, even when derived from cells not expressing MMPs, also have the ability to bind exogenous MMP-2. Furthermore, as an apparent consequence of this binding interaction, MMP-2 activity is stimulated by a direct effect on the active metalloproteinase, resulting in an increase in the catalytic efficiency of gelatin type I hydrolysis. The thermostable stimulatory activity appears to be a plasma membrane-associated protein. These data demonstrate that, together with the inhibitory TIMPs and the newly described membrane activator (MT-MMP), a cell surface MMP-2-binding protein also appears to participate actively in the regulation of MMP-2 activity. The presence of this activity on tumor cells suggests a potentially important role in the regulation of proteolysis associated with invasion and metastasis.


FOOTNOTES

*
This work was supported by Research Grants CA-58900 (to M. S. S.) from the NCI and HL-31932 and HL-43339 (to S. V. P.) from the NHLBI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Northwestern University Medical School, Tarry 4-755, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-908-8216; Fax: 312-908-8773.

(^1)
The abbreviations used are: PA, plasminogen activator; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases-2; APMA, p-aminophenylmercuric acetate; DCI, 3,4-dichloroisocoumarin; SBTI, soybean trypsin inhibitor; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Hideaki Nagase for the gift of TIMP-2-free MMP-2. We gratefully acknowledge Darcie Miller for technical assistance.


REFERENCES

  1. Liotta, L. A., Rao, C. N., and Wewer, U. M. (1986) Annu. Rev. Biochem. 55, 1037-57 [CrossRef][Medline] [Order article via Infotrieve]
  2. Dano, K., Andreasen, P. A., Grondahl-Hansen, J., Kristensen, P., Nielsen, L. S., and Skriver, L. (1985) Adv. Cancer Res. 44, 139-266 [Medline] [Order article via Infotrieve]
  3. Liotta, L. A., and Stetler-Stevenson, W. G. (1990) Semin. Cancer Biol. 1, 99-106 [Medline] [Order article via Infotrieve]
  4. Tryggvason, K., Hoyhtya, M., and Pyke, C. (1993) Breast Cancer Res. Treat. 24, 209-218 [Medline] [Order article via Infotrieve]
  5. Vassalli, J.-D., Baccino, D., and Belin, D. (1985) J. Cell Biol. 100, 86-92 [Abstract]
  6. Stoppelli, M. P., Tacchetti, C., Cubellis, M. V., Corti, A., Hearing, V. J., Cassani, G., Appella, E., and Blasi, F. (1986) Cell 45, 675-684 [Medline] [Order article via Infotrieve]
  7. Miles, L. A., and Plow, E. F. (1987) Thromb. Haemost. 58, 936-942 [Medline] [Order article via Infotrieve]
  8. Nielsen, L. S., Kellerman, G. M., Behrendt, N., Picone, R., Dano, K., and Blasi, F. (1988) J. Biol. Chem. 263, 2358-2363 [Abstract/Free Full Text]
  9. Stephens, R. W., Pollanen, J., Tapiovaara, H., Leung, K.-C., Sim, P.-S., Salonen, E.-M., Ronne, E., Behrendt, N., Dano, K., and Vaheri, A. (1989) J. Cell Biol. 108, 1987-1995 [Abstract]
  10. Zucker, S., Wieman, J. M., Lysik, R. M., Wilkie, D., Ramamurthy, N. S., Golub, L. M., and Lane, B. (1987) Cancer Res. 47, 1608-1614 [Abstract]
  11. Zucker, S., Moll, U. M., Lysik, R. M., DiMassimo, E. I., Stetler-Stevenson, W. G., Liotta, L. A., and Schwedes, J. W. (1990) Int. J. Cancer 45, 1137-1142 [Medline] [Order article via Infotrieve]
  12. Moll, U. M., Lane, B., Zucker, S., Suzuki, K., and Nagase, H. (1990) Cancer Res. 50, 6995-7002 [Abstract]
  13. Emonard, H. P., Remacle, A. G., Noel, A. C., Grimaud, J.-A., Stetler-Stevenson, W. G., and Foidart, J.-M. (1992) Cancer Res. 52, 5845-5848 [Abstract]
  14. Ward, R. V., Atkinson, S. J., Slocombe, P. M., Docherty, A. J. P., Reynolds, J. J., and Murphy, G. (1991) Biochim. Biophys. Acta 1079, 242-246 [Medline] [Order article via Infotrieve]
  15. Murphy, G., Willenbrock, F., Ward, R. V., Cockett, M. I., Eaton, D., and Docherty, A. J. P. (1992) Biochem. J. 283, 637-641 [Medline] [Order article via Infotrieve]
  16. Brown, P. D., Kleiner, D. E., Unsworth, E. J., and Stetler-Stevenson, W. G. (1993) Kidney Int. 43, 163-170 [Medline] [Order article via Infotrieve]
  17. Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039 [Abstract/Free Full Text]
  18. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65 [Medline] [Order article via Infotrieve]
  19. Moser, T. L., Young, T. N., Rodriquez, G. C., Pizzo, S. V., Bast, R. C., Jr., and Stack, M. S. (1994) Int. J. Cancer 56, 552-559 [Medline] [Order article via Infotrieve]
  20. Cawston, T. E., and Barrett, A. J. (1979) Anal. Biochem. 99, 340-345 [Medline] [Order article via Infotrieve]
  21. Stetler-Stevenson, W. G., Krutzsch, H. C., Wacher, M. P., Margulies, I. M. K., and Liotta, L. A. (1989) J. Biol. Chem. 264, 1353-1356 [Abstract/Free Full Text]
  22. Young, T. N., Rodriquez, G. C., Moser, T. L., Bast, R. C., Jr., Pizzo, S. V., and Stack, M. S. (1994) Am. J. Obstet. Gynecol. 170, 1285-1296 [Medline] [Order article via Infotrieve]
  23. Aronson, N. N., Jr., and Touster, O. (1974) Methods Enzymol. 31, 90-102 [Medline] [Order article via Infotrieve]
  24. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196-202 [Medline] [Order article via Infotrieve]
  25. Harris, E. D., Jr., and Krane, S. M. (1972) Biochim. Biophys. Acta 258, 566-576 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.