Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.060935
ISSN1525-2191
AutoresKazumitsu Cho, Toshiyuki Ishiwata, Eiji Uchida, Nando Nakazawa, Murray Korc, Zenya Naito, Takashi Tajiri,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoKeratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC. Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with few long-term survivors. Although the management and treatment of patients with PDAC have improved in the last few decades, the overall 5-year survival rate remains at less than 5%, underscoring the need for more effective therapeutic approaches. Clinically, this poor prognosis occurs because PDAC is often diagnosed at a late stage when the cancer is no longer resectable. Moreover, those patients that undergo resection frequently exhibit a high incidence of local recurrence, lymph node metastasis, hepatic metastasis, and peritoneal dissemination. At the molecular level, PDAC often harbors mutations in the K-ras oncogene, the p53 tumor suppressor gene, and the p16 cell cycle gene.1DiGiuseppe JA Hruban RH Pathobiology of cancer of the pancreas.Simin Surg Oncol. 1995; 11: 87-96Crossref Scopus (10) Google Scholar A high percentage of PDACs also overexpress a number of growth factors and their receptors, including the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-α, CRIPTO, transforming growth factor-β1, basic fibroblast growth factor (FGF), acidic FGF, and FGF-5.2Kornmann 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, 3Friess H Yamanaka Y Buchler M Kobrin MS 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, 4Friess H Yamanaka Y Buchler 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, 5Yamanaka Y Friess H Buchler 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, 6Friess H Yamanaka Y Buchler M Ebert M Beger HG Gold LI Korc M Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology. 1993; 105: 1846-1856Abstract PubMed Google Scholar, 7Korc M Chandrasekar B Yamanaka Y Friess H Buchier 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 alpha.J Clin Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (495) Google Scholar The multiple alterations in oncogenes and tumor suppressor genes in conjunction with the overexpression of mitogenic growth factors and their receptors may contribute to the biological aggressiveness of pancreatic cancers and to the formation of the abundant stroma that is characteristic of this malignancy.5Yamanaka Y Friess H Buchler 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, 6Friess H Yamanaka Y Buchler M Ebert M Beger HG Gold LI Korc M Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology. 1993; 105: 1846-1856Abstract PubMed Google Scholar Keratinocyte growth factor (KGF) is a member of the FGF group of heparin-binding polypeptides that was originally isolated from human embryonic lung fibroblasts.8Rubin JS Osada H Finch PW Taylor WG Rudikoff S 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 (738) Google Scholar, 9Finch 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 (818) Google Scholar KGF is synthesized by mesenchymal cells and T lymphocytes and acts predominantly on epithelial cells in a paracrine manner.10Rubin JS Bottaro DP Chedid M Miki T Ron D Cheon G Taylor WG Fortney E Sakata H Finch PW LaRochelle WJ Keratinocyte growth factor.Cell Biol Int. 1995; 19: 399-411Crossref PubMed Scopus (264) Google Scholar, 11Boismenu R Havran WL Modulation of epithelial cell growth by intraepithelial gamma delta T cells.Science. 1994; 266: 1253-1255Crossref PubMed Scopus (567) 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 acidic FGF or FGF-1, basic FGF or FGF-2, int-2 (FGF-3), hst/K-FGF (FGF-4), FGF-5, FGF-6, androgen-induced growth factor (FGF-8), glia activating factor (FGF-9), FGF-10, FGF-11 (FGF homologous factors-3), FGF-12 (FGF homologous factors-1), FGF-13 (FGF homologous factors-2), FGF-14 (FGF homologous factors-4), and FGF-16 to FGF-23.8Rubin JS Osada H Finch PW Taylor WG Rudikoff S 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 (738) Google Scholar, 9Finch 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 (818) Google Scholar, 12Itoh N Ornitz DM Evolution of the Fgf and Fgfr gene families.Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (873) Google Scholar, 13Ornitz DM Itoh N Fibroblast growth factors.Genome Biol. 2001; 2: 3005.3001-3005.3012Crossref Google Scholar KGF actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), also known as FGF receptor (FGFR) type II (FGFR-2IIIb).14Coulier F Pontarotti P Roubin R Hartung H Goldfarb M Birnbaum D Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families.J Mol Evol. 1997; 44: 43-56Crossref PubMed Scopus (185) Google Scholar This receptor possesses intrinsic tyrosine kinase activity and binds KGF and FGF-1 with high affinity but does not bind FGF-2.14Coulier F Pontarotti P Roubin R Hartung H Goldfarb M Birnbaum D Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families.J Mol Evol. 1997; 44: 43-56Crossref PubMed Scopus (185) 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.15Miki T Bottaro DP Fleming TP Smith CL Burgess 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 (659) Google Scholar KGFR is encoded by the fgfr-2 gene.15Miki T Bottaro DP Fleming TP Smith CL Burgess 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 (659) Google Scholar Because FGFR-2IIIc 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.15Miki T Bottaro DP Fleming TP Smith CL Burgess 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 (659) Google Scholar FGFR-2IIIc is mainly localized in mesenchymal cells, whereas KGFR is localized in epithelial cells. KGF is expressed in a variety of tissues including the lung, prostate, mammary gland, digestive tract, bladder, and skin and is implicated in organ development and homeostasis.10Rubin JS Bottaro DP Chedid M Miki T Ron D Cheon G Taylor WG Fortney E Sakata H Finch PW LaRochelle WJ Keratinocyte growth factor.Cell Biol Int. 1995; 19: 399-411Crossref PubMed Scopus (264) Google Scholar, 16Casey G Yamanaka Y Friess H Kobrin MS Lopez ME Buchler M Beger HG Korc M p53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis.Cancer Lett. 1993; 69: 151-160Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 17Koji T Chedid M Rubin JS Slayden OD Csaky KG Aaronson SA Brenner RM Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin.J Cell Biol. 1994; 125: 393-401Crossref PubMed Scopus (160) Google Scholar KGF expression is dramatically up-regulated in cutaneous wounds, where it can speed the repair process, and also in the hyperplastic skin disease psoriasis.18Pierce GF Yanagihara D Klopchin K Danilenko DM Hsu E Kenney WC Morris CF Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor.J Exp Med. 1994; 179: 831-840Crossref PubMed Scopus (167) Google Scholar, 19Werner S Peters KG Longaker MT Fuller-Pace F Banda MJ Williams LT Large induction of keratinocyte growth factor expression in the dermis during wound healing.Proc Natl Acad Sci USA. 1992; 89: 6896-6900Crossref PubMed Scopus (534) Google Scholar, 20Finch PW Murphy F Cardinale I Krueger JG Altered expression of keratinocyte growth factor and its receptor in psoriasis.Am J Pathol. 1997; 151: 1619-1628PubMed Google Scholar KGF also stimulates the growth of hair follicles and the thickening of the gastrointestinal tract mucosa.21Nguyen HQ Danilenko DM Bucay N DeRose ML Van GY Thomason A Simonet WS Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in polycystic kidneys and other organ malformations.Oncogene. 1996; 12: 2109-2119PubMed Google Scholar, 22Playford RJ Marchbank T Mandir N Higham A Meeran K Ghatei MA Bloom SR Goodlad RA Effects of keratinocyte growth factor (KGF) on gut growth and repair.J Pathol. 1998; 184: 316-322Crossref PubMed Scopus (43) Google Scholar Moreover, KGF-expressing transgenes exhibit pancreatic ductal hyperplasia, and KGF mRNA levels are elevated in PDAC.21Nguyen HQ Danilenko DM Bucay N DeRose ML Van GY Thomason A Simonet WS Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in polycystic kidneys and other organ malformations.Oncogene. 1996; 12: 2109-2119PubMed Google Scholar, 23Yi ES Yin S Harclerode DL Bedoya A Bikhazi NB Housley RM Aukerman SL Morris CF Pierce GF Ulich TR Keratinocyte growth factor induces pancreatic ductal epithelial proliferation.Am J Pathol. 1994; 145: 80-85PubMed Google Scholar, 24Siddiqi I Funatomi H Kobrin MS Friess H Buchler 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, 25Ishiwata T Naito Z Lu YP Kawahara K Fujii T Kawamoto Y Teduka K Sugisaki Y Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginine-induced acute pancreatitis.Exp Mol Pathol. 2002; 73: 181-190Crossref PubMed Scopus (25) Google Scholar Although we previously reported that KGFR and KGF were overexpressed in both the pancreatic cancer cells and the adjacent pancreatic parenchyma,26Ishiwata T Friess H Buchler MW Lopez ME Korc M Characterization of keratinocyte growth factor and receptor expression in human pancreatic cancer.Am J Pathol. 1998; 153: 213-222Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar the potential roles of KGFR and KGF in PDAC are still poorly understood. We now report that the coexpression of KGFR and KGF in PDAC is associated with a propensity for venous invasion, enhanced vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. The chemicals and reagents were purchased as follows: Isogen from Nippon Gene (Tokyo, Japan); a Takara RNA PCR kit (AMV) version 3.0 and pBAsi-hU6 Neo DNA vector from Takara Biotech (Tokyo, Japan); RNeasy mini kit from Qiagen GmbH (Hilden, Germany); Transcriptor First Strand cDNA Synthesis kit and LightCycler FastStart DNA Master SYBR Green I, FuGENE 6, and FuGENE HD transfection reagent from Roche Diagnostics GmbH (Mannheim, Germany); human VEGF Quantikine Colorimetric Sandwich enzyme-linked immunosorbent assay (ELISA) kit, goat polyclonal anti-FGF-7 antibodies, and recombinant human KGF (rhKGF) from R&D Systems Inc. (Westerville, OH); Immobilon P transfer membrane from Millipore (Yonezawa, Japan); M-PER Mammalian Protein Extraction reagent and Super Signal West Pico chemiluminescent substrates from Pierce (Rockford, IL); SERVA Blau G from Serva Electrophoresis GmbH (Heidelberg, Germany); Histofine Simple Stain Max PO (G) or (R) kit from Nichirei Biosciences, Inc. (Tokyo, Japan); anti-rabbit IgG-horseradish peroxidase secondary antibody and rabbit polyclonal anti-VEGF-A antibodies (A-20) from Santa Cruz Biotechnology (Santa Cruz, CA); Human Tissue Microarray 1 and Human Digestive Tissue Sets from Novagen (Darmstadt, Germany); fluorescein 5-isothiocyanate-conjugated anti-rabbit IgG and Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride from Vector Laboratories, Inc. (Burlingame, CA); silane-coated slides and a malinol mounting medium from Muto Pure Chemicals Co., Ltd. (Tokyo, Japan); and pIRES2-EGFP vector from Clontech (Palo Alto, CA). All other chemicals and reagents were purchased from Sigma Chemical Corp. (St. Louis, MO). Tissues from 53 patients with invasive PDAC were obtained for this study. These patients received treatment at Nippon Medical School Hospital (Bunkyo-ku, Tokyo, Japan) from 1995 to 2003. None of the patients received preoperative chemotherapy and radiotherapy. The patients consisted of 36 males and 17 females, whose median age was 64 years (range, 35 to 84 years). The clinicopathological stage was determined according to the TNM classification system of the International Union Against Cancer27Sobin LH Fleming ID TNM classification of malignant tumors, fifth edition (1997): Union Internationale Contre le Cancer and the American Joint Committee on Cancer.Cancer. 1997; 80: 1803-1804Crossref PubMed Scopus (987) Google Scholar and additionally characterized with the Japan Pancreas Society classification28Society JP General Rules for the Study of Pancreatic Cancer. Kanehara & Company Ltd., Tokyo2002Google Scholar (Table 1). Thirty-one patients did not receive postoperative chemotherapy, and 22 patients received adjuvant chemotherapy after surgery. Twelve patients received Uracil/Tegafur, and 10 patients received gemcitabine. The median follow-up period was 14.1 months. Paraffin-embedded specimens were prepared for immunohistochemical analysis as described previously.26Ishiwata T Friess H Buchler MW Lopez ME Korc M Characterization of keratinocyte growth factor and receptor expression in human pancreatic cancer.Am J Pathol. 1998; 153: 213-222Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar This study was performed in accordance with the principles embodied in the Declaration of Helsinki 1975, and informed consent for the usage of pancreatic tissues was obtained from each patient. Normal pancreatic tissues were obtained from Human Digestive Tissue Sets and Human Tissue Microarray 1.Table 1Correlation of Clinicopathological Features and KGFR, KGF, or Coexpression of KGFR and KGF in Pancreatic CancersKGFRKGFKGFR and KGFVariablesnn (%)Pn (%)Pn (%)PGender Male3617 (47)NS12 (33)NS9 (25)NS Female175 (29)6 (35)4 (24)Age <65248 (33)NS7 (29)NS3 (13)NS 65+2914 (48)11 (38)10 (34)UICC classification T-primary tumor T132 (67)NS1 (33)NS1 (33)NS T231 (33)1 (33)1 (33) T3157 (47)8 (53)5 (33) T43212 (36)8 (25)6 (19)N-Regional lymph nodes N01911 (58)NS8 (42)NS6 (32)NS N13411 (32)10 (29)7 (21)M-Distant metastasis M05121 (41)NS17 (33)NS12 (24)NS M121 (50)1 (50)1 (50)G-Histological grading G13114 (45)NS8 (26)NS6 (19)NS G2217 (33)9 (43)6 (29) G311 (100)1 (100)1 (100) G40000Stage I or II105 (50)NS1 (10)NS4 (40)NS III or IV4317 (40)17 (40)9 (21)Other tumor characteristics Lymphatic invasion Negative63 (50)NS2 (33)NS2 (33)NS Positive4719 (40)16 (34)11 (23) Venous invasion Negative3511 (31)0.0388 (23)0.0175 (14)0.016 Positive1811 (61)10 (56)8 (44) Nerve invasion (intrapancreatic) Negative116 (55)NS5 (45)NS3 (27)NS Positive4216 (38)13 (31)10 (24)UICC, International Union Against Cancer; NS, not significant. Open table in a new tab UICC, International Union Against Cancer; NS, not significant. PANC-1, MIA PaCa-2, KLM-1, and PK-1, -8, -9, and -59 pancreatic ductal adenocarcinoma cell lines were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan), and Capan-1 was purchased from American Type Culture Collection (Manassas, VA). The cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml kanamycin at 37°C under a humidified 5% CO2 atmosphere. Capan-1 was grown in the same medium containing 15% FBS. Total RNA was extracted from pancreatic cancer cell lines using Isogen according to the manufacturer's protocol. Then, cDNA synthesis and polymerase chain reaction (PCR) were performed using the Takara RNA PCR kit. The primer pair used for KGFR corresponded to nucleotides 1587 to 1606 (5′-CACTCGGGGATAAATAGTTC-3′) and nucleotides 1719 to 1736 (5′-CGCTTGCTGTTTTGGCAG-3′) (150 bp, accession no. NM022970). The primers used for KGF corresponded to nucleotides 765 to 784 (5′-TTGTGGCAATCAAAGGGGTG-3′) and nucleotides 905 to 927 (5′-CCTCCGTTGTGTGTCCATTTAGC-3′) of the human KGF cDNA (163 bp, accession no. NM00209). β-Actin mRNA, as the positive control, was amplified using the following primer pairs: nucleotides 331 to 353 (5′-GCACCACACCTTCTACAATGAGC-3′) and nucleotides 472 to 493 (5′-TAGCACAGCCTGGATAGCAACG-3′) (163 bp, accession no. NM001101). The authenticity of the PCR product was confirmed by the direct sequence method. Total RNA not subjected to reverse transcription was used as the negative control. Total RNA extraction from tumor cells was performed using the RNeasy Mini kit. cDNA synthesis was performed using the Transcriptor First Strand cDNA Synthesis kit following the manufacturer's protocol. Quantitative real-time PCR (Q-PCR) was performed using a LightCycler-FastStart DNA Master SYBR Green I system. The same KGFR, KGF, and β-actin primer pairs used for reverse transcription-PCR (RT-PCR) were used for real-time PCR analysis. PCR reaction mixture containing 2 μl of template cDNA, 3 mmol/L MgCl2, and 0.5 μmol/L of primers, and LightCycler-FastStart DNA Master SYBR Green I mix was applied into a capillary tube (Roche). Q-PCR was performed in a LightCycler (Roche), and the PCR products were analyzed by LightCycler Data Analysis software version 3.5 (Roche). The optimized program involved denaturation at 95°C for 10 minutes, followed by 50 cycles of amplification as follows: for KGFR, at 95°C for 10 seconds, at 60°C for 10 seconds, and at 72°C for 7 seconds; for KGF, at 95°C for 10 seconds, at 58°C for 10 seconds, and at 72°C for 8 seconds; and for β-actin, at 95°C for 10 seconds, at 64°C for 10 seconds, and at 72°C for 7 seconds. To confirm amplification specificity, PCR products were subjected to a melting-curve analysis. Results were expressed as target/β-actin as an internal standard concentration ratio. Gene expression measurements were performed in triplicate. Protein extraction was performed according to the protocol involving the use of the M-Per Mammalian Protein Extraction reagent. Briefly, the cultured pancreatic cancer cells were solubilized in M-Per reagent with Protease Inhibitor Cocktail for Mammalian Tissues. Lysates were centrifuged for 10 minutes at 13,000 rpm to pellet cell debris. The supernatants were collected, and protein concentration was measured by the Bradford method. The anti-KGFR antibody used in this study was an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acids of the human KGFR protein.25Ishiwata T Naito Z Lu YP Kawahara K Fujii T Kawamoto Y Teduka K Sugisaki Y Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginine-induced acute pancreatitis.Exp Mol Pathol. 2002; 73: 181-190Crossref PubMed Scopus (25) Google Scholar The cleared protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and the separated proteins were transferred to Immobilon P transfer membranes, which were then incubated for 16 hours at 4°C with the anti-KGFR antibody. The membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 60 minutes. After washing, the blot was visualized by enhanced chemiluminescence. The same anti-KGFR antibody used for the Western blot analysis was used for immunofluorescence staining of MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with the anti-KGFR antibody (1:100) in PBS containing 1% bovine serum albumin for 16 hours at 4°C. For negative control, MIA PaCa-2 cells were incubated with PBS containing 1% bovine serum albumin. The cells were washed with PBS and then incubated with fluorescein 5-isothiocyanate-conjugated anti-rabbit IgG. One hour after incubation, the cells were washed with PBS and then mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride. Fluorescent images were acquired using a Digital Eclipse TE 2000-E confocal laser scanning microscope (Nikon Insteck Co., Ltd., Tokyo, Japan) and a 100× immersion lens (Nikon Palm Apo VC) with blue diode and argon lasers and were analyzed using the confocal microscope and Digital Eclipse C1 control software EZ-C1 (version 2.30) (Nikon Insteck). The excitation wavelength for fluorescein 5-isothiocyanate was 488 nm, and emission was selected and recorded using a 500- to 530-nm band-pass filter. In addition, the excitation wavelength for 4′,6-diamidino-2-phenylindole dihydrochloride was 405 nm, and emission was selected and recorded using a 432- to 446-nm band-pass filter. Paraffin-embedded tissue sections (3.5 μm) were subjected to immunostaining using the Histofine Simple Stain Max PO (G) or (R) kit. After deparaffinization, endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 30 minutes, and the sections were incubated with the appropriate antibody for 16 hours at 4°C (1:1000 dilution for the anti-KGFR antibody, 1:50 dilution for the anti-KGF antibody, and 1:200 dilution for the anti-VEGF-A antibody) using PBS containing 1% bovine serum albumin. The anti-KGFR antibody used was the same antibody used in Western blot analysis and immunofluorescence staining. Bound antibodies were detected with Simple Stain Max PO (G) or (R) reagents using diaminobenzidine-tetrahydrochloride as the substrate, and the sections were counterstained with Mayer's hematoxylin. Negative control studies were performed by omitting the primary antibodies. The immunohistochemical results for KGFR, KGF, and VEGF-A were evaluated as follows: when staining was noted in the cytoplasm and/or membrane of more than 30% of the tumor cells, regardless of the intensity of staining, the cells were designated as positive. Two investigators (K.C. and T.I.) separately evaluated all of the specimens in a blinded manner. MIA PaCa-2 cells (1 × 105/well), plated in six-well plates, were grown in 2 ml of RPMI 1640 medium with 10% FBS for 24 hours and then cultured with serum-free medium for 48 hours. The cells were subsequently cultured in serum-free RPMI 1640 medium in the absence or presence of 10 ng/ml rhKGF for 1, 3, 6, and 12 hours. Expression of VEGF-A and RS-18 mRNA levels were examined by Q-PCR as previously described. The real-time PCR primers used for VEGF-A corresponded to nucleotides 1126 to 1148 (5′-GAGGAGGGCAGAATCATCACGAA-3′) and nucleotides 1348 to 1369 (5′-TGGTGAGGTTTGATCCGCATAA-3′) of the human VEGF-A cDNA (244 bp, accession no. NM003376). The primers used for RS-18 corresponded to nucleotides 184 to 207 (5′-AAAGCAGACATTGACCTCACCAAG-3′) and nucleotides 319 to 341 (5′-AGGACCTGGCTGTATTTTACC-3′) of the human RS-18 cDNA (158 bp, accession no. NM022551). The same KGF primer pairs used for RT-PCR were used for real-time PCR analysis. The optimized program involved denaturation at 95°C for 10 minutes, followed by 45 cycles of amplification at 95°C for 10 seconds, at 60°C for 10 seconds, and at 72°C for 10 seconds for VEGF-A; at 95°C for 10 seconds, at 65°C for 10 seconds, and at 72°C for 7 seconds for RS-18; and at 95°C for 10 seconds, at 58°C for 10 seconds, and at 72°C for 8 seconds for KGF. To confirm amplification specificity, PCR products were subjected to melting-curve analysis. Results were expressed as target/RS-18, as an internal standard concentration ratio. Each experiment was performed twice with gene expression measurements performed in triplicate. MIA PaCa-2 cells (2 × 103/well) were plated in 96-well plates, grown in 200 μl of RPMI 1640 medium with 10% FBS for 24 hours, and then cultured with serum-free medium for 48 hours. The cells were subsequently cultured in serum-free RPMI 1640 medium with 0, 10, or 100 ng/ml rhKGF for 48 hours. The culture supernatants were collected, and their VEGF-A levels were measured using ELISA kits. This study was performed by two separate experiments, each conducted in triplicate. Full-length KGF cDNA fragment was prepared by RT-PCR from human placental RNA. The primer pair used for the cDNA was 5′-GCTAGCAAGGAGATACCACCATGCACAAATGGATACTGACAT-3′ (including the NheI site) and 5′-CTGCAGCAATTAAGTTATTGCCATAG-3′ (including the PstI site). The full-length cDNA was digested with NheI and PstI and ligated to the 3′ end of the human cytomegalovirus early promoter/enhancer in pIRES2-EGFP eukaryotic expression vector. Proper insert orientation was verified by DNA sequencing. pIRES2-EGFP contains the internal ribosome entry site of the encephalomyocarditis virus between the KGF and the enhanced green fluorescent protein coding region. This permits both the KGF and the enhanced green fluorescent protein genes to be translated from a single bicistronic mRNA. Approximately 1 × 106/ml PANC-1 cells were transfected with 2 μg of DNA using FuGENE 6, and the cells were passaged and cultured with 500 μg/ml G-418. Independent colonies were isolated by ring cloning, transferred to micro titer wells, and expanded. KGF stably transfected PANC-1 cells (PANC-1-KGF) and mock-transfected PANC
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