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

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Cysteine Cathepsins Are Central Contributors of Invasion by Cultured Adenosylmethionine Decarboxylase-Transformed Rodent Fibroblasts [CANCER RESEARCH 64, 8831–8838, December 15, 2004] Cysteine Cathepsins Are Central Contributors of Invasion by Cultured Adenosylmethionine Decarboxylase-Transformed Rodent Fibroblasts Kirsi Ravanko,1 Kristiina Ja¨rvinen,1 Jari Helin,2 Nisse Kalkkinen,2 and Erkki Ho¨ltta¨1 1Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; and 2Institute of Biotechnology, University of Helsinki, Helsinki, Finland ABSTRACT including the capability to initiate a cascade leading to MMP activa- tion (4). In addition, cancer cells have been described as overexpress- Adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the ing several other invasion-related proteases encompassing members biosynthesis of polyamines, is often up-regulated in cancers. We have of the serine, cysteine, and aspartic proteinases (4–10). demonstrated previously that overexpression of AdoMetDC alone is suf- ficient to transform NIH 3T3 cells and induce highly invasive tumors in Previously, we have shown in rodent fibroblasts that overexpres- nude mice. Here, we studied the transformation-specific alterations in sion of S-adenosylmethionine decarboxylase (AdoMetDC), a key gene expression induced by AdoMetDC by using cDNA microarray and regulatory enzyme of polyamine biosynthesis, induces cell transfor- two-dimensional electrophoresis technologies. We specifically tried to mation and highly invasive tumors in nude mice (11), thus providing identify the secreted proteins contributing to the high invasive activity of a good model for studying the molecular mechanisms involved in cell the AdoMetDC-transformed cells. We found a significant increase in the invasion. Significantly, overexpressed AdoMetDC levels have also expression and secretion of procathepsin L, which was cleaved and acti- been detectable in various human cancers, and inhibition of tumor cell vated in the presence of glycosaminoglycans (heparin), and a smaller AdoMetDC activity prevents tumor formation and metastasis (12–14). increase in cathepsin B. Inhibition of the cathepsin L and B activity by Our intention was to identify fibroblast cell invasion-related factors by specific peptide inhibitors abrogated the invasive capacity of the use of cDNA microarray and proteome analyses. The results of the AdoMetDC transformants in Matrigel. The transformed cells also showed a small increase in the activity of gelatin-degrading matrix metallopro- studies indicate that the major proteases involved in the invasion of teinases (MMPs) and urokinase-type plasminogen activator activities, transformed rodent fibroblasts are cysteine cathepsins and, surpris- neither of which was sensitive to the inhibitors of cathepsin L and B. ingly, not MMPs or uPA. Furthermore, the invasive potency of the transformed cells remained unaffected by specific inhibitors of MMPs. The results suggest that cys- MATERIALS AND METHODS teine cathepsins are the main proteases contributing to the high invasive- ness of the AdoMetDC-transformed cells and that the invasion potential is Cell Culture. NIH 3T3 cells (ATCC CRL 1658; American Type Culture largely independent of activation of the MMPs. 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 INTRODUCTION previously (11). Both pools of transfectants and Ͼ10 individual Amdc-s clones were used for the experiments. A normal cell undergoes several changes during the course of its The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) transformation into a cancer cell. A fully transformed cell has usually containing penicillin, streptomycin, gentamicin, G418, and 5% (v/v) fetal or gained six new abilities: it can generate its own growth signals, newborn calf serum (Life Technologies, Inc., Paisley, Scotland, United King- acquire resistance to antigrowth signals, evade apoptosis, replicate dom), at 37°C in a humidified 5% CO2 atmosphere. with limitless potential, and induce neoangiogenic and invasive Metabolic Labeling and Concentration of Conditioned Medium. Cells growth (1). To escape the primary tumor and invade the adjacent were grown for 1 day to 80% confluence, and cultures were rinsed twice with tissue, the cancer cell has to degrade the surrounding basement mem- modified Eagle’s medium (MEM). The cells were incubated with methionine- 35 brane and extracellular matrix (ECM). This is a complex process that and cysteine-free MEM supplemented with 0.1 to 0.2 mCi/mL [ S]methi- likely involves multiple extracellular proteases and requires strictly onine/cysteine (Perkin-Elmer Life Sciences, Inc. Boston, MA) and one-twen- tieth volume of DMEM (to avoid depletion of methionine and cysteine) for 16 organized interaction of the cancer cell and the tumor microenviron- hours, after which the conditioned medium (CM) was collected. CM was ment. The most important regulators of this degradation process are concentrated to Յ one-fourteenth volume with the Ultrafree-15 concentration generally believed to be the matrix metalloproteinases (MMPs). These device MWCO 5000 (Millipore, Bedford, MA). Physiologic salts in the sam- are a family of zinc-containing endopeptidases comprising more than ples were removed by adding 10 mmol/L Tris-HCl (pH 7.4) to the initial 25 members capable of degrading various ECM components. Indeed, volumes and reconcentrating the samples. The protein concentrations of the several studies have shown a close correlation between stage of tumor samples were measured with the Protein Assay Kit (Bio-Rad Laboratories, progression and MMP expression (2, 3). Another extracellular prote- Hercules, CA). ase often associated with invasion is the urokinase-type plasminogen Two-Dimensional Electrophoresis. Equal amounts of proteins (50–100 activator (uPA). uPA is a serine protease with multiple actions, ␮g) from the CM were rehydrated on 18-cm NL DryStrips (pH 3–10; Amer- sham 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-cholamido- Received 9/25/03; revised 10/22/04; accepted 10/27/04. propyl)dimethylammonio]-1-propanesulfonic acid, 0.5% IPG buffer (pH 3–10) Grant support: The University of Helsinki, the Finnish Cancer Organizations, the Sigrid Juselius Foundation, the Academy of Finland, Helsinki University Central Hospital NL, and bromphenol blue for 12 hours at 20°C. Isoelectric focusing (IEF) was Research Funds, and the Ida Montin Foundation. K. Ravanko is a predoctoral fellow of the performed using Amersham Pharmacia Biotech IPGPhor at 500 V for 1 hour, Helsinki Graduate School in Biotechnology and Molecular Biology. 1,000 V for 2 hours, 4,000 V for 2 hours, and 8,000 V for 4 hours, with Imax The costs of publication of this article were defrayed in part by the payment of page per strip of 50 ␮A. In studying cathepsin L processing, 7-cm NL DryStrips (pH charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3–10) were used, and IEF was performed at 500 V for 30 minutes, 1,000 V for Note: K. Ravanko and K. Ja¨rvinen contributed equally to this work. 1 hour, 4,000 V for 1 hour, and 8,000 V for 2 hours. After IEF, the strips were Requests for reprints: Erkki Ho¨ltta¨, Department of Pathology, Haartman Institute, incubated in equilibration buffer [6 mol/L urea, 50 mmol/L Tris-HCl (pH 6.8), University of Helsinki and Helsinki University Central Hospital, P. O. Box 21, FIN-00014 2% SDS, 30% glycerol, 5 mmol/L tributylphosphine, and bromphenol blue] for Helsinki, Finland. Phone: 358-0-1912-6516; Fax: 358-0-1912-7765; E-mail: erkki.holtta@ helsinki.fi. 25 minutes at room temperature and run in 12% SDS-PAGE. The gels were ©2004 American Association for Cancer Research. then stained with silver (15) or blotted onto nitrocellulose membranes. For 8831 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. CYSTEINE CATHEPSINS IN CELL INVASION autoradiographic detection, the stained gels were dried and exposed to Fuji the cell lysates or CM were added up to 100-␮L final volume of lysis buffer BAS-2500 phosphorimager plates. supplemented or not supplemented with 200 ␮g/mL heparin. The different In-Gel Tryptic Digestion. Silver-stained spots of proteins were cut from cathepsin L inhibitors [cathepsin L inhibitor I (Calbiochem, Darmstadt, Germany), the gel and destained by incubating these gel pieces for 30 minutes at 37°C Z-Phe-Tyr(tBu)-dimethylketone, or Ac-Leu-Leu-Nle-aldehyde (Bachem, Buben- ␮ with 0.1 mol/L NH4HCO3 in 50% acetonitrile (ACN). The gel pieces were dorf, Switzerland)] at 0, 0.5, 15, 25, or 50 mol/L concentrations were incubated shrunk with 100% ACN, after which ACN was removed. Proteins were with the samples for 1 hour at 37°C. Thereafter, 100 ␮L of reaction buffer [0.4 reduced with 20 mmol/L dithiothreitol in 0.1 mol/L NH4HCO3 at 56°C for 30 mol/L sodium acetate (pH 5.5), 4 mmol/L EDTA, and 8 mmol/L dithiothreitol] minutes and shrunk again with ACN. To block disulfide formation, the and 150 ␮mol/L cathepsin L substrate (Z-Phe-Arg-para-nitroanilide; Bachem) sulfhydryl groups were alkylated by incubating the gel pieces with 55 mmol/L were added, the samples were incubated at 37°C, and A405 nm was measured as a iodoacetamide in 0.1 mol/L NH4HCO3 for 15 minutes at room temperature in function of time. the dark. The pieces were washed twice for 10 minutes with 0.1 mol/L Highly purified cathepsin L (specific activity, 6,036 milliunits/mg protein) NH4HCO3, shrunk with ACN, and dried. For tryptic digestion of the proteins, from human liver (Calbiochem) was used as a positive control in the assays. the pieces were rehydrated with 10 ␮L of 0.05 mg/mL modified trypsin The activity of the purified cathepsin L was also measured at physiologic pH (Promega, Madison, WI) in 0.1 mol/L NH4HCO3 and 10% ACN for 10 7.0 (HEPES buffer) and found to be only 16% of the activity at pH 5.5 (sodium minutes, after which 0.1 mol/L NH4HCO3 with 10% ACN was added to cover acetate buffer).
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