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(Pump-1), Secreted from Human Rectal Carcinoma Cell Line1

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(Pump-1), Secreted from Human Rectal Carcinoma Cell Line1 [CANCER RESEARCH 50, 7758-7764, December 15. 1990] Purification and Characterization of Extracellular Matrix-degrading Metalloproteinase, Matrin (Pump-1), Secreted from Human Rectal Carcinoma Cell Line1 Kaoru Miyazaki,2 Yasuhisa Hattori, Fuminori Umenishi, Hidetaro Yasumitsu, and Makoto Umeda Division of Cell Biology, Kifiara Institute for Biological Research, Yokohama City University, 2-120-3 Nakamura-cho, Minami-ku, Yokohama, Kanagawa 232, Japan ABSTRACT nases (14-18), serine proteinases (19-22), thiol proteinases (23-25), and aspartic proteinases (26). Among the four classes A metalloproteinase with M, 29,000 was purified to homogeneity as a of proteinases, the metalloproteinases appear to play a major latent proenzyme from the conditioned medium of a human rectal carci role in matrix degradation. Recent studies have revealed a noma cell line CaR-1. This enzyme hydrolyzed casein more potently than family of secreted zinc metalloproteinases capable of degrading gelatin embedded in polyacrylamide gels in zymography assay. Calcium ECM proteins. These include interstitial collagenase (27-29), ion was essential for the activity. It exerted the maximum activity at pH 7-9. Its activity was stimulated by organomercurials, such as p-amino- type IV collagenases (or gelatinases) with M, 72,000 (16, 17) phenyl mercuric acetate and p-chloromercuric benzoic acid, and was and M, 92,000 (18), and stromelysin (transin) (30-34). The inhibited by 1,10-phenanthroline but was hardly affected by diisopropyl interstitial collagenase preferentially hydrolyzes the interstitial fluorophosphate and pepstatin. When the purified proenzyme was acti collagens of types I, II, and III, whereas type IV collagenases vated by the organomercurials, it effectively hydrolyzed fibronectin, do hydrolyze type IV basement membrane collagen but not type laminin, type IV basement membrane collagen, and several types of I collagen (16-18, 27). Stromelysin has a more broad substrate gelatins but not interstitial type I and III collagens. The treatment of the specificity: it hydrolyzes fibronectin, laminin, type IV collagen, purified proenzyme with /7-aminophenyl mercuric acetate or trypsin and proteoglycans (30). These metalloproteinases are structur formed an active peptide with M, 20,000. The structural analysis indi cated that it was most likely identical to putative metalloproteinase-1, ally related to each other and are secreted as latent proenzymes (zymogens) from various types of cells including tumor cells the complementary DNA of which had been cloned from human tumor mRNAs capable of hybridizing to a rat transin complementary DNA. and normal connective tissue cells. In addition to these en Based on the fact that this enzyme is secreted extracellularly and degrades zymes, Woessner and Talpin (35) recently isolated a metallo the matrix proteins, we propose the name "matrin" for this newly proteinase with M, 28,000 from postpartum rat uterus, desig identified enzyme. nated MMP-7, which was capable of degrading casein, fibro nectin, and various types of gelatins. On the other hand, Muller et al. (36) cloned cDNAs of two kinds of putative metallopro INTRODUCTION teinases, stromelysin-2 and pump-1, from human tumors with ECM,3 which consists of fibrous structural proteins, glyco- the use of transin cDNA as a probe. Pump-1 has an exception ally small molecular size (267 amino acid residues) compared proteins, proteoglycans, and glycosaminoglycans, plays a fun to other mammalian metalloproteinases. Recently, Quantin et damental role in the formation and maintenance of tissue al. (37) expressed the pump-1 cDNA in COS cells and dem architecture. The major structural proteins, collagens, support onstrated that the recombinant pump-1 protein was a precursor the basic structure of ECM, while the major adhesive glycopro- form of a metalloproteinase with a substrate specificity similar teins, fibronectin and laminin, mediate the binding of cells to to that of MMP-7. They also showed that pump-1-related ECM (1-3). These ECM proteins affect proliferation, differ mRNA was expressed in postpartum rat uteri, suggesting that entiation, morphology, substrate attachment, and motility of pump-1 might correspond to rat MMP-7. However, the native cells in vitro (2, 4). There is increasing evidence indicating that form of pump-1 has not been identified in human sources. proteolytic degradation of ECM is required for tumor cells to invade basement membranes, stremai matrix, and cell-cell junc We previously found that transformation of a rat liver epi tions (5-7). Liotta et al. (8, 9) and other groups (10, 11) have thelial cell line, BRL, with Rous sarcoma virus induced marked secretion of a fibronectin-degrading metalloproteinase (38). demonstrated that the secretion of type IV collagen-degrading Recently, we surveyed culture media conditioned by more than enzymes by tumor cells is well correlated with the invasive 30 kinds of nonmalignant and malignant cell lines for secreted potentials. Furthermore, in vitro invasion experiments have suggested that a cascade of serine proteinases and metallopro- proteinases by the zymography technique. As a result, it has been found that a human rectal adenocarcinoma cell line se teinases is required for tumor invasion into the basement mem cretes a low molecular weight metalloproteinase capable of branes (12, 13). degrading ECM proteins. In the present paper, we report the It has been reported that tumor cells secrete metalloprotei- purification of this metalloproteinase, tentatively named "ma trin," and its characterization. The structural analysis of the Received 2/19/90; accepted 8/30/90. The costs of publication of this article were defrayed in part by the payment purified enzyme demonstrated that it might be identical to the of page charges. This article must therefore be hereby marked advertisement in putative metalloproteinase pump-1. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. 2To whom requests for reprints should be addressed. MATERIALS AND METHODS 3The abbreviations used are: ECM, extracellular matrix; APMA, p-amino- phenyl mercuric acetate; CBB, Coomassie brilliant blue R-250; DFP, diisopropyl Cells and Culture. A human rectal carcinoma cell line CaR-1 fluorophosphate; HPLC, high performance liquid chromatography; PAGE, poly (JCRB0207), which had been established from a metastatic lymph node acrylamide gel electrophoresis; PCMB, /7-chloromercuric benzoic acid; SDS, of a 70-year-old male patient with primary rectal adenocarcinoma by sodium dodecyl sulfate; MMP-7, matrix metalloproteinase-7; pump-1, putative metalloproteinase-1; cDNA, complementary DNA; DME, Dulbecco's modified Kaneko et al. (39), was used as the source of the low molecular weight Eagle's medium; FI2, Ham's F-12 medium. metalloproteinase matrin. Other human cancer cell lines tested for the 7758 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. METALLOPROTEINASE SECRETED BY HUMAN RECTAL CARCINOMA secretion of the metalloproteinase were HLE (hepatoma), A549 (lung Zymography of Proteinases. Caseinolytic activities of secreted pro- adenocarcinoma), T24 (bladder carcinoma), EJ-1 (bladder carcinoma), teinases were analyzed by zymography according to the methods of A431 (vulva epidermoid carcinoma), HeLa.S3 (cervix epitheloid carci Chin et al. (30) with some modifications. SDS-polyacrylamide gels noma), HSC-4 (tongue squamous carcinoma), T98G (glioblastoma), containing 1 mg/ml casein were prepared with 1.3 mg/ml ammonium YKG-1 (glioma), YST-2 (schwannoma), YST-3 (schwannoma), persulfate (2.7-fold the usual amount). Samples to be tested were mixed HT1080 (fibrosarcoma), NB-1 (neuroblastoma), VMRC-MELG (mel with an equal volume of the concentrated SDS sample buffer [4% (w/ anoma), and RPMI8226 (myeloma). These human cell lines, except v) SDS, 125 HIM Tris-HCl (pH 6.8), 10% (v/v) glycerol] and then A549, YKG-1, YST-2, YST-3, and NB-1, and an epithelial cell line electrophoresed on the casein-containing gels without heating the mix derived from an African green monkey kidney BSC-1 were provided by ture in boiling water. After electrophoresis, the proteinases separated the Japanese Cancer Research Resource Bank. on the gels were renatured by gently shaking the gels in 2.5% Triton These cells were cultured at 37°Cina humidified atmosphere of 5% X-100 containing 50 mM Tris-HCl (pH 7.5) and 0.1 M NaCl at room CO2 and 95% air. The basal medium (DME/F12) consisted of a 1:1 temperature for l h to remove SDS, followed by incubation in 250 ml mixture of Dulbecco's modified Eagle's medium (GIBCO, Grand Is of 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2 and 0.02% NaN3 land, NY) and Ham's F-12 medium (GIBCO), which was supplemented at 37°Cfor about 18 h. The resultant gels were stained with CBB and with 15 mM AL2-hydroxyethylpiperazine-A''-ethanesulfonic acid, 1.2 destained as described above. mg/ml of NaHCOj, 100 units/ml of penicillin G, and 0.1 mg/ml of Preparative SDS-PAGE. For structural analysis, the metalloprotei streptomycin sulfate. Cells were maintained in DME/F12 supple nase obtained from the Cellulofine GCL2000 column was further mented with 10% PCS (GIBCO) (10% PCS + DME/F12). Plastic purified by preparative SDS-PAGE on 10% polyacrylamide slab gels culture wares were obtained from Becton, Dickinson Labware (Oxnard, (160 mm long, 150 mm wide, 1 mm thick). After the electrophoresis, CA). the gels were briefly stained with CBB, destained, and soaked in water Preparation of Conditioned Medium. CaR-1 cells were grown to for 30 min. The CBB-stained band of the metalloproteinase was excised confluence in roller bottles (175 cm2) containing 200 ml of 10% PCS from the gel and cut into about 5 x 5-mm pieces. The gel pieces were + DME/F12. The cultures were then rinsed twice with Hanks' balanced placed in a dialysis bag with a small volume of 10 HIMTris-HCl (pH salt solution and incubated in serum-free DME/F12 overnight.
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