Title: MMP25 (MT6-MMP) Is Highly Expressed in Human Colon Cancer, Promotes Tumor Growth, and Exhibits Unique Biochemical Properties
Abstract: MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (Ki = 0.2 nm) over TIMP-2 (Ki = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface. MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (Ki = 0.2 nm) over TIMP-2 (Ki = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface. The matrix metalloproteinases (MMPs) 2The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane type-MMP; GPI, glycosylphosphatidylinositol; TIMP, tissue inhibitor of metalloproteinase; pAb, polyclonal antibody; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; PI-PLC, phosphatidylinositol-specific phospholipase C; PMN, polymorphonuclear; β-ME, β-mercaptoethanol; PI, protease inhibitors; MES, 2-(N-morpholino) ethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBS, Tris-buffered saline; FBS, fetal bovine serum; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; RT, reverse transcription; Dnp, 2,4-dinitrophenol.2The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane type-MMP; GPI, glycosylphosphatidylinositol; TIMP, tissue inhibitor of metalloproteinase; pAb, polyclonal antibody; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; PI-PLC, phosphatidylinositol-specific phospholipase C; PMN, polymorphonuclear; β-ME, β-mercaptoethanol; PI, protease inhibitors; MES, 2-(N-morpholino) ethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBS, Tris-buffered saline; FBS, fetal bovine serum; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; RT, reverse transcription; Dnp, 2,4-dinitrophenol. are multidomain, multifunctional zinc-dependent endopeptidases, which have been associated with the pathogenesis of a variety of human diseases, including cancer (1Nagase H. Visse R. Murphy G. Cardiovasc. Res. 2006; 69: 562-573Crossref PubMed Scopus (2279) Google Scholar, 2Deryugina E.I. Quigley J.P. Cancer Metastasis Rev. 2006; 25: 9-34Crossref PubMed Scopus (1609) Google Scholar). The MMP family includes secreted and membrane-anchored proteases (3Maskos K. Bode W. Mol. Biotechnol. 2003; 25: 241-266Crossref PubMed Scopus (98) Google Scholar), and thus they are mediators of proteolysis in the pericellular space and at the cell surface. The membrane-type MMP (MT-MMP) subfamily includes six members, four of which are anchored to the plasma membrane via a transmembrane domain (MMP14, MMP15, MMP16, and MMP24, also referred to as MT1-, MT2-, MT3-, and MT5-MMP, respectively) and two of which are membrane-anchored via a glycosylphosphatidylinositol (GPI) moiety (MMP17 and MMP25, referred to as MT4- and MT6-MMP, respectively) (4Hernandez-Barrantes S. Bernardo M. Toth M. Fridman R. Semin. Cancer Biol. 2002; 12: 131-138Crossref PubMed Scopus (148) Google Scholar, 5Zucker S. Pei D. Cao J. Lopez-Otin C. Curr. Top. Dev. Biol. 2003; 54: 1-74Crossref PubMed Google Scholar). Although there is a significant amount of information on the transmembrane MT-MMPs, little is known about the properties and functions of GPI-anchored MT-MMPs. MT6-MMP was originally cloned from human leukocytes (6Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (167) Google Scholar) and from a fetal liver cDNA library (7Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar). In human normal tissues, MT6-MMP mRNA is predominantly expressed in leukocytes (polymorphonuclear cells and monocytes) but is also detected in lung and spleen (6Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (167) Google Scholar, 7Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar, 8Bar-Or A. Nuttall R.K. Duddy M. Alter A. Kim H.J. Ifergan I. Pennington C.J. Bourgoin P. Edwards D.R. Yong V.W. Brain. 2003; 126: 2738-2749Crossref PubMed Scopus (284) Google Scholar). Because of its abundance in leukocytes, MT6-MMP is also known as leukolysin (6Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (167) Google Scholar). Functional studies with a recombinant catalytic domain showed that MT6-MMP can degrade various extracellular matrix components, including type IV collagen, gelatin, fibrin, fibronectin, chondroitin sulfate proteoglycan, and dermatan sulfate proteoglycan (9English W.R. Velasco G. Stracke J.O. Knauper V. Murphy G. FEBS Lett. 2001; 491: 137-142Crossref PubMed Scopus (70) Google Scholar), suggesting that MT6-MMP may contribute to pericellular extracellular matrix degradation. MT6-MMP was also shown to inactivate α1-proteinase inhibitor (10Nie J. Pei D. Exp. Cell Res. 2004; 296: 145-150Crossref PubMed Scopus (37) Google Scholar) and thus may play a role in inflammation (11Sosne G. Christopherson P.L. Barrett R.P. Fridman R. Investig. Ophthalmol. Vis. Sci. 2005; 46: 2388-2395Crossref PubMed Scopus (96) Google Scholar), consistent with its high expression in leukocytes. Several transmembrane MT-MMPs initiate a cascade of zymogen activation at the cell surface by promoting the activation of pro-MMP-2 (5Zucker S. Pei D. Cao J. Lopez-Otin C. Curr. Top. Dev. Biol. 2003; 54: 1-74Crossref PubMed Google Scholar). Some MT-MMPs can also partner with members of the tissue inhibitors of metalloproteinase (TIMP) family to accomplish pro-MMP-2 activation. This process, which has been demonstrated with MT1-MMP and MT3-MMP, involves the generation of a ternary complex on the cell surface, which is formed by an active MT-MMP, TIMP-2, or TIMP-3 and pro-MMP-2 (12Strongin 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 (1432) Google Scholar, 13Zhao H. Bernardo M.M. Osenkowski P. Sohail A. Pei D. Nagase H. Kashiwagi M. Soloway P.D. DeClerck Y.A. Fridman R. J. Biol. Chem. 2004; 279: 8592-8601Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The activation of pro-MMP-2 is then initiated by a neighboring TIMP-free MT-MMP. However, conflicting results were reported regarding the ability of MT6-MMP to accomplish pro-MMP-2 activation (9English W.R. Velasco G. Stracke J.O. Knauper V. Murphy G. FEBS Lett. 2001; 491: 137-142Crossref PubMed Scopus (70) Google Scholar, 14Kojima S. Itoh Y. Matsumoto S. Masuho Y. Seiki M. FEBS Lett. 2000; 480: 142-146Crossref PubMed Scopus (115) Google Scholar, 15Nie J. Pei D. Cancer Res. 2003; 63: 6758-6762PubMed Google Scholar). Furthermore, the role of TIMP-2 was not examined. MT6-MMP is inhibited by both TIMP-2 and TIMP-1 (9English W.R. Velasco G. Stracke J.O. Knauper V. Murphy G. FEBS Lett. 2001; 491: 137-142Crossref PubMed Scopus (70) Google Scholar), which sets it apart from other MT-MMPs, such as MT1-MMP, known to be insensitive to TIMP-1. TIMP-1 inhibition of MT6-MMP raises the question as to whether MT6-MMP can accomplish the surface activation of pro-MMP-9, which is known to bind TIMP-1, in a process analogous to the ternary complex mechanism of pro-MMP-2 activation. Evidence indicates that several MT-MMPs are highly expressed in cancer tissues and play a role in cancer progression (5Zucker S. Pei D. Cao J. Lopez-Otin C. Curr. Top. Dev. Biol. 2003; 54: 1-74Crossref PubMed Google Scholar). Early studies showed high levels of MT6-MMP mRNA expression in brain, colon, and urothelial and prostate cancers (7Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar, 16Riddick A.C. Shukla C.J. Pennington C.J. Bass R. Nuttall R.K. Hogan A. Sethia K.K. Ellis V. Collins A.T. Maitland N.J. Ball R.Y. Edwards D.R. Br. J. Cancer. 2005; 92: 2171-2180Crossref PubMed Scopus (153) Google Scholar, 17Wallard M.J. Pennington C.J. Veerakumarasivam A. Burtt G. Mills I.G. Warren A. Leung H.Y. Murphy G. Edwards D.R. Neal D.E. Kelly J.D. Br. J. Cancer. 2006; 94: 569-577Crossref PubMed Scopus (64) Google Scholar, 18Nuttall 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). However, the localization of MT6-MMP protein in cancer tissues and its role in cancer progression remain to be investigated. To gain more insight into the expression and properties of MT6-MMP, we examined the expression of MT6-MMP by immunohistochemistry in human colon cancer tissues, its role in tumorigenicity, and its properties when overexpressed in colon cancer cell lines. Cell Culture—Nonmalignant monkey kidney epithelial cells BS-C-1 (CCL-26) (13Zhao H. Bernardo M.M. Osenkowski P. Sohail A. Pei D. Nagase H. Kashiwagi M. Soloway P.D. DeClerck Y.A. Fridman R. J. Biol. Chem. 2004; 279: 8592-8601Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), human colorectal adenocarcinoma SW480 (CCL-228), HT-29 (HTB-38), and HCT-116 (CCL-247) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). FET human colon carcinoma cells were a generous gift from Dr. Bonnie Sloane (Wayne State University School of Medicine). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics. Human HeLa S3 cells were purchased from ATCC (CCL-2.2) and grown in suspension in MEM Spinner medium (Quality Biologicals, Inc., Gaithersburg, MD) supplemented with 5% horse serum and antibiotics. Recombinant Proteins and Antibodies—Human recombinant pro-MMP-2, pro-MMP-9, TIMP-2, and TIMP-1 were expressed in HeLa S3 cells infected with the appropriate recombinant vaccinia viruses and purified to homogeneity, as described previously (19Olson M.W. Bernardo M.M. Pietila M. Gervasi D.C. Toth M. Kotra L.P. Massova I. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Human TIMP-3 and TIMP-4 were purchased from R&D Systems (Minneapolis, MN). A recombinant catalytic domain of human MT6-MMP expressed in Escherichia coli was a generous gift from Dr. Alex Strongin (The Burnham Institute, San Diego). Human recombinant Clusterin was purchased from Axxora, LLC (San Diego). The rabbit polyclonal antibody (pAb) against the hinge region (Ab39031) of MT6-MMP was obtained from Abcam (Cambridge, MA). Rabbit pAb against the C-terminal end (RP2-MMP-25) or the prodomain (RP4-MMP-25) of human MT6-MMP were from Triple Point Biologics (Forest Grove, OR). The pAb against the hemopexin-like domain (Ab19089) of human MT6-MMP was from Chemicon (Temecula, CA). The mouse monoclonal antibody (mAb) against human MT6-MMP (mAb1142) with unknown epitope was from R&D Systems (Minneapolis, MN). A rabbit polyclonal antiserum raised against a synthetic peptide (107RYALSGSVWKKRTLT) from the catalytic domain of human MT6-MMP was produced by Invitrogen and referred to as pAb107. The specificity of pAb107 for MT6-MMP was tested by immunoblot analysis using purified recombinant catalytic domains of all human MT-MMPs (MT1-MMP to MT5-MMP) (supplemental Fig. 1), which were purchased from Calbiochem. Two mouse mAbs against human Clusterin were purchased from Research Diagnostics, Inc. (RDI-CLUSTabm-41D, Flanders, NJ), and Axxora, LLC (ALX-804-126-C100), respectively. Rabbit pAbs to human caveolin were from BD Biosciences. The mAb against human TIMP-1 (IM32L) was from Calbiochem. The mAb101 to human TIMP-2 was described previously (20Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The mAb to the catalytic domain of MT1-MMP (LEM-2/15) was a gift from Dr. A. Arroyo (Hospital de la Princesa, Madrid, Spain). The mAb IM50L against the catalytic domain of human MT3-MMP was from Calbiochem. The RP3-MMP17 pAb against the catalytic domain of human MT4-MMP was from Triple Point Biologics. The mAb MAB050 against polyhistidine was from R&D Systems. The mAb to human β-actin was from Sigma. cDNA Constructs—The full-length human MT6-MMP cDNA (a generous gift from Dr. D. Pei, University of Minnesota) was cloned into the expression plasmid pcDNA3.1™/myc-His(-)A (Invitrogen) to generate pcDNA3.1-MT6 for stable transfection. The human MT6-MMP cDNA was also cloned into the pTF7-EMCV-1 vector for expression in the vaccinia system (21Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar). The correct sequences of the insert and junctions in the vectors were verified by DNA sequencing of both strands. The pTF7-EMCV-1 expression vector containing human MT6-MMP cDNA under control of the T7 promoter was used to generate an MT6-MMP-expressing recombinant vaccinia virus (vTF-MT6) by homologous recombination, as described previously (21Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar). A chimeric GPI-anchored human pro-MT1-MMP (GPI-MT1) containing the GPI-anchoring sequence of MT6-MMP (Ala539–Arg562) cloned in the pTF7-EMCV-1 expression vector was described previously (22Toth M. Sohail A. Mobashery S. Fridman R. Biochem. Biophys. Res. Commun. 2006; 350: 377-384Crossref PubMed Scopus (8) Google Scholar). Tissue Samples and Immunohistological Analyses—Representative tissues were collected from archived human colon cancer resection specimens in the Department of Pathology at the University of Chicago under an IRB-approved protocol. Paired samples of invasive colonic adenocarcinoma and adjacent uninvolved benign colon from 61 patients were used to create tissue microarrays using from paraffin-embedded tissue using an ATA-27 automated arrayer (Beecher Systems, Sun Prairie, WI). Each patient's specimen was represented by four tissue cores, two each of tumor and benign tissue. Four-micron-thick sections of the arrays were deparaffinized, and antigen retrieval was performed by microwaving slides in 7.5 mm sodium citrate buffer, pH 6.0. After a brief rinse in Tris-buffered saline (TBS), pH 8.0, endogenous peroxidase activity and nonspecific background staining were blocked by incubating for 30 min in 3% hydrogen peroxide in methanol followed by 30 min in 0.3% bovine serum albumin in TBS. Slides were rinsed for 2 min each in TBS, TBS containing 0.01% Triton X-100, and TBS followed by incubation for 1 h at room temperature with rabbit antiserum pAb107 to human MT6-MMP (1:400 dilution). Slides were then rinsed in TBS and incubated for 30 min with goat anti-rabbit IgG conjugated to a horseradish peroxidase-labeled polymer (Envision+ System, DAKO, Carpinteria, CA). This incubation was followed by additional TBS rinses, visualization with diaminobenzidine chromogen (DAKO), and counterstained with hematoxylin. Negative controls used preimmune rabbit serum in place of the primary antibody. The sections were blindly analyzed for histopathological features by two pathologists (C. R. Weber and J. R. Turner). Intensity of staining in the epithelial cells of duplicate tumor and paired benign control samples was scored semi-quantitatively from 0 to2(0 = negative, 1 = weak staining, and 2 = strong staining). Staining scores for each patient were calculated as the mean score of the duplicate samples. Semiquantitative RT-PCR—Total RNA of SW480, HCT-116, FET, and HT-29 cells was extracted with RNeasy® mini kit (Qiagen, Valencia, CA). Total RNA from human polymorphonuclear (PMN) cells was kindly provided by Dr. Sosne (Wayne State University School of Medicine). RT-PCR was performed with 1 μg of each total RNA sample using SuperScript™ III reverse transcriptase (Invitrogen) and subsequently HotStar Taq® master mix kit (Qiagen) following the manufacturers' instructions. The sequences of the specific primers (IDT, Coralville, IA) for human MT6-MMP used are as follows: forward, 5′-ATG GCC TGC AGC AAC TCT AT-3′; reverse, 5′-AGG GGC CTT TGA AGA AGA AA-3′. Thirty two cycles of PCR were performed with 30 s at 94 °C, 30 s at 52 °C, and 30 s at 72 °C. The housekeeping gene GAPDH was also amplified and used as an internal control. The sequences of the human GAPDH primers (IDT) are as follows: forward, 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′; reverse, 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. The amplified genes were resolved by 1% agarose gels and detected by ethidium bromide staining. Generation of Stable HT-29 and HCT-116 Transfectants—HT-29 and HCT-116 colon cancer cells were grown until 60–80% confluence. The cells were then transfected with pcDNA3.1-MT6 or pcDNA3.1 without insert (referred to as EV). Twenty four hours later, the cells were grown in complete medium supplemented with geneticin (500 μg/ml, Invitrogen) for selection. Single clones were then selected from pooled populations by serial dilutions using standard protocols. In some studies, two to three independent clones from each transfectant (MT6 and EV) of HCT-116 and HT-29 cells were pooled to generate the MT6-MMP pools (referred to as MT6-HCT and MT6-HT) and the EV pools (referred to as EV-HCT and EV-HT). Co-infection and Infection-Transfection Procedures—vTF7-3, a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase was described previously (21Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar). BS-C-1 cells were co-infected with 1 plaque-forming unit/cell each of vTF-MT6 and vTF7-3 in infection medium (DMEM supplemented with 2.5% FBS and antibiotics) for 45 min. As a control, BS-C-1 cells in parallel wells were infected only with the vTF7-3 virus. In some experiments, BS-C-1 cells were co-infected to express MT6-MMP with pro-MMP-9 or pro-MMP-2 using the appropriate recombinant viruses (13Zhao H. Bernardo M.M. Osenkowski P. Sohail A. Pei D. Nagase H. Kashiwagi M. Soloway P.D. DeClerck Y.A. Fridman R. J. Biol. Chem. 2004; 279: 8592-8601Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). For infection-transfection, BS-C-1 cells were infected with vTF7-3 virus followed by transfection with plasmid vector encoding for MT6-MMP, MT1-MMP, or GPI-MT1 as described previously (20Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The cells were then analyzed for MT-MMP expression by immunoblot analyses or for ability to promote pro-gelatinase activation as described below. Tumorigenicity Assays—All experimental procedures were performed on 4-week-old female NCr nude mice (Taconic Farms, Germantown, NY) according to the Animal Welfare Regulations at Wayne State University. HCT-116 transfectants (MT6 clone M2 and EV clone E2 and pooled clones MT6-HCT and EV-HCT) were harvested and resuspended in phosphate-buffered saline (PBS, Invitrogen). Each mouse was inoculated subcutaneously with 3 × 106 cells in 100 μl of PBS. The volume of the tumor was measured three times a week with a caliper, using the formula V = LW2 × 0.4 (where V is volume (mm3); L is biggest diameter (mm); W is smallest diameter (mm)). Data were analyzed for statistical significance by unpaired t test with Welch correction using the GraphPad InStat® version 3.0 (GraphPad Software, San Diego). The differences were considered to be statistically significant at p < 0.05. The mice were euthanized 4–5 weeks after tumor was inoculated, and the tumors were harvested and fixed in formalin or frozen for histopathological analyses. Histopathological Analyses of Tumor Xenografts—Formalin-fixed tumors were embedded in paraffin. Four-micron-thick sections were stained with hematoxylin and eosin, and tumor invasiveness was calculated by determining the fraction of length along the tumor/stroma interface that showed an irregular invasive, rather than smooth pushing, border. The sections were blindly analyzed for histopathological features by two pathologists (C. R. Weber and J. R. Turner). To examine the expression of MT6-MMP in the tumor xenograft, frozen tumors (50 mg each) were homogenized in 500 μl of cold lysis buffer (1% Nonidet P-40, 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 60 mm β-octyl glucoside, and EDTA-free protease inhibitors (PI) mixture mix (Roche Applied Science)) using a Tissue Tearor™ (Biospec Products, Bartlesville, OK). The lysates were centrifuged (14,000 × g) for 15 min at 4 °C, and the protein concentration was determined by the BCA procedure (Pierce). The lysates (250 μg each) were pre-absorbed with immobilized protein A beads (Pierce) and centrifuged, and the supernatants were incubated with either 4 μg/ml pAb Ab39031 to the hinge region of MT6-MMP or rabbit IgG and protein A beads. The immunoprecipitates were mixed with Laemmli SDS-sample buffer, with or without β-mercaptoethanol (β-ME), resolved by SDS-PAGE, and subjected to immunoblot analyses using anti-MT6-MMP mAb1142. Preparation of Cell Lysates and Immunoblot Analyses—Cells were lysed with cold lysis buffer. In some experiments, the lysis buffer was supplemented with freshly added 20 mm N-ethylmaleimide (NEM) before cell solubilization. Briefly, the cells were incubated with lysis buffer for 1 h on ice and centrifuged (14,000 × g) for 15 min at 4 °C. The protein concentration in the lysates was determined by the BCA procedure (Pierce). An aliquot of each lysate was mixed with Laemmli SDS-sample buffer with or without 1% β-ME, boiled (95 °C, 5 min), and resolved by SDS-PAGE followed by immunoblot analyses using various antibodies. Crude Plasma Membrane Fractions—Confluent MT6-HCT cells from three 150-mm culture dishes were scraped in cold PBS supplemented with 2 mm EDTA. The cells were recovered by centrifugation (1000 × g, 5 min, 4 °C). The cells were then washed in PBS and resuspended in 25 mm Tris-HCl, pH 7.4, supplemented with 50 mm NaCl, and 8.5% sucrose supplemented with PI mixture mix. The cells were homogenized by passing though 22½-gauge needle, 20 times on ice. The homogenate was centrifuged (18,000 × g, 20 min, 4 °C) to remove cell organelles. The resulting supernatant was centrifuged (200,000 × g, 1 h, 4 °C), and the pellet was resuspended in 25 mm Tris-HCl buffer, pH 7.4, containing 50 mm NaCl and protease inhibitors and stored at -80 °C until used. The protein concentration was determined by the BCA procedure. PI-PLC Treatment—Confluent HT-29 and HCT-116 transfectants in 6-well plates were washed with cold PBS and treated with 0.3 units/well of phosphatidylinositol-specific phospholipase C (PI-PLC, Molecular Probes, Eugene, OR) in PBS for 30 min on ice. The supernatant was clarified and concentrated with Microcon® centrifugal filter devices (Millipore, Bedford, MA) in the presence of the PI mix. An aliquot of each concentrated supernatant was mixed with Laemmli SDS-sample buffer with or without β-ME, boiled, and resolved by SDS-PAGE followed by immunoblot analysis. PMN Cell Isolation—Lysates of human PMNs were kindly provided by Dr. Gabriel Sosne (Wayne State University School of Medicine). Briefly, freshly drawn blood in 8-ml EDTA vacutainers was centrifuged at ∼700 × g for 5 min at room temperature. The leukocyte-rich plasma and upper layer were transferred to a 50-ml centrifuge tube and subjected to gradient centrifugation through a layer of Histopaque®-1077 reagent over a layer of Histopaque®-1119 reagent (Sigma) (volume ratio blood, 1077:1119 = 2:1:1) at ∼700 × g for 30 min at room temperature. PMNs enriched at the interface of Histopaque®-1077 and -1119 were carefully collected and washed with PBS at room temperature and pelleted (∼700 × g, 5 min, room temperature). The lysate of PMNs was prepared by lysing ∼107 cells in 0.5 ml of Triton X-100 lysis buffer (1% Triton X-100 in PBS supplemented with PI mix). An aliquot of the lysate was mixed with Laemmli SDS-sample buffer with or without β-ME, boiled, and resolved by SDS-PAGE followed by immunoblot analysis. Successive Detergent Extraction of Lipid Rafts—The procedure was adapted from Solomon et al. (23Solomon K.R. Mallory M.A. Finberg R.W. Biochem. J. 1998; 334: 325-333Crossref PubMed Scopus (63) Google Scholar). Briefly, cells grown in 100-mm tissue culture dishes were scraped in PBS containing 2 mm EDTA. The cell pellet was resuspended in buffer A (25 mm MES, pH 6.5, 150 mm NaCl) on ice. An equal volume of the same buffer containing 2% Triton X-100 and the PI mix was added. The tube contents were gently mixed and incubated on ice for 30 min. Insoluble fractions were pelleted by centrifugation (14,000 × g) for 20 min at 4 °C. The Triton-soluble protein fraction was collected and labeled as nonlipid raft fraction. The pellet was resuspended in buffer B (10 mm Tris-HCl, pH 7.5, 1% Triton X-100, 500 mm NaCl, 60 mm β-octyl glucoside, and PI mix). After 30 min of incubation on ice, the cellular debris, insoluble fraction, was removed by centrifugation (14,000 × g), and β-octyl glucoside soluble protein was marked as lipid raft fraction. An aliquot of the nonlipid raft (referred to as S fraction) and lipid raft (referred to as I fraction) fractions were mixed with Laemmli SDS-sample buffer with or without β-ME, boiled, and resolved by SDS-PAGE followed by immunoblot analysis using various anti-MT6-MMP antibodies. The blots were reprobed with antibodies to human caveolin, a lipid raft protein. Pulse-Chase Analyses—MT6-HCT cells (80% confluent) in 6-well plates were incubated for 30 min with 1 ml/well of starving medium (DMEM without methionine and cysteine supplemented with 10 mm HEPES). The cells were then pulsed with 500 μCi/ml of [35S]methionine/cysteine in starvation medium supplemented with 1% dialyzed FBS (0.5 ml/well) for 15 min at 37 °C. After the pulse, the medium was aspirated, and the cells were washed twice with PBS before the addition of 1 ml/well chase medium (DMEM with 1% dialyzed FBS, 5 mm methionine, and 5 mm cysteine). At the end of the chase period (0–120 min at 37 °C), the medium was removed, and the cells were washed with cold PBS and lysed with cold lysis buffer as described before. The lysates were clarified by a brief centrifugation and were pre-absorbed with immobilized protein A beads (Pierce). The lysates were subjected to immunoprecipitation with either MT6-MMP antibodies or rabbit IgG (Pierce) and immobilized protein A beads. The immunoprecipitates were mixed with Laemmli SDS-sample buffer, with or without β-ME, and resolved by SDS-PAGE followed by autoradiography. Pro-gelatinase Activation Assays—These assays were carried out with infected BS-C-1 cells and with the HT-29 and HCT-116 transfectants. Briefly, BS-C-1 cells were co-infected to express wild type MT6-MMP and MT1-MMP or infected with control virus as described above. Four hour post-infection, the cells were incubated overnight in serum-free DMEM supplemented without or with various amounts (0–50 nm) of human recombinant TIMP-2 or TIMP-1. Then the media were aspirated and the cells were gently washed with DMEM to remove unbound TIMPs. The cells were then incubated (up to 8 h at 37 °C) with serum-free DMEM supplemented with 10 nm purified pro-MMP-2 or pro-MMP-9. The media were collected, and the cells were lysed with lysis buffer. The media and lysates wer