Enhanced Tumor Growth and Invasiveness in Vivo by a Carboxyl-Terminal Fragment of α1-Proteinase Inhibitor Generated by Matrix Metalloproteinases
1999; Elsevier BV; Volume: 154; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65292-3
ISSN1525-2191
AutoresHiroaki Kataoka, H Uchino, Takeshi Iwamura, Motoharu Seiki, Kazuki Nabeshima, Masashi Koono,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoMatrix metalloproteinases (MMPs) are believed to contribute to the complex process of cancer progression. They also exhibit an α1-proteinase inhibitor (αPI)-degrading activity generating a carboxyl-terminal fragment of ∼5 kd (αPI-C). This study reports that overexpression of αPI-C in S2–020, a cloned subline derived from the human pancreas adenocarcinoma cell line SUIT-2, potentiates the growth capability of the cells in nude mice. After stable transfection of a vector containing a chimeric cDNA encoding a signal peptide sequence of tissue inhibitor of metalloproteinase-1 followed by cDNA for αPI-C into S2–020 cells, three clones that stably secrete αPI-C were obtained. The ectopic expression of αPI-C did not alter in vitro cellular growth. However, subcutaneous injection of the αPI-C-secreting clones resulted in tumors that were 1.5 to 3-fold larger than those of control clones with an increased tendency to invasiveness and lymph node metastasis. These effects could be a result of modulation of natural killer (NK) cell-mediated control of tumor growth in nude mice, as the growth advantage of αPI-C-secreting clones was not observed in NK-depleted mice, and αPI-C-secreting clones showed decreased NK sensitivity in vitro. In addition, production of αPI and generation of the cleaved form of αPI by MMP were observed in various human tumor cell lines and in a highly metastatic subline of SUIT-2 in vitro. These results provide experimental evidence that the αPI-degrading activity of MMPs may play a role in tumor progression not only viathe inactivation of αPI but also viathe generation of αPI-C. Matrix metalloproteinases (MMPs) are believed to contribute to the complex process of cancer progression. They also exhibit an α1-proteinase inhibitor (αPI)-degrading activity generating a carboxyl-terminal fragment of ∼5 kd (αPI-C). This study reports that overexpression of αPI-C in S2–020, a cloned subline derived from the human pancreas adenocarcinoma cell line SUIT-2, potentiates the growth capability of the cells in nude mice. After stable transfection of a vector containing a chimeric cDNA encoding a signal peptide sequence of tissue inhibitor of metalloproteinase-1 followed by cDNA for αPI-C into S2–020 cells, three clones that stably secrete αPI-C were obtained. The ectopic expression of αPI-C did not alter in vitro cellular growth. However, subcutaneous injection of the αPI-C-secreting clones resulted in tumors that were 1.5 to 3-fold larger than those of control clones with an increased tendency to invasiveness and lymph node metastasis. These effects could be a result of modulation of natural killer (NK) cell-mediated control of tumor growth in nude mice, as the growth advantage of αPI-C-secreting clones was not observed in NK-depleted mice, and αPI-C-secreting clones showed decreased NK sensitivity in vitro. In addition, production of αPI and generation of the cleaved form of αPI by MMP were observed in various human tumor cell lines and in a highly metastatic subline of SUIT-2 in vitro. These results provide experimental evidence that the αPI-degrading activity of MMPs may play a role in tumor progression not only viathe inactivation of αPI but also viathe generation of αPI-C. It is generally accepted that a family of structurally related neutral metalloproteinases called matrix metalloproteinases (MMPs) have an important role in tumor progression, particularly in invasive/metastatic events via their degrading activity against various extracellular matrix (ECM) proteins.1Stetler-Stevenson WG Hewitt R Corcoran M Matrix metalloproteinase and tumor invasion: from correlation and causality to the clinic.Semin Cancer Biol. 1996; 7: 147-154Crossref PubMed Scopus (343) Google Scholar Recently, a number of researchers have reported that serine proteinase inhibitors (serpins) are also good substrates for MMPs.2Desrochers PE Jeffrey JJ Weiss SJ Interstitial collagenase (matrix metalloproteinase-1) expresses sepinase activity.J Clin Invest. 1991; 87: 2258-2265Crossref PubMed Scopus (108) Google Scholar, 3Winyard PG Zhang Z Chidwick K Blake DR Carrell RW Murphy G Proteolytic inactivation of human α1 antitrypsin by human stromelysin.FEBS Lett. 1991; 279: 91-94Crossref PubMed Scopus (55) Google Scholar, 4Zhang Z Winyard PG Chidwick K Murphy G Wardell M Carrell RW Blake DR Proteolysis of human native and oxidised α1-proteinase inhibitor by matrilysin and stromelysin.Biochim Biophys Acta. 1994; 1199: 224-228Crossref PubMed Scopus (51) Google Scholar, 5Sires UI Murphy G Baragi VM Fliszar CJ Welgus HG Senior RM Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of α1-antitrypsin.Biochem Biophys Res Commun. 1994; 204: 613-620Crossref PubMed Scopus (107) Google Scholar, 6Pei D Majmudar G Weiss SJ Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.J Biol Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar We have previously reported that many tumor cell lines produce and secrete serpins such as α1-proteinase inhibitor (αPI).7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar αPI, usually called α1-antitrypsin, is a major plasma serpin that has a broad inhibitory spectrum against serine proteinases and is the primary physiological inhibitor of leukocyte elastase. Among MMPs, interstitial collagenase (MMP-1, EC 3.4.24.7), neutrophil collagenase (MMP-8, EC 3.4.24.34), stromelysin-1 (MMP-3, EC 3.4.24.17), matrilysin (MMP-7, EC 3.4.24.23), and stromelysin-3 (MMP-11) effectively cleave αPI.2Desrochers PE Jeffrey JJ Weiss SJ Interstitial collagenase (matrix metalloproteinase-1) expresses sepinase activity.J Clin Invest. 1991; 87: 2258-2265Crossref PubMed Scopus (108) Google Scholar, 3Winyard PG Zhang Z Chidwick K Blake DR Carrell RW Murphy G Proteolytic inactivation of human α1 antitrypsin by human stromelysin.FEBS Lett. 1991; 279: 91-94Crossref PubMed Scopus (55) Google Scholar, 4Zhang Z Winyard PG Chidwick K Murphy G Wardell M Carrell RW Blake DR Proteolysis of human native and oxidised α1-proteinase inhibitor by matrilysin and stromelysin.Biochim Biophys Acta. 1994; 1199: 224-228Crossref PubMed Scopus (51) Google Scholar, 5Sires UI Murphy G Baragi VM Fliszar CJ Welgus HG Senior RM Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of α1-antitrypsin.Biochem Biophys Res Commun. 1994; 204: 613-620Crossref PubMed Scopus (107) Google Scholar, 6Pei D Majmudar G Weiss SJ Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.J Biol Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar Of these, matrilysin exhibits the most efficient αPI-degrading activity,4Zhang Z Winyard PG Chidwick K Murphy G Wardell M Carrell RW Blake DR Proteolysis of human native and oxidised α1-proteinase inhibitor by matrilysin and stromelysin.Biochim Biophys Acta. 1994; 1199: 224-228Crossref PubMed Scopus (51) Google Scholar, 5Sires UI Murphy G Baragi VM Fliszar CJ Welgus HG Senior RM Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of α1-antitrypsin.Biochem Biophys Res Commun. 1994; 204: 613-620Crossref PubMed Scopus (107) Google Scholar and stromelysin-3 is more potent than collagenases and stromelysin-1.6Pei D Majmudar G Weiss SJ Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.J Biol Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar Moreover, a matter of importance is that the mature forms of human stromelysin-3 exhibit a very limited substrate specificity and appear unable to degrade any major ECM component.6Pei D Majmudar G Weiss SJ Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.J Biol Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar The cleavages of αPI by these MMPs occur at peptide bonds within αPI's active-site loop,2Desrochers PE Jeffrey JJ Weiss SJ Interstitial collagenase (matrix metalloproteinase-1) expresses sepinase activity.J Clin Invest. 1991; 87: 2258-2265Crossref PubMed Scopus (108) Google Scholar, 3Winyard PG Zhang Z Chidwick K Blake DR Carrell RW Murphy G Proteolytic inactivation of human α1 antitrypsin by human stromelysin.FEBS Lett. 1991; 279: 91-94Crossref PubMed Scopus (55) Google Scholar, 4Zhang Z Winyard PG Chidwick K Murphy G Wardell M Carrell RW Blake DR Proteolysis of human native and oxidised α1-proteinase inhibitor by matrilysin and stromelysin.Biochim Biophys Acta. 1994; 1199: 224-228Crossref PubMed Scopus (51) Google Scholar, 5Sires UI Murphy G Baragi VM Fliszar CJ Welgus HG Senior RM Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of α1-antitrypsin.Biochem Biophys Res Commun. 1994; 204: 613-620Crossref PubMed Scopus (107) Google Scholar, 6Pei D Majmudar G Weiss SJ Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.J Biol Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar resulting in the inactivation of the inhibitory activity and release of a carboxyl-terminal (C-terminal) fragment of ∼5 kd (αPI-C) that is thought to be largely concealed in a native αPI. We have found that the tumor-cell-derived αPI and its cleaved form, which is ∼5 kd smaller than the noncleaved αPI, were concomitantly present in the serum-free culture conditioned media of the tumor cell lines.7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar These observations led to the postulation that the MMP-dependent hydrolysis of αPI and the subsequent generation of αPI-C may occur in a tumor-cell microenvironment. To date, little is known about the biological significance of αPI and the αPI-degrading activity of MMPs such as matrilysin and stromelysin-3 in tumors, and rather surprisingly, patients with immunohistochemically αPI-positive adenocarcinomas had worse prognosis than the negative ones.8Karashima S Kataoka H Itoh H Maruyama R Koono M Prognostic significance of α-1-antitrypsin in early stage of colorectal carcinomas.Int J Cancer. 1990; 45: 244-250Crossref PubMed Scopus (62) Google Scholar, 9Higashiyama M Doi O Kodama K Yokouchi H Tateishi R An evaluation of the prognostic significance of α-1-antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis.Br J Cancer. 1992; 65: 300-302Crossref PubMed Scopus (62) Google Scholar, 10Allgayer H Babic R Grutzner KU Beyer BCM Tarabichi A Schildberg FW Heiss MM Tumor-associated proteases and inhibitors in gastric cancer: analysis of prognostic impact and individual risk protease patterns.Clin Exp Metastasis. 1998; 16: 62-73Crossref PubMed Scopus (65) Google Scholar On the other hand, the ectopic expression of human stromelysin-3 in MCF-7 breast carcinoma cells resulted in enhanced tumorigenicity of the cells in nude mice.11Noël AC Lefebvre O Maquoi E VanHoorde L Chenard M-P Mareel M Foidart J-M Basset P Rio M-C Stromelysin-3 expression promotes tumor take in nude mice.J Clin Invest. 1996; 97: 1924-1930Crossref PubMed Scopus (82) Google Scholar Similarly, overexpression of matrilysin in a colon carcinoma cell line was found to increase its tumorigenicity in nude mice without modulation of the invasive property in vitro,12Witty JP McDonnell S Newell KJ Cannon P Navre M Tressler RJ Matrisian LM Modulation of matrilysin levels in colon carcinoma cell lines affects tumorigenicity in vivo.Cancer Res. 1994; 54: 4805-4812PubMed Google Scholar and intestinal tumorigenesis was suppressed in mice lacking matrilysin.13Wilson CL Heppner KJ Labosky PA Hogan BLM Matrisian LM Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin.Proc Natl Acad Sci USA. 1997; 94: 1402-1407Crossref PubMed Scopus (546) Google Scholar This evidence suggests that MMPs such as stromelysin-3 and matrilysin somehow favor the survival and growth of the cancer cells in a tissue microenvironment in vivo, possibly by functioning in an undefined capability independent of ECM degradation.14Chambers AF Matrisian LM Changing views of the role of matrix metalloproteinases in metastasis.J Natl Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1442) Google Scholar One of the key events in the survival and growth of cancer cells in vivo is a resistance to the host immune system. Natural cytotoxicity, mediated by natural killer (NK) cells and lymphokine-activated killer (LAK) cells, plays an important role in the host defense mechanism against cancer cells.15Brittenden J Heys SD Ross J Eremin O Natural killer cells and cancer.Cancer. 1996; 77: 1226-1243Crossref PubMed Scopus (304) Google Scholar As NK activity within tumors of patients with cancer is lower than that found in the peripheral blood, the presence of tumor-derived suppressor factors has been suggested.15Brittenden J Heys SD Ross J Eremin O Natural killer cells and cancer.Cancer. 1996; 77: 1226-1243Crossref PubMed Scopus (304) Google Scholar Cercek et al16Cercek L Cercek B Cancer-associated SCM-recognition, immune defence suppression, and serine protease protection petide. I. Isolation, amino acid sequence, homology, and origin.Cancer Detect Prev. 1992; 16: 305-319PubMed Google Scholar, 17Cercek L Cercek B Cancer-associated SCM-recognition, immune defence suppression, and serine protease protection peptide. II. Immunodefense suppressive effects of the CRISPPs peptide.Cancer Detect Prev. 1993; 17: 433-445PubMed Google Scholar have purified a peptide that could have immunosuppressive effects from sera of patients bearing various solid cancers. This peptide, designated as CRISPP (cancer recognition, immune defense suppression, and serine protease protection peptide), was reported to have unique reversible suppressive effects on NK and LAK cells in vitro.17Cercek L Cercek B Cancer-associated SCM-recognition, immune defence suppression, and serine protease protection peptide. II. Immunodefense suppressive effects of the CRISPPs peptide.Cancer Detect Prev. 1993; 17: 433-445PubMed Google Scholar Of particular interest is that the amino acid sequence of the CRISPP is highly homologous (83% to 100%) to that of the C-terminal part of αPI (Met358-Gln393).16Cercek L Cercek B Cancer-associated SCM-recognition, immune defence suppression, and serine protease protection petide. I. Isolation, amino acid sequence, homology, and origin.Cancer Detect Prev. 1992; 16: 305-319PubMed Google Scholar As this C-terminal part overlaps in αPI-C generated by the MMP-dependent hydrolysis of αPI, it can be hypothesized that αPI-C may have suppressive activity against NK and LAK cells and that MMPs, particularly matrilysin and stromelysin-3, may contribute to the survival of cancer cells in vivo through the generation of αPI-C from the tumor-cell-derived and/or host-derived αPI. However, the biological significance of αPI-C in vivo is yet to be clarified. To confirm the above hypothesis in vivo, we have attempted to construct an αPI-C expression/secretion vector to examine the effects of αPI-C in vivo. The generation of αPI-C by tumor cells enhanced the growth and invasiveness of the tumor cells in nude mice. All cultured cells are derived from cells of human origin. Cloned sublines S2–020, S2–007 and S2–028 were derived from a single pancreas adenocarcinoma cell line, SUIT-2.18Iwamura T Taniguchi S Kitamura N Yamanari H Kojima A Hidaka K Setoguchi T Katsuki T Correlation between CA19-9 production in vitro and histological grades of differentiation in vivo in clones isolated from a human pancreatic cancer cell line (SUIT-2).J Gastroenterol Hepatol. 1992; 7: 512-519Crossref PubMed Scopus (47) Google Scholar S2–020 formed poorly differentiated adenocarcinoma in the nude mouse.18Iwamura T Taniguchi S Kitamura N Yamanari H Kojima A Hidaka K Setoguchi T Katsuki T Correlation between CA19-9 production in vitro and histological grades of differentiation in vivo in clones isolated from a human pancreatic cancer cell line (SUIT-2).J Gastroenterol Hepatol. 1992; 7: 512-519Crossref PubMed Scopus (47) Google Scholar It was low metastatic in a spontaneous metastasis assay in nude mice but was highly invasive in vitro.19Taniguchi S Iwamura T Katsuki T Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines.Clin Exp Metastasis. 1992; 10: 259-266Crossref PubMed Scopus (64) Google Scholar, 20Kataoka H Seguchi K Iwamura T Moriyama T Nabeshima K Koono M Reverse-zymographic analysis of protease nexin-II/amyloid β protein precursor of human carcinoma cell lines, with special reference to the grade of differentiation and metastatic phenotype.Int J Cancer. 1995; 60: 123-128Crossref PubMed Scopus (16) Google Scholar S2–007 formed moderately differentiated adenocarcinoma and was highly metastatic in nude mice but was low invasive in vitro.18Iwamura T Taniguchi S Kitamura N Yamanari H Kojima A Hidaka K Setoguchi T Katsuki T Correlation between CA19-9 production in vitro and histological grades of differentiation in vivo in clones isolated from a human pancreatic cancer cell line (SUIT-2).J Gastroenterol Hepatol. 1992; 7: 512-519Crossref PubMed Scopus (47) Google Scholar, 19Taniguchi S Iwamura T Katsuki T Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines.Clin Exp Metastasis. 1992; 10: 259-266Crossref PubMed Scopus (64) Google Scholar, 20Kataoka H Seguchi K Iwamura T Moriyama T Nabeshima K Koono M Reverse-zymographic analysis of protease nexin-II/amyloid β protein precursor of human carcinoma cell lines, with special reference to the grade of differentiation and metastatic phenotype.Int J Cancer. 1995; 60: 123-128Crossref PubMed Scopus (16) Google Scholar S2–028 formed well differentiated adenocarcinoma and was nonmetastatic in nude mice and noninvasive in vitro.19Taniguchi S Iwamura T Katsuki T Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines.Clin Exp Metastasis. 1992; 10: 259-266Crossref PubMed Scopus (64) Google Scholar, 20Kataoka H Seguchi K Iwamura T Moriyama T Nabeshima K Koono M Reverse-zymographic analysis of protease nexin-II/amyloid β protein precursor of human carcinoma cell lines, with special reference to the grade of differentiation and metastatic phenotype.Int J Cancer. 1995; 60: 123-128Crossref PubMed Scopus (16) Google Scholar These clones were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS). S2–020 was used for the transfection study described below. In addition to the above-described SUIT-2 subclones, a normal fetal intestinal cell line (FHs74 int), a fibroblast cell line (Flow 2000), 7 colorectal adenocarcinoma cell lines (RCM-1, -2, and -3, CoCM-1, SW837, WiDr, and Colo 205), and 2 gastric adenocarcinoma cell lines (MKN-45 and -28), another SUIT-2 subline (S2–013), a renal cell carcinoma line (MRT-1), lung adenocarcinoma and squamous-cell carcinoma lines (LC-2/ad and LC-1/sq), 3 urinary bladder transitional-cell carcinoma lines (UMK-1 and -2 and T-24), a fibrosarcoma cell line (HT1080), an osteosarcoma cell line (OST), a primary culture of chordoma, and a glioblastoma cell line (MGM-1) were used. FHs74 int, WiDr, SW837, and Colo 205 were obtained from Dainihon Seiyaku (Osaka, Japan). MKN-45 and -28 were from IBL (Fujioka, Japan). Flow 2000 was from Japanese Cancer Research Resources Bank (Osaka, Japan). T-24 and OST were from RIKEN Cell Bank (Tsukuba, Japan). HT1080 was a kind gift from Dr. J. Suzumiya, Fukuoka University, Fukuoka, Japan. The other cell lines were established in our laboratory. To obtain serum-free conditioned medium (SFCM), confluent cells were washed three times with serum-free DMEM and cultured in it for 24 hours. To determine effects of tissue inhibitor of metalloproteinase-1 (TIMP-1), 2 μg/ml recombinant human TIMP-1 (rTIMP-1, Fuji Chemical Industries, Toyama, Japan) was added into the serum-free culture medium. Alternatively, the cells were transiently transfected with an expression plasmid pSG5 (Stratagene, La Jolla, CA) harboring a full-length cDNA for human TIMP-1 (pSG-TIMP), using lipofectamine reagent (Gibco-BRL, Gaithersburg, MD). Twenty-four hours after the transfection procedure, the cells were washed three times with serum-free medium, and SFCM was collected as described above. The enhanced secretion of TIMP-1 was confirmed by immunoblot analysis with anti-human TIMP-1 antibody (Fuji Chemical Industries) and by a TIMP-1 ELISA system (Amersham, Little Chalfont, UK). To examine in vitro growth characteristics, replicate 35-mm dishes were seeded at 2 × 105 cells/3 ml of growth medium. The number of viable cells was counted daily, and doubling time was determined during the log-phase of growth. In an attempt to generate an αPI-C expression/secretion vector, a chimeric cDNA encoding a signal peptide sequence of TIMP-1 followed by cDNA for αPI-C, which is the C-terminal region of αPI (Met358-Arg394), with a stop codon (TAA) at the 3′ terminus was constructed. First, cDNA corresponding to αPI-C including the stop codon was obtained by a reverse transcription polymerase chain reaction (RT-PCR) using poly A+ RNA obtained from cultured RCM-1 human rectal adenocarcinoma cell line that synthesizes and secretes αPI in vitro.21Kataoka H Nabeshima K Komada N Koono M New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro.Virchows Arch B Cell Pathol. 1989; 57: 157-165Crossref Scopus (45) Google Scholar A 37-mer forward primer (Atim 1), composed of a 17-mer that corresponded to the 3′ terminus of the signal sequence of TIMP-1 followed by a 20-mer corresponding to the 5′ terminus of αPI-C, and a 29-mer reverse primer (Atim 2), composed of a 9-mer carrying a SalI site followed by a 20-mer corresponding to the 3′ terminus of αPI-C, including the stop codon, were used. Sequences of Atim 1 and Atim 2 are 5′-ATAGCCCCAGCAGGGCCATGTCTATCCCCCCAGAGGT-3′ and 5′-GCGGTCGACTTATTTTTGGGTGGGATTCA-3′, respectively. Second, cDNA corresponding to the signal peptide sequence of TIMP-1 was amplified. A plasmid, pSG-TIMP, was used as the template. A 29-mer forward primer (Atim 3), composed of a 9-mer carrying an XhoI site followed by a 4-mer corresponding to the untranslated region upstream from the translational start site compatible with Kozak's rule and a 16-mer corresponding to the 5′ terminus of the signal peptide sequence of TIMP, and a 20-mer reverse primer (Atim 4) corresponding to the 3′ terminus of the signal peptide sequence of TIMP. Sequences of Atim 3 and Atim 4 are 5′-CCGCTCGAGCCACCATGGCCCCCTTTGAG-3′ and 5′-GGCCCTGCTGGGGGCTATCA-3′, respectively. Third, the gel-purified products from both of the above reactions were used as templates for fusion by overlap extension using PCR. For this reaction, Atim 3 and Atim 2 were used. The 206-bp DNA product, designated as ATIM, that was generated in this PCR was ethanol precipitated and agarose gel purified. In the fourth and final reaction, the product was double digested by SalI and XhoI, ethanol precipitated, agarose gel purified, and subcloned into pCI-neo (Promega, Madison, WI) expression vector to create plasmid pCI-neoATIM. The inserted sequence was confirmed by a double-strand DNA sequencing of the plasmid (see Figure 3B). S2–020 cells were transfected with pCI-neoATIM linearized by BamHI using lipofectamine reagent. Stable transfectants were selected with geneticin (0.5 mg/ml; Gibco-BRL), and isolated clones were obtained by ring cloning. For control, S2–020 cells were similarly transfected with linearized pCI-neo carrying no exogenous DNA, and the stable transfectants were cloned. Twenty clones of pCI-neoATIM-transfected (ATIM 1 to 20), and 10 clones of the mock-transfected control (pCI 1 to 10) were isolated. Each clone was cultured, and SFCM was harvested. The amount of αPI-C peptide in SFCM was measured by ELISA as described below. Three clones (ATIM 1, 8, and 11) secreted a notable amount of αPI-C and were used in the subsequent experiment. Polyclonal anti-αPI-C rabbit serum was obtained by immunizing synthetic peptide corresponding to Met358-Ile375 of αPI synthesized as multiple antigen peptide resin (Sawady Technology, Tokyo, Japan). The immunoglobulin fraction was purified (E-Z-SEP purification kit, Pharmacia, Uppsala, Sweden), and the antibody was further affinity purified. A 13-mer synthetic peptide corresponding to 12 amino acid residues of the amino terminus of αPI-C with an additional cystein residue at the C terminus (αPI-C13) was conjugated to activated 2-fluoro-1-methylpyridinium toluene-4-sulfonate cellulofine (Seikagaku Kogyo, Tokyo, Japan) and used for the affinity column preparation. The secreted αPI-C in the conditioned medium was measured by an antibody capture immunoassay. Briefly, 96-well microtiter plates (MaxiSorp, Nunc, Naperville, IL) were coated with samples or standard peptide (αPI-C13) solution in 20 mmol/L sodium carbonate buffer (pH 9.6) at 4°C overnight. The presence of adsorbed αPI-C was detected with an enzyme immunoassay using the rabbit anti-αPI-C IgG prepared as described above and peroxidase-conjugated swine anti-rabbit IgG (Dakopatts, Glostrup, Denmark). O-Phenylenediamine was used as the color reagent according to the standard technique.7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar Absorbance at 492 nm was measured using the microplate spectrophotometer, and values were analyzed using SOFT max PRO software (Molecular Devices, Sunnyvale, CA). Sandwich ELISA for human αPI was done according to the method described previously.7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar With this system, bovine αPI did not cross-react. Poly(A)+-enriched RNA was extracted using the Fast Track mRNA isolation kit (Invitrogen, San Diego, CA) from the cultured cells. Two micrograms of poly(A)+-enriched RNA was electrophoresed on 1% formaldehyde agarose gel and transblotted onto Hybond-N+ nylon membrane (Amersham), and RNA was ultraviolet cross-linked onto the membrane. Hybridization was performed in a mixed solution of 50% formamide, 5X Denhardt's solution, 25 mmol/L phosphate buffer (pH 6.5), 0.1% SDS, 100 μg/ml sonicated and heat-denatured salmon sperm DNA, and 5X standard saline citrate (SSC) at 42°C for 16 hours. The blots were washed as follows: three times in 0.1% SDS in 1X SSC for 15 minutes at room temperature and twice in the same solution for 20 minutes at 65°C. The membranes were autoradiographed with Kodak XR-5 film at −80°C for 6 hours or 24 hours. The αPI-C cDNA corresponding to the C-terminal region of αPI (Met358-Lys394) was synthesized by PCR using the pCI-neoATIM plasmid as a template. The 5′ end of the reverse primer was radiolabeled with [γ-32P]ATP, and the corresponding PCR product was gel purified and used as a probe. For internal control of loading, the blots were subsequently hybridized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Clontech, Palo Alto, CA). The GAPDH probes were radiolabeled by random priming with [α-32P]CTP. For gelatin zymography, each SFCM sample derived from the same cell number (3.6 × 104 cells) was applied to SDS-PAGE (10% separating gel) containing 0.1% gelatin as a substrate under nonreducing conditions without boiling. After electrophoresis, the gel was washed in 2.5% (v/v) Triton X-100 at room temperature for 60 minutes with two changes of the detergent solution to remove SDS. The gel was rinsed once with incubation buffer (5 mmol/L CaCl2 and 0.02% NaN3 in 50 mmol/L Tris/HCl, pH 7.6), incubated in the same buffer overnight at 37°C, and then stained with 2.5% Coomassie brilliant blue in 30% methanol and 10% acetate. Enzyme activity was detected as a clear band on the resulting blue background of undigested gelatin. Immunoblot analyses for matrilysin and αPI were done according to the method described previously.7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 22Kataoka H Meng J-Y Uchino H Nabeshima K Kihira Y Matuo Y Koono M Modulation of matrix metalloproteinase-7 (matrilysin) secretion in coculture of human colon carcinoma cells with fibroblasts from orthotopic and ectopic organs.Oncol Res. 1997; 9: 101-109PubMed Google Scholar For the detection of αPI, samples were pretreated with or without peptide N-glycosidase F (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.7Kataoka H Seguchi K Inoue T Koono M Properties of α1-antitrypsin secreted by human adenocarcinoma cell lines.FEBS Lett. 1993; 328: 291-295Abstract Full Text PDF PubMed Scopus (21) Google Scholar In addition, purified human αPI (12 μg/ml; Sigma Chemical Co., St. Louis, MO) treated with or without human recombinant matrilysin (0.3 μg/ml; Oriental Yeast Co., Shiga, Japan) was also subjected to the immunoblot analysis using rabbit anti-human αPI IgG (Dakopatts) to confirm the cleavage of αPI by matrilysin. The In vitro matrigel invasion assay was done according to the method described previously.20Kataoka H Seguchi K Iwamura T Moriyama T Nabeshima K Koono M Revers
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