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Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by in vivo and in vitro data. Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.

Gemcitabine is widely used as first-line chemotherapeutic drug in the treatment of pancreatic cancer. Our previous experimental chemotherapy studies have shown that treatment of human pancreatic carcinoma cells with 5-fluorouracil (5-FU) alters the cellular transporter expression profile and that modulation of the expression of multidrug resistance protein 5 (MRP5; ABCC5) influences the chemoresistance of these tumor cells. Here, we studied the influence of acute and chronic gemcitabine treatment on the expression of relevant uptake and export transporters in pancreatic carcinoma cells by reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunoblot analyses. The specific role of MRP5 in cellular gemcitabine sensitivity was studied by cytotoxicity assays using MRP5-overexpressing and MRP5-silenced cells. Exposure to gemcitabine (12 nM for 3 days) did not alter the messenger RNA (mRNA) expression of MRP1, MRP3, MRP5, and equilibrative nucleoside transporter 1 (ENT1), whereas high dosages of the drug (20 microM for 1 hour) elicited up-regulation of these transporters in most cell lines studied. In cells with acquired gemcitabine resistance (up to 160 nM gemcitabine), the mRNA or protein expression of the gemcitabine transporters MRP5 and ENT1 was upregulated in several cell lines. Combined treatment with 5-FU and gemcitabine caused a 5- to 40-fold increase in MRP5 and ENT1 expressions. Cytotoxicity assays using either MRP5-overexpressing (HEK and PANC-1) or MRP5-silenced (PANC1/shMRP5) cells indicated that MRP5 contributes to gemcitabine resistance. Thus, our novel data not only on drug-induced alterations of transporter expression relevant for gemcitabine uptake and export but also on the link between gemcitabine sensitivity and MRP5 expression may lead to improved strategies of future chemotherapy regimens using gemcitabine in pancreatic carcinoma patients.

Objectives: Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP., Methods: To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis., Results: Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen., Conclusions: These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.

Background: Helicobacter pylori has been suggested to be involved in pancreatic diseases, namely autoimmune pancreatitis and pancreatic carcinoma. We investigated the presence of conserved sequences of Helicobacter in pancreatic tissue and pancreatic juice from patients with chronic nonautoimmune and autoimmune pancreatitis as well as pancreatic ductal adenocarcinoma (PDAC)., Methods: 35 pancreatic juices collected during routine endoscopic retrograde cholangiopancreatography and 30 pancreatic tissues were studied. Nested PCR was used to detect H. pylori in the isolated DNA samples. In order to exclude a methodological bias, the samples were analyzed blindly in 2 different laboratories using either conventional or LightCycler PCR for H. pylori urease A and 16S ribosomal DNA., Results: In the pancreas of 11 patients with autoimmune pancreatitis, no H. pylori DNA could be detected. Further, in none of the other tissue samples of chronic pancreatitis or PDAC could we detect any Helicobacter sequences. Out of the pancreatic juice samples, none demonstrated either of the 2 Helicobacter gene sequences investigated., Conclusion: Despite good scientific reasoning for an involvement of Helicobacter in pancreatic diseases, a direct infection of the microbial agent seems unlikely. Rather, the pathomechanism must involve molecular mimicry in autoimmune pancreatitis, or the transformation of nitric food constituents to nitrosamines in pancreatic cancer. and IAP., (Copyright © 2010 S. Karger AG, Basel.)

Background: In spite of the increasing knowledge of the molecular pathology of pancreatic ductal adenocarcinoma (PDAC), treatment of this tumor still remains an unresolved problem. Thus, the identification of 'novel' genes involved in pancreatic tumor progression is essential for early diagnosis and new treatment regimens of PDAC. Ankyrin-B (ANK2) was identified as being overexpressed in PDAC in a previous study by our group. ANK2 overexpression has been described in several tumors; however, the function of ANK2 in pancreatic carcinoma has not been elucidated., Materials and Methods: In the present study, we confirmed ANK2 overexpression in PDAC and analyzed the effects of ANK2 knockdown in the pancreatic tumor cell line PANC-1., Results: ANK2 silencing reduced the activity of FAK, ERK1/2 and p38. Decreased ANK2 expression restrained migration and invasive potential of PANC-1 cells. Moreover, silencing of ANK2 decreased the proliferation of the pancreatic tumor cells and reduced their tumorigenicity in vitro and in vivo., Conclusion: Our results demonstrate that silencing of ANK2 expression reduced the malignant phenotype of pancreatic cancer cells, indicating that ANK2 represents a potential target for therapy of pancreatic cancer., (Copyright © 2010 S. Karger AG, Basel.)

Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-beta, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75(NTR) expression was weak in prPSC. In contrast to ihPSC TGF-beta activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-beta, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.

Background: Pancreatic cancer is characterized by high resistance to chemotherapy. Such chemoresistance can be mediated by multidrug resistance proteins (MRPs), breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein. However, the contribution of individual MRP isoforms to chemoresistance in pancreatic carcinoma is unclear. We studied ATP-binding cassette (ABC) transporter expression in human pancreatic carcinoma cell lines as compared to primary pancreatic duct cells, and analyzed the MRP expression profile in 5-fluorouracil-resistant cells., Methods: Transporter expression was analyzed by quantitative and qualitative RT-PCR, by immunoblot, and chemoresistance by cytotoxicity assay., Results: Primary pancreatic duct cells expressed MRP1, MRP3, MRP4, and MRP5, but not MRP2 mRNA. The established carcinoma cell lines expressed MRP1, MRP4, and MRP5, most of them also MRP2, MRP3, MRP7, and BCRP, but none contained detectable amounts of MRP6, MRP8, or MRP9 mRNA. Immunoblot analyses demonstrated presence of MRP1, MRP4, and MRP5 protein in all, but MRP3 and BCRP protein only in some of these cells. Compared to parental Capan-1 cells, Capan-1 cells with acquired chemoresistance towards 5-fluorouracil showed an upregulated mRNA and protein expression of MRP3, MRP4, and MRP5. In addition, silencing of MRP5 by RNA interference resulted in enhanced sensitivity of parental Capan-1 cells towards 5-fluorouracil cytotoxicity., Conclusion: MRP3, MRP4, and MRP5 are upregulated in 5-fluorouracil-resistant cells, and MRP5 contributes to 5-FU resistance in pancreatic carcinoma cells., (Copyright 2008 S. Karger AG, Basel and IAP.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesized that gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. We investigated the gene expression of eleven stromal tissues from PDAC, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified and validated using quantitative RT-PCR and immuno-histochemistry. We found 255 genes to be overexpressed and 61 genes to be underexpressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway of which the differential expression of SFRP1 and WNT5a was confirmed using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during cocultivation with a pancreatic carcinoma cell line. The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The overexpression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

The development of effective tools for the early detection of pancreatic cancer, or its precursors, in high-risk subjects could play a key role in reducing the burden of this disease, which is the most lethal among solid gastrointestinal tumors. Given the poor accessibility of the pancreas due to its anatomic site, and given the limitations of imaging modalities, biomarker screening might be a promising diagnostic option. This review focuses on the rationale of using stool markers for the early detection of pancreatic cancer, and systematically summarizes current evidence. Despite several potential advantages of stool testing for pancreatic cancer and its biological plausibility, only six studies investigating two genetic markers in stool (the K-ras and the p53 gene) could be identified. Even though these studies were limited in size and could hardly approximate the screening setting, both markers appear to lack sensitivity and, in particular, specificity. The investigation of further marker candidates (e.g., epigenetic markers) in adequately designed studies represents an important next step to explore the potential of stool testing for pancreatic cancer. Pertinent studies could greatly benefit from recent methodological advances gained in connection with stool testing for colorectal cancer.

A disintegrin and metalloproteinase (ADAM) molecules are known for their unique potential to combine adhesion, proteolysis, and signaling. To understand the role of ADAM17/tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) in pancreatic ductal adenocarcinoma (PDAC), we investigated its expression, function, and in vitro regulation. ADAM17/TACE mRNA was expressed in 3 of 10 normal pancreatic tissues, 6 of 8 samples from patients with chronic pancreatitis, 10 of 10 PDAC tissues, and 9 of 9 pancreatic cancer cell lines, but it was absent in primary duct epithelial cells. Immunohistochemical staining revealed positive cancer cells in 8 of 10 PDACs but no staining of ducts in normal pancreas. ADAM17/TACE was found in 0 of 16 pancreatic intraepithelial neoplasia (PanIN)-1A lesions, 1 of 30 PanIN-1B lesions, 2 of 13 PanIN-2 lesions but, in 13 of 15 PanIN-3 lesions, associated with PDAC. Western blot, flow cytometry, and confocal microscopy analyses showed the aberrant expression of ADAM17/TACE protein in pancreatic cancer cell lines. The proteolytic activity of ADAM17/TACE, assessed by the release of TNF-alpha, was inhibited by TNF-alpha protease inhibitor. ADAM17/TACE gene silencing using small interfering RNA technique in vitro reduced invasion behavior dramatically, whereas proliferation was unaffected. Furthermore, ADAM17/TACE mRNA expression was down-regulated in pancreatic cancer cells arrested in G2-M phase as well as in a time-dependent manner after TNF-alpha and interleukin-6 incubation. In conclusion, our findings provide evidence of aberrant expression of the proteolytically active ADAM17/TACE in advanced precursor lesions (PanIN-3) and PDAC while identifying its critical involvement in the invasion process.

Aim: To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism., Methods: TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF (asTF) was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma (PCa), 9 patients with chronic pancreatitis (CP) and 20 normal controls. Plasma samples (30 PCa-patients, 13 CP-patients and 20 controls) were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin III complex (TAT), prothrombin fragment 1 + 2 (F1 + 2)]., Results: All pancreatic carcinoma cell lines expressed TF (8/8) and most of them expressed asTF (6/8). TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF (17/19). In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F1 + 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 + 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients., Conclusion: We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase (hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor beta 1(TGFbeta1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of alpha-smooth muscle actin (alphaSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFbeta1 treatment upregulated the expression of alphaSMA, collagen type I (Col I), fibronectin and TGFbeta1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of alphaSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis.

Local therapy of pancreatic cancer with microencapsulated CYP2B1-producing cells and ifosfamide showed an effect both on the primary tumor and on distant metastatases. This possibly represents a consequence of the activation of immune response. Other studies have demonstrated that local tumor irradiation leads to the activation of the intratumoral lymphocyte infiltration. The aim of our study was to investigate the efficacy of the combined therapy with low-dose irradiation, ifosfamide and CYP2B1-producing cells. Syngenic pancreatic cancer was induced in 38 Lewis-rats by subcutaneous inoculation of 1 x 10(6) (DSL6A) tumor cells. Microencapsulated CYP2B1-producing cells were injected peritumorally 10--12 weeks after tumor implantation. Animals were randomized to the following groups: 1) control (NaCl, 1 ml i.p.), 2) ifosfamide (50 mg/kg, i.p., (3x/week), 3) local irradiation with 5 Gy and 4) ifosfamide plus irradiation. The tumor growth was monitored for 3 weeks. The tumor infiltration with CD4+, CD8+, NK-cells, microvessel density and proliferation rates were investigated by immunohistochemistry. Cytokine plasma level for TNF-alpha were measured by ELISA. Seven of 9 animals in the group of combined therapy showed an objective response to the therapy. The therapy with ifosfamide or radiation alone showed 5 and 3 responders, respectively. The mean tumor volume was significantly reduced after combined ifosfamide plus radiation therapy in the first week, whereas monotherapy with ifosfamide or radiation significantly decreased tumor growth earliest after 2 and 3 weeks, respectively. The high plasma level of TNF-alpha in the control group was significantly reduced after combined ifosfamide/irradiation treatment. The lymphocyte infiltration and tumor proliferation were not significantly different between the groups. Microvascular density was significantly increased after ifosfamide and ifosfamide plus irradiation therapy. The combination of ifosfamide/CYP2B1-producing cells and irradiation showed an earlier therapeutical effect on the growth of rat pancreatic cancer than the irradiation or ifosfamide alone. There was no evidence of late activation of lymphocyte infiltration and PCNA-positive tumor cells.

Background: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines., Methods: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells., Results: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change >3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin beta-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma., Conclusion: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development., (Copyright 2005 S. Karger AG, Basel.)

In order to define antigens that might be suitable as vaccines for pancreatic carcinoma, we investigated the composite expression of 10 cancer testis (CT) antigens (SCP-1, NY-ESO-1, SSX-1, SSX-2, SSX-4, GAGE, MAGE-3, MAGE-4, CT-7 and CT-8) by Reverse Transcriptase-PCR (RT-PCR) in fresh biopsies of human pancreatic adenocarcinoma, chronic pancreatitis and pancreatic carcinoma cell lines. While all CT genes were frequently expressed in cell lines derived from pancreatic cancer, no expression of MAGE-3, SSX-1, SSX-2, NY-ESO-1 and CT-7 was detected in fresh tumor biopsies, and MAGE-4 (1/52), SSX-4 (1/39) and CT-8 (2/41) were only rarely expressed. In contrast, HOM-TES-14/SCP-1 was expressed in 48% (29/61) and GAGE in 21% (13/61) of cases, respectively. One CT gene was expressed by 59% (75% in male, 46% in female patients; p = 0.05) and 2 or more CT genes by 15% of the samples. SCP-1 protein expression correlated well with mRNA expression. While SCP-1 and GAGE were absent in normal pancreas, they were found in 2/8 (SCP-1) and 1/8 (GAGE) samples of chronic pancreatitis, respectively, supporting the concept of chronic pancreatitis as a premalignant condition. SCP-1 and GAGE represent promising candidates for vaccine development in pancreatic carcinoma. Whether SCP-1 and GAGE expression identify cases of chronic pancreatitis with a high risk of malignant transformation remains to be shown., (Copyright 2004 Wiley-Liss, Inc.)

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies., (Copyright 2004 S. Karger AG, Basel and IAP.)

Introduction: K-ras mutations are present in most ductal adenocarcinomas (DACs) of the pancreas and may also be found in ductal precursor lesions and even in normal ductal epithelium. The question is addressed whether mutated K-ras interferes with the regulation of apoptosis or proliferation., Methodology: In 50 Whipple resection specimens, tissue adjacent to DACs was histologically screened for ductal lesions that were classified as pancreatic intraepithelial neoplasia (PanIN) according to WHO criteria. PanIN lesions were microdissected and analyzed for K-ras mutations by means of a nested PCR. Apoptosis was identified by the TUNEL method. Proliferation and the expression of p53 and Bcl-2 were immunohistochemically determined., Results: On average, 30% of PanIN-1A and B lesions showed mutated K-ras. In PanIN-2 and PanIN-3 lesions, the rate of mutated K-ras increased to 45% and 56%, respectively. Apoptosis was present only in 2 of 26 PanIN-3 lesions. There was a gradual increase in proliferative activity from PanIN-1 to PanIN-3. p53 expression was found in 11% of PanIN-2 and 44% of PanIN-3 lesions. Bcl-2 expression was lacking in PanIN lesions of all grades. In invasive DACs, the apoptotic rate correlated with the degree of tumor differentiation and proliferation, with grade 3 carcinomas showing the highest apoptotic rate., Conclusion: In view of the discrepancy between the considerable rate of K-ras mutations in PanIN-1 and PanIN-2 lesions and the lack of apoptosis and Bcl-2 expression, coupled with very low p53 immunoreactivity, it is unlikely that mutated K-ras affects the apoptotic activity in low grade PanINs. Instead, K-ras mutations may have an effect on proliferation in PanIN-1 and PanIN-2.

In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic

Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of several epithelial tumors including pancreatic adenocarcinoma. This tissue reaction may be deleterious or advantageous to the host or tumor. In a systematic analysis, we identified two growth factors expressed by human pancreatic carcinoma cells that are positively correlated with the ability to induce fibroblast proliferation both in vitro and in vivo, i.e., transforming growth factor (TGF)-beta1 and fibroblast growth factor-2. Here we demonstrate that the overexpression of TGF-beta1 induced up-regulation of matrix proteins and growth factors in the TGFbeta1-transfected pancreatic tumor cells. Furthermore, transfection of PANC-1 cells induces the same change in fibroblasts in either cocultivation experiments or when they are grown in conditioned medium from TGF-beta1-transfected PANC-1 cells. TGF-beta1-transfected pancreatic tumor cells induced a rich stroma after orthotopical transplantation in the nude mouse pancreas. The transfer of a single growth factor, TGF-beta1, conveys the ability to induce a fibroblast response similar to that seen in desmoplasia in human pancreatic adenocarcinoma. This effect cannot only be attributed to direct effects of TGF-beta1 but also results from the up-regulation of several other factors including collagen type I, connective tissue growth factor, and platelet-derived growth factor.

CD44 is an integral cell-surface glycoprotein. Overexpression of the CD44 standard (CD44st) and its variants (CD44v) has been implicated in transformation and progression of many cancer types. Here, we investigated expression of CD44st, CD44v3-7, CD44v7/8, and v10 in five human pancreatic tumor cell lines and normal human pancreatic duct cells transfected with the SV40 large T antigen. CD44st and its variant proteins were quantified using immunocytochemistry and flow cytometry. CD44v7 was expressed at low levels, whereas CD44st, CD44v3, CD44 v4, CD44v, and CD44v6 were expressed at moderate levels in all pancreatic tumor cell lines. In contrast, CD44v7/8 and CD44v10 were expressed at very low levels in two out of the five pancreatic tumor cell lines. Overall, staining of CD44st and CD44 variants was significantly weaker compared to another surface molecule, ICAM-1, reported to be overexpressed in pancreatic cancer cells. Furthermore, the SV40 large T transfected duct cells showed only a weak staining for CD44st, CD44v5, and CD44v6. To determine a possible mechanism for the regulation of surface expression of CD44st, v5 and v6, we incubated Panc-1 cells with bFGF, TGF-beta1, EGF, TNFalpha, and IFNgamma. Only IFNgamma affected the CD44 expression by down-regulation of CD44v6. The constitutive expression of CD44 variants seems to be associated with the malignant state of invasive carcinoma.

The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low-dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.

Conclusion: Preparation of DNA from pancreatic juice for subsequent polymerase chain reaction (PCR) is difficult, but manageable. The protocol presented offers a simple and fast solution. This method might be applicable to other complicated samples, such as saliva, would secretions, or stool washings., Background: Of all the biological samples used for PCR amplification, pancreatic juice is the most problematic because of the presence of potential inhibitory substances and the amount of nucleases. This demands a DNA preparation procedure that is suitable for routine diagnostic PCR, and is therefore efficient and safe. This is particularly true for pancreatic juice obtained during routine endoscopy., Methods: We describe here a simple method utilizing modified phenol/chloroform extraction and precipitation directly from native pancreatic juice suitable for diagnostic PCR applications, such as oncogenes., Results: DNA could be prepared in quantitative amounts from routine endoscopic specimens. DNA could also be prepared from samples kept several days at room temperature.

Preparation of pancreatic duct epithelial cells from adult organs is possible by limited digestion and outgrowth of cells. These primary cells are mitotically active for only a short period. Therefore transfection with SV40 large-T antigen is one method to obtain an immortalized cell clone. Because the transfection efficacy of primary cells with conventional vectors is comparatively low, our aim was to develop conditions with improved transfection rates. Best transfection rates (approximately 6% of the resting cells) were obtained by using the BES buffered saline (BBS) calcium phosphate (Ca-P) coprecipitation technique at low pH. By using these optimized transfection parameters, primary cultures of human pancreatic duct epithelial cells were successfully transfected with the plasmid pSV3neo, bearing the large- and small-T antigen of SV40. A G 418 resistant clone (E4) was maintained in culture for 14 months before reaching terminal crisis.