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• W2005285304 abstract "Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream. Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream. It is well established that the progression of metastatic cancer involves the interplay of the host environment with the malignant cells (1Bissell M.J. Radisky D. Nat. Rev. Cancer. 2001; 1: 46-54Crossref PubMed Scopus (1735) Google Scholar). Neoplasms employ multiple means to sustain themselves and to proliferate in vivo. Evidence has emerged that tumor immunity is an important defense mechanism protecting malignant cells from the host immune surveillance. The host immune system is an apparatus directed against foreign invading organisms and tumor cells (2Morgan B.P. Crit. Rev. Immunol. 1999; 19: 173-198Crossref PubMed Google Scholar, 3Morgan B. Methods Mol. Biol. 2000; 150: 1-13PubMed Google Scholar, 4Liszewski M.K. Farries T.C. Lublin D.M. Rooney I.A. Atkinson J.P. Adv. Immunol. 1996; 61: 201-283Crossref PubMed Google Scholar). Controlled activation of the complement system is a critical component of host immunity. Complement activation products stimulate a localized protective inflammation and are involved in both the inductive and effector phases of an immune response (5Jurianz K. Ziegler S. Garcia-Schuler H. Kraus S. Bohana-Kashtan O. Fishelson Z. Kirschfink M. Mol. Immunol. 1999; 36: 929-939Crossref PubMed Scopus (188) Google Scholar). In many cases malignant cells exhibit antigens that are not typically associated with normal cells. These antigens can be identified by the complement system via antibody recognition and, as a result, the recognized cells are attacked by the immune system (6Dachs G.U. Dougherty G.J. Stratford I.J. Chaplin D.J. Oncol. Res. 1997; 9: 313-325PubMed Google Scholar). The complement system is comprised of soluble proteins that interact in a stepwise manner. Complement can be activated via three different pathways: the classical pathway that is usually antibody-dependent, and the alternative and lectin pathways. In the classical pathway, immunoglobulin-coated targets bind and subsequently activate the complement component C1. This event starts the complement cascade, the propagation of which results in the generation of anaphylatoxins (C3a, C4a, and C5a) and ultimately the cytolytic C5b-9 membrane attack complex (MAC) 1The abbreviations used are: MAC, membrane attack complex; FBS, fetal bovine serum; HRP, horseradish peroxidase; MMP-2, matrix metalloproteinase-2; MT1-MMP, membrane type-1 matrix metalloproteinase; TIMP-2, tissue inhibitor of metalloproteinases-2; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter. (7Rus H.G. Niculescu F.I. Shin M.L. Immunol. Rev. 2001; 180: 49-55Crossref PubMed Scopus (108) Google Scholar). In the process of complement propagation, proteolytic cleavage of serum C3 and C4 creates transient soluble C3b and C4b products with an exposed reactive thioester group. Once exposed, the thioester group forms amide and ester bonds with the target cell surface molecules. This binding of C3b and C4b is critical for amplification of the cascade and for MAC formation. Subsequent proteolytic cleavage transforms the cell-bound C3b into the cleavage fragments iC3b, C3dg, and C3d, which remain covalently attached to the cell surface. These C3 fragments serve as ligands for receptors on phagocytic and NK cells. Opsonization of target cells with these C3 fragments promotes and enhances both antibody-dependent and complement-dependent cell cytotoxicity, two additional effector systems that play an important role in the elimination of neoplastic cells (8Delibrias C. Fischer E. Kazachtkine M. Rother K. Till G.O. Haensch G.M. The Complement System. 2nd Ed. Springer-Verlag, Berlin1997: 211-220Google Scholar, 9Cole D.S. Morgan B.P. Clin. Sci. (Lond.). 2003; 104: 455-466Crossref PubMed Scopus (155) Google Scholar). The deposition of C3b and C4b and the follow-up amplification of the complement cascade also results in the generation of soluble bioactive C3a and C5a peptides that may potentiate anti-tumor responses via their chemoattractant and proinflammatory activities. Thus, the inactivation of C3b and C4b and their removal from a cell surface represents an important immune evasion mechanism of tumor cells. An important role for complement resistance of tumor cells is indicated by the fact that many tumor cells overexpress one or more of the cell surface-associated complement regulatory proteins: CD46/MCP, CD55/DAF, and CD59/protectin. These regulatory proteins act at different stages of the complement propagation (10Caragine T.A. Okada N. Frey A.B. Tomlinson S. Cancer Res. 2002; 62: 1110-1115PubMed Google Scholar, 11Gorter A. Meri S. Immunol. Today. 1999; 20: 576-582Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 12Morgan B.P. Harris C.L. Complement Regulatory Proteins. Academic Press, San Diego1999Google Scholar, 13Carroll M.C. Fischer M.B. Curr. Opin. Immunol. 1997; 9: 64-69Crossref PubMed Scopus (83) Google Scholar). In addition to the cell-associated regulatory proteins, some tumor cells bind serum complement inhibitory proteins (14Fedarko N.S. Jain A. Karadag A. Van Eman M.R. Fisher L.W. Clin. Cancer Res. 2001; 7: 4060-4066PubMed Google Scholar, 15Fedarko N.S. Fohr B. Robey P.G. Young M.F. Fisher L.W. J. Biol. Chem. 2000; 275: 16666-16672Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), express proteases to clear the C3b protein from the cell surface, and inhibit the complement cascade (16Jean D. Hermann J. Rodrigues-Lima F. Barel M. Balbo M. Frade R. Biochem. J. 1995; 312: 961-969Crossref PubMed Scopus (28) Google Scholar, 17Jean D. Bar-Eli M. Huang S. Xie K. Rodrigues-Lima F. Hermann J. Frade R. Cancer Res. 1996; 56: 254-258PubMed Google Scholar, 18Jurianz K. Ziegler S. Donin N. Fishelson Z. Kirschfink M. Mol. Immunol. 1999; 36: 316Crossref Scopus (176) Google Scholar). Membrane type-1 matrix metalloproteinase (MT1-MMP) is the most common protease from the membrane-tethered enzyme subfamily of MMPs (19Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5134) Google Scholar). MT1-MMP plays an important, albeit insufficiently characterized, role in tissue remodeling and cell motility, and especially in tumor progression, metastasis, and angiogenesis (20Hotary K.B. Allen E.D. Brooks P.C. Datta N.S. Long M.W. Weiss S.J. Cell. 2003; 114: 33-45Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 21Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (510) Google Scholar). MT1-MMP is a cell surface activator of soluble pro-MMP-2 and pro-MMP-13 and has also been implicated in matrix proteolysis and turnover as well as in the proteolytic processing of cell surface-associated adhesion and signaling receptors (19Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5134) Google Scholar, 22Murphy G. Stanton H. Cowell S. Butler G. Knauper V. Atkinson S. Gavrilovic J. Acta Pathol. Microbiol. Immunol. Scand. 1999; 107: 38-44Crossref PubMed Scopus (391) Google Scholar, 23Sounni N.E. Devy L. Hajitou A. Frankenne F. Munaut C. Gilles C. Deroanne C. Thompson E.W. Foidart J.M. Noel A. FASEB J. 2002; 16: 555-564Crossref PubMed Scopus (241) Google Scholar). Although MT1-MMP is detectable in normal tissue, the expression of this protease is strongly associated with aggressive, invasive malignant cells (19Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5134) Google Scholar, 24Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3890) Google Scholar). Here, we report a novel, unexpected and highly significant function of MT1-MMP in the proteolysis of the opsonic complement components C3b and C4b. This proteolysis efficiently inhibits complement activation in cell-based models. We suspect that MT1-MMP is likely to make malignant cells more resistant to complement-mediated cytotoxicity in vivo. Our data indicate that this novel function of MT1-MMP is involved in the release of C3b from the tumor cell surface, and is likely to contribute to the survival and propagation of malignant cells. We believe that the proteolysis of the complement components by MT1-MMP is a powerful and efficient mechanism employed by aggressive malignant cells to protect themselves against host complement, immune surveillance, and destruction. Antibodies and Reagents—All reagents were purchased from Sigma unless otherwise indicated. Purified human C3b, iC3b, and C4b, goat anti-human C3 and C4 antibodies, normal human serum, and C5-depleted human serum were from Advanced Research Technologies. Monoclonal murine antibody H206 against human C3b α chain was purchased from Research Diagnostics. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG and HRP-conjugated F(ab′)2 fragment goat anti-mouse antibodies were obtained from Jackson ImmunoResearch Laboratories. Rabbit antibody AB815 against the hinge domain of MT1-MMP, HRP-conjugated rabbit anti-goat antibodies, the TMB/M and TMB/E substrates, and GM6001 (a broad-range hydroxamate inhibitor of MMPs) were from Chemicon. Murine anti-human CD59 monoclonal antibody BRIC229 and murine anti-human CD55 monoclonal antibody 1A10 are as described earlier (25Kinoshita T. Medof M.E. Silber R. Nussenzweig V. J. Exp. Med. 1985; 162: 75-92Crossref PubMed Scopus (301) Google Scholar, 26Maenpaa A. Junnikkala S. Hakulinen J. Timonen T. Meri S. Am. J. Pathol. 1996; 148: 1139-1152PubMed Google Scholar). Murine anti-human CD46 monoclonal antibody M75 was obtained from BD Pharmingen. The recombinant catalytic domain of human MT1-MMP (MT1-CAT) was expressed in Escherichia coli, purified from the inclusion bodies, and refolded to restore its native conformation as previously described (27Ratnikov B. Deryugina E. Leng J. Marchenko G. Dembrow D. Strongin A. Anal. Biochem. 2000; 286: 149-155Crossref PubMed Scopus (67) Google Scholar). Rabbit antiserum against the breast carcinoma MCF7 cell membranes was prepared by routine techniques and, where indicated, was used to sensitize MCF7 cells to human complement. Rabbit antibody against the recombinant catalytic domain of MT1-MMP was generated in our laboratory. Cell Lines—Human breast carcinoma MCF7 cells (ATCC) stably transfected with the empty pcDNA3-zeo vector (Invitrogen) (control MCF-zeo cells) and the full-length wild type MT1-MMP (MCF-MT cells) were constructed and extensively characterized in our prior work (28Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Human fibrosarcoma HT1080 cells, which synthesize MT1-MMP and MMP-2 naturally, were obtained from ATCC. Transfected cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM) (Irvine Scientific) supplemented with 10% fetal bovine serum (FBS) (Tissue Culture Biologicals) and 0.2 mg/ml zeocin. Human breast carcinoma BT549, MCF7, MDA-MB-231, T47D, and SK-Br-3 cells (ATCC) were propagated in DMEM supplemented with 5% FBS and penicillin-streptomycin (100 IU/ml and 100 μg/ml). MMP-2 Activation by MT1-MMP and Gelatin Zymography—TIMP-2-free pro-MMP-2 was isolated from a conditioned medium of p2AHT2A72 cells derived from an HT1080 fibrosarcoma cell line sequentially transfected with the E1A and MMP-2 cDNAs (29Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). MCF7-zeo and -MT cells (1 × 105 cells each) were incubated for 12 h in DMEM supplemented with pro-MMP-2 (20 ng/200 μl of medium). HT1080 cells (1 × 105 cells) were incubated 12 h in DMEM (200 μl) alone. Where indicated PMA (5 ng/ml) was added to the cells. Aliquots (10 μl) of medium conditioned by the cells were analyzed by gelatin zymography. Immunoprecipitation and Western Blotting—HT1080, MCF-zeo, and MCF-MT cells were grown in DMEM/FCS. Where indicated, PMA (5 ng/ml) was added to the cells. After incubation for 12 h, cells were washed with PBS and surface biotinylated with sulfo-NHS-LC-biotin (Pierce) according to the manufacturer's instructions. Next, cells were washed with ice-cold PBS and lysed with 50 mmN-octyl-β-d-glucopyranoside (Amresco) in PBS supplemented with 1 mm CaCl2, 1 mm MgCl2, and protease inhibitor mixture containing 1 mm phenylmethylsulfonyl fluoride and 1 μg/ml each of aprotinin, pepstatin, and leupeptin. The lysates were pre-cleared with Protein G-agarose beads (Calbiochem). The samples of cell lysates each containing 1.0 mg of protein were mixed with an MT1-MMP antibody (1 μg) and Protein G-agarose, and incubated at 4 °C overnight. After extensive washings, immune complexes were released by boiling the beads for 5 min in 2× SDS sample buffer containing 50 mm dithiothreitol. Solubilized proteins were subjected to SDS-PAGE and Western blotting (28Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). MT1-MMP Proteolysis of C3b and C4b in Vitro—Purified human C3b and C4b (200 ng each) were co-incubated with MT1-CAT (0.2–20 ng) at 37 °C in 15 μl of 50 mm HEPES, 10 mm CaCl2, 0.5 mm MgCl2, 50 μm ZnCl2, 0.01% Brij-35, pH 6.8. Where indicated, GM6001 (5 μm) was added to the reactions to block MT1-MMP activity. The cleavage samples were separated by SDS-PAGE in 10% gels followed by Western blotting with goat anti-human C3 and C4 antibodies (1 μg/ml each in PBS supplemented with 1% BSA and 0.1% Tween 20), HRP-conjugated rabbit anti-goat secondary antibodies (1:5,000 dilution), and the TMB/M substrate. MT1-MMP Proteolysis of the Cell Surface-bound C3b and C4b in MCF7 Cells—MCF-zeo and -MT cells (105 cells per well) were each grown for 48 h in DMEM/FBS in wells of a 48-well plate. The cells were then sensitized by incubation for 30 min at 37 °C in 20% rabbit anti-MCF7 heat-inactivated serum (56 °C, 30 min). The cell samples, in which sensitization with anti-MCF7 serum was omitted, were used as controls. Sensitized cells were additionally incubated for 5–60 min at 37 °C in 10 or 20% C5-depleted human serum to induce the deposition of the C3b and C4b onto the cell surface. Following extensive washing, cells were subjected for flow cytometry or additionally incubated at 37 °C for 2–12 h in DMEM/FBS (heat-inactivated) to induce the proteolytic shedding and the release of the cell-bound C3b and C4b into the extracellular milieu. Where indicated, GM6001 (50 μm) was added to DMEM/FBS (heat-inactivated) to block cellular MT1-MMP. The residual levels of cell surface-associated C3b and C4b were measured by Western blotting and flow cytometry. For flow cytometry analyses the cells were detached by an enzyme-free buffer (Specialty Media), washed, and stained for 1 h at 4 °C with the murine monoclonal antibody H206 against human C3b α chain (5 μg/ml) followed by incubation for 30 min with an fluorescein isothiocyanate-conjugated goat anti-mouse antibody. All incubation steps were performed in PBS supplemented with 1% BSA and 0.01% NaN3. After removal of unbound antibodies, cells were re-suspended in PBS supplemented with 3 μg/ml propidium iodide (Molecular Probes), 1% BSA, and 0.01% NaN3. Viable cells were analyzed on a FACScan flow cytometer (BD Biosciences). Where indicated, GM6001 (50 μm) was co-incubated with the cells for 24 h. For Western blotting cells were lysed for 1 h at 0 °C in 100 μl of 5 mm Tris-HCl buffer, pH 8.0, containing 0.5% SDS, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin. Aliquots (20 μl) were subjected to SDS-PAGE in 10% gels and Western blotting with goat anti-human C3 and C4 antibodies (1 μg/ml each in PBS supplemented with 1% BSA and 0.1% Tween 20), HRP-conjugated rabbit anti-goat secondary antibodies (1:5,000 dilution), and the TMB/M substrate. C3b liberated from the cell surface by proteolytic shedding and released into the extracellular milieu was identified by ELISA of the medium samples. For these purposes, MCF-zeo and -MT cells (105 cells each) were incubated for 1 h at 37 °Cin DMEM supplemented with 20% C5-depleted human serum to induce deposition of C3b on cell surfaces. Next, unbound material was removed by washing cells with PBS. A mixture of DMEM/FBS (200 μl) was added to the cells. Following incubation for 30–120 min to release the cell-bound C3b, the aliquots of medium were withdrawn for a subsequent analysis of the liberated C3b. Where indicated, GM6001 (50 μm) was co-incubated with the cells to block activity of the cellular MT1-MMP. ELISA of Soluble C3b—Wells of a 96-well plate (Corning) were coated with goat anti-human C3 antibody (5 μg/ml) and then blocked with 1% BSA. Medium aliquots (100 μl) were allowed to bind for 1 h at 37 °C with the antibody-coated wells. The bound C3b was detected with biotin-labeled goat anti-human C3 (1 μg/ml) followed by streptavidin-HRP and the TMB/E substrate. The absorbance of the samples was measured at 450 nm. Flow Cytometry of the Complement Regulatory Proteins—Flow cytometry was used to assess the cell surface expression of complement regulatory proteins CD46, CD55, and CD59 in MCF-zeo and -MT cells. For these purposes, cells were detached by the enzyme-free buffer (Specialty Media) and co-incubated with the respective primary antibody (5 μg/ml each) followed by incubation with fluorescein isothiocyanate-labeled secondary antibody (1:500 dilution). Population gates were set by using cells incubated with normal murine IgG. Cells were analyzed on a FACStar flow cytometer (BD Biosciences). Cytotoxicity Assay—MCF-zeo and -MT cells (1 × 105 each) were grown in DMEM/FBS in wells of a 48-well plate. To sensitize cells, 20% heat-inactivated rabbit MCF7 antiserum was co-incubated with the cells for 30 min at 37 °C. The sensitized cells were then placed for 1 h at 37 °C in 20% normal human serum to induce the activation of the complement pathway and lysis of the cells by the resulting membrane attack complex. The treatment with rabbit anti-MCF7 serum was omitted in control samples. The efficiency of the cell lysis was determined by using a Vybrant Cytotoxicity Assay Kit V-23111 (Molecular Probes) in accordance with the manufacturer's instructions. The cytotoxic effect of the complement was also assessed by microscopy. For this purpose, MCF-zeo and -MT cells (1 × 105 cells each) were grown in DMEM/FBS in wells of an 8-well Lab-Tek™ chamber glass slide (Nalge Nunc). Heat-inactivated 20% rabbit anti-MCF7 serum was co-incubated with the cells for 30 min at 37 °C. The sensitized cells were further incubated for 1 h at 37 °C in 20% normal human serum and then fixed with 4% glutaraldehyde for 1 h at ambient temperature and photographed. Dead cells were made visible with propidium iodide using a LIVE/DEAD Reduced Biohazard Viability/Cytotoxicity L-7013 Kit (Molecular Probes) and the images were taken by a Nikon Eclipse TE300 fluorescence microscope equipped with a SPOT Real-Time SP402–115 digital camera (Diagnostic Instruments). Purification and Iodination of TIMP-2—Human TIMP-2 was produced by Chinese hamster ovary cells stably transfected with the C-terminal His-tagged human TIMP-2. TIMP-2 was purified from conditioned medium by chelating chromatography and ion-exchange chromatography using the slightly modified protocol described in Ref. 30DeClerck Y.A. Yean T.D. Lu H.S. Ting J. Langley K.E. J. Biol. Chem. 1991; 266: 3893-3899Abstract Full Text PDF PubMed Google Scholar. TIMP-2 (10 μg) was labeled with Na125I (Amersham Biosciences) using the IODO-GEN iodination reagent (Pierce) and separated from unincorporated radioactivity by gel-filtration. Normally, the specific radioactivity of labeled TIMP-2 was 4–5 μCi/μg. Cell Surface Binding of Radiolabeled TIMP-2—To remove cell-bound endogenous TIMP-2, cells (1.5 × 105 cells per well of a 12-well plate) were washed prior to assays with 50 mm glycine-HCl buffer, pH 3.0, containing 100 mm NaCl, then neutralized with 0.5 m HEPES buffer, pH 7.5, containing 100 mm NaCl and, finally, equilibrated with DMEM supplemented with 20 mm HEPES, pH 7.5, and 0.2% BSA. Increasing concentrations of 125I-TIMP-2 (0.03–14 nm) were added to the cells. Following a 3-h incubation at 4 °C, cells were washed twice with ice-cold DMEM supplemented with 20 mm HEPES, pH 7.5, and 0.2% BSA, and lysed in 0.5 m NaOH containing 0.1% SDS; the radioactivity was then counted. The nonspecific background binding was determined using a 200-fold excess of unlabeled over highest concentration of 125I ligand. One well of each cluster was used to count the number of cells. Each experimental point was measured in duplicate. GraphPad Prism software was used to calculate the Scatchard plot and the Kd value. Isolation of RNA and Northern Blotting Analysis—The total RNA was isolated from the cells with a RNAzol B reagent (Tel-Tech). RNA (15 μg) was separated by electrophoresis on a 1% agarose/formaldehyde gel and then transferred to a Hybond-N membrane (Amersham Biosciences). The membrane was hybridized with the 32P-labeled MT1-MMP cDNA and with the 32P-labeled 28 S rRNA oligonucleotide probe (Clontech). Transfected Breast Carcinoma Cells Express Physiologically Relevant Levels of MT1-MMP—We selected breast carcinoma MCF7 cells for our studies because the parental cells are deficient in MT1-MMP and MMP-2 (28Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Here, we used MCF7 cells transfected with the wild type MT1-MMP cDNA (MCF-MT cells) as well as control cells transfected with the original pcDNA3-zeo plasmid (MCF-zeo cells). To exclude possible artifacts caused by MT1-MMP overexpression and to demonstrate that the transfected cells exhibit physiologically relevant, rather than aberrantly high levels of MT1-MMP, we compared MT1-MMP protease activity in MCF-MT and fibrosarcoma HT1080 cells. HT1080 cells produce MT1-MMP and MMP-2 naturally. The efficiency of the MT1-MMP-mediated activation of MMP-2 and the rate of conversion of the 68-kDa proenzyme into the 64-kDa intermediate and then into the active, mature 62-kDa enzyme were used as measures of the cell surface-associated MT1-MMP activity. Gelatin zymography was employed to visualize the levels of MMP-2 activation. MCF7 cells were supplemented in these assays with external pro-MMP-2 in amounts that were similar to those naturally synthesized by HT1080 cells. To promote the activation of MMP-2, HT1080 cells were stimulated with PMA (5 ng/ml) (31Bernardo M.M. Fridman R. Biochem. J. 2003; 374: 739-745Crossref PubMed Scopus (154) Google Scholar, 32Hernandez-Barrantes S. Bernardo M. Toth M. Fridman R. Semin. Cancer Biol. 2002; 12: 131-138Crossref PubMed Scopus (148) Google Scholar). Our data indicate that the efficiency of MMP-2 activation by the transfected MCF-MT cells is highly similar to that of HT1080 cells (Fig. 1A), suggesting similar levels of cell surface-associated mature MT1-MMP in these two cell types. To confirm these observations, we directly identified the levels of cell surface-associated MT1-MMP in MCF-MT and HT1080 cells. For these purposes, cells were surface-labeled with membrane-impermeable biotin and then analyzed. Biotin-labeled, cell surface-associated MT1-MMP was immunoprecipitated from cell lysates and detected by Western blotting with streptavidin-HRP (Fig. 1B). These studies demonstrated that cell surface-associated, full-length 60-kDa MT1-MMP was equally represented in HT1080 and MCF-MT cells. In addition, MCF-MT cells exhibit significant amounts of the 43–45-kDa catalytically inactive ectodomain forms of the protease. PMA treatment increased the levels of the degraded MT1-MMP in HT1080 cells. The existence of these full-length and degraded species of MT1-MMP is in agreement with our earlier findings (28Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and the results published by others (32Hernandez-Barrantes S. Bernardo M. Toth M. Fridman R. Semin. Cancer Biol. 2002; 12: 131-138Crossref PubMed Scopus (148) Google Scholar). Importantly, these data, which agree well with those of the MMP-2 activation studies, indicate similar levels of the full-length, catalytically potent MT1-MMP in HT1080 and MCF-MT cells. To extend these comparison studies further, we compared by Northern blotting the levels of the MT1-MMP mRNA in fibrosarcoma HT1080 cells with those in breast carcinoma BT549, MDA-MD-231, and MCF7 cells (Fig. 1C). In agreement with the data of Western blotting that did not detect the expression of the MT1-MMP protein in MCF7-zeo cells (Fig. 1B), Northern blotting did not identify any detectable amounts of the MT1-MMP mRNA in this cell type. In turn, the levels of the MT1-MMP mRNA were relatively low in MDA-MD-231 cells but significantly higher in BT549 cells and comparable with the levels of the messenger identified in HT1080 cells. To substantiate these observations even further, we identified the number of the TIMP-2-binding MT1-MMP cell surface sites in HT1080 and BT549 cells. For these purposes, we incubated the cells with increasing concentrations of radioactively labeled TIMP-2. An excess of unlabeled TIMP-2 fully blocked the binding of the labeled inhibitor with the cells. TIMP-1 (a poor inhibitor of MT1-MMP) was incapable of interfering with the labeled TIMP-2 ligand (data not shown). Following incubation with labeled TIMP-2 and washing to remove the unbound radioactivity, cells were lysed and the radioactivity was counted in cell lysates. Scatchard plot analysis of binding data demonstrated the existence of high affinity binding sites with KD = 3.9 nm and 235,000 sites per HT1080 cell (Fig. 1D). Similarly, BT549 cells exhibited 136,000 MT1-MMP sites/cell and KD = 1.34 nm. These data correlate well with the concentrations of the MT1-MMP mRNA identified by Northern blotting in HT1080 and BT549 cells (Fig. 1C). The data from literature also are in agreement with our results (33Nuttall R.K. Pennington C.J. Taplin J. Wheal A. Yong V.W. Forsyth P.A. Edwards D.R. Mol. Cancer Res. 2003; 1: 333-345PubMed Google Scholar, 34Zucker S. Drews M. Conner C. Foda H.D. DeClerck Y.A. Langley K.E. Bahou W.F. Docherty A.J. Cao J. J. Biol. Chem. 1998; 273: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Thus, real-time PCR studies identified that the levels of MT1-MMP expression in HT1080 cells are highly similar to those found in human umbilical vein endothelial cells, prostate carcinoma PC3, breast carcinoma BT549, and melanoma G361 cells (33Nuttall R.K. Pennington C.J. Taplin J. Wheal A. Yong V.W. Forsyth P.A. Edwards D.R. Mol. Cancer Res. 2003; 1: 333-345PubMed Google Scholar). We conclude from these findings and the data available from the literature that the levels of MT1-MMP in the MT1-MMP-transfected breast carcinoma MCF7 cell model are comparable with those existing naturally in fibrosarcoma HT1080 cells as well as in numerous other cell types including breast carcinoma BT549 cells, human umbilical vein endothelial cells, and melanoma G361 cells. Therefore, the effects of MT1-MM" @default.
• W2005285304 created "2016-06-24" @default.
• W2005285304 creator A5002939390 @default.
• W2005285304 creator A5005364645 @default.
• W2005285304 creator A5007896623 @default.
• W2005285304 creator A5013154347 @default.
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• W2005285304 creator A5042267819 @default.
• W2005285304 creator A5063647013 @default.
• W2005285304 date "2004-11-01" @default.
• W2005285304 modified "2023-10-17" @default.
• W2005285304 title "Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Cleaves C3b, an Essential Component of the Complement System" @default.
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