- Email: [email protected]
LY293111 improves gemcitabine efficacy in a new fluorescent orthotopic model of pancreatic cancer in athymic mice
regulation by these two isoforms. Conclusion: DNA replication is regulated by licensing proteins. The MCM2 protein has been found to have a novel isoform. These two isoforms are reciprocally related during senescence and proliferation. We conclude there is a molecular switch between these two isoforms that will dictate the rate of DNA replication during senescence versus proliferation.
patient with a type I tumor, 26 genes were sigmficantly (>2-fold) up-regulated and 48 genes down-regulated. Twenty-ninc genes were significantly up-regulated and the same number down-regulated in the type II ECL cell sample. Shared gene pathways up-regulated in each tumor type included immune function (35%) and hematological (35%). Other genes up-regulated in both samples include the EGR genes (early growth response, + 2.6 - + 4.6fold, p < 0.0002) and down-regulated trefoil factor 1 (-2.1 - -1.9-fold, p < 0.0002). Genes significantly altered in the type I tumor included beta-microseminoprotem (-2.8-fold, p < 0.0002) and somatostatin (-2.5-fold, p < 0.00001) and in the type 11 tumor, v-los (+39.3fold, p < 0.00002) and fos-B (+ 29-fold, p < 0.0003). Conclusions: These data demonstrate that human ECL cell tumorigenesis has an important immunological component and a hematological component irrespective of the tumor type. Gastrin-responsive (type 1) tumors have up-regulation of early response genes, while type II tumors (gastrin-responsive, genomic alteration in MEN-I), have in addition altered regulation of genes involved in cell proliferation.
M990 Altered Function of KLF5 Is Associated with Intestinal Tumor Progression Nicholas W. Bateman, Dongfeng Tan, Jennifer D. Black, Adrian R. Black Members of the kroppel-like family of transcription factors (KLFs) have been linked to the regulation of cell growth and tumorigenesis in a number of systems. In the normal intestinal epithelium, KLF5 (intestinal-kruppel like factor/BTEB2) is expressed m growing cells of the lower crypt but is downregulated in non-dividing ceils of the upper crypt and villus/surface mucosa, suggesting that it has a growth promoting role in this tissue. In support of this idea, a direct positive growth regulatory role for KLF5 in fibrobfasts and vascular smooth muscle ceils has been reported. To evaluate the expression of KLF5 during intestinal tumorigenesis, RT-PCR was performed on laser-capture microdissected samples from intestinal adenomas and normal epithelium. This analysis showed reduced expression of KLF5 mRNA in rain mouse and human adenomatons polyposis adenornas; thus, while KLF5 may be growth promoting in the normal intestinal epithelium, it may also have a tumor suppressive function in this tissue. This possibility was further investigated using a normalintestinal crypt epithelial cell line (1EC-18) and colon tumor cell lines (Ward and DLD-1). Overexpression of KLF5 inhibited colony formation in the colon tumor cell lines but enhanced colony number in IEC-18 cells, indicating that KLF5 has opposing effects on cell growth and/or survival in normal and tumor-derived intestinal epithelial cells. Differences in the effects of KLF5 were also seen in transient transfection assays, where KLF5 enhanced expression of growthrelated genes in [EC-18 cells but not DLD-1 or Ward cells. Furthermore, while phorbol ester-induced growth arrest of IEC-18 cells led to downregulation of KLF5, consistent with the posiuve relationship between this factor and cell growth in the normal intestinal epithelium, growth arrest of tumor cell lines led to an upregulation of KLF5, indicating that this factor is negatively associated with growth in tumor cells. Collectively, these data indicate that KLF5 is growth promoting in the normal intestinal epithelium but is associated with growth inhibition in tumor cells. Thus, intestinal tumor progression appears to be associated with a change in the growth-related functions of KLF5. Supported by the Roswell Park Alliance Foundation and by NIH grants CA16056 and DK54909.
M993 RASSF1A May Serve as Novel Tumor Suppressor Gene in Gastrointestinal Malignancies Regulating Apoptosis Juliocesar Bernabe Ortiz, Shahrooz Rabizadeh, Kazuhiro Ishiguro, Marco Lopez-Ilasaca, Andrei Khokhlatchev, Joseph Avruch, Brian Seed, Ramnik Xavier Background: A previous search for genes that map to regions of loss of heterozygosity on chromosome 3p21 as well as a yeast two hybrid screen led to the discovery of RASSF1A, a protein characterized by a Ras binding domain. RASSFIA was found to be silenced through promoter hypermethyfation in lung, prostate, colorectal, gastric and biliary tumors. This gene encodes a protein with several interesting domains, including one that potentially imeracts with the Ras family of proteins, a domain that is phosphorylated via the ataxiatelangiectasia (ATM) kinase pathway, and a DAG binding domain that may link it to tumor promoting activities. The aim of the present study was to elucidate the function of RASSF1A in intestinal epithelial cell lines. Methods and Results: In a screen of cancer cell lines, RASSFIA mRNA levels were found to be reduced when compared to normal human epithelial cells and fibroblasts. Functional analysis in epithelial cell lines revealed that RASSF1Aassociated complexes promote cell death. Microscopic studies showed that RASSF1A associates with microtubule structures to form an elaborate network in the cytosol of transformed intestinal epithelial cell lines. A yeast two-hybrid screen resulted in identification of a novel regulator of RASSF1A-mediated apoptosis. Structure-function studies demonstrated that key domains in RASSFIA mediate cell death. Conclusion: Taken together, these studies suggest that RASSEIA functions as a novel tumor suppressor in gastrointestinal epithehal malignancies.
M991 Nonsteroidal Anti-lnflammatory Drugs Inhibit Human Telomerase Reverse Transcriptase (hTERT) in Gastric and Colon Cancer Xiaohua Jiang, Marie C. M. Lin, Shiukum Lam, Hua He, Shuiping Tu, Hsianglu Kung, Benjamin C. Wong
M994 Recruitment of Claudin-2 to Tight Junctions by Inhibition of the Mitogenactivated Protein Kinase (MAPK) Pathway in ras-transforraed Pancreatic Cancer Cells Tillmann Bert, Stephanie Hoormann, Nadine Aurbek, Anskar 5chmidt, Babette Simon
Background and Aims: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase complex with reverse transcriptase activity, hTERT activation is observed in 90% of human cancers and is a critical step for carcinogenesis. The present study is aimed to investigate the effect of Nonsteroidal anti-inflammatory drugs (NSAIDs) on hTERT regulation. Materials and Methods: hTERT mRNA and protein expression in both gastric cancer and colon cancer cell lines were determined by RT-PCR and Western blotting. hTERT transcriptional activity was detected by transfection with different hTERT promoterluciferase constructs and luciferase assay. Results: We demonstrated that NSAIDs (including aspirin and the cyclooxygenase-2 specific inhibitors) suppressed hTERT gene expression at both mRNA and protein level NSAIDs also inhibited telomerase enzymatic activity in both human colon and gastric cancer cells. Using a promoter-luciferase construct containing a 283-bp upstream ATG in the hTERT promoter, we showed that NSAIDs st~ppressed hTERT transcriptional activity significantly, which suggested that the down-regulation of the telomerase activity and hTERT expression by NSAIDs was due to transcriptional repression of the hTERT gene. In addition, we used reporter plasmid which contained point mutations (CACGTG-TTTGTG) in each of the two E-box, located at -165 and -44 relative to the ATG initiation codon. Our results showed that mutation of E-box did not have any effect on NSA1Ds-mediated inhibition of bTERT promoter activity. Conclusion: NSAIDs regulate bTERT expression at transcriptional level. The inhibition was independent of E-box. The molecular mechanism(s) for NSAIDs-medlated inhibitory effect on hTERT needs further investigation
Background: To identify novel therapeutic targets for pancreatic cancer (PaTu) we isolated differentially expressed cDNA fragments of the metastasizing cell line PaTu8988S and nonmetastasizing cell line PaTu8988T using cDNA-represemational difference analysis (cDNARDA). Methods and Results: One of the isolated cDNA fragments revealed homology to cfaudin-2, a member of a tight junction (TJ) protein family. We cloned the full length gene with an ORF of 693 bp encoding a protein of 230 amino acids with a calculated MW of 24 kDa. RT-PCR and northern blotting verified dilferemial claudin-2 expression revealing a 3.5 kb primary transcript. Cfaudin-2 was expressed in 70% of human PaTu cell lines analysed. Genomic studies revealed an intronless claudin-2 gene and excluded homozygons deletion in PaTu8988T cells. Radiation hybrid mapping localized claudin-2 at Xq22.3-23. Expression profiling using a human multiple tissue array demonstrated a distinct organspecific distribution pattern of claudin-2. Important mechanisms of cellular regulation in epithelial tissues are mediated through intercellular junctional communication. Since little is known about junctional assembly during transformation we analysed the expression of distinct claudins (1 to 12) by RT-PCR. There was almost exclusive claudin-1 expression in PaTu8988T cells, while PaTu8988S cells highly expressed claudin-1, -2, -4, -7 and -12. Metastasis-associated expression was confirmed by studying the well defined PaTu SUIT-2 sublines characterized by differences in morphology, histological grade of differemiation and metastatic potentialin nude mouse xenografts. Immunlluorescence staining with cfaudin2 and TJ marker occludin antibodies localized daudin-2 primarily to the cytoplasm. Since activating K-ras mutations occur in a high percentage of PaTus and ras is thought to regulate the dynamics of cell-cell contacts, ras-transformed SUIT-2 cells were treated with PD98059 (50uM), a selective inhibitor of MEK1 activity and MAP kinase cascade.PD98059 treatment removed claudin-2 from intracellular granules and targeted claudin-2 to TJ sites. Conclusion: Interference in tight junctional communication might be an important molecular mechanism in the process of pancreatic cancer progression. This is supported by recent studies indicating that claudin-1 and -2 promote pro-MMP-2 activation.
M992 Genomic Analysis of Human Gastric ECL Cell Carcinoid Tumors Mark Kidd, Irvin M. Modbn, Shrikant Mane, Kevin D. Lye Introduction: The biological behavior of gastric ECL cell tumors remains an enigma and their clinical management a source of controversy and concern Biological strategies aimed at single pathways are not, however, effective in generating a molecular definition of disease processes. We postulated that examining global alterations in gene expression in different types of human ECL tissue would identify gene expression changes that occur during tumorigenesis. Methods: Gastric biopsy material from two patients was available for Affymetrix gene chip analysis One patient (male, 49 years) diagnosed with a gastrinoma and MENI syndrome (type II ECL tumor) and the second patient (female, 57 years) with a type I ECL cell tumor. Tissue samples from affected sites and normal adjacent mucosa were obtained. The patient with a type II disease had histologically confirmed ECL cell Dysplasia. Both patients were Helicobacter pylori negative and exhibited minimal gastritis. Total RNA was isolated from each sample (n = 4) using the RNeasy kit (Qiagen). Target cRNAs were generated and hybridized to human HU133A GeneChip sets (Affymetrix, Santa Clara, CA). The intensity was similarly scaled for each chip (intensity = 100) and those genes present in each sample identified using the GENECHIP software (MAS 5.0). Results: A range of 7,664-8,002 transcripts were identified as being present in the four cell samples. In the
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M995
LY293111 Improves Gemcitabine Efficacy in a New Fluorescent Orthotopic Model of Pancreatic Cancer in Athymic Mice Rene Hennig, Jacimhe Ventura, Rail Segersvard, Erin Ward, Xianahong Ding, Takeshi lwamura, Thomas E. Adrian Recently, we established a fluorescent orthotopic tumor model in nude mice that mimics the clinical behaviour of pancreatic cancer in humans. This model allows sensitive observation of primary tumor growth, metastases and angiogenesis over time. New therapeutic agents are desperately needed for this devastatmg disease. Leukotriene B4 (LTB4) receptors are overexpressed in human pancreatic cancer tissues and LTB4 stimulates pancreatic cancer growth. In contrast, the LTB4 receptor antagonist LY293111 markedly inhibits pancreatic
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cancer cell growth and induces apoptosis in vitro In the present study, we tested the effectiveness of LY293111 in vivo with dynamic monitoring of tumor progression. The welldifferentiated human pancreatic cancer cell line $2-0 I3, with stable expression of enhanced green fluorescent protein was used. Cells (Sx105) in 10 i~l MEM were injected into the duodenal lobe of the pancreas of athymic mice. The surgical procedure and weekly examinations were monitored using a stereo fluorescence microscope combined with an imaging system. Image quality was improved by a reversible skin-flap. We compared 4 groups with 8 mice/group: control (no treatment); LY293111; gemcitabine; LY293111 + gemcitabine. kY293111 was administered daily p.o. and gemcitabina injected 4 times i.p. Animals were euthanized 4 weeks after surgery. A staging system was developed to evaluate the tumor progression according to the TNM-classification. All 32 animals injected with $2-013 cells developed pancreatic cancer. Within 4 weeks, the control animals developed end-stage disease with invasive cancer obstructing the duodenum and bile duct, liver, lung and lymph node metastases, peritoneal carcinomatosis with malignant ascites and cachex~a. All treatment groups showed sigmficant inhibition of tumor growth and metastases. The combined treatment group with gemcitabine & LY293111 showed the greatest tumor suppression according to the scoring system, tumor weight and area. This cancer model allows sensitive detection of tumor growth, metastases and angiogenesis over time, mimics the clinical signs of pancreatic cancer in humans and has proven to be valuable for studying in vivo tumor behaviour and response to therapeutic agents. LY293111, especially in combination with gemcitahine, markedly inhibited cancer growth and prevented liver metastases. In conclusion, LY293111 is a new and promising therapeutic agent for adjuvant as well as palliative therapy of pancreatic adenocarcinoma.
M998 Comparison of Different Antiangiogenic Treatment Strategies for Experimental Human Pancreatic Cancer Hubert G. Hotz, Birgnt Hotz, Parkash S. Gill, Rizwan Masood, Oscar J. Hines, Howard A Rebel Heinz J. Bnhr Background: Inhibition of tumor ang~ogenesis is a novel treatment strategy for pancreatic cancer, which is almost resistant against radiocbemotherapy. This study compared three antiangaogenic therapies (blockade of vascular endothelial growth factor (VEGF) by an antisense oligonucleotide (AS-3), endothelial cell proliferation inhibition by the fumagilhn analogon TNP-470, and specific endothelial cell targeting by a diphtheria toxin-VEGF (DTVEGF) fusion protein) in pancreatic cancer in vitro and in vivo. Methods: In vitro: Human pancreatic cancer cells (AsPC-1) were exposed to increasing concentrations of the VEGF antisense oligonudeoride AS-3 (5'-TGG CTT GAA GAT GTA CTC GAT-T; 1 - i0 ~M), TNP-470 (1 pg/ml - 10fi ~g/ml), or DT-VEGF (VEGF165 fused with a 390 amino acid fragment of diphtheria toxin; 1 - 10000 ng/ml) Cell proliferation was assessed alter 3 days by cell count and MTT-assay. In vivo: 1 cubic mm fragments of se. AsPC-1 donor tumors were implanted into the pancreas of nude mice. Animals were randomized into control and 3 treatment groups: AS-3 (10 mg/kg, daily ip.), TNP-470 (30 mg/kg, every other day sc.), or DT-VEGF (200 Ixg/kg, every other day ip.) for 14 weeks or until death of the mice. Tumor volume (TU-Vol), local and metastatic spread (dissemination score; D-Score) were determined at autopsy. Microvessel density (MVD) was analyzed in CD31-stained tumor sections. Results: In vitro: Neither AS-3, nor TNP-470 affected proliferation of AsPC-1 ceils in physiologic concemrations. DT-VEGF inhibited AsPC-1 cell growth only at high (above 1000 ng/ml) concentrations. In vivo: table. Conclusions: All three therapies effect tumor growth, dissemination and surcival in a relevant orthmopic tumor model of human pancreatic cancer. In vitro data and reduced microvessel density indicate an antiangiogenic effect rather than a direct inhibition of pancreatic cancer ce]l growth Antiangiogenic therapy was not associated with systemic side effects such as weight loss in vivo.
M996 Treatment of Peritoneal Carcinomatosis using Genetically Modified Fibrohlasts to Express lnterleukin-12 in a Mouse Model of Secondary Pancreatic Cancer jean Marie Peron, Anny Souque, Christophe Bureau, Bettina Couderc
In vivo:
Background and aims : Pancreatic cancer is one of the most frequent cause of peritoneal carcinomatosis. Once ascites forms, the median survival time is less than 16 weeks. At this stage there is no curative treatment, lnterleukin- 12 (IL- 12) is one of the most potent antitumor cytokine. It is composed of 2 chains of 35 kD (p35) and 40 kD (p40). The aim of this study was to examine the antitumor effect of intraperitonea[ delivery of Interleukln- 12 using an ex vivo gene therapy approach in a murine model of peritoneal carcinomatosis of pancreatic origin. Methods : Syngenic fihrublasts (BALB/c) were genetically modified in vitro to express intefleukin-12 using a polycistmnic TFG murine interleukin-12 retroviral vector (TFGmIk12) coding for both p35 and p40 murine intefleukin-12 subunits. Neo transfected fibrublasts were used as controls. A clone of fibroblasts expressing 2 ng/million cellsf24h was used. Peritoneal carcinomatosis was generated by direct intraperitoneal inoculation of 500 000 Capan-1 cells (human pancreatic cancer tumor cell line) in Swiss nu/nu athymic mice. Ex vivo gene therapy consisted in injection of the genetically modified fibmblasts, BALWc-IL12 or BAkB/c-neo (5 million cells per injection) that were administered intraperitonealy twice a week. The treatment started 8 days after Capan-1 tumor cell injection as the peritoneal carcinomatosis was confirmed in control mice at that time. Fibroblasts injections were continued from then on until animals were sacrificed 41 days after initial Capan-1 cell injection. Results : A total of 22 animals were injected in 2 independent experiments, 11 in each group. There was no treatment related liver toxicity : serum liver enzymes levels (Bilirubine, AST, ALT, g-glutamyl transpeptidase, alkaline phosphatase) at the end of treatment were similar in the 2 groups. There was no ascites in animals treated by the IL-12 producing fibroblasts. On the contrary, 10 out of 11 animals treated with the Neo producing fibroblasts developped ascites (mean volume was 622 +/- 873 ml). Peritoneal carcinomatosis as measured by the weight of the peritoneal tumor nodules was significantly reduced in the animals treated with the IL-12 expressmg fibroblasts (0.93 g +/- 0.21 vs 3.52 g +/- 0.47 p < 0.05). Two animals died in the control group before the endpoint of the study.Conclusions : Experimental peritoneal carcinomatosis of pancreatic origin can be efficiently treated by ex vivo interle"ttkin-12 gene therapy.
"p
Control 1404+t-149 16.7+1-0.9 1/8 64.1+/- 4.4
AS.3 1046 +/- 81 6.5+/-0.8" 6/8' 33.2 +/- 2.3"
TNP470 918+/-152" 7.5+/-1.4" 3/8 26.0 +/- 3.4~
DT.VEGF 652+/-73" 8,344-1.1 ~ 6/8' 26,6 a'4-3,8*
M999 Suppression of the Malignant Phenotype in Pancreatic Cancer Cells by the Overexpression of Glutathione Peroxidase (GPx) Jingru Liu, Christine Weydert, Justine Ritchie, Larry Oberley, Joseph Cullen Background: Pancreatic cancer has low levels of antioxidant enzymes including glutathione peroxidase (GPx) (Pancreas, in press). Recent studies have demonstrated that overexpression of manganese superomde dismutase (MnSOD) has a tumor suppressive effect in pancreatic cancer (Cancer Res. in press). However, GPx overexpression has been shown to reverse the tumor cell growth inhibition caused by MnSOD overexpression in other types of cancer (Cancer Res. 60:3927, 2000). Aims: To determine if overexpression of GPx alters in vitro tumor cell behavior and if delivering the GPx gene directly to tumor xenografts alters growth and survival. Methods: M1A PaCa-2 cells were transfected with an adenovirus-GPx construct (AdGPx) (0 - 200 MOI), a adenovirus-MnSOD construct (AdMnSOD) (100 MOO, an AdGPx + AdMnSOD construct (100 MOI), or with AdLacZ (100 MOI), and cell growth and plating efficiency determined. 3% sucrose (controls), Adbgllf, AdGPx, AdMnSOD, or AdGPx + AdMnSOD were delivered by direct injection to tumor xenografts in nude mice. Results: The AdGPx and AdMnSOD adenoviral constructs increased immunoreactive protein and activity for GPx and MnSOD, respectively. In vitro, AdGPx and AdMnS,0D slowed tumor growth and decreased plating efficiency compared to AdLacZ (P<0.01). The AdGPx + AdMnSOO construct had the greatest effect on in vitro tumor growth and plating efficiency (P<0.00] vs AdLacZ, P < 0 0 5 vs AdGPx and AdMnSOD). In vivo, AdGPx and AdMnSOD groups had slower tumor growth when compared to controls (P<0.05). The AdGPx + AdMnSOD group had the greatest tumor suppresswe effect (P<0.0001) and had the longest survival (P<0.01 vs controls). Conclusions: GPx may be a tumor suppressor gene in pancreatic cancer. Delivery of the GPx gene alone or in combination with the MnSOD gene may prove beneficial for pancreatic cancer. Support: NIH grams DK 60618 and CA 66081.
M997 Decrease of Human Pancreatic Cancer Cell Tumorigenicity by AI,3galactosyltransferase Gene Transfer Muriel Aubert, Christian Crotte, Jean-Paul Bernard, Dominique Lombardo, Marie-Odile Sadoulet, Eric Mas The enzyme al,3galactosyltransferase synthesizes the aGal epitope, a carbohydrate structure (Gal al,3Gall3,4GlcNAc-R), on glycoconjugates in lower mammals. The enzyme is absent in humans but large amounts of natural antibodies which recognize aGal epitopes are present in human serum These antibodies likely contribute to the host defense and participate in the hyperacute rejection of xenograft. Previous studies indicated that the glycosyltransferase gene transfer into tumoral cells can modify the structure of gtycoconjugates at the cell surface and, as a consequence, modulates the metastatic and tumorigenic behaviors of these cells. The aim of this study was to determine whether the expression of aGal epitope can modify the tumorigenicity of human pancreatic cancer cells. The expression of ctGal epitopes in the human pancreatic cancer cell lines BxPC-3 and Pane-1 was obtained by selecting stable cell clones transfected with murine al,3galactosyhransferase gene. The expression of the enzyme activity in BxPC-3 and Pane-1 cells resulted in the formation at the cell surface of aGal epitopes which are recognized by human anti-aGal antibodies, aGal epitope expression at the surface of pancreatic cancer cells was associated with the fixation of complement lq to human anti-aGal antibodies. The aGal epitope expression also resulted in a delay in the tumoral development of BxPC-3 and Panc-i ceils in vivo after xenograft transplantation of nude mice. In addition to the impairment of the metastatic potential of routine tumor cell lines and the activation of immune response, our study provides evidence that the cell surface expression of aGal epitopes also modulates the tumorigenic behavior of human pancreatic cancer cells.
M1000 Adeno-Associated Virus Mediated Geae Transfer of Endostatin Inhibits Angiogenesis and Tumor Growth in u Christian Teschendorf, Wenyin Shi, Nicholas Muzyczka, Wolff Schmiegel, Dietmar W. Siemann Targeting tumor angiogenesis including the use of the antiangiogeinc protein endostatin is an attractive and virtually universally applicable treatment strategy for malignant tumors. Crucial for this type of approach is the extended suppression of angiogenensis. Employing endostatin or angiostatin for this purpose has been hampered by the difficulty to produce large amounts of biologically active protein. In the present study we evaluated whether f endostatm secreted from skeletal muscle transduced with recombinant adeno-associated virus (rAAV) can suppress angiogenesis and tumor growth in vivo. AAV is known to stably transduce several tissues including muscle and subsequently to lead to sustained expression of therapeutic proteins. In vitro human endostatin (HuEndo) suppressed both the proliferation and migration of endothelial cells at media endostatin concentrations of 50 ng/m]. In vivo, clones of the human colon cancer cell line HT29 engineered to express high levels of endostatin were found to induce significantly fewer blood vessels than did parental cells. Similarly, HT29 cells induced far fewer vessels in mice with elevated serum endostatin levels after injection of an endostatin producer cell line (293) than did tumor cells inoculated into normal mice The intramuscular injection of rAAV carrying a HuEndo expression cassette
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patient with a type I tumor, 26 genes were sigmficantly (>2-fold) up-regulated and 48 genes down-regulated. Twenty-ninc genes were significantly up-regulated and the same number down-regulated in the type II ECL cell sample. Shared gene pathways up-regulated in each tumor type included immune function (35%) and hematological (35%). Other genes up-regulated in both samples include the EGR genes (early growth response, + 2.6 - + 4.6fold, p < 0.0002) and down-regulated trefoil factor 1 (-2.1 - -1.9-fold, p < 0.0002). Genes significantly altered in the type I tumor included beta-microseminoprotem (-2.8-fold, p < 0.0002) and somatostatin (-2.5-fold, p < 0.00001) and in the type 11 tumor, v-los (+39.3fold, p < 0.00002) and fos-B (+ 29-fold, p < 0.0003). Conclusions: These data demonstrate that human ECL cell tumorigenesis has an important immunological component and a hematological component irrespective of the tumor type. Gastrin-responsive (type 1) tumors have up-regulation of early response genes, while type II tumors (gastrin-responsive, genomic alteration in MEN-I), have in addition altered regulation of genes involved in cell proliferation.
M990 Altered Function of KLF5 Is Associated with Intestinal Tumor Progression Nicholas W. Bateman, Dongfeng Tan, Jennifer D. Black, Adrian R. Black Members of the kroppel-like family of transcription factors (KLFs) have been linked to the regulation of cell growth and tumorigenesis in a number of systems. In the normal intestinal epithelium, KLF5 (intestinal-kruppel like factor/BTEB2) is expressed m growing cells of the lower crypt but is downregulated in non-dividing ceils of the upper crypt and villus/surface mucosa, suggesting that it has a growth promoting role in this tissue. In support of this idea, a direct positive growth regulatory role for KLF5 in fibrobfasts and vascular smooth muscle ceils has been reported. To evaluate the expression of KLF5 during intestinal tumorigenesis, RT-PCR was performed on laser-capture microdissected samples from intestinal adenomas and normal epithelium. This analysis showed reduced expression of KLF5 mRNA in rain mouse and human adenomatons polyposis adenornas; thus, while KLF5 may be growth promoting in the normal intestinal epithelium, it may also have a tumor suppressive function in this tissue. This possibility was further investigated using a normalintestinal crypt epithelial cell line (1EC-18) and colon tumor cell lines (Ward and DLD-1). Overexpression of KLF5 inhibited colony formation in the colon tumor cell lines but enhanced colony number in IEC-18 cells, indicating that KLF5 has opposing effects on cell growth and/or survival in normal and tumor-derived intestinal epithelial cells. Differences in the effects of KLF5 were also seen in transient transfection assays, where KLF5 enhanced expression of growthrelated genes in [EC-18 cells but not DLD-1 or Ward cells. Furthermore, while phorbol ester-induced growth arrest of IEC-18 cells led to downregulation of KLF5, consistent with the posiuve relationship between this factor and cell growth in the normal intestinal epithelium, growth arrest of tumor cell lines led to an upregulation of KLF5, indicating that this factor is negatively associated with growth in tumor cells. Collectively, these data indicate that KLF5 is growth promoting in the normal intestinal epithelium but is associated with growth inhibition in tumor cells. Thus, intestinal tumor progression appears to be associated with a change in the growth-related functions of KLF5. Supported by the Roswell Park Alliance Foundation and by NIH grants CA16056 and DK54909.
M993 RASSF1A May Serve as Novel Tumor Suppressor Gene in Gastrointestinal Malignancies Regulating Apoptosis Juliocesar Bernabe Ortiz, Shahrooz Rabizadeh, Kazuhiro Ishiguro, Marco Lopez-Ilasaca, Andrei Khokhlatchev, Joseph Avruch, Brian Seed, Ramnik Xavier Background: A previous search for genes that map to regions of loss of heterozygosity on chromosome 3p21 as well as a yeast two hybrid screen led to the discovery of RASSF1A, a protein characterized by a Ras binding domain. RASSFIA was found to be silenced through promoter hypermethyfation in lung, prostate, colorectal, gastric and biliary tumors. This gene encodes a protein with several interesting domains, including one that potentially imeracts with the Ras family of proteins, a domain that is phosphorylated via the ataxiatelangiectasia (ATM) kinase pathway, and a DAG binding domain that may link it to tumor promoting activities. The aim of the present study was to elucidate the function of RASSF1A in intestinal epithelial cell lines. Methods and Results: In a screen of cancer cell lines, RASSFIA mRNA levels were found to be reduced when compared to normal human epithelial cells and fibroblasts. Functional analysis in epithelial cell lines revealed that RASSF1Aassociated complexes promote cell death. Microscopic studies showed that RASSF1A associates with microtubule structures to form an elaborate network in the cytosol of transformed intestinal epithelial cell lines. A yeast two-hybrid screen resulted in identification of a novel regulator of RASSF1A-mediated apoptosis. Structure-function studies demonstrated that key domains in RASSFIA mediate cell death. Conclusion: Taken together, these studies suggest that RASSEIA functions as a novel tumor suppressor in gastrointestinal epithehal malignancies.
M991 Nonsteroidal Anti-lnflammatory Drugs Inhibit Human Telomerase Reverse Transcriptase (hTERT) in Gastric and Colon Cancer Xiaohua Jiang, Marie C. M. Lin, Shiukum Lam, Hua He, Shuiping Tu, Hsianglu Kung, Benjamin C. Wong
M994 Recruitment of Claudin-2 to Tight Junctions by Inhibition of the Mitogenactivated Protein Kinase (MAPK) Pathway in ras-transforraed Pancreatic Cancer Cells Tillmann Bert, Stephanie Hoormann, Nadine Aurbek, Anskar 5chmidt, Babette Simon
Background and Aims: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase complex with reverse transcriptase activity, hTERT activation is observed in 90% of human cancers and is a critical step for carcinogenesis. The present study is aimed to investigate the effect of Nonsteroidal anti-inflammatory drugs (NSAIDs) on hTERT regulation. Materials and Methods: hTERT mRNA and protein expression in both gastric cancer and colon cancer cell lines were determined by RT-PCR and Western blotting. hTERT transcriptional activity was detected by transfection with different hTERT promoterluciferase constructs and luciferase assay. Results: We demonstrated that NSAIDs (including aspirin and the cyclooxygenase-2 specific inhibitors) suppressed hTERT gene expression at both mRNA and protein level NSAIDs also inhibited telomerase enzymatic activity in both human colon and gastric cancer cells. Using a promoter-luciferase construct containing a 283-bp upstream ATG in the hTERT promoter, we showed that NSAIDs st~ppressed hTERT transcriptional activity significantly, which suggested that the down-regulation of the telomerase activity and hTERT expression by NSAIDs was due to transcriptional repression of the hTERT gene. In addition, we used reporter plasmid which contained point mutations (CACGTG-TTTGTG) in each of the two E-box, located at -165 and -44 relative to the ATG initiation codon. Our results showed that mutation of E-box did not have any effect on NSA1Ds-mediated inhibition of bTERT promoter activity. Conclusion: NSAIDs regulate bTERT expression at transcriptional level. The inhibition was independent of E-box. The molecular mechanism(s) for NSAIDs-medlated inhibitory effect on hTERT needs further investigation
Background: To identify novel therapeutic targets for pancreatic cancer (PaTu) we isolated differentially expressed cDNA fragments of the metastasizing cell line PaTu8988S and nonmetastasizing cell line PaTu8988T using cDNA-represemational difference analysis (cDNARDA). Methods and Results: One of the isolated cDNA fragments revealed homology to cfaudin-2, a member of a tight junction (TJ) protein family. We cloned the full length gene with an ORF of 693 bp encoding a protein of 230 amino acids with a calculated MW of 24 kDa. RT-PCR and northern blotting verified dilferemial claudin-2 expression revealing a 3.5 kb primary transcript. Cfaudin-2 was expressed in 70% of human PaTu cell lines analysed. Genomic studies revealed an intronless claudin-2 gene and excluded homozygons deletion in PaTu8988T cells. Radiation hybrid mapping localized claudin-2 at Xq22.3-23. Expression profiling using a human multiple tissue array demonstrated a distinct organspecific distribution pattern of claudin-2. Important mechanisms of cellular regulation in epithelial tissues are mediated through intercellular junctional communication. Since little is known about junctional assembly during transformation we analysed the expression of distinct claudins (1 to 12) by RT-PCR. There was almost exclusive claudin-1 expression in PaTu8988T cells, while PaTu8988S cells highly expressed claudin-1, -2, -4, -7 and -12. Metastasis-associated expression was confirmed by studying the well defined PaTu SUIT-2 sublines characterized by differences in morphology, histological grade of differemiation and metastatic potentialin nude mouse xenografts. Immunlluorescence staining with cfaudin2 and TJ marker occludin antibodies localized daudin-2 primarily to the cytoplasm. Since activating K-ras mutations occur in a high percentage of PaTus and ras is thought to regulate the dynamics of cell-cell contacts, ras-transformed SUIT-2 cells were treated with PD98059 (50uM), a selective inhibitor of MEK1 activity and MAP kinase cascade.PD98059 treatment removed claudin-2 from intracellular granules and targeted claudin-2 to TJ sites. Conclusion: Interference in tight junctional communication might be an important molecular mechanism in the process of pancreatic cancer progression. This is supported by recent studies indicating that claudin-1 and -2 promote pro-MMP-2 activation.
M992 Genomic Analysis of Human Gastric ECL Cell Carcinoid Tumors Mark Kidd, Irvin M. Modbn, Shrikant Mane, Kevin D. Lye Introduction: The biological behavior of gastric ECL cell tumors remains an enigma and their clinical management a source of controversy and concern Biological strategies aimed at single pathways are not, however, effective in generating a molecular definition of disease processes. We postulated that examining global alterations in gene expression in different types of human ECL tissue would identify gene expression changes that occur during tumorigenesis. Methods: Gastric biopsy material from two patients was available for Affymetrix gene chip analysis One patient (male, 49 years) diagnosed with a gastrinoma and MENI syndrome (type II ECL tumor) and the second patient (female, 57 years) with a type I ECL cell tumor. Tissue samples from affected sites and normal adjacent mucosa were obtained. The patient with a type II disease had histologically confirmed ECL cell Dysplasia. Both patients were Helicobacter pylori negative and exhibited minimal gastritis. Total RNA was isolated from each sample (n = 4) using the RNeasy kit (Qiagen). Target cRNAs were generated and hybridized to human HU133A GeneChip sets (Affymetrix, Santa Clara, CA). The intensity was similarly scaled for each chip (intensity = 100) and those genes present in each sample identified using the GENECHIP software (MAS 5.0). Results: A range of 7,664-8,002 transcripts were identified as being present in the four cell samples. In the
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M995
LY293111 Improves Gemcitabine Efficacy in a New Fluorescent Orthotopic Model of Pancreatic Cancer in Athymic Mice Rene Hennig, Jacimhe Ventura, Rail Segersvard, Erin Ward, Xianahong Ding, Takeshi lwamura, Thomas E. Adrian Recently, we established a fluorescent orthotopic tumor model in nude mice that mimics the clinical behaviour of pancreatic cancer in humans. This model allows sensitive observation of primary tumor growth, metastases and angiogenesis over time. New therapeutic agents are desperately needed for this devastatmg disease. Leukotriene B4 (LTB4) receptors are overexpressed in human pancreatic cancer tissues and LTB4 stimulates pancreatic cancer growth. In contrast, the LTB4 receptor antagonist LY293111 markedly inhibits pancreatic
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cancer cell growth and induces apoptosis in vitro In the present study, we tested the effectiveness of LY293111 in vivo with dynamic monitoring of tumor progression. The welldifferentiated human pancreatic cancer cell line $2-0 I3, with stable expression of enhanced green fluorescent protein was used. Cells (Sx105) in 10 i~l MEM were injected into the duodenal lobe of the pancreas of athymic mice. The surgical procedure and weekly examinations were monitored using a stereo fluorescence microscope combined with an imaging system. Image quality was improved by a reversible skin-flap. We compared 4 groups with 8 mice/group: control (no treatment); LY293111; gemcitabine; LY293111 + gemcitabine. kY293111 was administered daily p.o. and gemcitabina injected 4 times i.p. Animals were euthanized 4 weeks after surgery. A staging system was developed to evaluate the tumor progression according to the TNM-classification. All 32 animals injected with $2-013 cells developed pancreatic cancer. Within 4 weeks, the control animals developed end-stage disease with invasive cancer obstructing the duodenum and bile duct, liver, lung and lymph node metastases, peritoneal carcinomatosis with malignant ascites and cachex~a. All treatment groups showed sigmficant inhibition of tumor growth and metastases. The combined treatment group with gemcitabine & LY293111 showed the greatest tumor suppression according to the scoring system, tumor weight and area. This cancer model allows sensitive detection of tumor growth, metastases and angiogenesis over time, mimics the clinical signs of pancreatic cancer in humans and has proven to be valuable for studying in vivo tumor behaviour and response to therapeutic agents. LY293111, especially in combination with gemcitahine, markedly inhibited cancer growth and prevented liver metastases. In conclusion, LY293111 is a new and promising therapeutic agent for adjuvant as well as palliative therapy of pancreatic adenocarcinoma.
M998 Comparison of Different Antiangiogenic Treatment Strategies for Experimental Human Pancreatic Cancer Hubert G. Hotz, Birgnt Hotz, Parkash S. Gill, Rizwan Masood, Oscar J. Hines, Howard A Rebel Heinz J. Bnhr Background: Inhibition of tumor ang~ogenesis is a novel treatment strategy for pancreatic cancer, which is almost resistant against radiocbemotherapy. This study compared three antiangaogenic therapies (blockade of vascular endothelial growth factor (VEGF) by an antisense oligonucleotide (AS-3), endothelial cell proliferation inhibition by the fumagilhn analogon TNP-470, and specific endothelial cell targeting by a diphtheria toxin-VEGF (DTVEGF) fusion protein) in pancreatic cancer in vitro and in vivo. Methods: In vitro: Human pancreatic cancer cells (AsPC-1) were exposed to increasing concentrations of the VEGF antisense oligonudeoride AS-3 (5'-TGG CTT GAA GAT GTA CTC GAT-T; 1 - i0 ~M), TNP-470 (1 pg/ml - 10fi ~g/ml), or DT-VEGF (VEGF165 fused with a 390 amino acid fragment of diphtheria toxin; 1 - 10000 ng/ml) Cell proliferation was assessed alter 3 days by cell count and MTT-assay. In vivo: 1 cubic mm fragments of se. AsPC-1 donor tumors were implanted into the pancreas of nude mice. Animals were randomized into control and 3 treatment groups: AS-3 (10 mg/kg, daily ip.), TNP-470 (30 mg/kg, every other day sc.), or DT-VEGF (200 Ixg/kg, every other day ip.) for 14 weeks or until death of the mice. Tumor volume (TU-Vol), local and metastatic spread (dissemination score; D-Score) were determined at autopsy. Microvessel density (MVD) was analyzed in CD31-stained tumor sections. Results: In vitro: Neither AS-3, nor TNP-470 affected proliferation of AsPC-1 ceils in physiologic concemrations. DT-VEGF inhibited AsPC-1 cell growth only at high (above 1000 ng/ml) concentrations. In vivo: table. Conclusions: All three therapies effect tumor growth, dissemination and surcival in a relevant orthmopic tumor model of human pancreatic cancer. In vitro data and reduced microvessel density indicate an antiangiogenic effect rather than a direct inhibition of pancreatic cancer ce]l growth Antiangiogenic therapy was not associated with systemic side effects such as weight loss in vivo.
M996 Treatment of Peritoneal Carcinomatosis using Genetically Modified Fibrohlasts to Express lnterleukin-12 in a Mouse Model of Secondary Pancreatic Cancer jean Marie Peron, Anny Souque, Christophe Bureau, Bettina Couderc
In vivo:
Background and aims : Pancreatic cancer is one of the most frequent cause of peritoneal carcinomatosis. Once ascites forms, the median survival time is less than 16 weeks. At this stage there is no curative treatment, lnterleukin- 12 (IL- 12) is one of the most potent antitumor cytokine. It is composed of 2 chains of 35 kD (p35) and 40 kD (p40). The aim of this study was to examine the antitumor effect of intraperitonea[ delivery of Interleukln- 12 using an ex vivo gene therapy approach in a murine model of peritoneal carcinomatosis of pancreatic origin. Methods : Syngenic fihrublasts (BALB/c) were genetically modified in vitro to express intefleukin-12 using a polycistmnic TFG murine interleukin-12 retroviral vector (TFGmIk12) coding for both p35 and p40 murine intefleukin-12 subunits. Neo transfected fibrublasts were used as controls. A clone of fibroblasts expressing 2 ng/million cellsf24h was used. Peritoneal carcinomatosis was generated by direct intraperitoneal inoculation of 500 000 Capan-1 cells (human pancreatic cancer tumor cell line) in Swiss nu/nu athymic mice. Ex vivo gene therapy consisted in injection of the genetically modified fibmblasts, BALWc-IL12 or BAkB/c-neo (5 million cells per injection) that were administered intraperitonealy twice a week. The treatment started 8 days after Capan-1 tumor cell injection as the peritoneal carcinomatosis was confirmed in control mice at that time. Fibroblasts injections were continued from then on until animals were sacrificed 41 days after initial Capan-1 cell injection. Results : A total of 22 animals were injected in 2 independent experiments, 11 in each group. There was no treatment related liver toxicity : serum liver enzymes levels (Bilirubine, AST, ALT, g-glutamyl transpeptidase, alkaline phosphatase) at the end of treatment were similar in the 2 groups. There was no ascites in animals treated by the IL-12 producing fibroblasts. On the contrary, 10 out of 11 animals treated with the Neo producing fibroblasts developped ascites (mean volume was 622 +/- 873 ml). Peritoneal carcinomatosis as measured by the weight of the peritoneal tumor nodules was significantly reduced in the animals treated with the IL-12 expressmg fibroblasts (0.93 g +/- 0.21 vs 3.52 g +/- 0.47 p < 0.05). Two animals died in the control group before the endpoint of the study.Conclusions : Experimental peritoneal carcinomatosis of pancreatic origin can be efficiently treated by ex vivo interle"ttkin-12 gene therapy.
"p
Control 1404+t-149 16.7+1-0.9 1/8 64.1+/- 4.4
AS.3 1046 +/- 81 6.5+/-0.8" 6/8' 33.2 +/- 2.3"
TNP470 918+/-152" 7.5+/-1.4" 3/8 26.0 +/- 3.4~
DT.VEGF 652+/-73" 8,344-1.1 ~ 6/8' 26,6 a'4-3,8*
M999 Suppression of the Malignant Phenotype in Pancreatic Cancer Cells by the Overexpression of Glutathione Peroxidase (GPx) Jingru Liu, Christine Weydert, Justine Ritchie, Larry Oberley, Joseph Cullen Background: Pancreatic cancer has low levels of antioxidant enzymes including glutathione peroxidase (GPx) (Pancreas, in press). Recent studies have demonstrated that overexpression of manganese superomde dismutase (MnSOD) has a tumor suppressive effect in pancreatic cancer (Cancer Res. in press). However, GPx overexpression has been shown to reverse the tumor cell growth inhibition caused by MnSOD overexpression in other types of cancer (Cancer Res. 60:3927, 2000). Aims: To determine if overexpression of GPx alters in vitro tumor cell behavior and if delivering the GPx gene directly to tumor xenografts alters growth and survival. Methods: M1A PaCa-2 cells were transfected with an adenovirus-GPx construct (AdGPx) (0 - 200 MOI), a adenovirus-MnSOD construct (AdMnSOD) (100 MOO, an AdGPx + AdMnSOD construct (100 MOI), or with AdLacZ (100 MOI), and cell growth and plating efficiency determined. 3% sucrose (controls), Adbgllf, AdGPx, AdMnSOD, or AdGPx + AdMnSOD were delivered by direct injection to tumor xenografts in nude mice. Results: The AdGPx and AdMnSOD adenoviral constructs increased immunoreactive protein and activity for GPx and MnSOD, respectively. In vitro, AdGPx and AdMnS,0D slowed tumor growth and decreased plating efficiency compared to AdLacZ (P<0.01). The AdGPx + AdMnSOO construct had the greatest effect on in vitro tumor growth and plating efficiency (P<0.00] vs AdLacZ, P < 0 0 5 vs AdGPx and AdMnSOD). In vivo, AdGPx and AdMnSOD groups had slower tumor growth when compared to controls (P<0.05). The AdGPx + AdMnSOD group had the greatest tumor suppresswe effect (P<0.0001) and had the longest survival (P<0.01 vs controls). Conclusions: GPx may be a tumor suppressor gene in pancreatic cancer. Delivery of the GPx gene alone or in combination with the MnSOD gene may prove beneficial for pancreatic cancer. Support: NIH grams DK 60618 and CA 66081.
M997 Decrease of Human Pancreatic Cancer Cell Tumorigenicity by AI,3galactosyltransferase Gene Transfer Muriel Aubert, Christian Crotte, Jean-Paul Bernard, Dominique Lombardo, Marie-Odile Sadoulet, Eric Mas The enzyme al,3galactosyltransferase synthesizes the aGal epitope, a carbohydrate structure (Gal al,3Gall3,4GlcNAc-R), on glycoconjugates in lower mammals. The enzyme is absent in humans but large amounts of natural antibodies which recognize aGal epitopes are present in human serum These antibodies likely contribute to the host defense and participate in the hyperacute rejection of xenograft. Previous studies indicated that the glycosyltransferase gene transfer into tumoral cells can modify the structure of gtycoconjugates at the cell surface and, as a consequence, modulates the metastatic and tumorigenic behaviors of these cells. The aim of this study was to determine whether the expression of aGal epitope can modify the tumorigenicity of human pancreatic cancer cells. The expression of ctGal epitopes in the human pancreatic cancer cell lines BxPC-3 and Pane-1 was obtained by selecting stable cell clones transfected with murine al,3galactosyhransferase gene. The expression of the enzyme activity in BxPC-3 and Pane-1 cells resulted in the formation at the cell surface of aGal epitopes which are recognized by human anti-aGal antibodies, aGal epitope expression at the surface of pancreatic cancer cells was associated with the fixation of complement lq to human anti-aGal antibodies. The aGal epitope expression also resulted in a delay in the tumoral development of BxPC-3 and Panc-i ceils in vivo after xenograft transplantation of nude mice. In addition to the impairment of the metastatic potential of routine tumor cell lines and the activation of immune response, our study provides evidence that the cell surface expression of aGal epitopes also modulates the tumorigenic behavior of human pancreatic cancer cells.
M1000 Adeno-Associated Virus Mediated Geae Transfer of Endostatin Inhibits Angiogenesis and Tumor Growth in u Christian Teschendorf, Wenyin Shi, Nicholas Muzyczka, Wolff Schmiegel, Dietmar W. Siemann Targeting tumor angiogenesis including the use of the antiangiogeinc protein endostatin is an attractive and virtually universally applicable treatment strategy for malignant tumors. Crucial for this type of approach is the extended suppression of angiogenensis. Employing endostatin or angiostatin for this purpose has been hampered by the difficulty to produce large amounts of biologically active protein. In the present study we evaluated whether f endostatm secreted from skeletal muscle transduced with recombinant adeno-associated virus (rAAV) can suppress angiogenesis and tumor growth in vivo. AAV is known to stably transduce several tissues including muscle and subsequently to lead to sustained expression of therapeutic proteins. In vitro human endostatin (HuEndo) suppressed both the proliferation and migration of endothelial cells at media endostatin concentrations of 50 ng/m]. In vivo, clones of the human colon cancer cell line HT29 engineered to express high levels of endostatin were found to induce significantly fewer blood vessels than did parental cells. Similarly, HT29 cells induced far fewer vessels in mice with elevated serum endostatin levels after injection of an endostatin producer cell line (293) than did tumor cells inoculated into normal mice The intramuscular injection of rAAV carrying a HuEndo expression cassette
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