Jörg Ringel

The MUC gene family: Their role in diagnosis and early detection of pancreatic cancer

The early diagnosis of pancreatic cancer, as well as distinguishing between chronic pancreatitis and malignant pancreatic disease, remains still a clinical problem. Presently, there is no specific tumor marker for diagnosing pancreatic cancer. Mucin-associated marker like CA 19-9 are the most widely available pancreatic cancer tumor marker, but its value as a screening marker is limited by its reduced specificity.Mucins (MUCs) are heavily glycosylated, high molecular weight glycoproteins with an aberrant expression profile in various malignancies.This review considers briefly the potential use of the mucin expression pattern in diagnosis of pancreatic neoplasm. The overview will point out the present knowledge about changes in the mucin gene expression in pancreatic intraepithelial neoplasia (PanINs) as precursor lesions and in pancreatic adenocarcinoma, compared to normal pancreas and chronic pancreatitis and the potential role for differentiating chronic pancreatitis from pancreatic cancer.Furthermore, the potential use of MUCs in the diagnosis and differentiation of intraductal papillary-mucinous neoplasms (IPMNs) will be discussed.

Pancreatic Adenocarcinoma Cell Lines Show Variable Susceptibility to TRAIL-Mediated Cell Death

Background and Aims Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy. Methods Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well. Results Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis. Conclusions The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

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 β 1(TGFβ1) 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 α-smooth muscle actin (αSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFβ1 treatment upregulated the expression of αSMA, collagen type I (Col I), fibronectin and TGFβ1. 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 αSMA, 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.

Aberrant Expression of a Disintegrin and Metalloproteinase 17/Tumor Necrosis Factor-α Converting Enzyme Increases the Malignant Potential in Human Pancreatic Ductal Adenocarcinoma

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.

DNA Microarray Analysis of Pancreatic Malignancies

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.

Ribosomal highly basic 23-kDa protein as a reliable standard for gene expression analysis.

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.

CD44 in Normal Human Pancreas and Pancreatic Carcinoma Cell Lines

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.

Combined therapy of experimental pancreatic cancer with CYP2B1 producing cells: low-dose ifosfamide and local tumor irradiation.

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 × 106 (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., (3×/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‐α 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‐α 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.

Apoptotic molecules in pancreatic carcinoma cell lines.

Recently, a novel mechanism of immune evasion by tumor cells has been described, namely “the tumor counter-attack model.” In this model tumor cells kill activated lymphocytes through functional expression of Fas ligand (FasL).1–3 FasL and TRAIL are two highly homologous tumor necrosis factor (TNF) family members with the ability to induce apoptosis in susceptible cells through interaction with their membrane receptors Fas and DR4, DR5, respectively.4,5 To study the role of apoptosis in pancreatic malignancy, we determined the expression pattern of several apoptosis receptors and ligands, FasL/Fas, sFas (an alternatively spliced soluble form of Fas that was shown to protect cells from apoptosis),6 Trail/DR4 and DR3 in five human adenocarcinoma lines, BxPC3, Panc1, AsPC, PaCa 44, and PancTu using three different methods—reverse transcriptase polymerase chain reaction (RT/PCR), flow cytometry and Western blotting.

CD44, bFGF and hyaluronan in human pancreatic cancer cell lines.

Altered expression of cell surface molecules and the overexpression of growth factors and their receptors may be involved in malignant progression of human exocrine pancreatic cancer. CD44 is a transmembrane glycoprotein postulated to play a functional role in tumor cell metastasis. A “standard” form of CD44 (CD44st) binds hyaluronan (HA) and is reported to be overexpressed in pancreatic tumors.2 Alternative splicing gives rise to numerous CD44 isoforms. Variants containing the exon v6 appear to be of major importance for metastatic behavior in a rat pancreas carcinoma model.1 The published data about the expression of CD44st and CD44 isoforms in pancreatic cancer are contradictive.2–4 The regulation of CD44 expression is nearly unknown. Recently, a study showed a possible role of basic fibroblast growth factor (bFGF) in endothelial cells. It has been reported that bFGF and its high-affinity receptor (FGF-R) is abundantly present in pancreatic cancer.5 bFGF is one of the heparin-binding growth factors (FGF, epidermal growth factor (EGF)). The interaction with heparin protects bFGF from proteolytic degradation. Glycosaminoglycan heparin influences the binding of bFGF to the high-affinity receptor, too. Heparin and heparin derivatives can regulate tumor growth and inhibit metastatic behavior. The mechanisms of these regulation processes are poorly understood. Heparin is found exclusively in mast cells. Interestingly, mast cells accumulate by surrounding various neoplastic tissues. We asked whether bFGF alone, bFGF in combination with heparin, heparin alone or the CD44-ligand HA—a member of the glycosaminoglycans—modulates the expression of CD44. Therefore, we used the pancreatic adenocarcinoma cell lines PancTu and Panc-1 (both grade II–III). Furthermore, we investigated the effects of bFGF/heparin on proliferation behavior.