The expression of notch family genes in human pancratic cancer

reintroduction of p16 in the human pancreatic cancer cell line Capanl reversed anoikisresistance and tumorigenicity in nude mice, properties commonly attributed to the transforming potential of oncogenic Ras. In an attempt to delineate events that link p16 restitution to anoikis, we here addressed the role of K-Ras. Methods: Anoikis was measured by flowcytometry. Ras isoforms were then detected by immunoblotting with isoform-specific antibodies and GTP-bound Ras was selectively precipitated to determine Ras activity. Ras function was modulated via stable overexpression of K-ras or transfection of K-ras antisense-oligonucleotides. Results: K-Ras activity accounted for most of overall Ras activity in Capanl cells. When subjected to suspension culture for 20 hours, Capan-1 control cells (wild-type, mock) displayed a pronounced increase in K-Ras activity. In contrast, no K-Ras activity could be detected in p16 reconstituted cells (Capan-1~16) grown either in suspension or on substrate. The loss of K-Ras activity corresponded to a dramatic reduction of K-Ras expression in p16 restituted cells. To address the functional relevance of K-Ras regulation for anoikis induction, Capanl cells were treated with K-ras antisense-oligonucleotides. This approach produced a moderate reduction of K-Ras expression and increased anoikis. Inversely, restitution of K-Ras in Capan-1/p16 cells reversed the anoikis-sensitive phenotype. Conclusion: Suppression of K-Ras activity is required for p16 mediated anoikis induction in Capan-1 cells. These results establish a functional link between the two most common genetic alterations in pancreatic cancer and provide a basis for the high frequency of joint alterations of p16 and K-ras

this work was to demonstrate that following the intracellular degradation of FAPP, the Cterminal domain of the protein was exposed to the surface of pancreatic tumoral cells. In the present study, we have shown that monoclonal antibodies to oncofctal glycotopes of the mucin-fike C-terminal domain of FAPP decorated the surface of SOJ-6 cells. These glycotopes were associated with a membrane 32 kDa peptide which did not react with polyclonal antibodies to the N-terminal domain of FAPP. This peptide represented the endproduct of cellular FAPP degradation and corresponded to the O-glycosylated C-terminal domain of FAPP. Geldauamycin, a drug that disrupts the interaction between the glucoseregulated 94 kDa protein and its substrate increased the amount of 32 kDa peptide presented at the cell surface. These results demonstrate that the 32 kDa mucin-like domain of FAPP is presented at the surface of pancreatic tumoral cells. The possibility to obtain recombinant structures of this mucin-like peptide and the availability of monoclonal antibodies to these structures may be promiaing tools in immunological approaches to cure pancreatic cancers.

M1012

15-deoxy-A~2,~4-prostaglandin J~ Stimulates Cathepsin-L Expression In Human Pancreatic Cancer Cells through Multiple Pathways Guido Eibl, Howard A. Reber, Yuji Okada, John P. Dully, Oscar J. Hines Elevated serum levels of the cysteine proteinase cathepsiu-L can be found in patients with ductal adenoearciuoma of the pancreas. We have previously shown that 15-deoxy-A L2'14prostaglandin J2 (PGJ2) stimulates expression of cathepsiu-L in the human pancreatic cancer (PaCa) cell line MIA PaCa-2 independent of PPAR-'y. in the present study we investigated the effect of PGJ2 on various PaCa cell lines and the intracellular pathways involved. Methods: The effects of PGJ2 on cathepsin-L protein expression in human pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1) were determined by immunoblottiug. In another series cells were pre-treated with PD98059 (MEK-1 inhibitor), SB203580 (p38 inhibitor), PG-490 (NF-KB transactivation inhibitor), H-89 (PKA inhibitor), and Ro 31-8220 (PKC inhibitor) followed by PGJ2. Cathepsin-L protein expression was quantified by laser densitometry and ImageQuaNT software. Results: Pro-eathepsin-L (~40kDa) and active cathepsin-L (~25kDa) were detectable in all 6 PaCa cell lines. PGJ2 dose-dependently stimulated the expression of pro-cathepsin-L after 6 hours, while after 24 hours both the pro-form and the mature cathepsiu-L were increased in all 6 cell lines (3.0 to 8.4 fold with IO~M PGJ2). Pre-incubation with PD98059 (20~M) attenuated the effect of PGJ2 on catbepsin-k protein expression (relative increase in cathepsin-k expression in M1A PaCa-2 cells compared to control: i.4 fold with PD90059+ IO~,M PGJ2 vs 3.7 fold with vehicle + 10~M PGJ2). Cathepsin-L protein was barely detectable in M1A PaCa-2 cells pre-treated with Ro 31-8220 (201zM) or PG-490 (200ng/ml). The PKA inhibitor H-89 (10~M) and the p38 inhibitor SB203580 (10g,M) had no effect. Conclusion: PGJ2 doseand time dependently stimulates cathepsin-L expression independent of PPAR-"/ through multiple intracellular pathways, including p42/44 MAPK, PKC and NF-gB. PKA and p38 MAPK appear not to be involved.

MI015

The Expression of Notch Family Genes in Human Pancreatic Cancer Noriaki Suzuki, Kenji Kimura, Tooru Shimosegawa Background: Notch signaling is implicated in regulating differentiation, proliferation and apoptosis in various mammalian cells. Notch receptors have been reported to be upregulated in embryonic developing pancreas. On the other hand, the extra cellular deletion of Notch receptors, truncated Notch, has crucial role in neoplastic pathway in T-cell leukemia, mammary cancer and colon cancer. However, the role of Notch family genes in pancreatic cancer is largely unknown. Aims: To clarify the role of Notch-related genes in pancreatic cancer. Methods: MIA Paca-2 and BxPC-3, human pancreatic cell lines were cultured in condition media containing retinoic acid (RA), known as a differentiation promoting factor of various tumor cells. The mRNA expression of Notch 1, 2, 4, Jagged 1 and HES 1 were examined by RT-PCR. Protein expression of these molecules was examined by immunohistochemistry on human pancreatic cancer tissues. Results: Notch i and 2 mRNAs were expressed in both M1A Paca-2 and BxPC-3 grown in the absence of RA, but Notch 4 mRNA was not expressed in these. Jagged 1 and HES 1 mRNA were also expressed in these cells. The morphology of BxPC-3 was converted to duct like appearance by adding RA in the condition media. Carbonic anhydrase l[ mRNA, a ductal phenotype, was expressed in the ductal cell-like BxPC-3. Notch i and 2 mRNA were decreased in this condition. Notch ] protein was expressed in dysplastic ductal epithebal cells and tubular complexes in human pancreatic cancer tissues, fmmunoreactivity of Notch 1 was observed even in tubular complexes in non-tumorous fibrotic area. Notch 2 immunoreactivity was also observed in dysplastic ductal epithelial ceils but it was much weaker than that of Notch 1. Notch 2 was intensely expressed in islets of Langerhans. Notch 4 immunoreactivity was very faint or absent. Normal ductal cells and acinar cells showed undetectable or barely detectable Notch 1, 2 and 4 staining. Conclusion: Notch signaling is activated in neoplastic ductal epithelial cells in the pancreas. Notch could be implicated in cell fate decision from benign to malignant phenotype and regenerative processes in pancreatic cancer. Our data suggest that Notch-related gene might be a novel therapeutic target in pancreatic cancer.

M1013

Proliferative Effects of ANP on Pancreatic Cancer Cells Are Mediated by Natriuretic Peptide Receptor Subtypes A and C William R. Gower Jr., Suzanne M. Solivan, Quentin W. McAfee, Gay M. Carter Background: Atrial natriuretic peptide (ANP) acting through its specific receptor subtypes A (NPR-A) and C (NPR-C) is known to affect the proliferation of a variety of cell types. Recent evidence suggests that both NPR-A and NPR-C are expressed in pancreas. The aims of this study were to determine if receptors for ANP are expressed in pancreatic cancer cells and to examine the effect of ANP on pancreatic cancer cell proliferation. Methods: Pancreatic cancer cell lines (AR42J, HPAC, HPAF, PANC-1, MiaPaCa-2, AsPc-1, CAPAN-1) were examined for the expression of NPR-A and NPR-C transcripts by RT-PCR and protein by Western blot and immunohistochemical analyses. Cyclic GMP (cGMP) was measured by RIA. Cell proliferation was measured by 3H-thym]dine (3H-Thd) incorporation. Results: Human ductal adenocarcinoma cell lines differentially expressed NPR-A and NPR-C gene products. MiaPaCa-2 and AsPc-1 expressed predominantly NPR-A. HPAF expressed both NPR-A and NPR-C. CAPAN, PANC-1 and HPAC expressed primarily NPR-C. The rat pancreatic acinar cell line AR42J expressed primarily NPR-C. ANP (0.1nM-I.0~M) caused a dose-dependant inhibition of 3H-Thd incorporation in HPAF, AsPc-1, MiaPaCa-2, PANC-1 and HPAC cells. Maximal effect was observed at 1.0g~M and ranged from i8 to 32% inhibition vs. control (P
M1016 Novel Mechanism of Nitrosarive Stress from Dietary Nitrate Relevant to Gastroesophageal Junction Cancer Katsunori Iijima, Jeanette Grant, Kenny McElroy, Sharon Anderson, Valerie Fyfe, Stuart Paterson, Kenneth E. L. McColl Abstract: High concentrations of nitric oxide are generated at the gastroesophageal junction due to the reduction of salivary nitrite to nitric oxide by acidic gastric juice containing ascorbic acid. Salivary nitrite is derived from the enterosalivary recirculation of dietary nitrate and its reduction to nitrite by buccal bacteria. Aims: To determine whether nitric oxide generated in the above way will exert nitrosative stress on the adjacent epithelium. Methods: A benchtop model was constructed reproducing the chemistry occurring at the gastroesophageal junction including a lumen and an adjacent epithelial compartment. The latter was maintained at pH7.4 and separated from the acidic lumen by a thin hydrophobic barrier with the permeability properties of the epithelial cell membrane. The secondary amine morpholine was added to each compartment and N-nitrosomorpholine formation at 15 min measured. Results: Adding 100uM nitrite to the acidic (pill.5) luminal compartment in the absence of ascorbic acid generated 6.2 + 2.0uM (mean + SE) N-nitrosomorpholine in that compartment and 2.2 + O.luM in the adjacent epithelial compartment. When lOOuM nitrite was added to the acidic luminal compartment (pH 1.5) containing ascorbic acid, all the nitrite was immediately converted to nitric oxide and no N-iutrusomorpholine was formed within that compartment. However, the nitric oxide rapidly diffused into the adjacent epithelial compartment (pH 7.4) where it generated very high concentrations of N-nitrosomorpholine (137 + 5.6uM). The concentrations of N-nitrosomorpholine generated in the epithelial compartment decreased with increasing luminal pH being 9SuM at pH2.5, 65uM at pH3.5 and 12uM at pH4.5. The concentration of N-nitrosomorpholine generated in the epithelial compartment was directly related to the nitric oxide concentration in the lumen. The addition of ascorbic acid or ghitathione to the epithelial compartment could only reduce this nitric oxide induced nitrosation within the epithelial compartment by 40%. Conclusion: Ascorbic acid in gastric juice prevents acid-catalysed nitrosation within the gastric lumen. However, in doing so, it generates nitric oxide which exerts a far higher nitrosative stress on the adjacent epithelium. This mechanism is likely to be relevant to the aetiology of mutagenesis and neopfasia at the gastroesophageal junctio

M1014 K-Ras Downregulation Is Required for p16 jNK~Mediated Anoikis Induction in Capan-I Pancreatic Cancer Cells Anja Rabien, Bertram Wiedenmann, Stefan Rosewicz, Katharina M. Detjen Background and Aim: Pancreatic cancer is characterized by a set of genetic alterations. Most common are the oncogcnic activation of K-Ras and the inactivation of the cell cycle inhibitor p16rNK~(p16), which occur in over 90% of cases. We have previously demonstrated that

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