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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 µM 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.

Cell encapsulation represents an innovative technique. However, clinical applications are sparse. Most experiments and clinical studies have been performed with either alginate or cellulose sulfate capsules, containing several cell lines and a broad variety of applications, ranging all the way from substitution for impaired organ function and release of cytokines or growth factors to gene-directed enzyme prodrug therapy. A few clinical studies have been conducted and/or are under way.

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.

Recent evidence suggests that Runt-related transcription factors play a role in different human tumours. In the present study, the localisation of the Runt-related transcription factor-2 (Runx2), its transcriptional activity, as well as its regulation of expression was analysed in human pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time PCR and immunohistochemistry were used for Runx2 expression and localisation analysis. Runt-related transcription factor-2 expression was silenced using specific siRNA oligonucleotides in pancreatic cancer cells (Panc-1) and immortalised pancreatic stellate cells (IPSCs). Overexpression of Runx2 was achieved using a full-length expression vector. TGF-beta1, BMP2, and other cytokines were assessed for their potential to regulate Runx2 expression. There was a 6.1-fold increase in median Runx2 mRNA levels in PDAC tissues compared to normal pancreatic tissues (P0.0001). Runt-related transcription factor-2 was localised in pancreatic cancer cells, tubular complexes, and PanIN lesions of PDAC tissues as well as in tumour-associated fibroblasts/stellate cells. Coculture of IPSCs and Panc-1 cells, as well as treatment with TGF-beta1 and BMP2, led to increased Runx2 expression in Panc-1 cells. Runt-related transcription factor-2 overexpression was associated with decreased MMP1 release as well as decreased growth and invasion of Panc-1 cells. These effects were reversed by Runx2 silencing. In conclusion, Runx2 is overexpressed in PDAC, where it is regulated by certain cytokines such as TGF-beta1 and BMP2 in an auto- and paracrine manner. In addition, Runx2 has the potential to regulate the transcription of extracellular matrix modulators such as SPARC and MMP1, thereby influencing the tumour microenvironment.

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-α (TNF-α) 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-α, was inhibited by TNF-α 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-α 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. (Cancer Res 2006; 66(18): 9045-53)

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.

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, NYESO-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. © 2004 Wiley-Liss, Inc.

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.

The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

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.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.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.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.

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.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.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.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.

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.

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.Transporter expression was analyzed by quantitative and qualitative RT-PCR, by immunoblot, and chemoresistance by cytotoxicity assay.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.MRP3, MRP4, and MRP5 are upregulated in 5-fluorouracil-resistant cells, and MRP5 contributes to 5-FU resistance in pancreatic carcinoma cells.

Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDlα), up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation.

To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism.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)].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.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.

The prognosis of peritoneal spread from gastrointestinal cancer and subsequent malignant ascites is poor, and current medical treatments available are mostly ineffective. Targeted chemotherapy with intraperitoneal prodrug activation may be a beneficial new approach. L293 cells were genetically modified to express the cytochrome P450 enzyme 2B1 under the control of a cytomegalovirus immediate early promoter. This CYP2B1 enzyme converts ifosfamide to its active cytotoxic compounds. The cells are encapsulated in a cellulose sulfate formulation (Capcell). Adult Balb/c mice were inoculated intraperitoneally with 1 x 10(6) colon 26 cancer cells, previously transfected with GFP to emit a stable green fluorescence, by injection into the left lower abdominal quadrant. Two or five day's later animals were randomly subjected to either i.p. treatment with ifosfamide alone or ifosfamide combined with microencapsulated CYP2B1-expressing cells. Peritoneal tumor volume and tumor viability were assessed 10 days after tumor inoculation by means of fluorescence microscopy, spectroscopy and histology. Early i.p. treatment with ifosfamide and CYP2B1 cells resulted in a complete response. Treatment starting on day 5 and single-drug treatment with ifosfamide resulted in a partial response. These results suggest that targeted i.p. chemotherapy using a combination of a prodrug and its converting enzyme may be a successful treatment strategy for peritoneal spread from colorectal cancer.

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 [β-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. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

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. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

Although pancreatic adenocarcinoma has become one of the best characterized malignant diseases, severe diagnostic and therapeutic problems are still associated with this disease. The establishment of a molecular model of pancreatic carcinogenesis may provide tools that could result in earlier diagnosis of this disease and, in turn, improves prognosis. Since pancreatic adenocarcinoma seems to originate in epithelial cells in the pancreatic ducts, cultivation of native pancreatic duct epithelial cells (PDEC) is the initial step in the establishment of an in vitro model of pancreatic carcinogenesis. As these native cells survive only a short period in culture, the aim of this study was to establish a stable pancreatic duct cell line by immortalization with the SV40 large T antigen. Furthermore, initial steps in pancreatic carcinogenesis should possibly be imitated by additional transfections of mutated ki-ras and/or mutated p53 genes. By optimization of the isolation protocol and the culture medium, yield as well as proliferative activity of isolated PDEC was increased considerably. Transfection of SV40 large T antigen resulted in an increase in the proliferative lifetime of the isolated cells, but no real immortal phenotype was obtained. Moreover, one step in the transformation from the normal to the malignant phenotype was imitated successfully by additional transfection of mutated ki-ras.

Matthias Lohr (principal investigator) · Zoltan Tibor Bago · Helga Bergmeister · Manfred Ceijna · Mathias Freund · Wolfgang Gelbmann · Walter H. Gunzburg · Ralf Jesnowski · Johannes Hain · Karlheinz Hauenstein Wolfgang Henninger · Anne Hoffmeyer · Peter Karle · Jens-Christian Kroger · Gunther Kundt · Stefan Liebe Udo Losert · Petra Muller · Alexander Probst · Katrin Puschel · Matthias Renner · Renate Renz · Robert Saller Brian Salmons · Maximilian Schuh · Ilse Schwendenwein · Kerstin von Rombs · Thomas Wagner · Ingrid Walter (coinvestigators)

The prognosis of pancreatic adenocarcinoma is poor and current treatment is for the most part ineffective. We describe here a novel treatment strategy using a mouse model system for pancreatic cancer. Human embryonic epithelial cells have been genetically modified to express the cytochrome P450 2B1 enzyme under the control of a CMV immediate-early promoter. This CYP2B1 gene converts oxazaphosphorines (ifosfamide or cyclophosphamide) to their active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. A number of assays were performed to demonstrate the CYP2B1 gene function as well as toxic effects on neighbouring cells (bystander effect). The cells were then encapsulated in a cellulose sulphate formulation shown to be well tolerated in the pancreas of immunocompetent mice, and injected 1 cm away from pre-established tumours derived from a human pancreatic tumour cell line (PaCa-44). Intraperitoneal administration of low-dose ifosfamide to tumour bearing mice that received the encapsulated cells results in partial or even complete tumour ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immuno-protected environment may prove useful for solid tumour therapy in man.

Background/Aims: Analysis of gene expression is dependent on normalization using housekeeping genes. However, many of these housekeeping genes (e.g. GAPDH, β-actin) are upregulated in chronic pancreatitis and pancreatic cancer, and cannot be used for normalization. For this reason we tried to identify a housekeeping gene useful for expression analysis in pancreatic diseases. Methods: RNA isolated from various tissues and states of disease was subjected to reverse transcription and subsequently amplified by PCR using primers for GAPDH and for the ribosomal highly basic 23-kDa (rb 23-kDa, RPL13A) protein. Results: As anticipated, expression of GAPDH varied markedly in the different tissues, whereas the expression of rb 23-kDa was constant in all samples investigated. Conclusion: We recommend the use of the ribosomal highly basic 23-kDa protein as a standard for normalization at least for the pancreas and the prostate.