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• W2020140319 abstract "We have previously shown coexpression of hepatocyte growth factor (HGF) and its receptor Met in the invasive tumor front of human breast carcinomas. We have also demonstrated secretion of HGF, constitutive activation of Met, and increased invasion in a murine breast carcinoma cell line, SP1. These observations suggest the presence of an HGF autocrine loop in some breast carcinoma cells, which confers increased survival, growth, and invasiveness during tumor progression and metastasis. c-Src tyrosine kinase, which is critical in regulating the expression of many genes, is activated in SP1 carcinoma cells, as well as in most human breast cancers. We therefore examined the role of c-Src kinase in HGF expression in breast carcinoma cells. Expression of activated c-Src in SP1 cells increased transcription from the HGF promoter and expression of HGF mRNA and protein, while dominant negative c-Src had the opposite effect. Using deletion analysis, we showed that the region between −254 and −70 base pairs was required for c-Src responsiveness of the HGF promoter. This region contains two putative consensus sequences (at −110 and −149 base pairs) for the Stat3 transcription factor, which bind protein complexes containing Stat3 (but not Stat1, -5A, or -5B). Coexpression of activated c-Src and Stat3 synergistically induced strong HGF promoter activity in SP1 cells, as well as in a nonmalignant epithelial cell line, HC11 (HGF negative). c-Src kinase activity correspondingly increased the tyrosine 705 phosphorylation and DNA binding affinity of Stat3 (but not Stat1, -5A, or -5B). Collectively, our data indicate a cooperative effect of c-Src kinase and Stat3 in the activation of HGFtranscription and protein expression in breast carcinoma cells. This process may be important in overriding the strong repression ofHGF expression in nonmalignant epithelium, and thereby promote tumorigenesis. We have previously shown coexpression of hepatocyte growth factor (HGF) and its receptor Met in the invasive tumor front of human breast carcinomas. We have also demonstrated secretion of HGF, constitutive activation of Met, and increased invasion in a murine breast carcinoma cell line, SP1. These observations suggest the presence of an HGF autocrine loop in some breast carcinoma cells, which confers increased survival, growth, and invasiveness during tumor progression and metastasis. c-Src tyrosine kinase, which is critical in regulating the expression of many genes, is activated in SP1 carcinoma cells, as well as in most human breast cancers. We therefore examined the role of c-Src kinase in HGF expression in breast carcinoma cells. Expression of activated c-Src in SP1 cells increased transcription from the HGF promoter and expression of HGF mRNA and protein, while dominant negative c-Src had the opposite effect. Using deletion analysis, we showed that the region between −254 and −70 base pairs was required for c-Src responsiveness of the HGF promoter. This region contains two putative consensus sequences (at −110 and −149 base pairs) for the Stat3 transcription factor, which bind protein complexes containing Stat3 (but not Stat1, -5A, or -5B). Coexpression of activated c-Src and Stat3 synergistically induced strong HGF promoter activity in SP1 cells, as well as in a nonmalignant epithelial cell line, HC11 (HGF negative). c-Src kinase activity correspondingly increased the tyrosine 705 phosphorylation and DNA binding affinity of Stat3 (but not Stat1, -5A, or -5B). Collectively, our data indicate a cooperative effect of c-Src kinase and Stat3 in the activation of HGFtranscription and protein expression in breast carcinoma cells. This process may be important in overriding the strong repression ofHGF expression in nonmalignant epithelium, and thereby promote tumorigenesis. Scatter factor, also known as hepatocyte growth factor (HGF),1 is a multifunctional cytokine. Through binding to its receptor (Met), HGF can induce cell survival (1Qiao H. Saulnier R. Patrzykat A. Rahimi N. Raptis L. Rossiter J.P. Tremblay E. Elliott B.E. Cell Growth Differ. 2000; 11: 123-133PubMed Google Scholar), growth (2Rubin J.S. Chan A.M. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirschfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (470) Google Scholar), differentiation (3Montesano R. Matsumoto K. Nakamura T. Orci L. Cell. 1991; 67: 901-908Abstract Full Text PDF PubMed Scopus (1085) Google Scholar), and motility (4Gherardi E. Gray J. Stoker M. Perryman M. Furlong R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5844-5848Crossref PubMed Scopus (358) Google Scholar). It has been shown that both HGF and Met are essential for embryo development. Disruption of HGF expression in mice results in lethality in early development (5Schmidt C. Bladt F. Goedecke S. Brinkmann V. Zschiesche W. Sharpe M. Gherardi E. Birchmeier C. Nature. 1995; 373: 699-702Crossref PubMed Scopus (1223) Google Scholar), while deletion of Met causes underdevelopment of limb buds (6Bladt F. Riethmacher D. Isenmann S. Aguzzi A. Birchmeier C. Nature. 1995; 376: 768-771Crossref PubMed Scopus (1091) Google Scholar). During development of the mammary gland, HGF is expressed by stromal cells, whereas epithelial cells express Met, but not HGF (7Andermarcher E. Surani M.A. Gherardi E. Dev. Genet. 1996; 18: 254-266Crossref PubMed Scopus (104) Google Scholar). Paracrine stimulation of normal breast epithelium with HGF, in cooperation with other growth factors (e.g. neuregulin), promotes branching morphogenesis (8Yang Y.M. Spitzer E. Meyer D. Sachs M. Niemann C. Hartmann G. Weidner K.M. Birchmeier C. Birchmeier W. J. Cell Biol. 1995; 131: 215-226Crossref PubMed Scopus (279) Google Scholar). The tissue-specific suppression of HGF expression in normal epithelial cells provides a tightly controlled regulation of mammary ductal morphogenesis (9Liu Y. Beedle A.B. Lin L. Bell A.W. Zarnegar R. Mol. Cell. Biol. 1994; 14: 7046-7058Crossref PubMed Scopus (28) Google Scholar). In contrast to normal breast epithelium, HGF and Met are frequently overexpressed in breast carcinomas (10Ghoussoub R.A.D. Dillon D.A. D'Aquila T. Rimm B.E. Fearon E.R. Rimm D.L. Cancer. 1998; 82: 1513-1520Crossref PubMed Scopus (174) Google Scholar, 11Yamashita J. Ogawa M. Yamashita S. Nomura K. Kuramoto M. Saishoji T. Shin S. Cancer Res. 1994; 54: 1630-1633PubMed Google Scholar, 12Tuck A.B. Park M. Sterns E.E. Boag A. Elliott B.E. Am. J. Pathol. 1996; 148: 225-232PubMed Google Scholar) as well as many other cancer types (10Ghoussoub R.A.D. Dillon D.A. D'Aquila T. Rimm B.E. Fearon E.R. Rimm D.L. Cancer. 1998; 82: 1513-1520Crossref PubMed Scopus (174) Google Scholar, 11Yamashita J. Ogawa M. Yamashita S. Nomura K. Kuramoto M. Saishoji T. Shin S. Cancer Res. 1994; 54: 1630-1633PubMed Google Scholar, 13Toi M. Taniguchi T. Ueno T. Asano M. Funata N. Sekiguchi K. Iwanari H. Tominaga T. Clin. Cancer Res. 1998; 4: 659-664PubMed Google Scholar, 14Di Renzo M.F. Poulsom R. Olivero M. Comoglio M. Lemoine N.R. Cancer Res. 1995; 55: 1129-1138PubMed Google Scholar). This high level of HGF and Met expression has been identified as a possible independent predictor of poor survival in breast cancer patients (11Yamashita J. Ogawa M. Yamashita S. Nomura K. Kuramoto M. Saishoji T. Shin S. Cancer Res. 1994; 54: 1630-1633PubMed Google Scholar). Our laboratory has previously shown that invasive human carcinoma cells coexpress HGF and Met, particularly at the migrating tumor front (12Tuck A.B. Park M. Sterns E.E. Boag A. Elliott B.E. Am. J. Pathol. 1996; 148: 225-232PubMed Google Scholar). We have also found that breast carcinoma cell lines frequently express HGF and Met, whereas most nonmalignant epithelial cell lines express Met, but not HGF. 2W. Hung, J. Gin, and B. Elliott, unpublished results.2W. Hung, J. Gin, and B. Elliott, unpublished results. Furthermore, overexpression of HGF or a constitutively active mutant form of Met (Tpr-Met) in transgenic mice (15Liang T.J. Reid A.E. Xavier R. Cardiff R.D. Wang T.C. J. Clin. Invest. 1996; 97: 2872-2877Crossref PubMed Scopus (88) Google Scholar, 16Takayama H. LaRochelle W.J. Sharp R. Otsuka T. Kriebel P. Anver M. Aaronson S.A. Merlino G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 701-706Crossref PubMed Scopus (374) Google Scholar) or in transformed cell lines (17Fixman E.D. Holgado-Madruga M. Nguyen L. Kamikura D.M. Fournier T.M. Wong A.J. Park M. J. Biol. Chem. 1997; 272: 20167-20172Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,18Jeffers M. Rao M.S. Rulong S. Reddy J.K. Subbarao V. Hudson E. Vande Woude G.F. Resau J.H. Cell Growth Differ. 1996; 7: 1805-1813PubMed Google Scholar) promotes tumorigenesis and metastasis. Together, these results suggest that establishment of an autocrine HGF loop and sustained activation of the Met signal transduction pathway in carcinoma cells may promote tumor progression. However, the mechanisms leading to aberrant expression of HGF in carcinoma cells are not known. A number of signaling molecules, such as c-Src (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), Grb2/Ras (17Fixman E.D. Holgado-Madruga M. Nguyen L. Kamikura D.M. Fournier T.M. Wong A.J. Park M. J. Biol. Chem. 1997; 272: 20167-20172Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and phosphatidylinositol 3-kinase (1Qiao H. Saulnier R. Patrzykat A. Rahimi N. Raptis L. Rossiter J.P. Tremblay E. Elliott B.E. Cell Growth Differ. 2000; 11: 123-133PubMed Google Scholar), have been shown to be part of the HGF/Met signaling pathway. Activation of Met through binding of HGF causes autophosphorylation of two specific tyrosine residues in the cytoplasmic tail of the receptor tyrosine kinase (20Fixman E. Fournier T. Kamikura D. Naujokas M. Park M. J. Biol. Chem. 1996; 271: 13116-13122Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These phosphorylated tyrosine residues act as multifunctional docking sites that bind the SH2 domain of specific cytoplasmic signaling molecules and causes their activation. The c-Src nonreceptor tyrosine kinase is expressed in many cell types, and its activity is increased in response to HGF and binding to Met (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Increased activation of the tyrosine kinase c-Src occurs in many human cancer cells, and c-Src plays a critical role in breast cancer. Overexpression of an activated form of c-Src in transgenic mice induces mammary hyperplasia (21Webster M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7849-7853Crossref PubMed Scopus (71) Google Scholar). Furthermore, c-Src kinase is required in polyoma middle T-induced mammary tumorigenesis in transgenic mice (22Guy C.T. Muthuswamy S.K. Cardiff R.D. Soriano P. Muller W.J. Genes Dev. 1994; 8: 23-32Crossref PubMed Scopus (182) Google Scholar). We have shown previously that c-Src kinase is constitutively activated in a mouse breast carcinoma cell line, SP1, which expresses both HGF and tyrosine-phosphorylated Met and which exhibits spontaneous invasion through matrigel (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B.E. Cell Growth Differ. 1996; 7: 263-270PubMed Google Scholar,24Rahimi N. Tremblay E. Elliott B.E. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Furthermore, c-Src kinase activity is required for HGF-dependent cell motility and anchorage-independent growth of SP1 cells (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Collectively, these findings indicate that c-Src kinase is an important requirement, but is not sufficient, for mammary tumorigenesis. Activation of c-Src kinase can lead to increased expression of many genes, including growth factors such as vascular endothelial growth factor (25Mukhopadhyay D. Tsiokas L. Sukhatme V.P. Cancer Res. 1995; 55: 6161-6165PubMed Google Scholar, 26Mukhopadhyay D. Tsiokas L. Zhou X.M. Foster D. Brugge J.S. Sukhatme V.P. Nature. 1995; 375: 577-581Crossref PubMed Scopus (540) Google Scholar) and parathyroid hormone-related peptide (27Karaplis A.C. Lim S.K. Baba H. Arnold A. Kronenberg H.M. J. Biol. Chem. 1995; 270: 1629-1635Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We therefore hypothesized that elevated c-Src activity can promote increased HGF expression and the establishment of an HGF autocrine loop in SP1 cells. We observed that the c-Src tyrosine kinase inhibitor PP2 causes a 2-fold reduction in HGF transcription in SP1 cells. In addition, expression of a dominant negative mutant of c-Src (SRC-RF) in SP1 cells leads to similar levels of reduction in HGF mRNA and functional protein. Using deletion mutants of the HGFpromoter, we have located a region (between −254 and −70) of theHGF promoter responsive to increased c-Src kinase activity in SP1 cells. This region contains two putative consensus binding sites for Stat3. Stat3 is a transcription factor originally described as the target of interferon receptors (28Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Crossref PubMed Scopus (271) Google Scholar), but recent reports have indicated that Stat3 can be activated by c-Src kinase via platelet-derived growth factor (29Wang Y. Wharton W. Garcia R. Kraker A.J. Jove R. Pledger W. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (100) Google Scholar) and HGF receptors (30Boccaccio C. Ando M. Tamagnome L. Bardelli A. Michieli P. Battistini C. Comoglio P. Nature. 1998; 391: 285-288Crossref PubMed Scopus (452) Google Scholar), and is important in mammary differentiation (30Boccaccio C. Ando M. Tamagnome L. Bardelli A. Michieli P. Battistini C. Comoglio P. Nature. 1998; 391: 285-288Crossref PubMed Scopus (452) Google Scholar). We therefore examined the role of Stat3 in c-Src-dependent regulation of HGF transcription. The results indicate that while expression of Stat3 alone increasedHGF promoter activity, simultaneous expression of Stat3 and activated c-Src led to strong cooperative activation of HGFtranscription in both nonmalignant epithelial and carcinoma cells. Expression of mutant c-Src kinases in breast carcinoma cells altered both the tyrosine phosphorylation status and DNA binding activity of Stat3. While activated c-Src induced Stat3 tyrosine phosphorylation and DNA binding activity, a dominant negative mutant of c-Src reduced tyrosine phosphorylation and DNA binding. Together these data suggest that c-Src kinase and Stat3 act cooperatively in the activation of HGF expression in breast carcinoma cells, and may be important in overriding the strong repression of HGF expression in nonmalignant epithelial cells. Rabbit anti-c-Src IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody EC10 against chicken c-Src was a gift from Dr. S. Parsons. Rabbit anti-sheep IgG conjugated with horseradish peroxidase was from Jackson ImmunoResearch Laboratories (West Grove, PA). Sheep anti-HGF IgG was a gift from Genentech (San Francisco, CA). Rabbit anti-HGF antibody was generated against recombinant glutathioneS-transferase-HGF-(1–120) protein in our laboratory at Queen's University, this antibody recognizes only the N-terminal portion of HGF (data not shown). Anti-Stat1, -Stat3, -Stat5A, and -Stat5B and anti-phospho-Stat3 (Y705) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). c-Src family kinase inhibitor PP2 was obtained from Calbiochem (La Jolla, CA). c-Src expression plasmids were constructed by subcloning activated (Y527F) and dominant negative (K295R,Y527F) chicken c-src cDNAs (gift from Drs. J. Brugge and D. Shalloway) into the EcoRI site of DNA polymerase I (Klenow fragment)-treated pBabePuro plasmid to generate pBabe Y527F and pBabe Src-RF. A reporter construct containing the full-length HGF promoter region fused to luciferase (2.7 HGF-luc) was constructed by ligating theHindIII/XbaI fragment (treated with DNA polymerase I (Klenow fragment)) of 2.8 HGF-CAT (gift from Dr. R. Zarnegar) into the HindIII site of pGL2-Basic (Promega), also treated with DNA polymerase I (Klenow fragment). Further deletions were constructed by cutting 2.7 HGF-luc with SmaI,SacI, and BglII, followed by re-ligation to generate 0.5 HGF-luc, 0.3 HGF-luc, and 0.1 HGF-luc, respectively. The 1.2 HGF-luc was constructed by ligating the 1.4-kb SalI fragment from 2.7 HGF-luc into the XhoI site of pGL2-Basic. An internal deletion mutant 0.5Δ HGF-luc was constructed by digestion of 0.5 HGF-luc with PvuII/BglII and treatment with DNA polymerase I (Klenow fragment) before re-ligation. The Δ1 HGF-luc was constructed by ligating the SmaI fragment of 2.7 HGF-luc into the same site of 0.5Δ HGF-luc. The Δ2 HGF-luc was constructed by ligating the SmaI fragment of 2.7 HGF-luc into 0.8 HGF-luc. The ΔΔ HGF-luc was made by ligating theSmaI fragment of Δ2 HGF-luc into the same site of 0.5Δ HGF-luc. For normalization of transfection efficiency of each sample, pSG5βgal (a gift from Dr. M. Petkovich) or pCHCβgal (a gift from Dr. F. Kern) (31McLeskey S.W. Kurebayashi J. Honig S.F. Zwiebel J. Lippman M.E. Dickson R.B. Kern F.G. Cancer Res. 1993; 53: 2168-2177PubMed Google Scholar), which expresses β-galactosidase under the control of SV40 and cytomegalovirus promoters, respectively, was used. The SP1 tumor cell line is derived from a spontaneous poorly metastatic murine mammary intraductal adenocarcinoma, and expresses both HGF and Met. The characterization of the SP1 cell line has been described previously (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B.E. Cell Growth Differ. 1996; 7: 263-270PubMed Google Scholar, 24Rahimi N. Tremblay E. Elliott B.E. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Maintenance medium for SP1 cells was RPMI 1640 supplemented with 7% fetal bovine serum. HC11 is a mammary epithelial cell line (32Doppler W. Groner B. Ball R.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 104-108Crossref PubMed Scopus (217) Google Scholar) and was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, insulin (5 μg/ml), and epidermal growth factor (10 ng/ml). All transfections were carried out with LipofectAMINE Plus reagent (Canadian Life Technology, Burlington, ON, Canada) according to manufacturer's instructions. Cells (15,000) were seeded in a 24-well plate and transfected with 0.4 μg of reporter plasmid, 0.1 μg of pSG5βgal, and up to 0.4 μg of expression plasmids (such as c-Src) as indicated. After 48 h, transfected cells were harvested and lysed. One-fifth of the cell lysate was used to assay for β-galactosidase activity, an equal amount of lysate was used for a luciferase assay using PharMingen Luciferase Substrates (BD PharMingen, Mississauga, ON). Luciferase activity was measured using a luminometer with wavelength at 562 nm. Luciferase activity of each sample was normalized to the corresponding β-galactosidase activity. For immunoprecipitation and in vitro c-Src kinase assays, 2.5 × 105 cells were seeded in a 100-mm tissue culture plate and transfected with 4 μg of reporter plasmid, 1 μg of pSG5-β-galactosidase, and up to 4 μg of expression plasmids as indicated. One-tenth of the cells was used for a luciferase assay, and the remaining cells were lysed and used for immunoprecipitation. To obtain stably transfected cells, SP1 cells were plated at 70% confluence in 60-mm plates and transfected with 2 μg of plasmids expressing various mutants of c-Src. Puromycin (2 μg/ml, Sigma, Oakville, ON) was added to cells 24 h following transfection, and was maintained until all cells in the mock transfection were killed. Puromycin-resistant cells were then collected and used as pooled cell lines. Expression and activity of c-Src mutants in transfected cells were checked using Western blotting analysis and a c-Src kinase assay. Total c-Src protein was immunoprecipitated with an excess amount of anti-c-Src (pan) antibody to maximize the amount of antibody-protein complex formed. We have previously found that these c-Src mutants are quite effective, and that relatively small levels of expression can result in significant phenotypes (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Cells grown to 80% confluence on a 100-mm dish were washed and lysed with TriZol reagent (Canadian Life Technology). Phase separation was achieved by addition of chloroform and centrifugation at top speed in a microcentrifuge for 10 min. Aqueous phase containing total RNA was removed to a new tube and precipitated with an equal volume of isopropyl alcohol for 10 min at room temperature. The RNA pellet was recovered by centrifugation and washed with 70% ethanol. After brief drying, the RNA pellet was resuspended in diethyl pyrocarbonate-treated water. RNA concentration was determined by spectrophotometry. An aliquot (1 μg) of total RNA was used for reverse transcription with avian myeloblastosis reverse transcriptase at 42 °C for 15 min. One-tenth of the reaction was used in PCR analysis with end-labeled oligonucleotides specific forHGF (5′-TGTCGCCATCCCCTATGCAG-3′ and 5′-GGAGTCACAAGTCTTCAACT-3′) and β-glucuronidase (GUSB) sequences, as previously described (33Ivanchuk S.M. Myers S.M. Mulligan L.M. Oncogene. 1998; 16: 991-996Crossref PubMed Scopus (31) Google Scholar). The PCR reaction conditions were 2 min at 95 °C, followed by 25 cycles of 1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C, and a final cycle of 10 min at 72 °C. The reaction was then analyzed on a 2% agarose gel by electrophoresis. The bands corresponding to theHGF and GUSB products were excised and the amount of radioactivity was determined by scintillation counting. Conditioned media were collected and HGF was partially purified using copper (II) affinity column chromatography, as described previously (34Rahimi N. Etchells S. Elliott B. Protein Expression Purif. 1996; 7: 329-333Crossref PubMed Scopus (9) Google Scholar). Cells were grown to 80% confluence. The cell monolayer was washed with fresh Dulbecco's modified Eagle's medium and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. Conditioned media were collected, and cell debris was removed by centrifugation. Conditioned medium (10 ml) from each cell line was then loaded onto a copper (II) affinity column. The copper (II) affinity column was prepared by chelating Cu2+ ions on a 1-ml HiTrap Chelating column (Amersham Pharmacia Biotech, Baie d'Urfe, PQ), and equilibrated with equilibration buffer (20 mm sodium phosphate, pH 7.2, 1 m NaCl, 1 mm imidazole). The conditioned medium was recycled through the column 5 times to ensure binding of all HGF proteins, and the column was washed thoroughly with 15 volumes of equilibration buffer. HGF protein was eluted from the column with equilibration buffer containing 80 mm imidazole at a flow rate of 1 ml/min. Fractions of 1 ml each were collected; previous experiments have determined that essentially all HGF was eluted in fraction 2 (Ref. 34Rahimi N. Etchells S. Elliott B. Protein Expression Purif. 1996; 7: 329-333Crossref PubMed Scopus (9) Google Scholar and data not shown). The fraction containing HGF was concentrated by centrifugation with Microcon centrifugal filter devices (Millipore Corp., Bedford, MA) with a 10-kDa molecular mass cut off. The samples were analyzed on a denaturing 10% SDS-PAGE gel, followed by Western blotting with anti-HGF antibody. Cells were grown to confluence and treated as indicated. After three washes with cold phosphate-buffered saline, cells were lysed in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 1 mmNa3VO4, 50 mm NaF, 2 mmEGTA, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride. Cell debris was removed by centrifugation and protein concentrations were determined by a bicinchoninic acid protein assay (Pierce, Rockford, IL). For immunoprecipitation, equal amounts of lysate were incubated with the indicated antibodies at 4 °C for 2 h or overnight. Immunoprecipitates were collected on protein A-Sepharose (Amersham Pharmacia Biotech), washed three times with lysis buffer, separated by SDS-PAGE gel, and transferred to a nitrocellulose membrane. Western blotting analysis was performed as described previously (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In vitro c-Src kinase assays were performed as described previously (19Rahimi N. Hung W. Saulnier R. Tremblay E. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Briefly, each lysate was immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above. One-half of each immunoprecipitate was subject to SDS-PAGE under nondenaturing conditions and Western blot analysis to confirm the amount of c-Src protein present. The other half of each immunoprecipitate was assayed for c-Src kinase activity by incubating with 10 μl of reaction buffer (20 mm PIPES, pH 7.0, 10 mm MnCl2, 10 μm Na3VO4), 1.4 μg of freshly prepared acid-denatured enolase (Sigma), and 10 μCi of [γ-32P]ATP. After a 10-min incubation at 30 °C, reactions were terminated by the addition of 2 × SDS sample buffer, and samples were subjected to 8% SDS-PAGE. Serine and threonine phosphorylations were hydrolyzed by incubating the acrylamide gel in 1 m KOH at 45 °C for 30 min, followed by fixing in 45% MeOH and 10% acetic acid for 30 min at room temperature. The gel was dried under vacuum. Autoradiograms were produced and analyzed with a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Oligonucleotides used for electrophoretic mobility shift assay binding were Stat3-110F (5′-GGGCTGTTGTTAAACAGT-3′), Stat3-110R (5′-AGAACTGTTTAACAACAG-3′), Stat3-149F (5′-GGGGTTGAGGAAAGGAAG-3′), and Stat3-149R (5′-CCCCTTCCTTTCCTCAAC-3′). Complementary oligonucleotides were annealed by boiling equal molar amounts of each oligonucleotide for 10 min and then cooling slowly to room temperature. The annealed oligonucleotides (20 pmol) were labeled by a filling-in reaction with Klenow enzyme and [α-32P]dCTP. Nuclear extracts were prepared as described previously (35Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2210) Google Scholar). Briefly, 107 cells were washed once with phosphate-buffered saline before resuspension in cold buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm sodium orthovanadate). Cells were allowed to swell on ice for 10 min before lysis by brief vortexing. Nuclei were pelleted and resuspended in buffer C (20 mm HEPES, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm dithiothreitol, 0.2 mmphenylmethylsulfonyl fluoride, 0.5 mm sodium orthovanadate). High salt extraction was performed by incubation on ice for 30 min in buffer C and centrifugation at 4 °C. The protein content of the supernatant (nuclear extract) was determined using a Bradford protein assay (Bio-Rad, Mississauga, ON). Electrophoretic mobility shift assays were performed as described by Mohan et al. (36Mohan W.S. Chen Z.Q. Zhang X. Khalili K. Honjo T. Deeley R.G. Tam S.P. J. Lipid Res. 1998; 39: 255-267Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly the binding reaction was performed by incubating 5 μg of nuclear extracts with 0.1 pmol of32P-labeled oligonucleotide probe in the presence of binding buffer (10 mm HEPES, pH 7.9, 60 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol), 9% glycerol, and 4 μg of poly(dI-dC) (Amersham Pharmacia Biotech). Binding was allowed to proceed at room temperature for 10 min before analysis on 5% nondenaturing PAGE gel in Tris glycine buffer (40 mm Tris-HCl, pH 8.4, 266 mm glycine). When unlabeled oligonucleotides were added, 10-fold molar excess was included in the binding reaction. For supershifting experiments, nuclear extracts were incubated with 2 μg of the indicated antibody at room temperature for 20 min prior to the binding reaction. After electrophoresis, the gel was fixed in 7% acetic acid, 40% methanol for 30 min, and dried under vacuum. The gel was then exposed to a PhosphorImager screen, and analyzed using a Storm PhosphorImager. To study the regulation of HGF expression in breast carcinoma cells, we used the mouse mammary carcinoma cell line SP1, which coexpresses HGF and tyrosine-phosphorylated Met (23Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B.E. Cell Growth Differ. 1996; 7: 263-270PubMed Google Scholar). Semi-quantitative RT-PCR was performed to determine the levels of HGF mRNA in SP1 cells. We first examined the dose-dependent effect of an inhibitor of c-Src family kinases, PP2 (37Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). Total RNA was isolated from SP1 cells treated with different concentrations of PP2 and used for cDNA synthesis by reverse transcription. Relative HGF mRNA levels were determined" @default.
• W2020140319 created "2016-06-24" @default.
• W2020140319 creator A5078874538 @default.
• W2020140319 creator A5086392967 @default.
• W2020140319 date "2001-04-01" @default.
• W2020140319 modified "2023-10-17" @default.
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