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[Cancer Research 64, 8831-8838, December 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Cysteine Cathepsins Are Central Contributors of Invasion by Cultured Adenosylmethionine Decarboxylase-Transformed Rodent Fibroblasts

Kirsi Ravanko1, Kristiina Järvinen1, Jari Helin2, Nisse Kalkkinen2 and Erkki Hölttä1

1 Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; and 2 Institute of Biotechnology, University of Helsinki, Helsinki, Finland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of polyamines, is often up-regulated in cancers. We have demonstrated previously that overexpression of AdoMetDC alone is sufficient to transform NIH 3T3 cells and induce highly invasive tumors in nude mice. Here, we studied the transformation-specific alterations in gene expression induced by AdoMetDC by using cDNA microarray and two-dimensional electrophoresis technologies. We specifically tried to identify the secreted proteins contributing to the high invasive activity of the AdoMetDC-transformed cells. We found a significant increase in the expression and secretion of procathepsin L, which was cleaved and activated in the presence of glycosaminoglycans (heparin), and a smaller increase in cathepsin B. Inhibition of the cathepsin L and B activity by specific peptide inhibitors abrogated the invasive capacity of the AdoMetDC transformants in Matrigel. The transformed cells also showed a small increase in the activity of gelatin-degrading matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator activities, neither of which was sensitive to the inhibitors of cathepsin L and B. Furthermore, the invasive potency of the transformed cells remained unaffected by specific inhibitors of MMPs. The results suggest that cysteine cathepsins are the main proteases contributing to the high invasiveness of the AdoMetDC-transformed cells and that the invasion potential is largely independent of activation of the MMPs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A normal cell undergoes several changes during the course of its transformation into a cancer cell. A fully transformed cell has usually gained six new abilities: it can generate its own growth signals, acquire resistance to antigrowth signals, evade apoptosis, replicate with limitless potential, and induce neoangiogenic and invasive growth (1) . To escape the primary tumor and invade the adjacent tissue, the cancer cell has to degrade the surrounding basement membrane and extracellular matrix (ECM). This is a complex process that likely involves multiple extracellular proteases and requires strictly organized interaction of the cancer cell and the tumor microenvironment. The most important regulators of this degradation process are generally believed to be the matrix metalloproteinases (MMPs). These are a family of zinc-containing endopeptidases comprising more than 25 members capable of degrading various ECM components. Indeed, several studies have shown a close correlation between stage of tumor progression and MMP expression (2 , 3) . Another extracellular protease often associated with invasion is the urokinase-type plasminogen activator (uPA). uPA is a serine protease with multiple actions, including the capability to initiate a cascade leading to MMP activation (4) . In addition, cancer cells have been described as overexpressing several other invasion-related proteases encompassing members of the serine, cysteine, and aspartic proteinases (4, 5, 6, 7, 8, 9, 10) .

Previously, we have shown in rodent fibroblasts that overexpression of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulatory enzyme of polyamine biosynthesis, induces cell transformation and highly invasive tumors in nude mice (11) , thus providing a good model for studying the molecular mechanisms involved in cell invasion. Significantly, overexpressed AdoMetDC levels have also been detectable in various human cancers, and inhibition of tumor cell AdoMetDC activity prevents tumor formation and metastasis (12, 13, 14) . Our intention was to identify fibroblast cell invasion-related factors by use of cDNA microarray and proteome analyses. The results of the studies indicate that the major proteases involved in the invasion of transformed rodent fibroblasts are cysteine cathepsins and, surprisingly, not MMPs or uPA.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
NIH 3T3 cells (ATCC CRL 1658; American Type Culture Collection, Manassas, VA) stably transfected with the neomycin resistance gene (4N) or cotransfected with human AdoMetDC cDNA (Amdc-s) and Rat-1 cells stably transfected with the AdoMetDC cDNA have been described previously (11) . Both pools of transfectants and >10 individual Amdc-s clones were used for the experiments.

The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin, streptomycin, gentamicin, G418, and 5% (v/v) fetal or newborn calf serum (Life Technologies, Inc., Paisley, Scotland, United Kingdom), at 37°C in a humidified 5% CO2 atmosphere.

Metabolic Labeling and Concentration of Conditioned Medium.
Cells were grown for 1 day to 80% confluence, and cultures were rinsed twice with modified Eagle’s medium (MEM). The cells were incubated with methionine- and cysteine-free MEM supplemented with 0.1 to 0.2 mCi/mL [35S]methionine/cysteine (Perkin-Elmer Life Sciences, Inc. Boston, MA) and one-twentieth volume of DMEM (to avoid depletion of methionine and cysteine) for 16 hours, after which the conditioned medium (CM) was collected. CM was concentrated to ≤ one-fourteenth volume with the Ultrafree-15 concentration device MWCO 5000 (Millipore, Bedford, MA). Physiologic salts in the samples were removed by adding 10 mmol/L Tris-HCl (pH 7.4) to the initial volumes and reconcentrating the samples. The protein concentrations of the samples were measured with the Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA).

Two-Dimensional Electrophoresis.
Equal amounts of proteins (50–100 µg) from the CM were rehydrated on 18-cm NL DryStrips (pH 3–10; Amersham Pharmacia Biotech, San Francisco, CA) with a buffer containing 7 mol/L urea, 2 mol/L thiourea, 2 mmol/L tributylphosphine, 3.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 0.5% IPG buffer (pH 3–10) NL, and bromphenol blue for 12 hours at 20°C. Isoelectric focusing (IEF) was performed using Amersham Pharmacia Biotech IPGPhor at 500 V for 1 hour, 1,000 V for 2 hours, 4,000 V for 2 hours, and 8,000 V for 4 hours, with Imax per strip of 50 µA. In studying cathepsin L processing, 7-cm NL DryStrips (pH 3–10) were used, and IEF was performed at 500 V for 30 minutes, 1,000 V for 1 hour, 4,000 V for 1 hour, and 8,000 V for 2 hours. After IEF, the strips were incubated in equilibration buffer [6 mol/L urea, 50 mmol/L Tris-HCl (pH 6.8), 2% SDS, 30% glycerol, 5 mmol/L tributylphosphine, and bromphenol blue] for 25 minutes at room temperature and run in 12% SDS-PAGE. The gels were then stained with silver (15) or blotted onto nitrocellulose membranes. For autoradiographic detection, the stained gels were dried and exposed to Fuji BAS-2500 phosphorimager plates.

In-Gel Tryptic Digestion.
Silver-stained spots of proteins were cut from the gel and destained by incubating these gel pieces for 30 minutes at 37°C with 0.1 mol/L NH4HCO3 in 50% acetonitrile (ACN). The gel pieces were shrunk with 100% ACN, after which ACN was removed. Proteins were reduced with 20 mmol/L dithiothreitol in 0.1 mol/L NH4HCO3 at 56°C for 30 minutes and shrunk again with ACN. To block disulfide formation, the sulfhydryl groups were alkylated by incubating the gel pieces with 55 mmol/L iodoacetamide in 0.1 mol/L NH4HCO3 for 15 minutes at room temperature in the dark. The pieces were washed twice for 10 minutes with 0.1 mol/L NH4HCO3, shrunk with ACN, and dried. For tryptic digestion of the proteins, the pieces were rehydrated with 10 µL of 0.05 mg/mL modified trypsin (Promega, Madison, WI) in 0.1 mol/L NH4HCO3 and 10% ACN for 10 minutes, after which 0.1 mol/L NH4HCO3 with 10% ACN was added to cover the pieces. Digestions were continued overnight at 37°C. The peptides formed were extracted from the gels once with 25 mmol/L NH4HCO3 for 10 minutes and twice with 5% HCOOH (15 minutes each).

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.
Peptides were desalted by reversed phase material (OligoR3; PerSeptive Biosystems, Farmingham, MA) packed into an Eppendorf gel-loading pipette tip. Peptides were eluted from the column directly onto the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) target plate with the matrix ({alpha}-cyano-4-hydroxycinnamic acid, a saturated solution in 0.1% trifluoroacetic acid; 60% ACN) together with internal calibration standards (angiotensin II and adrenocorticotropic hormone-clip 18–39; Sigma-Aldrich Chemie GmbH, Steinhein, Germany). MALDI-TOF mass spectrometry (MS) was performed on a BiflexTM time-of-flight instrument (Bruker Daltonik, Bremen, Germany) equipped with a nitrogen laser operating at 337 nm. For protein identification, the mass spectrometric data generated were analyzed by the public domain search program ProFound3 with the NCBInr databases.

Complementary DNA Microarray Analysis.
Polyadenylated mRNA was isolated by oligo(dT) cellulose chromatography. The mRNA expression levels were examined with the Atlas cDNA expression array, mouse 1.2 (Clontech Laboratories, Palo Alto, CA).4 The poly(A)+ RNA samples were treated with DNase I as described in the Clontech expression array user manual before probe synthesis by reverse transcription. Complementary DNA probes were synthesized with [{alpha}-32P]dATP (Amersham Pharmacia Biotech). The membranes were prehybridized, hybridized, and washed in a hybridization oven, according to the manufacturer’s instructions. The imaging plates were scanned with a Fuji BAS-2500 phosphorimager using MacBas 2.5 software. AtlasImage 2.0 software, in the global normalization mode, was used to normalize and compare signal intensities on the arrays.

Immunoblotting.
CM proteins were separated on 12% SDS-PAGE and transferred to nitrocellulose filters (Bio-Rad) using the trans-Blot cell (Bio-Rad). The filters were incubated in blocking buffer [25 mmol/L Tris (pH 8.0), 125 mmol/L NaCl, 0.1% Tween 20, 2% bovine serum albumin, and 0.1% NaN3] overnight at room temperature and then incubated with a specific antibody to cathepsin L (D-20; Santa Cruz Biotechnology, Santa Cruz, CA), diluted in the blocking buffer, for 2 hours at room temperature. Filters were rinsed five times in washing buffer [10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.05% Nonidet P-40, and 0.05% Tween 20] and incubated with horseradish peroxidase-conjugated rabbit antigoat IgGs (DAKO, Carpinteria, CA) for 30 minutes at room temperature. Finally, the filters were rinsed five times in the washing buffer, rinsed for 15 minutes in high-salt buffer [10 mmol/L Tris (pH 8.0) and 300 mmol/L NaCl], and rinsed three times in Tris-buffered saline [10 mmol/L Tris (pH 8.0) and 150 mmol/L NaCl]. The proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL) exposing filters to the Fuji RX film.

Determination of Cathepsin L Activity.
Cells were grown for 2 days, harvested by centrifugation, washed twice with PBS, and suspended in lysis buffer [0.1 mol/L sodium acetate (pH 5.0), 1 mmol/L EDTA, and 0.01% Triton X-100]. Lysis was completed by freezing and thawing the samples three times, after which the lysates were clarified by centrifugation at maximal speed in an Eppendorf microcentrifuge at 4°C for 10 minutes. Next, 10 µg of proteins from the cell lysates or CM were added up to 100-µL final volume of lysis buffer supplemented or not supplemented with 200 µg/mL heparin. The different cathepsin L inhibitors [cathepsin L inhibitor I (Calbiochem, Darmstadt, Germany), Z-Phe-Tyr(tBu)-dimethylketone, or Ac-Leu-Leu-Nle-aldehyde (Bachem, Bubendorf, Switzerland)] at 0, 0.5, 15, 25, or 50 µmol/L concentrations were incubated with the samples for 1 hour at 37°C. Thereafter, 100 µL of reaction buffer [0.4 mol/L sodium acetate (pH 5.5), 4 mmol/L EDTA, and 8 mmol/L dithiothreitol] and 150 µmol/L cathepsin L substrate (Z-Phe-Arg-para-nitroanilide; Bachem) were added, the samples were incubated at 37°C, and A405 nm was measured as a function of time.

Highly purified cathepsin L (specific activity, 6,036 milliunits/mg protein) from human liver (Calbiochem) was used as a positive control in the assays. The activity of the purified cathepsin L was also measured at physiologic pH 7.0 (HEPES buffer) and found to be only 16% of the activity at pH 5.5 (sodium acetate buffer). The specificity of cathepsin L inhibitor I toward the highly purified cathepsin L and cathepsin B was tested [purity by SDS-PAGE, 95%; specific activity, 32.7 units/mg protein (Calbiochem)].

Assay of Secreted Urokinase-Type Plasminogen Activator Activity.
Cells were grown for 1 day, after which they were rinsed twice with DMEM and incubated for 16 hours with serum-free DMEM. The CM was centrifuged to remove cell debris and concentrated as described above. The hydrolysis of H-D-Nle:HHT-Lys-para-nitroanilide in 11 mmol/L Tris-HCl (pH 7.4) buffer supplemented with 0.0024% Tween 20, 155 µg/mL bovine plasminogen, 1 mmol/L Spectrozyme PL (American Diagnostica, Greenwich, CT), 1.36 mmol/L 6-aminohexanoic acid, and the sample containing 10 µg of protein was monitored by following the absorbance at 405 nm at 37°C (16) .

Gelatin Zymography.
Cells were cultured for 1 or 2 days, after which they were rinsed with DMEM and incubated overnight in DMEM with or without 200 µg/mL heparin, and in both cases with or without 25 µmol/L cathepsin L inhibitor I (Calbiochem). CM was concentrated as described above. Equal amounts of proteins (1 µg) from CM were suspended in nonreducing Laemmli sample buffer and incubated at 37°C for 1 hour. Proteins were separated on 8% SDS-PAGE containing 2 mg/mL gelatin (Sigma-Aldrich Chemie GmbH). To activate gelatin-degrading MMPs, the gel was incubated in buffer 1 [50 mmol/L Tris-HCl (pH 7.6), 5 mmol/L CaCl2, 1 µmol/L ZnCl2, and 2.5% Triton X-100] at room temperature twice for 15 minutes, in buffer 2 [50 mmol/L Tris-HCl (pH 7.6), 5 mmol/L CaCl2, and 1 µmol/L ZnCl2] at room temperature for 5 minutes, and in buffer 3 [50 mmol/L Tris-HCl (pH 7.6), 5 mmol/L CaCl2, 1 µmol/L ZnCl2, 1% Triton X-100, and 0.02% NaN3] at 37°C overnight. Finally, the gel was stained with Coomassie Blue (Novex, San Diego, CA).

Invasion Assay.
Twenty four-well plates were overlaid with 300 µL of growth factor-reduced Matrigel (Becton Dickinson Biosciences, Bedford, MA) diluted 1:3 in DMEM. Matrigel was allowed to polymerize for 30 minutes at 37°C. Thereafter, 10,000 cells were plated on top of the Matrigel in 100 µL of DMEM and allowed to adhere for 1 hour at 37°C. Excess DMEM was removed, and a second 250-µL Matrigel layer was cast above the cells. Finally, 500 µL of DMEM containing 10% fetal calf serum were added on top of the Matrigel matrix. The experiment was performed without or with 0.5, 5, or 25 µmol/L cathepsin L inhibitor I (Calbiochem) or MMP inhibitors (Ilomastat GM6001 or its negative control and Marimastat BB2516; Calbiochem) included in the Matrigel and growth medium. The cells were grown in three-dimensional Matrigel for the indicated times, and the growth medium was replenished every third day. The growth patterns and morphology of the cells were examined daily by phase-contrast microscopy and photographed. To also count the cells, the Matrigel was allowed to liquify on ice overnight, and the number of the cells was determined with a Coulter particle counter (particles {phi} ≥ 6 µm counted). MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cell lines served as a reference.

Bovine Capillary Endothelial Cell Migration Assay.
The endothelial cell migration assay was performed essentially as described previously (17) . Six-well plates were coated with 800 µL of 1.9 mg/mL collagen I (Upstate Biotechnology, Charlottesville, VA) made in MEM containing 5% newborn calf serum (Life Technologies, Inc.). A 1-cm–diameter cylinder was placed in the middle of the well, and 200,000 bovine capillary endothelial cells were allowed to adhere to the area inside the cylinder. The cylinder was removed, and the cells were rinsed with MEM. Then another collagen layer was spread with a concomitant casting of a 2-mm–diameter sample well at an approximately 3-mm distance from the endothelial cells. Finally, a 3-mL layer of 1.5% agar in MEM with 5% newborn calf serum was spread on top of the collagen. The chemoattractant samples were prepared as follows: 18 µg of proteins from the CM of Amdc-s or 4N cells with 0 or 25 µmol/L cathepsin L inhibitor I or 20 ng of specific antibody to cathepsin L (D-20; Santa Cruz Biotechnology) were incubated at 37°C for 10 minutes, 200 µg/mL heparin was added, and the incubation was continued for 1 hour to activate the samples. Ten micrograms of basic fibroblast growth factor was used as a positive control, and mere DMEM was used as a negative control. The cell cultures were grown at 37°C and 5% CO2, and fresh chemoattractants were added to the sample wells every second day. Migration of the bovine capillary endothelial cells toward the sample wells was monitored daily and visualized by photography after 7 to 10 days.

Immunohistochemical Staining of Cathepsin L.
Paraffin-embedded tissue sections of AdoMetDC-induced tumors in nude mice were cut at 5 µm, deparaffinized, and heated in microwave oven at 900 W for 2 minutes and 400 W for 8 minutes in 10 mmol/L citric acid buffer (pH 6). Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30 minutes. The sections were incubated with the primary antibody to cathepsin L (D-20; Santa Cruz Biotechnology) at 4°C overnight followed by processing using the StreptABComplex/horseradish peroxidase duet, mouse/rabbit kit (DAKO) according to the manufacturer’s protocol. The chromogen used was 3-amino-9-ethylcarbazole (AEC) (Sigma-Aldrich Inc.). The sections were gently counterstained with Harris hematoxylin (Polysciences, Inc.). Sections incubated without the primary antibody or with normal goat IgGs were used as negative controls, and they gave no reactions. All of the stainings were repeated at least three times. Images were obtained using a Nikon Eclipse E800M microscope equipped with a digital camera system, and the images were captured using the Nikon Capture software package.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cathepsin L Was the Major Secreted Protein of AdoMetDC-Transformed Cells.
As a first attempt to identify the proteins responsible for the high invasiveness of the AdoMetDC-transformed fibroblasts, we performed a comparative analysis of the secreted proteins between the normal and transformed cells. The normal and AdoMetDC-transformed NIH 3T3 (Fig. 1A)Citation and Rat-1 (Fig. 1B)Citation cells were metabolically labeled with [35S]methionine and [35S]cysteine, and the proteins secreted into the media were analyzed by two-dimensional electrophoresis. For visualization of the proteins, the gels were stained with silver and exposed to phosphorimager plates. The metabolic labeling of the proteins allowed more accurate determination of the differences in protein expression than did silver staining, which is limited by the narrow linear range in which silver quantitatively binds to proteins. The labeling also confirmed that the proteins were synthesized by the cells and did not represent serum proteins differentially bound to the cell surfaces of the normal and transformed cells.



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  		Fig. 1. Analysis of secreted proteins from the normal and AdoMetDC-transformed NIH 3T3 (A) and Rat-1 (B) cells. Equal amounts of proteins (100 µg) from conditioned media of the metabolically labeled normal NIH 3T3 (4N) and Rat-1 fibroblasts and their human AdoMetDC cDNA transfectants (Amdc-s) were separated by two-dimensional electrophoresis using 18-cm IPG strips. Gels were stained with silver and exposed to phosphorimager plates to analyze the total and radioactively labeled proteins, respectively. Spots chosen for further investigations are circled. Molecular mass standards (in kDa) are indicated on the left.

 
The AdoMetDC-transformed cells (pools of transfectants and individual clones) consistently showed a markedly increased expression of a set of five proteins at about 39 kDa (pI ranging from 6.5 to 8) as compared with expression in normal cells (Fig. 1)Citation . The five protein spots were individually excised from the gels, digested "in-gel" with trypsin, and analyzed by MS. The MALDI-TOF mass fingerprint spectra obtained from all of the spots were highly similar; one of them is shown in Fig. 2Citation . The peptide mass data were then used to identify the proteins from the NCBInr databases. All of the peptide fingerprints from the five proteins individually gave the cathepsin L precursor their top score.



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  		Fig. 2. MS spectrum of peptides obtained by digestion of one of the circled proteins (39 kDa, pI 6.5–8) in Fig. 1Citation by trypsin. Protein spots were cut from the silver-stained gels, digested with trypsin, and analyzed by MALDI-TOF MS.

 
Immunoblotting was then additionally used to confirm that the five spots identified by MALDI-TOF MS analysis represented different forms of cathepsin L. Equal amounts of secreted proteins from the normal (4N) and AdoMetDC-transformed NIH 3T3 and Rat-1 cells were separated by two-dimensional electrophoresis, transferred to nitrocellulose membrane, and immunoblotted with a specific antibody to cathepsin L. By standard enhanced chemiluminescence detection, signals were detected only in the immunoblots of the transformed cells, and they were at three distinct locations: at 39 kDa, with isoelectric point (pI) ranging from about pH 6.5 to 8; at 25 to 30 kDa, with pI around 4 to 5; and at ≤10 kDa, with pI about 6.5 (Fig. 3Citation ; data not shown). The five 39-kDa signals thus represent differentially charged procathepsin L forms. The immunologically detected 25- to 30-kDa molecular mass spots were then subjected to reciprocal MALDI-TOF MS analyses (spectra not shown) and identified as cathepsin L, the mature forms.



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  		Fig. 3. Secreted cathepsin L is found in different forms in AdoMetDC-transformed NIH 3T3 and Rat-1 cells. CM of the normal fibroblasts (4N) and AdoMetDC transformants was resolved on two-dimensional electrophoresis, transferred to nitrocellulose, and immunoblotted with a specific antibody to cathepsin L. Signals were visualized by enhanced chemiluminescence. Molecular mass markers are shown on the left.

 
In parallel with the proteomic analyses, we examined the gene expression patterns of the normal and AdoMetDC-transformed NIH 3T3 cells by Atlas cDNA expression array (Clontech). The overall hybridization patterns of the microarrays were remarkably similar, with only about 1% of the genes showing a ≥2-fold change (data not shown). We found, however, that the mRNA level of cathepsin L was highly up-regulated (approximately 10-fold) in the AdoMetDC-transformed cells. Moreover, the mRNA levels of cathepsins B and C in the transformed cells were slightly elevated, whereas the cathepsins A, H, W, and D showed no transformation-specific change (Fig. 4Citation ; data not shown). The increase in cathepsin L mRNA was also one of the greatest changes in mRNA expressions when {approx}9,500 genes were analyzed by Incyte MouseGEM arrays.5 It is also noteworthy that our global gene expression analyses with the Affymetrix Mouse Expression Set 430 arrays (containing over 34,000 genes) have not revealed any additional cathepsins, except cathepsin K (low expression), to be significantly increased in the AdoMetDC-transformed cells.6



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  		Fig. 4. Complementary DNA microarray analysis of cathepsin gene expression in normal and AdoMetDC-transformed NIH 3T3 cells. Representative sections of the cDNA expression arrays shown were scanned with a phosphorimager. Expression levels were normalized and quantified densitometrically. The Atlas arrays include single spots of each cDNA. ß-Actin is included as a control.

 
Because protease expression in cultured cells may differ from that in vivo, we wanted to verify that cathepsin L is also expressed in AdoMetDC-induced tumors in nude mice (11) . Significantly, immunohistochemical analyses showed high levels of cathepsin L expression in the tumor tissue (Fig. 5)Citation . Histologically, the tumors were composed of a fairly homogenous population of typical fibrosarcoma cells. The specimens were also stained with anti-macrophage L1 protein (ab 8740; Abcam); no macrophages were detected inside the tumors (data not shown).



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  		Fig. 5. Immunohistochemical analysis of cathepsin L expression in AdoMetDC-induced tumors in nude mice. Paraffin-embedded tumor specimens were incubated with or without anti-cathepsin L antibody, and the signals were detected by using biotinylated antigoat immunoglobulins as a secondary antibody with avidin-biotin amplification and 3-amino-9-ethylcarbazole (AEC) as a chromogen (red). The slides were counterstained with hematoxylin (blue). Magnification, x400.

 
Cathepsin L Activity Was Elevated in the Growth Medium but not Intracellularly in the AdoMetDC-Transformed Cells.
We then measured cathepsin L activity in cell lysates and in CM of the normal and AdoMetDC-transformed NIH 3T3 cells. The amount and activity of the intracellular cathepsin L remained unchanged (data not shown), whereas the activity of cathepsin L in the medium of the AdoMetDC-transformed cells was higher than that in the normal cells (Fig. 6A)Citation . Direct analysis of the culture medium, however, showed the enzymatic activity toward the synthetic substrate to be very low, but preincubation of the CM with glycosaminoglycans such as heparin at acidic pH resulted in recovery of high activity in CM of the transformed cells. Instead, the activity of intracellular cathepsin L was not enhanced by the addition of heparin. The activity of secreted cathepsin L could be blocked by addition to the culture medium of cathepsin L inhibitor I (Z-FF-FMK), with ≥15 µmol/L Z-FF-FMK causing a complete inhibition (Fig. 6A)Citation . Analysis of the inhibitor specificity toward highly purified enzymes from human liver in a test tube showed that Z-FF-FMK inhibited not only cathepsin L but also cathepsin B. At inhibitor concentrations of 0.5, 5, and 25 µmol/L, the activity of human liver cathepsin L was inhibited by 96%, 98%, and 99% and the activity of cathepsin B was inhibited by 50%, 79%, and 85%, respectively. During the cell culturing, part of the inhibitor was found to be precipitated, resulting in a decrease in the active inhibitor concentration in the medium. Secreted cathepsin L activity was also inhibited with the other inhibitors tested [Z-Phe-Tyr(tBu)-dimethylketone or Ac-Leu-Leu-Nle-aldehyde (data not shown)]. In the presence of heparin and acidic pH, the 39-kDa procathepsin L secreted was processed to 25- to 30-kDa forms with a more acidic pI, and the cathepsin L inhibitor Z-FF-FMK inhibited this processing, at least in part, as shown by the one- and two-dimensional electrophoresis and immunoblotting analyses (Fig. 6B and C)Citation . There are studies indicating that cathepsin L can promote DNA synthesis and cell growth (see ref. 18 ). However, growth of the AdoMetDC-transformed cells remained undisturbed or was only slightly inhibited (≤15%) in the presence of the cathepsin L inhibitor I during a culture for 3 days (data not shown).



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  		Fig. 6. A, activity of secreted cathepsin L of AdoMetDC-transformed cells compared with their normal counterparts. CM was preincubated with heparin at acidic pH for 1 hour in the absence or presence of cathepsin L inhibitor I at the concentrations indicated. Cathepsin L substrate was then added, the samples were incubated at 37°C, and A405 nm was measured in the linear range of the enzyme reaction. Variations in the activity measurements in duplicate samples were <1.5%. B, procathepsin L is processed to a mature form (30 kDa) in the presence of heparin at acidic pH as shown by SDS-PAGE and immunoblotting analyses with specific antibody to cathepsin L. C, processing of secreted cathepsin L is sensitive to cathepsin L inhibitors. Heparin-treated CM and untreated CM of AdoMetDC transformants were analyzed by two-dimensional electrophoresis using 7-cm IPG strips and immunoblotted with anti-cathepsin L antibody. Molecular mass markers (in kDa) are shown on the left.

 
Cathepsins L and B Did Not Promote the Activation of Gelatinases or uPA in AdoMetDC Transformants.
A recent suggestion is that cathepsins can initiate a proteolytic cascade, resulting in the activation of uPA and MMs (for a review, see ref. 7 ). To study the possible role of cathepsins L and B in MMP activation, we performed zymographic analysis of gelatinase activities in the CM of normal and AdoMetDC-transformed NIH 3T3 cells cultured with and without cathepsin L inhibitor I. Results showed that the AdoMetDC-transformed cells displayed elevated MMP-2 content and activity and that inhibition of the cathepsin L and B activity by Z-FF-FMK did not affect the MMP-2 activity (Fig. 7ACitation ; data not shown). Furthermore, we analyzed the activity of uPA in the CM of the normal and transformed cells, and the effect of the cathepsin L inhibitor I on uPA, which activates plasminogen to plasmin, a broad spectrum serine protease able to also activate MMPs. The uPA activity appeared to be only slightly higher in the AdoMetDC transformants as compared with that in the normal cells (Fig. 7B)Citation , and it was not affected by the cathepsin L inhibitor I.



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  		Fig. 7. A. The inhibitor of cathepsins L and B fails to prevent activation of gelatinases in AdoMetDC-transformed NIH 3T3 cells. Equal amounts of proteins (10 µg) from the CM of normal (4N) and AdoMetDC-transformed NIH 3T3 cells cultured with or without cathepsin L inhibitor I (25 µmol/L) were separated on nonreducing SDS-polyacrylamide gel impregnated with gelatin. The gel was assayed for gelatinase activity as described in Materials and Methods. The mobilities of the gelatinases expressed by HT-1080 cells used as a reference are shown on the left. B. Cathepsin L inhibitor has no significant effect on uPA activity. Equal amounts of proteins from CM of cells, cultured with or without 25 µmol/L cathepsin L inhibitor, were incubated at 37°C with plasminogen and its synthetic substrate. Progression of the enzymatic reactions was monitored by measuring A405 nm, and the results are from a linear range.

 
Invasive Capacity of AdoMetDC-Transformed NIH 3T3 and Rat-1 Cells Was Dependent on Cathepsin L and B Activity.
The invasive potential of the AdoMetDC transformants was then studied in vitro by using a reconstituted basement membrane model: culturing the cells between two layers of three-dimensional Matrigel matrix. We used growth factor-reduced Matrigel to avoid any possible interference by exogenous growth factors. We found that, in contrast to normal cells, the AdoMetDC transformants were capable of rapidly (in a day) invading the matrix, forming a more extended phenotype than in a culture on plastic tissue culture dishes. The presence of cathepsin L inhibitor I inside the matrix completely inhibited this invasion by AdoMetDC-transformed cells (Fig. 8ACitation ; data not shown). Counting the number of the invasive and proliferated cells revealed that 5 µmol/L cathepsin L inhibitor I reduced invasive cell growth by ≥30%, 10 µmol/L cathepsin L inhibitor I reduced invasive cell growth by 85%, and 25 µmol/L cathepsin L inhibitor I totally prevented invasive growth (Fig. 8B)Citation . Because activity of MMPs (specifically, MMP-2) was activated in the AdoMetDC-transformed cells, we also tested the effects of various MMP inhibitors, including Ilomastat GM6001, Marimastat BB2516 (Calbiochem), and BB3302 (British Biotech Pharmaceuticals) on this cellular invasion. Inhibition of MMPs somewhat reduced the invasive potential of AdoMetDC-transformed cells at the higher inhibitor concentrations tested, but the same effect was also observable with a control peptide (GM 6001, negative control; Calbiochem), suggesting that the effect was nonspecific (Fig. 8BCitation ; data not shown).



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  		Fig. 8. Inhibition of cathepsin L and B activity reduces invasive growth of AdoMetDC-transformed NIH 3T3 cells in the Matrigel matrix. A. Normal (4N) and AdoMetDC-transformed NIH 3T3 cells were cultured for 1 or 7 days in growth factor-reduced Matrigel matrix with or without cathepsin L inhibitor I (25 µmol/L) and analyzed by phase-contrast microscopy and photography. B, the number of transformed cells grown in Matrigel matrix in the absence or presence of cathepsin L or MMP inhibitors (0.5, 5, or 25 µmol/L) for 7 days. Cell numbers (counted in a Coulter counter) are expressed relative to the AdoMetDC cell cultures grown without the inhibitors (control).

 
As a reference, we also cultured MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cell lines, which were previously shown to be highly invasive, in the Matrigel matrix. Parallel analyses showed that the invasive capacity of the AdoMetDC-transformed cells is much higher than that of the MDA-MB-231 or HT-1080 cells. Invasion by the MDA-MB-231 and HT-1080 cells was also inhibited by the cathepsin L inhibitor I (data not shown).

Cathepsin L Activity Did Not Induce Migration of Capillary Endothelial Cells.
Recent studies indicate that cathepsin L may also play a role in the regulation of angiogenesis (19) . Our group has noticed that proteins/factors secreted by the AdoMetDC-transformed NIH 3T3 cells induced migration of bovine capillary endothelial cells in a three-dimensional collagen gel.7 To test whether secreted cathepsin L contributes to this phenomenon, we studied the capability of CM of AdoMetDC-transformed NIH 3T3 cells, treated or not treated with cathepsin L inhibitor or specific antibody to cathepsin L, to induce the migration of bovine capillary endothelial cells. Elimination of cathepsin L activity had no effect on this migration (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, our comparative cDNA microarray and two-dimensional electrophoresis analyses of normal and AdoMetDC-transformed fibroblast models revealed only a limited number of proteins whose expression was consistently altered to a significant degree. One of the most marked changes in the AdoMetDC-transformed cells was increased expression and secretion of procathepsin L, which, most likely, is one important factor contributing to the high invasiveness of these transformants.

Cathepsin L is a lysosomal cysteine protease, which is ubiquitously expressed. It is translated as preprocathepsin L, which is then processed to procathepsin L with a molecular mass of 39 kDa and further processed to the mature cathepsin L, which exists in either single-chain (30 kDa) or two-chain (25 and 5 kDa) forms (9 , 20, 21, 22) . Cathepsin L is suggested to participate in turnover of intracellular or endocytosed proteins (8) , degradation of ECM proteins, antigen processing and presentation (23) , bone resorption (24 , 25) , and various other processes. Many growth factors and oncogenes can stimulate cathepsin L expression (26, 27, 28, 29) , and increased expression of cathepsin L is commonly associated with malignant transformation (30 , 31) . The overexpressed procathepsin L in transformed cells is often secreted. However, why it is secreted even though it has a lysosomal targeting signal, mannose-6-phosphate (8) , is not yet known. On the other hand, a recent report notes that a significant proportion of the overexpressed cathepsin L remains intracellularly packed into multivesicular endosomes (32) .

Our studies showed that AdoMetDC-transformed cells displayed highly increased levels of cathepsin L mRNA, with no increase in intracellular cathepsin L protein content or activity, whereas a high increase in extracellular cathepsin L level and activity was observed. These data might suggest that the main function(s) of cathepsin L regarding transformation is executed extracellularly. The procathepsin L secreted by the AdoMetDC transformants existed in five forms with different isoelectric points, which may result from differential phosphorylation of the oligosaccharide moieties (33) .

The secreted cathepsin L in CM of the AdoMetDC-transformed cells was predominantly in the 39-kDa precursor form. The enzyme activity measurements revealed that the inactive precursor was converted to the mature active enzyme in the presence of acidic pH and glycosaminoglycans such as heparin, as also shown by others (34 , 35) . Purified 39-kDa procathepsin L is known to be first cleaved to 31- to 34-kDa intermediate forms and then further cleaved to 30-kDa single-chain and 25- and 5-kDa two-chain forms in vitro, and in vivo it is reported to be similarly processed intracellularly in lysosomes (21 , 22) , but less is known about the processing of extracellular procathepsin L in vivo. Our two-dimensional electrophoresis Western blot and MALDI-TOF analyses of the CM proteins from AdoMetDC-transformed cells showed that in addition to 39-kDa procathepsin L, 25- to 30-kDa cathepsin L forms were present. Exposure of the CM in vitro to heparin at low pH resulted in complete conversion of procathepsin L to the mature 30- and 25-kDa cathepsin L forms, as confirmed by the two-dimensional electrophoresis and activity measurements. These data suggest that proteolytic cleavage of secreted procathepsin L can occur to a small extent without added heparin but that extracellular glycosaminoglycans and acidic pH are essential for activation. Thus the extracellular glycosaminoglycans that occur at the cell surface, in the ECM, and in basement membrane proteins may play an important role in the regulation and targeting of cathepsin L activity.

Most interestingly, our data suggest that cathepsin L is a major determinant of the invasive capacity of the AdoMetDC-transformed NIH 3T3 cells because its expression was highly increased, and the invasiveness of the cells in a reconstituted basement membrane (a three-dimensional Matrigel matrix) was totally blocked in the presence of the cathepsin L inhibitor I. However, it cannot be said that the invasive ability of the AdoMetDC transformants is solely attributable to cathepsin L because the inhibitor at higher concentrations was also found to inhibit cathepsin B, which is also up-regulated in these transformants. Also, it cannot be excluded that Z-FF-FMK might still inhibit some of the as yet uncharacterized cysteine cathepsins present in the mouse genome (36) . Considering the possible therapeutic applications of these inhibitors, this may yet be an advantage because any possible concomitant or compensatory increase in other cathepsins (e.g., cathepsin B) after specific cathepsin L inhibition would be blocked as well. At any rate, there are several studies indicating that cathepsin L by itself may also play an important role in invasion of different malignant cell lines (31 , 37, 38, 39) .

Recent studies suggest that the cysteine proteases cathepsin L and B may act upstream of uPA and MMPs and initiate the protease cascade involved in the degradation of ECM (refs. 40 and 41 ; reviewed in ref. 7 ). The AdoMetDC-transformed cells were also found to display a small increase in the activities of their uPA and MMP, which may have been triggered by cathepsin L. However, the activities of uPA and MMP-2 were not affected by the cathepsin L and B inhibitor (Z-FF-FMK). It is also noteworthy that treatment of the AdoMeDC-transformed cells with specific inhibitors of MMPs failed to block the invasion. Hence, instead of activating other proteases, cathepsins L and B probably affect invasiveness through other mechanisms, possibly by directly degrading the ECM proteins. Indeed, cathepsin L has been found to degrade several key ECM proteins, such as collagens I and IV, fibronectin, and laminin (19 , 34 , 42) , whose degradation is essential for cancer cell invasion.

In conclusion, in transformed rodent fibroblasts overexpressing AdoMetDC, we found a large increase in cathepsin L expression and secretion and a smaller increase in cathepsin B expression. Cathepsin L expression was found to be elevated also in AdoMetDC-induced tumors in nude mice. Inhibition of the activity of these cathepsins by selective peptide inhibitors blocked the invasive capacity of the cells in three-dimensional Matrigel. These results indicate that the activity of the cathepsin L, alone or together with cathepsin B (or some as yet uncharacterized cysteine cathepsin), may be responsible for the invasion potential of the transformants. Increased cathepsin L expression is also known to occur in many human tumors, such as metastatic bone tumors (43) , melanomas (44) , malignant gliomas (45) , non–small-cell lung (46) , ovarian (47) , colorectal (48, 49, 50) , and breast cancers (48) . It is also noteworthy that our preliminary immunohistochemical studies of human fibrosarcomas have shown cathepsin L to be highly expressed by the tumor cells in invasive areas of the tumors.8 It is possible that because cathepsins L and B may be of general importance for cellular invasion, they may offer potentially good targets for future cancer therapies in attempts to prevent cancer cell metastasis.


   ACKNOWLEDGMENTS
 
After submission of our manuscript, Joyce et al. (51) have interestingly reported that inhibition of cathepsin cysteine proteases with a broad spectrum inhibitor impairs the invasive growth of pancreatic islet tumors in mice.


   FOOTNOTES
 
Grant support: The University of Helsinki, the Finnish Cancer Organizations, the Sigrid Juselius Foundation, the Academy of Finland, Helsinki University Central Hospital Research Funds, and the Ida Montin Foundation. K. Ravanko is a predoctoral fellow of the Helsinki Graduate School in Biotechnology and Molecular Biology.

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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: K. Ravanko and K. Järvinen contributed equally to this work.

Requests for reprints: Erkki Hölttä, Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, P. O. Box 21, FIN-00014 Helsinki, Finland. Phone: 358-0-1912-6516; Fax: 358-0-1912-7765; E-mail: erkki.holtta{at}helsinki.fi

3 http://prowl.rockefeller.edu. Back

4 www.atlas.clontech.com. Back

5 P. Nummela and E. Hölttä, unpublished data. Back

6 M. Yin and E. Hölttä, unpublished data. Back

7 A. Paasinen-Sohns and E. Hölttä, unpublished data. Back

8 K. Ravanko, E. Kääriäinen and E. Hölttä, unpublished data. Back

Received 9/25/03. Revised 10/22/04. Accepted 10/27/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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