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• W2016156871 abstract "Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situhybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo. Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situhybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo. Pancreatic ductal adenocarcinoma is the fifth leading cause of cancer death in the Western world with an overall 5-year survival rate of less than 1% and a median survival after diagnosis of 4 months.1Silverberg E Lubera J Cancer statistics.Cancer J Clin. 1989; 3: 3-39Google Scholar, 2Gudjonsson B Cancer of the pancreas: 50 years of surgery.Cancer. 1987; 60: 2284-2303Crossref PubMed Scopus (619) Google Scholar Histologically, the cancer cells exhibit well to poorly differentiated ductal-like structures, often surrounded by an extensive desmoplastic reaction and infiltration with inflammatory cells.3Reyman TA The Pancreas.in: Toledo-Pereyra LH Principles of Medical and Surgical Practice. Wiley, New York1985: 87-89Google Scholar The adjacent pancreatic parenchyma harbors regions of acinar cell degeneration and ductal cell proliferation.4Klöppel G Pancreatic Pathology.in: Klöppel G Heitz PU Churchill Livingstone, New York1984: 93-95Google Scholar A high percentage of these cancers overexpress a number of growth factors and their receptors, including the epidermal growth factor (EGF) receptor, EGF, transforming growth factor (TGF)-α, CRIPTO, TGF-β1, basic fibroblast growth factor (bFGF), acidic FGF (aFGF), and FGF-5.5Korc M Chandrasekar B Yamanaka Y Friess H Büchler M Beger HG Overexpression of the epidermal growth factor receptor in human pancreatic cancer is associated with concomitant increases in the levels of epidermal growth factor and transforming growth factor α.J Clin Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (492) Google Scholar, 6Friess H Yamanaka Y Büchler M Kobrin M Tahara E Korc M Cripto, a member of the epidermal growth factor family, is over-expressed in human pancreatic cancer, and chronic pancreatitis.Int J Cancer. 1994; 56: 668-674Crossref PubMed Scopus (100) Google Scholar, 7Yamanaka Y Friess H Büchler M Beger HG Uchida E Onda M Kobrin MS Korc M Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.Cancer Res. 1993; 53: 5289-5296PubMed Google Scholar, 8Friess H Yamanaka Y Büchler M Beger HG Do DA Kobrin MS Korc M Increased expression of acidic and basic fibroblast growth factors in chronic pancreatitis.Am J Pathol. 1994; 144: 117-128PubMed Google Scholar, 9Friess H Yamanaka Y Büchler M Ebert M Beger HG Gold LI Korc M Enhanced expression of transforming growth factor β isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology. 1993; 105: 1846-1856Abstract PubMed Google Scholar, 10Kornmann M Ishiwata T Beger HG Korc M Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.Oncogene. 1997; 15: 1417-1424Crossref PubMed Scopus (98) Google Scholar The overexpression of these mitogenic growth factors may contribute to the biological aggressiveness of pancreatic cancers and to the formation of the abundant stroma that is characteristic of this malignancy.7Yamanaka Y Friess H Büchler M Beger HG Uchida E Onda M Kobrin MS Korc M Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.Cancer Res. 1993; 53: 5289-5296PubMed Google Scholar, 9Friess H Yamanaka Y Büchler M Ebert M Beger HG Gold LI Korc M Enhanced expression of transforming growth factor β isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology. 1993; 105: 1846-1856Abstract PubMed Google ScholarKeratinocyte growth factor (KGF) is a member of the FGF group of heparin-binding polypeptides that was originally isolated from human embryonic lung fibroblasts.11Finch PW Rubin JS Miki T Ron D Aaronson SA Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755Crossref PubMed Scopus (812) Google Scholar, 12Rubin J Osada H Finch PW Taylor WG Rudikoff SA Aaronson SA Purification and characterization of a newly identified growth factor specific for epithelial cells.Proc Natl Acad Sci USA. 1989; 86: 802-806Crossref PubMed Scopus (735) Google Scholar It shares 30 to 70% amino acid sequence homology with other FGFs. In addition to KGF, which is also known as FGF-7, this family includes aFGF, or FGF-1; bFGF, or FGF-2; int-2 (FGF-3); hst/K-FGF (FGF-4); FGF-5; FGF-6; androgen-induced growth factor (AIGF, or FGF-8); glia activating factor (GAF, or FGF-9); FGF-10; and FGF-like molecules termed FGF-11–14.11Finch PW Rubin JS Miki T Ron D Aaronson SA Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755Crossref PubMed Scopus (812) Google Scholar, 12Rubin J Osada H Finch PW Taylor WG Rudikoff SA Aaronson SA Purification and characterization of a newly identified growth factor specific for epithelial cells.Proc Natl Acad Sci USA. 1989; 86: 802-806Crossref PubMed Scopus (735) Google Scholar, 13Yamasaki M Miyake A Tagashira S Itoh N Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family.J Biol Chem. 1996; 271: 15918-15921Crossref PubMed Scopus (263) Google Scholar, 14Smallwood PM Munoz-Sanjuan I Tong P Macke JP Hendry SHC Gilbert DJ Copeland NG Jenkins NA Nathans J Fibroblast growth factor (FGF) homologous factors.Proc Natl Acad Sci USA. 1996; 93: 9850-9857Crossref PubMed Scopus (329) Google Scholar KGF actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR).15Coulier F Pontarotti P Roubin R Hartung H Goldfarb M Birnbaum D Of worms and men: an evoluationary perspective on the fibroblast growth factor (FGF) and FGF receptor families.J Mol Evol. 1997; 4: 43-57Crossref Scopus (184) Google Scholar This receptor possesses intrinsic tyrosine kinase activity and binds KGF and aFGF with high affinity but does not bind bFGF.15Coulier F Pontarotti P Roubin R Hartung H Goldfarb M Birnbaum D Of worms and men: an evoluationary perspective on the fibroblast growth factor (FGF) and FGF receptor families.J Mol Evol. 1997; 4: 43-57Crossref Scopus (184) Google Scholar The extracellular domain of KGFR consists of two or three immunoglobulin-like (Ig-like) regions, whereas its intracellular domain contains a tyrosine kinase region that is interrupted by a nonkinase intervening sequence.16Miki T Bottaro DP Fleming TP Smith CL Bergess WH Chan AM Aaronson SA Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.Proc Natl Acad Sci USA. 1992; 89: 246-250Crossref PubMed Scopus (652) Google Scholar KGFR is encoded by the FGF receptor type II (FGFR-2) gene.16Miki T Bottaro DP Fleming TP Smith CL Bergess WH Chan AM Aaronson SA Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.Proc Natl Acad Sci USA. 1992; 89: 246-250Crossref PubMed Scopus (652) Google Scholar Because FGFR-2 and KGFR derive from the same gene, the two receptors are homologous in their intracellular domains and most of their extracellular domains. However, they differ from each other in the carboxyl-terminal half of the third Ig-like region of the extracellular domain, as a consequence of alternative mRNA splicing.16Miki T Bottaro DP Fleming TP Smith CL Bergess WH Chan AM Aaronson SA Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.Proc Natl Acad Sci USA. 1992; 89: 246-250Crossref PubMed Scopus (652) Google ScholarKGF mRNA levels are elevated in human pancreatic cancers.17Siddiqi I Funatomi H Kobrin MS Friess H Büchler MW Korc M Increased expression of keratinocyte growth factor in human pancreatic cancer.Biochem Biophys Res Commun. 1995; 215: 309-315Crossref PubMed Scopus (59) Google Scholar It is not known, however, whether the cancer cells within the pancreatic tumor mass express KGF, KGFR, or FGFR-2. Therefore, in the present study, we examined the expression of KGF, KGFR, and FGFR-2 in the normal human pancreas and in human pancreatic cancers. We now report that KGF, KGFR, and FGFR-2 are overexpressed in pancreatic cancer and that this overexpression occurs to a variable degree in the cancer cells and in the adjoining acinar, ductal, and islet cells.Materials and MethodsMaterialsThe following were purchased: pGEM3Zf and pGEM7Zf vectors from Promega Biotechnology (Madison, WI); Genius 3 (nonradioactive nucleic acid detection kit), Genius 4 (nonradioactive RNA labeling kit), and proteinase K from Boehringer Mannheim (Indianapolis, IN); yeast tRNA from GIBCO BRL (Gaithersburg, MD); RPA II kit for ribonuclease protection assays from Ambion (Austin, TX); aqueous mounting medium from Dako Corp. (Carpinteria, CA); Tween-20 from Bio-Rad Laboratories (Hercules, CA); glycine and formamide from Fisher Scientific (Fair Lawn, NJ); goat anti-human KGF (FGF-7, N-14) and rabbit anti-human FGFR-2 (Bek, C-17) polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); Immobilon-P nitrocellulose membranes from Millipore Corp. (Bedford, MA); enhanced chemiluminescence (ECL) substrate from Pierce (Rockford, IL); HistoMark Biotin/Streptavidin-peroxidase kit and biotinylated goat anti-guinea pig IgG secondary antibodies from Kirkegaard & Perry Laboratories (Gaithersburg, MD); Vectastain Universal ABC Elite Peroxidase kit from Vector Laboratories (Burlingame, CA); RPMI medium, fetal bovine serum (FBS), penicillin G, and streptomycin from Irvine Scientific (Irvine, CA). All other chemicals and reagents were purchased from Sigma Chemical Corp. (St. Louis, MO). In addition, a highly specific anti-human FGFR-2 monoclonal antibody was a gift from Prizm Pharmaceuticals (San Diego, CA), and T3M4 human pancreatic cancer cells were a gift from Dr. R. S. Metzgar, Duke University (Durham, NC).Tissue SamplesPancreatic carcinoma samples (four male, six female; mean age, 55.7 years; range, 32 to 68 years) were obtained from patients undergoing surgery for pancreatic cancer. Normal pancreatic tissues (four male, one female; mean age, 41.8 years; range, 18 to 55 years) were obtained from organ donors through an organ donor program. Tissues were fixed in Bouin's solution or 10% paraformaldehyde solution (PFA) for 18 to 20 hours and embedded in paraffin. All studies were approved by the Human Ethics Committees of the University of California, Irvine, and the University of Bern, Switzerland.Probe PreparationA 297-bp BamHI-HindIII cDNA fragment, corresponding to nucleotides 461 to 764 of the human KGF cDNA sequence11Finch PW Rubin JS Miki T Ron D Aaronson SA Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755Crossref PubMed Scopus (812) Google Scholar was generated by polymerase chain reaction (PCR) amplification of single-stranded cDNA that was reverse transcribed (RT) from human placental RNA, as previously described.18Ebert M Yokoyama M Kobrin MS Friess H Lopez ME Büchler MW Johnson GR Korc M Induction and expression of amphiregulin in human pancreatic cancer.Cancer Res. 1994; 54: 3959-3962PubMed Google Scholar The primers used for KGF cDNA preparation corresponded to nucleotides 461 to 481 (5′-CTGACATGGTCCTGCCAAC-3′) and 745 to 764 (5′-GAGAAGCTTCCAACTGCCACTGTCCTG-3′) of the human KGF cDNA. A 168-bpBamHI-HindIII cDNA fragment, corresponding to nucleotides 1349 to 1516 of the human KGFR cDNA sequence16Miki T Bottaro DP Fleming TP Smith CL Bergess WH Chan AM Aaronson SA Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.Proc Natl Acad Sci USA. 1992; 89: 246-250Crossref PubMed Scopus (652) Google Scholar was similarly generated by RT-PCR. The primer pair used for KGFR cDNA preparation was derived from sequences located on either side of exon IIIb of the human KGFR cDNA, corresponding to nucleotides 1349 to 1367 (5′-GCGGATCCGTTCTCAAGCACTCGGGGA-3′) and 1498 to 1516 (5′-GCAAGCTTCCAGG-CGCTTGCTGT-3′). A cDNA encoding sequences corresponding to the human FGFR-2 receptor cDNA19Dionne CA Crumley G Bellot F Kaplow JM Searfoss G Ruta M Burgess WH Jaye M Schlessinger J Cloning and expression of two distinct high affinity receptors cross-reacting with acidic and basic fibroblast growth factor.EMBO J. 1990; 9: 2685-2692Crossref PubMed Scopus (544) Google Scholar was generated by RT-PCR from PANC-1 human pancreatic cancer cells. The primers used for FGFR-2 were derived from sequences located on either side of the human FGFR-2 cDNA that are specific for exon IIIc, corresponding to nucleotides 1103 to 1122 (5′-GCGGATCCTCAAGGTTCTCAAGGCCG-3′) and 1254 to 1273 (5′-GTAAGCTTCCAG-GCGCTGGCAGAAC-3′). The 297-bp KGF cDNA fragment and the 171-bp FGFR-2 cDNA fragment were subcloned separately into the pGEM3Zf vector, and the 168-bp KGFR cDNA was subcloned into pGEM7Zf. Authenticity of the three fragments was confirmed by sequencing. The probes were labeled with digoxigenin-UTP by SP6 or T7 RNA polymerase using the Genius 4 RNA labeling kit.In Situ HybridizationIn situ hybridization was performed as previously reported20Martinez-Montero JC Herrington CS Stickland J Sawyer H Evans M Flannery DMJ McGee JO'D Model system for optimizing mRNA non-isotopic in situ hybridization: riboprobe detection of lysozyme mRNA in archival gut biopsy specimens.J Clin Pathol. 1991; 44: 835-839Crossref PubMed Scopus (22) Google Scholar, 21Ishiwata T Bergmann U Kornmann M Lopez M Beger HG Korc M Altered expression of insulin-like growth factor II receptor in human pancreatic cancer.Pancreas. 1997; 15: 367-37320Crossref PubMed Scopus (34) Google Scholar with minor modifications. Briefly, tissue sections (4 μm thick) were placed on 3-aminopropyl-methoxysilane-coated slides, deparaffinized, and incubated at 23°C for 20 minutes with 0.2 N HCl and at 37°C for 15 minutes with 20 μg/ml proteinase K. The sections were then post-fixed for 5 minutes in phosphate-buffered saline (PBS) containing 4% paraformaldehyde, incubated briefly twice with PBS containing 2 mg/ml glycine and once in 50% (v/v) formamide/2X SSC for 1 hour before initiation of the hybridization reaction by the addition of 100 μl of hybridization buffer. The hybridization buffer contained 0.6 mol/L NaCl, 1 mmol/L EDTA, 10 mmol/L Tris/HCl (pH 7.6), 0.25% SDS, 200 μg/ml yeast tRNA, 1X Denhardt's solution, 10% dextran sulfate, 40% formamide, and 100 ng/ml of the indicated digoxigenin-labeled riboprobe. Hybridization was performed in a moist chamber for 16 hours at 42°C. The sections were then washed sequentially with 50% formamide/2X SSC for 30 minutes at 50°C, 2X SSC for 20 minutes at 50°C, and 0.2X SSC for 20 minutes at 50°C. For immunological detection, the Genius 3 nonradioactive nucleic acid detection kit was used. The sections were washed briefly with buffer 1 solution (100 mmol/L Tris/HCl and 150 mmol/L NaCl, pH 7.5) and incubated with 1% (w/v) blocking reagents in buffer 1 solution for 60 minutes at 23°C. The sections were then incubated for 30 minutes at 23°C with a 1:2000 dilution of alkaline-phosphatase-conjugated polyclonal sheep anti-digoxigenin Fab fragment containing 0.2% Tween 20. The sections were then washed twice for 15 minutes at 23°C with buffer 1 solution containing 0.2% Tween 20 and equilibrated with buffer 3 solution (100 mmol/L Tris/HCl, 100 mmol/L NaCl, 50 mmol/L MgCl2, pH 9.5) for 2 minutes. The sections were then incubated with color solution containing nitroblue tetrazolium and X-phosphate in a dark box for 2 to 3 hours. After the reaction was stopped with TE buffer (10 mmol/L Tris/HCl, 1 mmol/L EDTA, pH 8.0), the sections were mounted in aqueous mounting medium.ImmunohistochemistryA highly specific goat anti-human KGF and two different anti-human FGFR-2 antibodies were used for immunohistochemistry. The anti-KGF antibody was an affinity-purified goat polyclonal antibody raised against a peptide corresponding to amino acids 33 to 46 mapping at the amino terminus of the KGF precursor of human origin. This antibody reacts with KGF of human origin by immunoblotting and ELISA but does not react with any other member of the FGF family (Santa Cruz Biotechnology). The C-17 anti-FGFR-2 antibody from Santa Cruz was an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acids 789 to 802 mapping at the carboxy terminus of the FGFR-2 precursor of human origin. This antibody reacts principally with FGFR-2 and KGFR and may cross-react to a limited extent with FGFR-1, -3, or -4 (Santa Cruz Biotechnology). Therefore, a second anti-FGFR-2 antibody from Prizm Pharmaceuticals was also used. This mouse monoclonal antibody is directed against the acid box region (TDGAEDFVSEN) located in the extracellular domain of FGFR-2 and shared by both FGFR-2 and KGFR but not by other FGF receptors. Therefore, it is highly specific for FGFR-2 and KGFR and does not cross-react with other FGF receptors.22Larocca D Witte A Gonzalez AM Houston LL Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.Hybridoma. 1998; 17: 21-31Crossref PubMed Scopus (9) Google Scholar Its specificity has been previously demonstrated in immunoblotting studies and ELISAs.22Larocca D Witte A Gonzalez AM Houston LL Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.Hybridoma. 1998; 17: 21-31Crossref PubMed Scopus (9) Google Scholar Because both the polyclonal and monoclonal anti-FGFR-2 antibodies recognize KGFR in addition to FGFR-2, positive immunostaining obtained with either antibody was reported as reflecting KGFR/FGFR-2 immunoreactivity.Paraffin-embedded sections (4 μm) were subjected to immunostaining using the streptavidin-peroxidase technique.23Yamanaka Y Friess H Büchler M Beger HG Gold LI Korc M Synthesis and expression of transforming growth factor-β1, β2, and β3 in the endocrine and exocrine pancreas.Diabetes. 1993; 42: 746-756Crossref PubMed Scopus (0) Google Scholar, 24Naef M Ishiwata T Friess H Büchler MW Gold LI Korc M Differential localization of transforming growth factor-β isoforms in human gastric mucosa and overexpression in gastric carcinoma.Int J Cancer. 1997; 71: 131-137Crossref PubMed Scopus (56) Google Scholar Endogenous peroxidase activity was blocked by incubation for 30 minutes with 0.3% hydrogen peroxide in methanol. Tissue sections were incubated for 15 minutes (23°C) with 10% normal rabbit serum for the KGF antibody and 10% normal goat serum for the polyclonal FGFR-2 antibody and then incubated for 16 hours at 4°C with the KGF (1:500 dilution) and polyclonal FGFR-2 antibody (1:500 dilution) in PBS containing 1% bovine serum albumin (BSA). To perform immunostaining with the monoclonal anti-FGFR-2 antibody, sections were incubated for 20 minutes (23°C) with 5% normal horse serum and then incubated for 30 minutes at 23°C with the antibody (1:500 dilution) in PBS containing 1% bovine serum albumin. Bound antibodies were detected with biotinylated rabbit anti-goat IgG secondary antibodies for KGF staining, goat anti-rabbit IgG secondary antibodies for staining with the polyclonal anti-FGFR-2 antibody, and biotinylated universal antibody for staining with the monoclonal anti-FGFR-2 antibody. For insulin staining, guinea pig polyclonal anti-porcine insulin antibodies (1:3000 dilution in PBS containing 1% bovine serum albumin), cross-reactive with human insulin, and biotinylated goat anti-guinea pig IgG secondary antibodies were used after incubation with 10% normal goat serum. Sections were then treated with streptavidin-peroxidase complex, using diaminobenzidine tetrahydrochloride as the substrate, and counterstained with Mayer's hematoxylin. However, in the case of the monoclonal anti-FGFR-2 antibody, the avidin-peroxidase complex was used. Some sections were incubated with nonimmunized goat anti-IgG for KGF and rabbit anti-IgG for FGFR-2 or without primary antibodies, which did not yield positive immunoreactivity.Cell CultureT3M4 human pancreatic cancer cells were grown in RPMI medium supplemented with 10% fetal bovine serum, penicillin G (100 U/ml), and streptomycin (100 μg/ml) and maintained in monolayer culture at 37°C in humidified air with 5% CO2.Immunoblotting and Ribonuclease Protection AssayWe previously reported that T3M4 human pancreatic cancer cells express exceedingly low levels of KGFR that are detectable only after amplification by PCR.17Siddiqi I Funatomi H Kobrin MS Friess H Büchler MW Korc M Increased expression of keratinocyte growth factor in human pancreatic cancer.Biochem Biophys Res Commun. 1995; 215: 309-315Crossref PubMed Scopus (59) Google Scholar To determine whether these cells express FGFR-2, immunoblotting10Kornmann M Ishiwata T Beger HG Korc M Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.Oncogene. 1997; 15: 1417-1424Crossref PubMed Scopus (98) Google Scholar was carried out using the monoclonal anti-FGFR-2 antibody that does not cross-react with other FGF receptors.22Larocca D Witte A Gonzalez AM Houston LL Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.Hybridoma. 1998; 17: 21-31Crossref PubMed Scopus (9) Google Scholar In addition, a ribonuclease protection assay was carried out using total RNA isolated from these cells. For this purpose, RNA (10 μg/sample) was hybridized overnight (42°C) with a [α-32P]CTP-labeled FGFR-2 riboprobe (100,000 cpm/sample). Single-stranded RNA was then digested with RNAse A/T1, size-fractionated on a 6% polyacrylamide/8 mol/L urea gel, and subjected to autoradiography.10Kornmann M Ishiwata T Beger HG Korc M Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.Oncogene. 1997; 15: 1417-1424Crossref PubMed Scopus (98) Google ScholarResultsIn the normal pancreas, KGF immunoreactivity was present in a focal but intense pattern in a few islet cells (Figure 1A, arrowheads). Many ductal cells in the small ducts exhibited faint KGF immunoreactivity (Figure 1A, arrows). Occasional acinar cells and vascular smooth muscle cells (VSMCs) also exhibited faint KGF immunoreactivity (not shown). Using the polyclonal anti-FGFR-2 antibody, intense and abundant KGFR/FGFR-2 immunoreactivity was present in many islet cells (Figure 1B, arrowheads) but was faint in the ductal cells (arrows) and completely absent in the acinar cells and VSMCs. With the monoclonal anti-FGFR-2 antibody, KGFR/FGFR-2 immunoreactivity was diffuse but faint in the islet cells (Figure 2A, arrowheads), of moderate intensity in the ductal cells (Figure 2A, arrow), and absent in the acinar cells (Figure 2A). As expected, many islet cells were strongly positive for insulin (Figure 1C, arrowheads).Figure 2Immunohistochemistry of KGFR/FGFR-2 using a monoclonal antibody.A: In the normal pancreas, mild to moderate KGFR/FGFR-2 was present in the endocrine islet cells (outlined by arrowheads) and ductal cells (arrow). B: In the pancreatic cancers, abundant KGFR/FGFR-2 immunoreactivity was present in the ductal-like cancer cells. C: Moderate to strong KGFR/FGFR-2 immunoreactivity was present in the small ductal cells and endocrine islets adjacent to the cancer cells (outlined byopen arrowheads). D: Moderate to strong KGFR/FGFR-2 immunoreactivity was present in the ductal cells of the large ducts adjacent to the cancer cells. Magnification, ×600 (A), ×400 (B andD), and ×200 (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In 6 of 10 pancreatic cancer samples, KGF immunoreactivity was present in many of the cancer cells in a diffuse cytoplasmic pattern that was of moderate intensity (Figure 3A; Table 1). Moderate to strong KGF immunoreactivity was also present in the endocrine islets and in some fibroblasts and VSMCs (not shown). Using the polyclonal anti-FGFR-2 antibody, faint to moderate KGFR/FGFR-2 immunoreactivity was present in the cancer cells in 7 of the same 10 cancer samples (Figure 3B). Using the monoclonal anti-FGFR-2 antibody, strong KGFR/FGFR-2 immunoreactivity was present in the cancer cells (Figure 2B). Most KGF-positive cancers were also positive for KGFR/FGFR-2 (Table 1). Thus, all six cancer samples that were strongly positive for KGF exhibited KGFR/FGFR-2 immunoreactivity, whereas three of the four cancers that were negative for KGF were also negative for KGFR/FGFR-2.Figure 3Expression of KGF, KGFR, and KGFR/FGFR-2 in human pancreatic cancer tissues. Immunostaining revealed moderate to strong KGF (A) immunoreactivity in the cytoplasm of the cancer cells and faint to moderate KGFR/FGFR-2 immunoreactivity (B) in these cells. In situ hybridization analysis of serial sections revealed moderate KGF (C), FGFR-2 (D), and KGFR (E) mRNA signals in the cancer cells. Hybridization with the sense KGFR probe (F) did not yield any specific signals. Magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Summary of KGF and KGFR/FGFR-2 ImmunohistochemistryNumber of casesKGFKGFR/FGFR-24++++2++1−+3−−In 6 of 10 cases, the cancer cells were positive for both KGF and KGFR/FGFR-2 immunoreactivity. In 4 of 10 cases, the cancer cells were negative for KGF immunoreactivity. In 3 of 10 cases, the cancer cells were negative for KGFR/FGFR-2 immunoreactivity. Immunoreactivity was scored as follows: −, <10% of the cancer cells exhibited positive immunoreactivity and were defined as negative; +, 11 to 80% of cancer cells were positive; ++, >80% of the cancer cells were positive. Open table in a new tab Previously, we reported that cultured pancreatic cancer cell lines express variable levels of KGFR.17Siddiqi I Funatomi H Kobrin MS Friess H Büchler MW Korc M Increased expression of keratinocyte growth factor in human pancreatic cancer.Biochem Biophys Res Commun. 1995; 215: 309-315Crossref PubMed Scopus (59) Google Scholar To determine whether pancreatic cancer cell lines express FGFR-2, we next characterized FGFR-2 expression in T3M4 cells by immunoblotting and ribonuclease protection. Immunoblotting with the monoclonal anti-FGFR-2 antibody revealed a distinct band of" @default.
• W2016156871 created "2016-06-24" @default.
• W2016156871 creator A5023861100 @default.
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• W2016156871 date "1998-07-01" @default.
• W2016156871 modified "2023-09-27" @default.
• W2016156871 title "Characterization of Keratinocyte Growth Factor and Receptor Expression in Human Pancreatic Cancer" @default.
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