CELL REGULATION, VOI. 1, 895-905, November 1990

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of a2-macroglobulin secretion

Jozef Bizik,*t Alena Lizonov6,*t* Ross W. Stephens,* Marta Gr6fovi,t and Antti Vaheri,*§ *Department of Virology University of Helsinki Helsinki 00290, Finland tDepartment of Viral Oncogenesis Cancer Research Institute Bratislava 81232, Czechoslovakia Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only __

Mrx 103 AL

94

67 -AlmimL

I 43

30 20

14 Figure 1. Zymography of PAs secreted by exponentially growing melanoma cell lines. Enzyme-containing samples of serum-free conditioned media corresponding to 15 x 1 3 cells were electrophoresed in 90/o SDS-PAGE. After electrophoresis, the resolved enzyme bands were detected by incubation on a casein-agarose layer containing plasminogen. Plasminogen, when activated by PAs present in the medium sample, degraded casein and formed a clear disc of caseinolysis in the gel during the sample diffusion, proportional to the PAs activity of the sample and time of diffusion. The photograph was taken with dark ground illumination after 16 h. Human u-PA and t-PA were used as standards.

Inhibition of PAs by conditioned media The inhibitory activities present in conditioned media of the cell lines were quantitated by measuring their ability to inhibit t-PA- or u-PA-mediated conversion of plasminogen to plasmin, as described in Materials and Methods. As seen in Figure 2, conditioned medium from the Bowes cell line did not inhibit t-PA. Conditioned media from M2Ge, M1Do, and M3Beu cells gave only a minimal inhibition of t-PA, whereas culture media from the HMB-2 cell line and its clones 15 and 33 contained higher inhibitory activity to tPA. Culture medium conditioned by M3Dau cells exhibited the strongest inhibitory activity for tPA of all cell lines tested. Aliquots of the same conditioned media were tested in a similar way for their capacity to inhibit u-PA (Figure 2). Conditioned medium from the Bowes cell line did not inhibit u-PA; media of all other cell lines inhibited u-PA, and much more strongly than t-PA. The M3Dau cell line appeared, in repeated experiments, to secrete the most inhibitor of u-PA of all cell lines studied. Vol. 1, November 1990

Cell-surface localization of t-PA We used acid elution and microplate immunocapture assay to investigate whether cultured human melanoma cells have PAs bound on their surface. It should be noted that the term "cell bound," used in the present report for bound PA and plasmin, may include enzyme bound to pericellular matrix or substratum. The cultures were rinsed seven times with serum-free medium, and endogenous cell-bound PAs were released from the cell surface with glycine-HCI buffer, pH 3. The buffer was rapidly neutralized and the eluates tested for their activity by the immunocapture method. As shown in Table 2, t-PA was recovered from all cell lines; the highest t-PA activity was detected in the eluates of the cell lines HMB-2 and M3Dau. No u-PA activity was detected (Table 2). The results suggest that melanoma cells possess binding sites for t-PA on their surface. Binding of exogenous u-PA onto the surface of human melanoma cells As previously reported, plasma membrane receptors for u-PA have been identified on a num897

J. Bizik et al.

Table 2. Activity of u-PA and t-PA in cell surface eluates of monolayer cultures of melanoma cell lines

Inhibition of t-PA ~~~~a

c

9

Cell line

0,8

0

m1

096

M3Dau M2Ge M1Do M4Beu HMB-2 RD

/;S /H--MB--a2M

'2~~~~ ,/ M Dau M-2 c. 1 W. / / --*I ---+-~Bowes M 2 Ge --- 0f-Ml Do a M 4Beu - --D - -HMB-2 * HMB-2 cl. 15 6 HMB-2 cl. 33 2 4 6 8 I

0,

1 0

Dilution (-log 2)

t-PA activity

0.001* 0.001 0.001 0.001 0.001 0.550

0.230 0.114 0.110 0.150 0.248 0.005

The melanoma cell lines shown were cultured for 24 h in serum-free medium, the monolayers thoroughly rinsed, and the bound plasminogen activator activities recovered from the cells by elution at pH 3.0 for 5 min. Cultures of human RD rhabdomyosarcoma cells were used as a control. The plasminogen activator activities in the neutralized eluates were measured by immunocapture assay. * The values are given in international units per milliliter for acid eluates of 5 x 105 cells/ml.

washing, and acid elution, the activity of eluates

E

was analyzed by microplate immunocapture assay. The results are summarized in Table 3. As shown, the recovery of bound u-PA activity

0

.0

.0 4

Dilution (-log 2)

Figure 2. The PAI content in conditioned media of the melanoma cell lines. Wells of microtests were precoated with goat anti-human u-PA (A) or anti-human t-PA antibodies (B). Unbound antibodies were washed out, and wells were filled with human u-PA or t-PA or t-PA solution. After intensive washing, serum-free conditioned media, estimated and adjusted to the same cell number for each cell line in starting aliquot or plasma a2M (2.5 mg/ml), were serially diluted across the plate. The plates were washed, and then each well was incubated with human plasminogen and the plasmin activity measured by assay of thioesterase activity.

ber of cells (Vassalli et al., 1985), including human tumor cells (Stoppelli et al., 1986). In most u-PA-producing cells, the receptors are at least partly occupied by the endogenously produced enzyme, but in some cells the receptor is synthesized in the absence of u-PA production (Blasi, 1988). To investigate whether our human melanoma cell lines possess binding sites for u-PA, we incubated monolayer cultures with 10 lU/mi of exogenous u-PA. After incubation, 898

u-PA activity

detected in the eluates of human melanoma cell lines was very low, whereas there was a considerable amount of activity in similar eluates of RD human rhabdomyosarcoma cells after incubation with exogenous u-PA. The amount of u-PA bound by melanoma cells was insignificant in terms of its inability to produce bound plasmin (see Table 4). These results indicate that functional binding sites for u-PA are either poorly expressed or absent on the surface of the above human melanoma cell lines.

Table 3. Binding of exogenous u-PA onto the surface of human melanoma cells

Cell line

M3Dau M2Ge

M,Do M4Beu HMB-2 RD (rhabdomyosarcoma)

u-PA activity in eluates of monolayer cell cultures

0.080* 0.045 0.050 0.068 0.030 0.800

Monolayers of melanoma cell lines and RD rhabdomyosarcoma were rinsed with serum-free medium and then treated with human u-PA (10 lU/ml) in serum-free medium for 2 h. After rinsing, the bound u-PA was recovered by acid elution and the activity measured by immunocapture assay. * The values are given in international units per milliliter of the eluate.

CELL REGULATION

Plasminogen activation on melanoma cell surface

Table 4. Effect of anti-catalytic monoclonal antibodies and aprotinin on formation of bound plasmin on the surface of HMB-2 melanoma cells Incubation of cell monolayer with

1. +plasminogen (40 jg/ml) 2. -plasminogen 3. +plasminogen + aprotinin 4. +plasminogen + anti-t-PA monoclonal

antibodyt 5. +plasminogen + anti-u-PA monoclonal antibodyt

Bound plasmin activity (%)

100* 10 12 18 92

Monolayers of HMB-2 cells were incubated with MEM medium containing 10% heat-inactivated and plasminogendepleted FCS with the effectors shown. Aprotinin was used at 200 klU/ml, and the monoclonal antibodies (protein Apurified IgG) at 5 ,g/ml. Bound plasmin formed on the cells was recovered by elution with 1 mM tranexamic acid and assay by a thioesterase method. * The plasmin activity after incubation of cell monolayer with 40 ug/ml of plasminogen was used as the 100% control. t The same antibody preparations were previously used to establish the role of u-PA on HT-1 080 and RD sarcoma cells (Stephens et al., 1989; Pollanen et al., 1990).

Activation of plasminogen on the surface of human melanoma cells After addition of purified human plasminogen to cultures of human HMB-2 melanoma cells growing in a medium with 100/% plasminogendepleted fetal calf serum (FCS), plasmin activity could be recovered as a bound fraction from the cell layer. After the culture was rinsed with phosphate-buffered saline (PBS), the plasmin on the cell surface could be released by 1 mM tranexamic acid and its activity measured as described in Materials and Methods. As we varied the concentration of added plasminogen, the bound plasmin activity increased in a dosedependent manner (Figure 3). To test whether the cell-surface plasmin might have been either derived from preformed plasmin, added as a trace contaminant with the plasminogen preparations, or derived from plasmin formed in the medium and subsequently bound to the cells, we added plasmin to the culture medium of HMB-2 cells. As shown in Figure 4, virtually no plasmin activity was detected on the cell surface when the medium contained 100% FCS. Plasmin added to the serum medium did not reach the cell surface, or at least its activity was abolished by the serum protease inhibitors. There was considerable dose-dependent binding of plasmin in the absence of serum. These findings indicate that Vol. 1, November 1990

the observed cell-bound plasmin activity is formed by activation of plasminogen on the surface of HMB-2 melanoma cells and could not have been taken up from the medium. Inhibition of formation of bound plasmin on HMB-2 cells in serum culture To confirm that t-PA was responsible for the cell surface plasminogen activation, the cells were incubated with plasminogen in the presence of anti-catalytic monoclonal antibodies to human t-PA. The results, in Table 4, show that inhibition of the enzymatic activity of t-PA resulted in a strong reduction in plasmin activity on the cell surface, whereas incubation with an anti-catalytic monoclonal antibody to human uPA only slightly decreased the amount of plasmin activity. Bound plasmin activity was also rapidly reduced when cells were incubated with aprotinin (Table 4).

Expression of a1IM-specific mRNAs and production of mature protein in melanoma cell lines We have previously reported on a2M production by human melanoma cells (Bizik et al., 1986, 1989). Now we analyzed the a2M expression in more detail. Northern blot hybridization analysis 30 HMB-2 3

_MI 3 OaU

CL

0

20

/

M4BOU

1

CL 0

Plasminogen added

(p.g

per well)

Figure 3. Activation of plasminogen on melanoma cell surface. Confluent layers of cultured human HMB-2 (O), M3Dau (m), M2Ge (0), M4Beu (0), or M1Do (-) melanoma cells were incubated with MEM containing 10% heat-inactivated and plasminogen-depleted FCS and human plasminogen as indicated. After 2 h incubation, plasmin activity was recovered from the cell surface by elution with 1 mM tranexamic acid, and assays were made with a thioester substrate.

899

J. Bizik et al.

1-

a)

present in conditioned medium of Bowes cells. This experiment and those above showed that TA-a2M accounted for the majority of PAI activity secreted by human melanoma cells and that this is specific for u-PA.

20

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CU

*0m C

0

Plasmin added ( p.g per well) Figure 4. Binding of plasmin onto HMB-2 cells from serum-free and serum-containing medium. Confluent layers of HMB-2 cells were incubated for 2 h with serum-free medium (0) or medium with 100/% FCS (-) and human plasmin as indicated. Plasmin was then assayed in the cell-bound fraction after elution with 1 mM tranexamic acid.

of equal amounts of poly(A)+mRNA isolated from the analyzed cell cultures was performed with a human a2M cDNA. As can be seen in Figure 5, there is a striking variation in the expression of a2M mRNAs. Four of the six melanoma cell lines M3Dau, M1Do, M4Beu, HMB-2 and both HMB-2 cell clones contained significant amounts of 4.6 kb a2M-specific transcript. To determine whether the a2M mRNAs found in melanoma cells are translated to produce mature a2M, we labeled the cell lines with [35S]methionine and analyzed the culture media by SDS-PAGE. The results are presented in Figure 6. In previous studies we have shown that the polypeptide band in the region of molecular mass 180 kDa is a2M. There were similar relative intensities of a2M bands in the autoradiogram corresponding to those detected in the Northern blot for individual cell cultures. Inhibition activity of conditioned media after removal of TA-a2M Samples of conditioned media after immunoabsorption with rabbit anti-TA-a2M coupled to Protein A-Sepharose CL-4B were analyzed for the residual inhibitory activity to u-PA. As can be seen in Table 5, after elimination of TA-a2M from the tested samples, the values of inhibition were similar or identical with inhibitory activity 900

Discussion The results obtained in this study provide evidence for the presence of bound t-PA on the surface of human melanoma cells and for its ability to function in producing bound, functionally active plasmin. Thus, active t-PA, not u-PA, was recovered in acid eluates of melanoma cells, and insignificant binding of exogenous uPA could be demonstrated. On addition of plasminogen, bound active plasmin was formed on the cells, and this process was sensitive to inhibition by antibodies to t-PA and not antibodies to u-PA. Binding of t-PA has been documented previously for both endothelial cells (Hajjar et aL, 1987; Barnathan et al., 1988) and human fibroblasts (Hoal et al., 1983). However, clear characterization of a specific receptor protein, as in the case of u-PA (Nielsen et al., 1988), has not been accomplished. In this study, we showed that t-PA could be recovered in acid eluates of thoroughly washed melanoma cells, providing some evidence that a high-affinity binding site exists. The putative site also exhibited specificity in that exogenous u-PA did not bind. Because we were able to demonstrate t-PAdependent formation of bound plasmin on serum-cultured cells, we propose that human melanoma cells may employ a t-PA system to produce surface-localized proteolysis instead of u-PA. This possibility has precedent in the process of ovulation in rodents, where t-PA has been shown to be triggered on resumption of meiotic maturation (Huarte et al., 1985). t-PAdependent proteolysis was observed around live secondary oocytes, demonstrating a possible role in tissue destruction. Elevated levels of tPA have also been found in superficial keratinizing cells of psoriatic skin (Gr0ndahl-Hansen et al., 1987), a condition that is also associated with tissue destruction. As yet, we can only speculate why t-PA may be employed in certain tissues in preference to u-PA. As two conditions of the skin, viz., melanoma and psoriasis, both appear to involve tPA, this could indicate local conditions that favor t-PA activity over u-PA activity. Of particular relevance may be our earlier finding of a2M in human melanomas (Matoska et al., 1988), which can inhibit u-PA (Straight et al., 1985) but not tCELL REGULATION

Plasminogen activation on melanoma cell surface

'O

10

Figure 5. Expression of TA-a2M specific mRNAs in melanoma cell lines as detected by Northern blot analysis. Ten micrograms of poly(A)+mRNA extracted from the cell cultures was fractionated on a 1.2% agarose/formaldehyde gel and transferred to nitrocellulose membrane. The blotted mRNAs were hybridized to a r32PI-labeled plasmid pa2Ml, which contained the coding sequence of a2M. Ribosomal mRNAs were separated on the same gel and were used as markers.

TA -2M

PA. The present results with immunoabsorption of a2M established that this was the principal inhibitor present in melanoma cell culture media. Although we have detected traces of PAI2 in the culture media with immunoblotting, PAI1 has not been detected (J. Bizik, unpublished results), and no PAls were present in concentrations producing significant inhibition of PA activity. Human melanoma cell lines have previously been shown to secrete mostly t-PA. In a survey of melanoma cell lines, Hoal-Van Helden et aL (1986) found that only 1 out of 7 cell lines studied was a u-PA producer. Consistently, Wagner and Binder (1986) screened a panel of 24 melanoma cell cultures and found that 22 cell lines secreted predominantly t-PA; only 4 cell lines also produced u-PA. In our study also, a marked characteristic of the melanoma cell lines was that the PA secreted was almost entirely of the tissue type. The Bowes cell line, included in our study, is in fact widely used as a source material for the purification of t-PA (Rijken and Collen, 1981). Vol. 1, November 1990

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Thus, our findings strongly suggest that melanoma cells in vitro are able to form active plasmin on their surface by the activation of plasminogen with surface-bound t-PA. This proteolytic system occurs in the presence of concomitant secretion of TA-a2M, which, we show, does not regulate plasminogen activation by t-PA. The TA-a2M may, however, inhibit uPA in vivo if this enzyme is produced by tumor stroma. The significance of such a t-PA system in an invasive human tumor, and the underlying reason for its use of t-PA rather than u-PA, awaits further investigation. Materials and methods Cell cultures The human melanoma cell line HMB-2 was established from a lymph node metastasis and was kindly provided by Dr. J. Svec, Cancer Research Institute, Bratislava, Czechoslovakia (Siracjky et al., 1982; Svec et al., 1988). The parental HMB2 cell line and clones 15 and 33, also used in this study, have been described in detail (Bizik etal., 1989). The Bowes cell line was a gift from Dr. Desir6 Collen, Katholieke Universiteit, Leuven, Belgium. The human melanoma lines M3Dau, M,Do, M4Beu, and M2Ge were originally provided

901

J. Bizik et al.

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TA -2 M

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Figure 6. SDS-PAGE of proteins secreted by metabolically labeled melanoma cell lines. Semiconfluent cell layers of melanoma cell lines cultivated in MEM with 5%o FCS were labeled by [35S]methionine for 24 h. After this time interval, the medium was changed to serum-free medium. This medium was collected after 24 h, and detached cells were removed by centrifugation. The conditioned media were dialyzed against PBS, and aliquots corresponding to 5 x 105 cells for each cell line were analyzed by SDS-PAGE and fluorography. The position of TA-a2M is indicated by an arrow. by Dr. J. F. Dor6, Centre Leon B6rard, Lyon, France (Jakubovich et al., 1985). The human RD rhabdomyosarcoma cell line (CCL 136) was obtained from the American type Culture Collection, Rockville, MD. All these cell lines were monitored for mycoplasma contamination and were found to be negative. The cultures were grown in Eagle's minimal essential medium (MEM), supplemented with 10% FCS and antibiotics; were harvested by brief exposure to 0.25% trypsin/ 0.02% EDTA; and were subcultured weekly. After reaching confluence, cells were rinsed three times with serum-free MEM and then changed to either serum-free medium or medium containing 100% heat-inactivated and plasminogendepleted (i.e., absorbed with lysine-Sepharose; Pharmacia Fine Chemicals, Uppsala, Sweden) FCS, as indicated in the experiments.

Preparations of conditioned media and cell lysates After 3 d of cultivation in MEM supplemented with 10% FCS, the cells were washed three times with serum-free medium and incubated with fresh medium for 2 h. This medium was discarded and replaced by fresh medium. After a further 24-h incubation, the conditioned serum-free medium was harvested and passed through a 0.22-lim pore size nitrocellulose filter (Millipore, Bedford, MA) and then tested for u-PA and t-PA activity. Aliquots of the media were

902

adjusted by addition of serum-free medium so as to represent the same number of cells for each cell line. To prepare cell lysates, we treated 5 x 105 cells with 50 mM glycine pH 7.8, 5 mM iodoacetamide, 5 mM EDTA, 0.5% Triton X100. After lysis, the samples were centrifuged at 5000 x g for 10 min and supernatants were used for the assays of uPA and t-PA activity.

u-PA assay Cell culture supernatants and cell lysates were assayed for u-PA activity by the following modification of an immunocapture method (Stephens et al., 1987). This assay is capable of detecting 0.09 IU/mI of u-PA. Microtiter wells of polystyrene immunoplates (type 269620; Nunc, Roskilde, Denmark) were coated overnight at 370C with 50 lA of a solution of goat IgG to human u-PA (catalog No. 398; American Diagnostica, New York, NY). The coating solution contained 2.5 ,qg IgG/ml in 0.1 M sodium carbonate (pH 9.8). After rinsing, the wells were treated with conditioned media (50 1J) for 2 h at 230C, then rinsed again. To assay the enzyme activity bound to the wells, we added 40 jl of a solution of plasminogen (Deutsch and Mertz, 1970)(1 00 ,g/ml in u-PA assay buffer consisting of 50 mM glycine pH 7.8, 0.10/o Triton X100, 0.1Y% gelatin and 10 mM 6-aminocaproic acid) and incubated the mixture for 30 min at 370C. The plasmin produced was then assayed by its thioesterase activity (Green CELL REGULATION

Plasminogen activation on melanoma cell surface Table 5. Total and residual inhibition activity to u-PA of conditioned media after removal of TA-a2M

0/0 of inhibitiont Cell line M3Dau Bowes M2Ge M1Do M4Beu HMB-2 HMB-2 c1.15 HMB-2 cl.33

TA-a2M* ++++

+ ++ ++++ +++++ ++++ +++++

Untreated conditioned media

Media after immunoabsorption$

90 15 30 45 67 82 62 89

20 15 15 17 12 21 19 12

* TA-a2M levels are given in arbitrary units, - to +++++, estimated from Northern blot and PAGE. t 9/ of inhibition was calculated from absorbance of the wells at 405 nm. * Rabbit anti-TA-a2M coupled to protein A-Sepharose CL4B was used for immunoabsorption.

and Shaw, 1979) after the addition of 150 pl of a solution containing 200 mM potassium phosphate (pH 7.5), 200 mM KCI, 0.1%o Triton X-100, 200 MM z-lysine thiobenzyl ester (Peninsula Laboratories, San Carlos, CA) and 220,MM 5,5'dithiobis(2-nitrobenzoic) acid (DTNB; Sigma, St. Louis, MO). This mixture was incubated for 30 min at 370C, and the absorbancies of the wells were measured in a Labsystems Multiskan MCC/340 plate reader at 405 nm. High-molecularweight two-chain u-PA (American Diagnostica; 80 000 IU/ mg protein) was used as a standard.

t-PA assay Microtiter plate wells coated with tissue activator antibody (goat IgG to human t-PA, catalog no. 387; American Diagnostica) were treated with the test sample (50 Ml) as above for u-PA. The assay of bound enzyme was carried out as for u-PA, but with an assay buffer containing 50 mM tris(hydroxymethyl)aminomethane-HCI (Tris-HCI), pH 7.8, 60 mM NaCI, 0.01 Yo Triton X-100, and 20 Mg/ml poly-D-lysine as a stimulator of enzyme activity (Allen, 1982). The sensitivity was 0.03 lU/ml. Active t-PA and u-PA bound to the cell layer were recovered for immunocapture assay by acid elution. Each culture well (2 cm2), with -5 x 105 cells, was rinsed with serumfree medium and eluted with 150 Al of 50 mM glycine/HCI pH 3.0 containing 0.10 M NaCI (Stoppelli et al., 1986), for 5 min at 230C. The eluate was neutralized with 50 Ml of 0.5 M Tris-HCI and tested for activity by immunocapture assay as above. Two-chain t-PA (American Diagnostica; 700 000 lU/mg protein) was used as a standard.

Plasmin assay Plasmin bound to the cell layer was recovered and assayed as follows. After harvest of culture medium, the cells were rinsed three times with PBS, and then the bound plasmin was specifically eluted with a solution of 1 mM tranexamic

acid (Cyklokapron; Kabi Vitrum, Stockholm, Sweden) in the rinsing solution (150 MI/well). Plasmin activity was assayed in 50-Ml samples of eluate by incubation with thioester subVol. 1, November 1990

strate (Green and Shaw, 1979) solution (see above) (200 Mll well) for 3 h at 37°C. An estimate of the amount of active plasmin present was made from calibration curves using human plasmin (Kabi Vitrum) diluted in serum-free medium covering an appropriate range of activity. Tranexamic acid at 1 mM had no effect on the thioesterase activity of plasmin in these assays.

Binding of u-PA on the cell surface Cells were plated on tissue culture multiwell plates with 24 flat-bottom wells (Linbro) in 100/ FCS containing MEM. One day later, cells were rinsed with serum-free medium, and the monolayer cultures (-5 x 105 cells) were incubated with 10 lU/ml u-PA (human u-PA 54 kDa; American Diagnostica; 80 000 lU/mg protein) in serum-free medium. After 2 h incubation at 37°C, cell cultures were rinsed three times with serum-free medium and activator eluted with 150 Ml of 50 mM glycine-HCI pH 3 containing 0.1 M NaCI. After 5 min the eluate was collected, neutralized, and tested for activity by the use of immunocapture assay, as above.

Inhibition of PA activity by monoclonal antibodies Monolayer cultures of 5 x 105 melanoma cells growing in MEM containing 10%o heat-inactivated and plasminogendepleted FCS were incubated with 1) native human plasminogen (40 Mg/ml); 2) plasminogen and anti-catalytic murine monoclonal IgG antibody to human u-PA (5 Mg/ml; American Diagnostica); 3) plasminogen and anti-catalytic monoclonal antibody to human t-PA (5 Mg/ml; American Diagnostica); and 4) plasminogen and aprotinin (Trasylol, 200 klU/ml; Bayer, Leverkusen, FRG). The cultures were incubated for 3 h at 370C before assay of cell-bound plasmin. The incubation with plasminogen was used as the 1 00%o control for bound plasmin.

RNA hybridization and DNA probe Total cellular RNA was extracted using the urea-LiCI method (Auff ray and Rougeon, 1980), poly(A)+RNA was selected by oligo(dT)-cellulose (Type 77F; Pharmacia, Uppsala, Sweden), electrophoresed in 1 Y/o agarose gels containing 2.2 M formaldehyde, transferred (Towbin et al., 1979) to nitrocellulose (Schleicher & Schuell, Dassel, FRG), and hybridized to 32PDNA probes according to established procedures (Sambrook et al., 1989). When hybridization was completed, the filters were washed under nonstringent (1 x NaCI/citrate, 0.50/o SDS, 650C) and/or stringent (0.1 x NaCI/citrate, 0.50/% SDS, 65°C) conditions. Filters were exposed to Kodak XOmat AR film at -750C. The DNA probe was labeled by nick translation (Sambrook et al., 1989) using a nick-translation kit (Amersham, Arlington Heights, IL). The a2M cDNA probe used in this study represented the unfragmented plasmid pa2Ml covering the complete a2M gene (Kan et al., 1985).

Radiolabeling of cultured cells The conditioned culture medium from a semiconfluent cell layer was replaced with MEM supplemented with 50/o FCS, and [5S]methionine (1200 mCi/Mumol; Amersham) was added to a final activity of 30 MCi/ml. After 24 h of incubation at 350C, the medium was changed for serum-free medium. This medium was collected after 18 h and any detached cells were removed by centrifugation (3000 x g for 10 min). The conditioned medium was then filtered through a 0.22Am-pore-size nitrocellulose filter (Millipore) dialyzed against PBS, lyophilized, and analyzed by SDS-PAGE.

903

J. Bizik et a/.

SDS-PAGE Samples dissolved in 1.25 M Tris-HCI, pH 6.8, containing 40/ SDS, 20% glycerol, 100/ 2-mercaptoethanol and 0.005Yo bromphenol blue were heated for 2 min in boiling water and analyzed by the use of vertical polyacrylamide slab gels with a 3.5% acrylamide spacer gel as described (Laemmli, 1970). Gel with labeled material was impregnated with 2,5-diphenyloxazole (Bonner and Laskey, 1974), dried, and placed in contact with Kodak X-Omat AR film at -700C.

Zymography After electrophoresis in a 9% polyacrylamide slab gel under nonreducing conditions, and without heating the specimen, SDS was removed from the gels with extensive washing on a gyratory shaker for 6 h in PBS with 2.5/o Triton X-100 (Granelli-Piperno and Reich, 1978), and indicator agarose gel containing casein and plasminogen was placed in contact with the polyacrylamide gel. An agarose gel lacking plasminogen was used as a control, and the gel sandwich was incubated overnight at 37°C in a humidified atmosphere to develop lysis zones.

Immunoabsorption of TA-a2M Rabbit anti-TA-a2M IgG (Bizik et aL., 1989) was coupled to protein A-Sepharose CL-4B (Pharmacia) as described by Hudson and Hay (1980). One milliliter of protein A-Sepharose was incubated with the same volume of conditioned media for 3 h at room temperature. After incubation, the samples were centrifuged and supernatants were tested for residual inhibiting activity. The colorimetric assay described above was used to test the activity of conditioned media.

Acknowledqments This work was supported by grants from the Sigrid Juselius Foundation, Helsinki, the Medical Research Council of the Academy of Finland, and the Finnish Cancer Organizations.

Received: June 7, 1990. Revised and accepted: August 28, 1990.

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