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• W2083955716 abstract "We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, akt, and CD171, which are overexpressed by Panc1 cells. Loss of PDAC syk expression in culture is due to promoter methylation, and reversal of promoter methylation caused reexpression of syk and concomitant down-regulation of CD171. Moreover, suppression of syk expression in BxPC3 cells caused de novo CD171 expression, consistent with the reciprocal expression of syk and CD171 we observe in situ. Importantly, Panc1/syk cells demonstrated dramatically reduced invasion in vitro. Affymetrix analysis identified statistically significant regulation of >2000 gene products by syk in Panc1 cells. Of these, matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 were down-regulated, suggesting that the MMP2 axis might mediate Panc1/mock invasion. Accordingly, MMP2 inhibition suppressed the in vitro invasion of Panc1/mock cells without effect on Panc1/syk cells. This study demonstrates a prominent role for syk in regulating the differentiation state and invasive phenotype of PDAC cells. We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, akt, and CD171, which are overexpressed by Panc1 cells. Loss of PDAC syk expression in culture is due to promoter methylation, and reversal of promoter methylation caused reexpression of syk and concomitant down-regulation of CD171. Moreover, suppression of syk expression in BxPC3 cells caused de novo CD171 expression, consistent with the reciprocal expression of syk and CD171 we observe in situ. Importantly, Panc1/syk cells demonstrated dramatically reduced invasion in vitro. Affymetrix analysis identified statistically significant regulation of >2000 gene products by syk in Panc1 cells. Of these, matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 were down-regulated, suggesting that the MMP2 axis might mediate Panc1/mock invasion. Accordingly, MMP2 inhibition suppressed the in vitro invasion of Panc1/mock cells without effect on Panc1/syk cells. This study demonstrates a prominent role for syk in regulating the differentiation state and invasive phenotype of PDAC cells. Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates of all cancers.1National Cancer Institute Strategic plan for addressing the recommendations of the Pancreatic Cancer Progress Review Group. National Cancer Institute, Bethesda, MD2002Google Scholar Despite this, the biology of PDAC remains poorly understood. Studies have identified key factors in the etiology of the disease, which have been incorporated into a genetic model of PDAC development.2Bardeesy N DePinho RA Pancreatic cancer biology and genetics.Nat Rev. 2002; 2: 897-909Crossref Scopus (950) Google Scholar Although such a timeline is significant in the definition of factors contributing to disease onset, a similar timeline has been difficult to address with regard to disease progression. Syk is a nonreceptor tyrosine kinase central to the signaling of many hematopoietic cell types.3Sada K Takano T Yanagi S Yamamura H Structure and function of syk protein-tyrosine kinase.J Biochem. 2001; 130: 177-186Crossref PubMed Scopus (239) Google Scholar Syk has also been implicated in the signaling processes of nonhematopoietic cell types,4Yanagi S Inatome R Takano T Yamamura H Syk expression and novel function in a wide variety of tissues.Biochem Biophys Res Commun. 2001; 288: 495-498Crossref PubMed Scopus (153) Google Scholar and syk has further been identified as a putative breast cancer (BC) suppressor in humans, based in part on the reduced expression of syk in a progression-related manner and on the fact that ectopic expression of syk in syk-negative BC cells retarded their growth in vivo, whereas suppression of endogenous syk activity reciprocally enhanced BC tumorigenicity.5Coopman PJ Do MTH Barth M Bowden ET Hayes AJ Basyuk E Blancato JK Vezza PR McLeskey SW Mangeat PH Mueller SC The syk tyrosine kinase suppresses malignant growth of human breast cancer cells.Nature. 2000; 406: 742-747Crossref PubMed Scopus (289) Google Scholar More recent studies have shown that syk is a negative regulator of BC mitosis,6Zyss D Montcourrier P Vidal B Anguille C Merezegue F Sahuquet A Mangeat PH Coopman PJ The syk tyrosine kinase localizes to the centrosomes and negatively affects mitotic progression.Cancer Res. 2005; 65: 10872-10880Crossref PubMed Scopus (59) Google Scholar transcription,7Wang L Devarajan E He J Reddy SP Dai JL Transcription repressor activity of spleen tyrosine kinase mediates breast cancer suppression.Cancer Res. 2005; 65: 10289-10297Crossref PubMed Scopus (44) Google Scholar motility,8Mahabeleshwar GH Kundu GC Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor κB-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3′-kinase activity in breast cancer cells.J Biol Chem. 2003; 278: 6209-6221Crossref PubMed Scopus (71) Google Scholar, 9Zhang X Shrikhande U Alicie BM Zhou Q Geahlen RL Role of the protein tyrosine kinase Syk in regulating cell-cell adhesion and motility in breast cancer cells.Mol Cancer Res. 2009; 7: 634-644Crossref PubMed Scopus (60) Google Scholar and invasion,10Wang L Duke L Zhang PS Arlinghaus RB Symmans WF Sahin A Mendez R Dai JL Alternative splicing disrupts a nuclear localization signal in spleen tyrosine kinase that is required for invasion suppression in breast cancer.Cancer Res. 2003; 63: 4724-4730PubMed Google Scholar as well as anchorage-independent growth and tumorigenicity of melanoma cells.11Muthusamy V Duraisamy S Bradbury CM Hobbs C Curley DP Nelson B Bosenberg M Epigenetic silencing of novel tumor suppressors in malignant melanoma.Cancer Res. 2006; 66: 11187-11193Crossref PubMed Scopus (142) Google Scholar In patients, loss of syk correlates with poor survival and tumor metastasis in breast,12Toyama T Iwase H Yamashita H Hara Y Omoto Y Sugiura H Zhang Z Fujii Y Reduced expression of the Syk gene is correlated with poor prognosis in human breast cancer.Cancer Lett. 2003; 189: 97-102Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Repana K Papazisis K Foukas P Valeri R Kortsaris A Deligiorgi E Kyriakidis D Expression of Syk in invasive breast cancer: correlation to proliferation and invasiveness.Anticancer Res. 2006; 26: 4949-4954PubMed Google Scholar, 14Ding YB Wu ZY Wang S Fan P Zha XM Zheng W Liu XA Expression of tyrosine kinase Syk in breast cancer and their clinical significance.Zhonghua Wai Ke Za Zhi. 2004; 42: 137-139PubMed Google Scholar bladder,15Kunze E Wendt M Schlott T Promoter hypermethylation of the 14-3-3σ: SYK and CAGE-1 genes is related to the various phenotypes of urinary bladder carcinomas and associated with progression of transitional cell carcinomas.Int J Mol Med. 2006; 18: 547-557PubMed Google Scholar liver,16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar and gastrointestinal tract tumors.17Wang S Ding YB Chen GY Xia JG Wu ZY Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma.World J Gastroenterol. 2004; 10: 1815-1818Crossref PubMed Scopus (47) Google Scholar, 18Nakashima H Natsugoe S Ishigami S Okumura H Matsumoto M Hokita S Aikou T Clinical significance of nuclear expression of spleen tyrosine kinase (Syk) in gastric cancer.Cancer Lett. 2006; 236: 89-94Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Yang ZL Wang L Kang L Peng JS Xiang J Huang MJ Wang JP Association between hypermethylation of Syk gene and clinicopathological characteristics in colorectal cancer patients.Zhonghua Wei Chang Wai Ke Za Zhi. 2008; 11: 458-461PubMed Google Scholar Together these data clearly implicate syk as a potential tumor suppressor in epithelial tissues. However, it is important to note that higher levels of syk expression were observed in squamous cell carcinomas of the head and neck and their lymph node metastases than normal tissue (high syk expression correlated significantly with poor survival), and further that syk promoted migration and invasion of squamous cell carcinoma of the head and neck cells in vitro.20Luangdilok S Box C Patterson L Court W Harrington K Pitkin L Rhys-Evans P Ocharoenrat P Eccles S Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.Cancer Res. 2007; 67: 7907-7916Crossref PubMed Scopus (64) Google Scholar Indeed, the inappropriate activation of syk in breast epithelial cells contributes to cellular transformation,21Katz E Lareef MH Rassa JC Grande SM King LB Russo J Ross SR Monroe JG MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture.J Exp Med. 2005; 201: 431-439Crossref PubMed Scopus (101) Google Scholar enhanced nuclear factor-κB activation, and resistance to tumor necrosis factor-induced apoptosis in mouse mammary tumor virus-mediated tumorigenesis.22Zhou Q Geahlen RL The protein-tyrosine kinase syk interacts with TRAF-interacting protein TRIP in breast epithelial cells.Oncogene. 2009; 28: 1348-1356Crossref PubMed Scopus (43) Google Scholar These data highlight the complex nature of syk activity in regulating processes associated with tumorigenesis, even within the same tissue (ie, breast epithelium), and thus it is not a foregone conclusion that syk will function as a tumor suppressor in all epithelial cells. In this study, we demonstrate the expression of syk in ductal epithelial cells of the normal pancreas, and the loss of syk during PDAC “dedifferentiation.” Our data demonstrate that syk regulates gene expression and a myriad of phenotypic parameters responsible for maintaining a more differentiated epithelial state, demonstrating for the first time a role for syk in regulating the phenotype of PDAC cells. AsPC1, CAPAN1, CAPAN2, CFPAC1, HPAFII, SU.86.86, BxPC3, MIAPaCa2, and Panc1 cells were originally from the American Type Culture Collection (Manassas, VA) and were cultured as recommended by the American Type Culture Collection. Leukemic HL60 cells were a generous gift of A. Raz (Detroit, MI), and cultured according to the American Type Culture Collection. COLO357 cells were generously provided by M. Korc (Irvine, CA) and cultured in Dulbecco’s modified Eagle’s medium/10% fetal bovine serum (FBS). Serum-free medium consisted of all media components except serum, as appropriate for the cell line, supplemented with 0.5% bovine serum albumin. Anti-syk-LR polyclonal antibody (pAb), 4D10 monoclonal antibody (mAb), and anti-extracellular-regulated kinase-2 (Erk2) pAb (C14) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin was from Sigma-Aldrich (St. Louis, MO). Anti-pan-Akt pAb was from Cell Signaling Technologies (Beverly, MA). Anti-CD171 mAb UJ127 was from Neomarkers/LabVision (Fremont, CA). Anti-cyclin D1 was a kind gift from M. Just (eBioscience, San Diego, CA). pEGFP-N1 and anti-green fluorescent protein (GFP) pAb were from BD Clontech (Palo Alto, CA). 5-Aza-2′-deoxycytidine (5AzaC) was from Invivogen (San Diego, CA). N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine, 2-aminoethyl amide (TAPI1) was from EMD (San Diego, CA). Tissue inhibitor of metalloproteinase-2 (TIMP2) was from Novus (Littleton, CO). pEMCV/syk23Zoller KE MacNeil IA Brugge JS Protein tyrosine kinases syk and ZAP-70 display distinct requirements for src family kinases in immune response receptor signal transduction.J Immunol. 1997; 158: 1650-1659PubMed Google Scholar was a generous gift of S. Shattil (University of California, San Diego [UCSD], La Jolla, CA). The wild-type human pp72syk CDS encoding sykA was excised from pEMCV/syk and ligated into a pCDNA3.1(zeo) vector into which an IRES sequence had been introduced 5′ to a hygromycin phosphotransferase gene. Panc1 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), hygromycin-selected, and assessed for protein expression. For small interfering RNA (siRNA) studies, a sense and antisense-containing 60 mer of the syk-6 siRNA sequence24Ruschel A Ullrich A Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells.Cell Signal. 2004; 16: 1249-1261Crossref PubMed Scopus (24) Google Scholar was inserted into pSuper.25Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3973) Google Scholar BxPC3 cells (4 × 106) were pulsed (270 V, 550 μF) with a Bio-Rad GenePulser II in basal media/10% glucose containing 20 μg of DNA (15 μg of syk-6/pSuper and 5 μg of pEGFP) and replated in full growth medium. Samples (n = 92) were obtained under the institutional review board protocol from the UCSD Department of Pathology archives. Normal tissue was from patients who died of nonpancreatic disease or trauma and are not included in the survival analysis. Patient demographics (including sex, age, and race) and tissue characterization (including tumor size, differentiation, node status, margin involvement, and perineural or vascular invasion status) were described in detail previously.26Bouvet M Gamagami RA Gilpin EA Romeo O Sasson A Easter DW Moossa AR Factors influencing survival after resection for periampullary neoplasms.Am J Surg. 2000; 180: 13-17Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Katz MHG Bouvet M Al-Refaie W Gilpin EA Moossa AR Non-pancreatic periampullary adenocarcinomas: an explanation for favorable prognosis.Hepatogastroenterology. 2004; 51: 842-846PubMed Google Scholar Tissue differentiation grade was categorized as the highest grade present (ie, a patient whose tumor contained elements of G2 and G3 was classified as G3). Samples were deparaffinized, rehydrated, and incubated with 1% H2O2. Slides were blocked with 2% horse serum/5% bovine serum albumin/PBS, pH 7.4, and renatured using DAKO Target Retrieval Solution (UJ127) or DAKO High-pH Target Retrieval Solution (4D10), before incubation with 0.5 to 2.0 μg/ml 4D10 or UJ127. Slides were washed and biotinylated-anti-mouse was applied according to the VectaStain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Sections were developed with diaminobenzidine, counterstained with hematoxylin, dehydrated, and mounted. Lysates (250 μg) were incubated overnight at 4°C with 20 μl of anti-syk LR-AC pAb (agarose conjugate). Beads were washed with lysis buffer and prepared for immunoblotting. Samples were prepared and analyzed as described previously.28Silletti S Yebra M Perez B Cirulli V McMahon M Montgomery AMP The cell adhesion molecule L1 promotes a motile and invasive phenotype via sustained MAP-kinase activation, gene transcription and induction of integrin-dependent migration.J Biol Chem. 2004; 279: 28880-28888Crossref PubMed Scopus (127) Google Scholar For cyclin D1, cells were harvested at subconfluence. Cells (5 × 102/well) were seeded into a 48-well plate. After 24 hours (and every 72 hours thereafter), fresh growth medium was replaced, and the initial time point was fixed with 1% paraformaldehyde/PBS, pH 7.4. Additional triplicate wells were fixed at 24-hour intervals, stained with 1% crystal violet, and compared with a standard curve of cells. Dye was extracted with 10% acetic acid and quantitated at 550 nm. A top layer containing 5 × 103 cells in 0.5% agar/Dulbecco’s modified Eagle’s medium/10% FBS was seeded onto a base layer of 0.7% agar/Dulbecco’s modified Eagle’s medium containing 10% FBS in a six-well plate. Cultures were incubated at 37°C, medium was replaced every third day, and the assay was stopped on day 10. Cultures were stained with 0.01% crystal violet. Colonies were enumerated on a Bio-Rad GelDoc XR system using QuantityOne Software (sensitivity = 8.1, average = 5). A total of 107 cells were injected into the flanks of 6-week old nu−/nu− mice, and tumors were grown for 4 weeks, at which time they were harvested, fixed in formalin, and weighed wet. Subconfluent cells (2.5 × 105) were seeded in serum-free media into BioCoat Growth Factor-Reduced Matrigel Invasion Chambers (BD Biosciences, Bedford, MA) in wells containing serum-free media or media containing 10% FBS. Chambers were incubated at 37°C for 24 hours before fixing, staining with 1% toluidine blue, removal of uninvaded cells, and manual enumeration. TAPI1 (40 μmol/L) or TIMP2 (8 nmol/L) assays involved preincubating cells for 15 minutes before seeding into inserts seated in wells with equal concentrations of TAPI1 (or dimethyl sulfoxide control) or TIMP2 in Dulbecco’s modified Eagle’s medium/10% FBS. Cells were plated in serial twofold dilutions to achieve similar densities at harvest, beginning with 1.5 × 106 cells/well, and allowed to adhere for 24 hours before growth medium was replaced, and cells were treated daily with dimethyl sulfoxide or 2 μmol/L 5AzaC in full growth medium. DNA, RNA, and protein were collected at the indicated times. For the density analysis, cells were plated at 2 × 105 or 4 × 104 cells/well (to achieve maximal density for “confluent” and subconfluence for “subconfluent” cultures at 120 hours) and daily received basal media containing 10, 3, or 1% FBS with dimethyl sulfoxide or 2 μmol/L 5AzaC. Methylation-specific PCR was performed as described by Yuan et al.16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar Genomic DNA was modified using the MethylDetector Bisulfite Kit (Active Motif, Carlsbad, CA). cDNA was synthesized from 1 μg of total RNA using oligo(dT) primer. PCR was performed on 1 μl of total cDNA using the primers described in Table 1.16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar, 29Hegedüs L Cho H Xie X Eliceiri GL Additional MDA-MB-231 breast cancer cell matrix metalloproteinases promote invasiveness.J Cell Physiol. 2008; 216: 480-485Crossref PubMed Scopus (74) Google Scholar, 30Yagi S Suzuki K Hasegawa A Okumura K Ra C Cloning of the cDNA for the deleted syk kinase homologous to zap-70 from human basophilic leukemia cell line (KU812).Biochem Biophys Res Commun. 1994; 200: 28-34Crossref PubMed Scopus (30) Google Scholar, 31Christopoulos TA Papageorgakopoulou N Ravazoula P Mastronikolis NS Papadas TA Theocharis DA Vynios DH Expression of metalloproteinases and their tissue inhibitors in squamous cell laryngeal carcinoma.Oncol Rep. 2007; 18: 855-860PubMed Google ScholarTable 1PCR Primers and SourcesGeneForward primer Reverse primerSourceβ-Actin5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′Stratagene, La Jolla, CA5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′GAPDH5′-CCACCCATGGCAAATTCCATGGCA-3′Stratagene, La Jolla, CA5′-TCTAGACGGCAGGTCAGGTCCACC-3′MMP25′-GCTGGCTGCCTTAGAACCTTTC-3′Hegedus et al29Hegedüs L Cho H Xie X Eliceiri GL Additional MDA-MB-231 breast cancer cell matrix metalloproteinases promote invasiveness.J Cell Physiol. 2008; 216: 480-485Crossref PubMed Scopus (74) Google Scholar5′-GAACCATCACTATGTGGGCTGAGA-3′MMP95′-GCACGACGTCTTCCAGTACC-3′Hegedus et al29Hegedüs L Cho H Xie X Eliceiri GL Additional MDA-MB-231 breast cancer cell matrix metalloproteinases promote invasiveness.J Cell Physiol. 2008; 216: 480-485Crossref PubMed Scopus (74) Google Scholar5′-GCACTGCAGGATGTCATAGGT-3′SYK5′-AAAGAAGTTCGACACGCTCTGG-3′ (F20)Yagi et al30Yagi S Suzuki K Hasegawa A Okumura K Ra C Cloning of the cDNA for the deleted syk kinase homologous to zap-70 from human basophilic leukemia cell line (KU812).Biochem Biophys Res Commun. 1994; 200: 28-34Crossref PubMed Scopus (30) Google Scholar5′-GCAAGTTCTGGCTCATACGGA-3′ (R19)External SYK MS-PCR5′-TTTAGGGAATATGTTATGTAGTG-3′Yuan et al16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar5′-CACATAATTTCAACACTTTTACC-3′Internal SYK MS-PCR methylated5′-CGATTTCGCGGGTTTCGTTC-3′Yuan et al16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar5′-AAAACGAACGCAACGCGAAAC-3′Internal SYK MS-PCR unmethylated5′-ATTTTGTGGGTTTTGTTTGGTG-3′Yuan et al16Yuan Y Wang J Li J Wang L Li M Yang Z Zhang C Dai JL Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma.Clin Cancer Res. 2006; 12: 6687-6695Crossref PubMed Scopus (71) Google Scholar5′-ACTTCCTTAACACACCCAAAC-3′TIMP15′-CCTTCTGCAATTCCGACCTCGTC-3′Christopoulos et al31Christopoulos TA Papageorgakopoulou N Ravazoula P Mastronikolis NS Papadas TA Theocharis DA Vynios DH Expression of metalloproteinases and their tissue inhibitors in squamous cell laryngeal carcinoma.Oncol Rep. 2007; 18: 855-860PubMed Google Scholar5′-CGGGCAGGATTCAGGCTATCTGG-3′TIMP25′-TGGAAACGACATTTATGGCAACC-3′Christopoulos et al31Christopoulos TA Papageorgakopoulou N Ravazoula P Mastronikolis NS Papadas TA Theocharis DA Vynios DH Expression of metalloproteinases and their tissue inhibitors in squamous cell laryngeal carcinoma.Oncol Rep. 2007; 18: 855-860PubMed Google Scholar5′-ACAGGAGCCGTCACTTCTCTTGAT-3′GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SYK, spleen tyrosine kinase; MS, methylation-specific. Open table in a new tab GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SYK, spleen tyrosine kinase; MS, methylation-specific. Analysis was performed as described previously.32Goodison S Nakamura K Iczkowski KA Anai S Boehlein SK Rosser CJ Exogenous mycoplasmal p37 protein alters gene expression, growth and morphology of prostate cancer cells.Cytogenet Genome Res. 2007; 118: 204-213Crossref PubMed Scopus (26) Google Scholar Total RNA was prepared for hybridization according to the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). RNA was extracted (RNeasy, Qiagen, Valencia, CA) and quality-assessed on a bioanalyzer (RNA 6000 Nano Chip, Agilent Technologies, Palo Alto, CA), and 5 μg was used as a template for cDNA synthesis (SuperScript, Invitrogen Life Technologies, Gaithersburg, MD). First-strand synthesis was primed with a T7-(dT) 24-oligonucleotide primer containing a T7 RNA polymerase promoter sequence on the 5′ end. Second-strand products were cleaned (GeneChip Sample Cleanup Module, Affymetrix) and in vitro-transcripted with biotin-labeled nucleotides (Bioarray Labeling Kit, Enzo Diagnostics, Farmingdale, NY). cRNA product was cleaned and 20 μg was heated at 94°C for 35 minutes in fragmentation buffer (Affymetrix). Adjusted cRNA (15 μg) was hybridized for 16 hours at 45°C to an Affymetrix U133 Plus 2.0 GeneChip (47,000 transcripts). Each array was then stained with a streptavidin-phycoerythrin conjugate (Molecular Probes/Invitrogen), washed, and visualized with a microarray scanner (Genearray Scanner, Agilent Technologies, Santa Clara, CA). Images were inspected visually for hybridization artifacts. In addition, quality assessment metrics were generated for each scanned image and evaluated based on empirical data from previous hybridizations and the signal intensity of internal standards. Microarray Suite (version 5, Affymetrix), was used to generate *.cel files, and Probe Profiler (version 1.3.11, Corimbia Inc., Berkeley, CA), developed specifically for the GeneChip system, was used to convert intensity data into quantitative estimates of gene expression. A probability statistic was generated for each probe set (gene) with the null hypothesis being that the expression level is equal to zero (background). Genes not significantly expressed above background in at least two samples (P < 0.05) were considered absent. Statistical tests were performed using BioConductor statistical software.33Gentleman RC Carey VJ Bates DM Bolstad B Dettling M Dudoit S Ellis B Gautier L Ge Y Gentry J Hornik K Hothorn T Huber W Iacus S Irizarry R Leisch F Li C Maechler M Rossini AJ Sawitzki G Smith C Smyth G Tierney L Yang JY Zhang J Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar The raw data were normalized by the Robust Multichip Analysis approach implemented in the Affy package.34Bolstad BM Irizarry RA Astrand M Speed TP A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6492) Google Scholar The fold change was computed based on the normalized data. A significant P value was computed by a statistical test based on a probe level analysis using the affyPLM package.35Bolstad BM Collin F Brettschneider J Simpson K Cope L Irizarry RA Speed TP Quality assessment of Affymetrix GeneChip data.in: Gentleman R Carey V Huber W Irizarry R Dutoit S Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Springer, Heidelberg2005Crossref Google ScholarP values were further adjusted using the Benjamini and Hochberg method.36Benjamini Y Hochberg Y Controlling the false discovery rate: a practical and powerful approach to multiple testing.J Roy Stat Soc B. 1995; 57: 289-300Google Scholar Genes with P < 0.05 were considered as differentially expressed genes at a statistically significant level. Gene Ontology annotations were obtained from Affymetrix. Biological network relationships among significantly regulated genes were explored using KEGG and GenMapp pathways using AnalyzeIt Tools. Panc1/mock and Panc1/syk cells were plated at equal densities, grown 3 days, and serum-starved (24 hours), and supernatant was collected. Equal amounts of clarified supernatants and serum-free media (control) were processed using gelatin-embedded SDS-polyacrylamide gel electrophoresis gels as described previously.37Silletti S Kessler T Goldberg J Boger DL Cheresh DA Disruption of matrix metalloproteinase 2 binding to integrin αvβ3 by an organic molecule inhibits angiogenesis and tumor growth in vivo.Proc Natl Acad Sci USA. 2001; 98: 119-124PubMed Google Scholar Images of ethidium bromide-stained agarose gels were captured with Quantity One software on a Bio-Rad Gel Doc XR using the appropriate filter and transmitted UV light. Chemiluminescence-exposed films and printouts of agarose gels were scanned on an Epson Perfection 4490 Photo flatbed scanner. Images were imported into Adobe Photoshop for removal of unused levels and cropping. Minimal alterations to brightness and contrast were used for a subset of images to improve the visual nature of the image. Nonlinear adjustments were not used. Immunohistochemical images were acquired as 24-bit RGB (.tif) and phase-contrast images as 8-bit gray scale (.tif) using SpotBasic with Nikon TE2000-S or TE300 microscopes, respectively, each fitted with a model 3.2.0 charge-coupled device camera and used at the Moores UCSD Cancer Center Microscopy Shared Resource. Final images were compiled in Adobe InDesign, rasterized, and converted to jpeg format at a minimum of 300 dpi. Survival was evaluated according to the Kaplan-Meier method using a univariate log-rank test. Variables were coded as 1 for 100% syk-positive tumors and 0 for those that evinced heterogeneity. Colony-forming and invasion were analyzed by a two-tailed Student’s t test. Tumor growth was compared using the Mann-Whitney U test. A panel of tissue samples was analyzed by immunohistochemistry with the anti-syk mAb 4D10. Uniform syk expression was observed in both the cytoplasm and nucleus of cells of normal ducts and ductules adjacent to acini (Figure 1A).26Bouvet M Gamagami RA Gilpin EA Romeo O Sasson A Easter DW Moossa AR Factors influencing survival after resection for periampullary neoplasms.Am J Surg. 2000; 180: 13-17Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Katz MHG Bouvet M Al-Refaie W Gilpin EA Moossa AR Non-pancreatic periampullary adenocarcinomas: an explanation for favorable prognosis.Hepatogastroenterology. 2004; 51: 842-846PubMed Google Scholar Well differentiated malignant ducts also demonstrated strong uniform syk expression; however, a progressive loss of syk was noted in more pleomorphic, moderately differentiated PDAC cells. Poorly differentiated cells exhibiting distinct nuclear atypia and dramatic cellular pleomorphism were essentially devoid of syk expression. Overall, 100% of normal ducts and G1 tumors demonstrated homogeneous syk expression, whereas 71% of G2 tumors and no G3/sarcomatoid tumors retained normal syk expression (n = 92) (Figure 1B). K" @default.
• W2083955716 created "2016-06-24" @default.
• W2083955716 creator A5030813046 @default.
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• W2083955716 date "2009-12-01" @default.
• W2083955716 modified "2023-10-14" @default.
• W2083955716 title "Syk Tyrosine Kinase Acts as a Pancreatic Adenocarcinoma Tumor Suppressor by Regulating Cellular Growth and Invasion" @default.
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