Degradation of Extracellular Matrix Tumor-associated ...

Tumor-associated Trypsin Participates in Cancer Cell-mediated Degradation of Extracellular Matrix Erkki Koivunen, Ari Ristimäki, Outi Itkonen, et al. Cancer Res 1991;51:2107-2112. Published online April 1, 1991.

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(CANCER RESEARCH 51. 2107-2112, April 15. 1991]

Tumor-associated Trypsin Participates in Cancer Cell-mediated Degradation of Extracellular Matrix1 Erkki Koivunen,2 Ari RistimÃ¤ki,Outi Itkonen, Sirpa Osman, Matti Vuento, and Ulf-HÃ¡kan Stenman Department of Obstetrics and Gynecology, Helsinki L'nirersity Central Hospital, Haartmaninkatu 2, SF-00290 Helsinki [E. K., A, Râ€žO. I., S. O,, U-H. SJ, and Department of Biochemistry, L'nirersity of Helsinki, L'nioninkatu 35, SF-OOI70 Helsinki [M. V.I.Finland

ABSTRACT We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumorassociated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pan creatic carcinoma, and HT I080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([-'Miserine, |3H)proline, and (35S|sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H|serine-labeled ECM. Plasminogen added exogenously to the cul ture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cellmediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.

(11), and cathepsin B (4) have been found to correlate with invasiveness and the metastatic ability of tumor cell lines. In extracts of tumor tissues the levels of urokinase-type plasmin ogen activator (u-PA) generally correlate with the degree of malignancy of the tumors (12). It is interesting that TAT-2, the major TAT isoenzyme, also shows close association with ma lignancy; the levels are higher in malignant and borderline ovarian tumor cyst fluids than in benign ones (13). In addition, purified TAT-1 and TAT-2 activate pro-u-PA in vitro (8). These results suggest that TAT-2 may promote the dissemination of tumor cells. TAT-2 zymogen is produced by many cancer cell lines, and it has been purified from the culture medium of COLO 205 colon adenocarcinoma and HT 1080 fibrosarcoma cell lines (14). In the present study we have examined the role of TAT-2 in cell-mediated degradation of extracellular matrix. We devel oped a method for determining degradation of fibronectin based on analysis of immunoreactive fibronectin fragments liberated by tumor cells growing on protein-coated surfaces. We also analyzed the degradation of isotopically labeled ECM. The results show that tumor cell-mediated proteolysis can be signif icantly abrogated by inhibiting TAT-2. MATERIALS AND METHODS

INTRODUCTION

Chemicals. Reagents were obtained as follows: aprotinin from Sigma Chemical Co. (St. Louis, MO); EACA from Fluka AG (Buchs, Swit Production of proteolytic enzymes by malignant tumor cells zerland); RPMI 1640, Medium 199, PBS (without magnesium and is believed to be essential to the ability of the tumor to invade calcium), and fetal bovine serum from Flow Laboratories (Irvine, Scot and degrade extracellular matrix. Of the proteases secreted by land); pooled human serum from the Finnish Red Cross Blood Trans tumor cells, collagenases (1), plasminogen activators (2), tranfusion Service (Helsinki, Finland); L-glutamine and antibiotics from sin/stromelysin (3), and cathepsins (4-6) have been the most GIBCO Laboratories (Grand Island, NY); and gelatin from Merck thoroughly studied. We have recently characterized a novel (Darmstadt, Germany). tumor-associated protease, TAT,3 (7, 8). It was initially identi Proteinases. Human plasminogen and plasmin were obtained from fied in the cyst fluid of mucinous ovarian tumors during a Kabi Vitrum (Stockholm, Sweden). Porcine enteropeptidase and bovine trypsin were obtained from Sigma. Enteropeptidase was dissolved in search of the target protease for TATI, which is a marker for PBS and passed through an affinity column of TATI-Sepharose 4B to mucinous ovarian cancer (9). TAT occurs as two isoenzymes, remove contaminating trypsin-like enzymes. TAT-2 was purified by a TAT-1 and TAT-2. They are identical to the corresponding monoclonal antibody affinity column from serum-free culture medium pancreatic trypsins with respect to amino-terminal amino acid of COLO 205 cells as described (14). TAT-2 was obtained in zymogen sequence, molecular weight, and immunoreactivity, but they form and autoactivated by neutralizing the pH. differ with respect to substrate specificity, enzyme stability, and Purification of TATI. TATI was purified by a novel affinity chroma elution pattern in ion-exchange chromatography. At present it tography method from the urine of cancer patients. The pH of the urine is not known whether these differences are explained by tissue- was adjusted to 7.4, and it was dialyzed against water with a hollow specific modification of trypsin or whether distinct genes code fiber dialyzer (15) until the conductivity corresponded to that of 50 mM Tris, pH 7.4. Dialyzed urine was then applied at a flow rate of 60 ml/ for trypsinogens derived from tumors and pancreas. h at 4Â°Cto a Q Sepharose anion-exchange column (Pharmacia, Upps The levels of type IV collagenase (10), plasminogen activators ala, Sweden) and an affinity column of bovine trypsin-Sepharose 4B previously equilibrated with Tris buffer. Most urine proteins were Received 7/31/90; accepted 2/4/91. removed by the first anion-exchange column. After the trypsin-Sepha The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in rose column was washed with Tris buffer containing l M NaCI, 0.1% accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Brij 35, and 1% 2-propanol, TATI was eluted with 0.1 % trifluoroacetic 1This work was supported by grants from the Academy of Finland, the Finnish acid. TATI was finally purified by reversed-phase chromatography on Cancer Society, the Finnish Social Security Institution, the Jenny and Antti Wihuri Foundation, and the Ida Montin Foundation. a C,Â«column with a linear acetonitrile gradient (from 10 to 60% in 20 2 To whom requests for reprints should be addressed. min) in 0.1% trifluoroacetic acid. By this method, 2 mg of TATI were 3 The abbreviations used are: TAT, tumor-associated trypsin(ogen); TATI, obtained from 1 liter of urine. tumor-associated trypsin inhibitor; u-PA, urokinase-type plasminogen activator; Anticatalytic Antibodies. The IgG fraction was prepared from a rabbit ECM, extracellular matrix: EACA. Â«-aminocaproicacid; PBS. phosphate-buffered saline. preimmune serum and antiserum against human pancreatic trypsin 1 2107


TUMOR CELL TRYPSIN AND MATRIX DEGRADATION

(8) by sequential affinity Chromatograph) on Protein G-Sepharose and protein A-Sepharose (Pharmacia). Anti-u-PA IgG was prepared from goat antiserum against human low-molecular-weight u-PA (Biopool AB, UmeÃ , Sweden) by using protein A-Sepharose. Anti-trypsin IgG and anti-u-PA IgG were anticatalytic. because they inhibited the amidolytic activity of TAT-2 and u-PA, respectively. Cell Culture. Human cancer cell lines COLO 205 colon adenocarcinoma (ATCC CCL 222). HT 1080 fibrosarcoma (ATCC CCL 121), K562 erythroleukemia (ATCC CCL 243), and CAPAN-1 pancreatic adenocarcinoma (ATCC HTB 79) were obtained from the American Type Culture Collection (Rockville. MD). Cells were cultured in 800ml Nunclon flasks (Roskilde, Denmark) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM Lglutamine. 100 units/ml penicillin, and 100 ^g/ml streptomycin. Treat ment with trypsin was not required for detachment of the adherent cells, possibly because the cells produce their own trypsin. After serum was removed, and the flasks were washed twice with PBS, cells were incubated for 10-15 min in PBS at room temperature. The flasks were vigorously shaken until all cells detached. Preparation of Wells Coated with ECM and Kibronectin. Endothelial cells were isolated from human umbilical cord veins by the method originally described by Jaffe et al. (16), and ECM was prepared essen tially as described (17. 18). Endothelial cells were seeded in Nunclon 24-well multidishes, which were previously coated with gelatin, and cultured in Medium 199 containing 20% human serum. Labeling medium containing 5 Â¿iCi/inlpHlserine, 5 ^Ci/ini ['Hjproline, or 20 fiCi/ml [100 kDa) than those mediated by en Immune and preimmune IgG were used at concentrations of 10-25 /jg/ teropeptidase (Fig. 2, Lanes 4 and 5). A similar difference in ml. The concentrations of TATI and aprotinin were 1-10 ^g/ml, and fragment pattern was found when purified fibronectin was that of EACA was 330 Â¿ig/ml.Aliquots of 50 and 200 ÃŸ\ were collected digested by plasmin and TAT-2, respectively (Fig. 2, Lanes 1 at various time points, centrifuged at 9000 rpm for 5 min, and analyzed and 2). In addition, enteropeptidase treatment and TAT-2 by fibronectin immunoassay and ^-scintillation counting, respectively. Detection of Fibronectin Immunoreactivity. Polyclonal antibodies digestion yielded a characteristic fragment migrating at 85 kDa. were prepared from rabbit antiserum against fibronectin (20) by Na2SO4 Proteolysis mediated by untreated cells predominantly gener precipitation and used to develop a time-resolved immunofluorometric ated large (>100 kDa) fibronectin fragments (Fig. 2, Lane 6). assay according to methods previously described (21). In a typical assay The plasminogen-mediated proteolysis of fibronectin was of cell culture media, the samples were incubated for 60 min at 25Â°C in microtiter wells coated with the fibronectin antibodies. After wash ing, the wells were incubated for 30 min with fibronectin antibodies that had been labeled with a europium chelate (Wallac Oy, Turku, Finland) (22). After washing of the wells and addition of an "enhance ment solution" (22), the bound europium was measured with an Arcus 1230 time-resolved fluorometer (Wallac). Using a 50-^1 sample volume, the linear measuring range of the assay was 0.5-250 ng/ml of fibronec tin, and the coefficient of variation was below 10% in this concentration range. Characterization of Fibronectin Fragments. The culture media were dialyzed against 5 mM NH4HCO, for 2 h. lyophilized, and analyzed by gel filtration and immunoblotting. Gel filtration was performed on a SuperÃ³se 12 column (Pharmacia) at a flow rate of 30 ml/h. using 50 mM Tris-HCI buffer (pH 7.4) containing 0.1 M NaCI and 0.02% NaN3. Fractions of 400 n\ were collected and analyzed by fibronectin immu noassay. Sodium dodecyl sulfate gel electrophoresis was performed on 7.5% polyacrylamide gels under nonreducing conditions (23). Proteins were electrotransferred to nitrocellulose membranes (24), and fibronec tin fragments were detected by incubating with polyclonal fibronectin antibodies followed by secondary peroxidase-conjugated swine antirabbit antibodies (Dakopatts, Glostrup, Denmark). Peroxidase staining was performed with 3.3'-diaminobenzine tetrahydrochloride (Fluka).

200 no additions -f enteropeptidase

o a

150

E E

100

4) w O C 3

+ plasminogen

20

Time(h)

40

60

Fig. I. Time course of COLO 205 cell-mediated release of fibronectin frag ments from fibronectin-coated wells. Cells were plated at a density of 2 X IO*/ well in 1 ml serum-free medium in the absence (X) or presence of plasminogen (O) (6 fjg/ml) and enteropeptidase (D) (l Â¿ig/ml).Aliquots of culture media were collected for analysis by fibronectin immunoassay at the time points indicated. Points, means for quadruplicate wells; bars. SD.

2108



inhibited by aprotinin, EACA, and anticatalytic polyclonal uPA IgG, but not by TATI or anticatalytic polyclonal trypsin IgG (Table 1). By contrast, TATI and anti-trypsin IgG inhibited both the enteropeptidase-mediated proteolysis and the proteolysis mediated by untreated cells (Table 1). TATI and antitrypsin IgG caused 81 and 92% inhibition on the enteropepti dase-mediated proteolysis, respectively, and 75 and 32% inhi bition on the proteolysis mediated by untreated cells, respec tively. Preimmune IgG had no effect. When COLO 205 cells were plated on ECM deposited by human umbilical vein endothelial cells, proteolysis of fibronectin was very rapid (Fig. 3). Fibronectin immunoreactivity was detected in the medium after a 1-h culture and further increased for 24 h. After a 6-h culture, large (>200 kDa) fibronectin fragments predominated in the culture medium, as detected by

5

6

500

246 Time (h)

Fig. 3. Inhibition of COLO 205 cell-mediated degradation of ECM fibronectin by trypsin antibodies. Cells were plated on ECM-coated wells at a density of 2 x lO'/well and incubated in 1 ml serum-free medium in the absence (O) or presence of anti-trypsin IgG (â€¢)and preimmune IgG (Ãœ).Control wells were incubated without cells (X). The concentrations of immune and preimmune IgG were 25 Mg/ml. Aliquots of culture media were collected for analysis by fibronectin immunoassay at 2-h intervals. Points, means for quadruplicate measurements; ears. SD.

i

800

200 o

600-

E

400

_

200

COLO 205

K-562

CAPAN-1

Cancer cell line

30

Fig. 2. Immunoblot analysis of fibronectin fragments released by cells from coated wells or generated by digestion of purified fibronectin with TAT-2 and plasmin. Lane I, fibronectin digested for l h with plasmin (0.025 casein unit); Lane 2, fibronectin digested for l h with TAT-2 (IO ng); Lane 3, untreated fibronectin; Lane 4, culture medium of plasminogen-treated cells; Lane 5, culture medium of enteropeptidase-treated cells; Lane 6. culture medium of untreated cells. Asterisks, 85-kDa fragment characteristic of enteropeptidase- and TAT-2mediated proteolysis.

Table 1 Effect of protease inhibitors and anticatalytic protease antibodies on COLO 205 cell-mediated release of fibronectin fragments from fibronectin-coated wells ci of inhibition with following additions to serum-free culture medium" Inhibitor"

None

TATIAprotininEACAAnti-trypsin

Enteropeptidase

Plasminogen 3.589 Â± 6.968 Â± 0.40Â±0.8ND49 Â±

IgGPreimmune IgGAnti-u-PA IgG7580N32306.2'8.4D"5.91.28.08182N92041.22.4D5.56.23.92 Â±1.5 Â°TATI and aprotinin Â»ereused at a concentration of 1 ^g/ml. EACA at a concentration of 330 fig/ml, and prcimmune and immune IgG at a concentration of 10 fig/ml. * Fibronectin immunoreactivity released into the culture medium was deter mined by the fibronectin immunoassay after culturing for 16-24 h. The inhibition is given as the decrease of the immunoreactivity compared with that observed in the absence of inhibitors. Cells (2 x 10*) were plated on fibronectin-coated wells in the presence or absence of plasminogen and enteropeptidase in serum-free medium. c Mean Â±SD for at least 2 experiments with 2 wells/experiment. d ND. not determined.

Fig. 4. Prevention of cancer cell-mediated degradation of ECM fibronectin by TATI. COLO 205. K-562. and CAPAN-I cells were seeded at densities of 2 x 10'. 5 x IO5,and 2.5 x 105/well. respectively, in 0.5 ml serum-free medium. Cells were cultured in the absence (â€¢)or presence (EH)of TATI, and the culture media were analyzed for fibronectin immunoreactivity. COLO 205 cells were incubated for 4 h, and K-562 and CAPAN-1 cells were incubated overnight. The concentra tion of TATI was 10 ^g/ml. Columns, means from triplicate wells; bars, SD. Untreated control wells did not release fibronectin immunoreactivity.

immunoblotting (not shown). TATI and anti-trypsin IgG inhib ited degradation by 51 and 40%, respectively (Figs. 3 and 4). The proteolysis caused by two other cell lines which produce TAT-2, K-562 (14) and CAPAN-1 was similarly inhibited by TATI (Fig. 4). TATI prevented K-562 and CAPAN-1 cellmediated proteolysis by 47 and 54%, respectively. TATI was also effective in inhibiting the destruction of isotopically labeled ECM, when cells were cultured under serumfree conditions in the absence of plasminogen. TATI added at a concentration of 10 Mg/ml reduced the release of ['H]proline label from ECM by all three cell lines studied. After a 24-h incubation, TATI prevented the proteolysis mediated by COLO 205, CAPAN-1, and HT 1080 by 40, 48, and 35%, respectively (Fig. SB). The degradation of ECM labeled with ['Hjserine could be inhibited by TATI in COLO 205 and in CAPAN-1 cells (Fig. 5A). The percentage of inhibition was 57 and 51%, respectively. However, TATI did not prevent HT 1080 cellmediated degradation of ['Hjserine-labeled ECM (data not shown). It is notable that of the three cell lines studied, HT 1080 hydrolyzed the matrix most weakly, and it secreted the lowest amount of TAT-2 as analyzed by TAT-2 immunoassay (not shown). The degradation of [35S]sulfate-labeled ECM could

2109


TUMOR CELL TRYPSIN AND MATRIX DEGRADATION 2000

1500-

1000

500

COLO205

CAPAN-1

COLO 205

CAPAN 1

HT 1080

COLO205

Cancer cell line

Fig. 5. Inhibition of cancer cell-mediated destruction of isotopically labeled matrices by TATI. A, Â¡'HJserine-labeled ECM: B. [3H|proline-labeIed ECM: C. |35S]sulfate-labeled ECM. COLO 205 and HT 1080 cells were seeded at a density of 5 x lO'/well, and CAPAN-1 cells at a density of 2.5 x lO'/well in 0.5 ml serum-free RPMI medium. Cells were cultured for 24-48 h in the absence (â€¢)or presence (D) of TATI, and the isotope label released into the medium was measured by Â¿-scintillation counting. The concentration of TATI was 10 ng/ml. Columns, means from triplicate wells; bars. SD. Untreated control wells released 229 Â±10, 123 Â±21, and 73 Â±4 cpm |'H]serine, |'H]proline, and [35S]sulfate label, respectively. 4000 Control + plasminogen IH plasminogen + TATI

serine

sulfate Labeled ECM

Fig. 6. Effect of plasminogen on degradation of isotopically labeled ECM by COLO 205 cells. Cells (5 x 10') were incubated without plasminogen (â€¢).with plasminogen (6 Mg/ml) (D), or with plasminogen (6 /jg/ml) and TATI (10 ng/ml) (D) in 0.5 ml serum-free medium. After culturing for 16 h, the released radioac tivity was measured by /Â¿-scintillationcounting. Columns, means from triplicate wells; bars, SD. Control wells treated with plasminogen alone released 386 Â±29, 177 Â±49, and 108 Â±9 cpm [3H]serine, [3H|proline, and ("S]sulfate label, respectively.

be prevented by TATI in COLO 205 cells (Fig. 5C), but not in CAPAN-1 or HT 1080 cells (not shown). The inhibition of TATI in COLO 205 cells was 63%. As in the case of fibronectin proteolysis, addition of plasmin ogen to the culture medium substantiated the degradation of all three isotopically labeled matrices by COLO 205 cells. In contrast to the proteolysis of fibronectin, TATI weakly but significantly prevented proteolysis in the presence of plasmin ogen (Fig. 6). The TATI-mediated inhibition (expressed in cpm) corresponded to the radioactivity released in the plasminogenindependent proteolysis. Therefore, TATI may have inhibited the portion of proteolysis that was exerted by TAT-2 but not by plasmin. Active TAT-2 purified from COLO 205 culture medium (100 ng) hydrolyzed all three labeled matrices, as demonstrated by release of label into the medium (not shown).

mediated degradation of ECM. To demonstrate TAT-2 activity in vitro, it was essential to culture cells in serum-free medium to avoid the presence of serum trypsin inhibitors and plasmin ogen. Under these conditions, the specific inhibitor TATI pre vented the proteolysis of fibronectin or isotopically labeled ECM by tumor cell lines that produce TAT-2. Further evidence for involvement of TAT-2 in proteolysis was the finding that anticatalytic trypsin antibodies were inhibitory but preimmune antibodies had no effect. In addition, the cell-mediated degra dation of fibronectin was augmented by adding exogenous enteropeptidase, an activator of trypsinogen, to the culture medium. Analysis of fibronectin fragments generated indicated that enteropeptidase-induced proteolysis yielded fragments similar to those formed by digestion of purified fibronectin with TAT-2. Previous studies have indicated that cell-mediated degrada tion of ECM or protein substrates is enhanced by addition of plasminogen (17,18, 25-28). This was also found in the present study. Addition of plasminogen to serum-free medium in creased up to 10-fold the degradation of labeled ECM and fibronectin by COLO 205 cells. Plasminogen-dependent tumor cell-mediated proteolysis can be inhibited by protease nexin I (29), plasminogen activator inhibitor 1 (30), or plasminogen activator inhibitor 2 (18), indicating an important role for uPA in the protease cascade. However, several investigators have found that there is also a basal cell-mediated proteolytic activity in the absence of plasminogen (25-28). Our results suggest that in the cell lines studied a major portion of this is due to the activity of TAT-2. In addition to degrading ECM by itself, TAT-2 can also participate in the activation of pro-u-PA (8). This could be an explanation for the rapid activation of plas minogen exogenously added to cell culture. Perhaps surprisingly, TATI prevented the degradation of [3H]proline-labeled ECM by all three tumor cell lines studied: COLO 205; CAPAN-1; and HT 1080. As the [3H]proline label is assumed to be biosynthetically incorporated primarily into the collagen component of newly synthesized subendothelial matrix (26), our results suggest that TAT-2 could mediate the degradation of collagenous material. This is also supported by the finding that purified TAT-2 released ['H]proline label from the matrix. It remains to be shown whether TAT-2 can degrade native collagens in ECM or whether it degrades other trypsinsensitive proline-containing matrix components. It is also pos sible that TAT-2 can activate latent collagenase, because trypsin is a known activator of procollagenase (1). TATI inhibited the degradation of ['Hjserine-labeled ECM by COLO 205 and CAPAN-1 cells and the degradation of ["Sjsulfate-labeled ECM by COLO 205 cells. The ['Hjserine label would be biosynthetically incorporated into most matrix proteins (18), and [35S]sulfate label into sulfated proteoglycans

and proteins (25). These results suggest that depending on tumor cell line, TAT-2 contributes to the degradation of matrix (glyco)proteins and proteoglycans. Because TATI did not in hibit the destruction of all labeled matrices by CAPAN-1 and HT 1080 cells, other proteinases not characterized in this study are also involved in the proteolysis. When proteolysis by COLO 205 cells was studied in plasminogen-supplemented medium, TATI caused an inhibition similar to that observed in the absence of plasminogen, apparently that portion that was due to TAT-2. These results suggest that ECM degradation is DISCUSSION accomplished by the concerted action of plasmin, plasminogen These studies demonstrate that the novel trypsinogen isoen- activators, TAT-2, and other proteases. In COLO 205 cells zyme TAT-2 produced by cancer cells contributes to the cell- plasminogen was activated by u-PA, inasmuch as the proteolysis 2110



of fibronectin could be partially inhibited by anticatalytic urokinase antibodies. Recently, COLO 205 cells have been shown to express u-PA and its mRNA (18, 31). Because TAT-2 is secreted in trypsinogen form from the cells (14), an obvious question is how it is activated. Part of TAT-2 is in active form in cell culture as indicated by inhibition of proteolysis by TATI and trypsin antibodies. Enteropeptidase (32) and cathepsin B (33) are activators of pancreatic trypsinogen. Purified TAT-2 is activated by enteropeptidase (14), and we show here that it potentiates COLO 205 cell-mediated degradation of fibronectin. The potentiation is due to activation of TAT-2, as the proteolysis can be inhibited by TATI and trypsin antibodies. It remains to be shown whether an enteropeptidase-like protease-activating TAT-2 exists in tumor cells. This study demonstrates that TATI, also known previously as pancreatic secretory trypsin inhibitor (34), is a potential inhibitor of tumor cell-mediated degradation of ECM. Even though the plasminogen-dependent proteolysis was poorly or not at all inhibited by TATI, apparently because it is only a weak inhibitor of plasmin with a KÂ¡of 9.7 nivi (35), TATI may play an important matrix-stabilizing role by controlling the activation of TAT-2 zymogen. Coexpression of TATI with TAT-2 in the cyst fluids of ovarian tumors (13) and cancer cell lines COLO 205 (14) and CAPAN-1 (36) further suggests a specific role for TATI in controlling TAT-2. TATI was initially identified as a tumor marker for ovarian cancer (37, 38), but the levels in serum and urine are also elevated in patients with other types of advanced cancer (9, 37, 39). We suggest that the elevation of TATI is a reaction to TAT-2 expression with the aim of controlling TAT-2 activity and ECM degradation. How ever, TATI may also have other functions. TATI (or pancreatic secretory trypsin inhibitor) at concentrations present in serum is mitogenic on human fibroblasts (40, 41) and endothelial cells (42). Furthermore, it has been demonstrated that TATI binds specifically to various cultured cells. A 140-kDa cell surface receptor protein mediating the binding could be characterized by chemical cross-linking (43). It is not known whether cell surface proteases are involved in the cell binding and mitogenic activities of TATI. Degradation of ECM and basement membrane is an essential step at several stages of cancer invasion (44). The proteolysis assay used in the present study can be envisaged as a simple model of endothelial basement membrane degradation occurring during intra- and extravasation of tumor cells. Our studies suggest that TAT-2 may have a significant role in the degradation of basement membranes by tumor cells. TAT-2 may be one component of the protease cascade that cells utilize for migration through tissue barriers. ACKNOWLEDGMENTS We thank Dr. Olii Saksela for helpful discussions and Lusa Airas for technical assistance.

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correlation with metastatic potential. Science (Washington DC), 212: 11511153, 1981. 5 Denhardt, D. T., Greenberg, A. H., Egan, S. E., Hamilton, R. T., and Wright, J. A. Cysteine protease cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts. Oncogene, 2: 55-59, 1987. 6. Spyratos, F., Brouillet, J-P., Defrenne, A., Macene, K., RouÃ©sse,J., Maudelonde. T., Brunei, M.. Andrieu, C., Desplaces, A., and Rochefort, H. Cathep sin D: an independent prognostic factor for metastasis of breast cancer. Lancet, 2: 1115-1118, 1989. 7. Stenman, U-H., Koivunen, E., and Vuento, M. Characterization of a tumorassociated serine protease. Biol. Chem. Hoppe-Seyler, 369: 9-14, 1988. Koivunen, E., Huhtala, M-L., and Stenman, U-H. Human ovarian tumorassociated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase. J. Biol. Chem., 264: 14095-14099, 1989. 9 Halila. 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