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Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425–34]

Supplementary Figure and Table Legends from Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Supplementary Figures 1-3, Table 1 from Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425–34]

Background & Aims: Inhibition of cell-cell adhesion between epithelial cells represents an early step during tumor metastasis. Down-regulation or perturbation of E-cadherin-mediated adherens junctions is an essential requirement in this process. Methods: The interaction between polysialylated neural cell adhesion molecule (PSA-NCAM) and the E-cadherin adhesion complex was studied by coimmunoprecipitation assays. The presence of PSA-NCAM was correlated with tumor invasion by using cell-cell aggregation and cell migration assays. The importance of polysialic acid (PSA) in the interaction of NCAM with E-cadherin and inhibition of cell-cell adhesion was confirmed by enzymatic removal of PSA from NCAM and down-regulation of PSA-transferases by siRNA. Results: Expression of oncogenic K-Ras(V12) in pancreatic carcinoma cells resulted in induction of PSA-NCAM expression and reduced E-cadherin-mediated cellular adhesion. The association of PSA-NCAM with the E-cadherin adhesion complex correlated with decreased cell-cell aggregation and elevated cell migration of pancreatic carcinoma cells. Enzymatic removal of PSA from NCAM or reduction of polysialyltransferase expression led to reduced association between NCAM and E-cadherin and subsequently increased E-cadherin-mediated cell-cell aggregation and reduced cell migration. Conclusions: Our data suggest the induction of PSA-NCAM by oncogenic K-Ras as a novel molecular mechanism by which E-cadherin-mediated cellular adhesion is reduced and dissemination of tumor cells is facilitated.

Background: Endobronchial forceps biopsies are often small and are associated with a relevant extent of artifacts. To overcome these limitations is an important task. Especially when considering predictive factors for pharmacological therapies of lung cancer (ERCC1, RRM1) a development of biopsy techniques seems to be essential. This is the first report on a new endobronchial biopsy technique called cryobiopsy. Objectives: In this study the feasibility and the potential advantages of applying cryoprobes for harvesting samples for histological examination in flexible bronchoscopies will be focused on. Methods: In 12 patients suffering from exophytic endobronchial malignancies, a modified flexible cryoprobe was used for immediate recanalization. The extracted tissue was examined histologically regarding sample quality and sample size. Results: Tissue samples obtained using the cryoprobe showed an extraordinary good quality in terms of size (median diameter of 6.7 mm, range 4.2–13 mm) and artifact-free sample area (75% of the samples showed an artifact-free sample area of more than 75%). Additionally molecular markers were shown to be well preserved. Conclusions: The new technique termed cryobiopsy might widen the chest physician’s range of tools for diagnostic bronchoscopies.

Ductal concretions in chronic pancreatitis (CP) are one of the causes of ductal obstruction, resulting in pancreatic ductal hypertension (PDH) and duct ectasia. Ductal epithelium subjected to chronic stress by PDH may undergo molecular alterations, thereby not only initiating and sustaining the inflammatory process but also activating molecules that have transforming potential. Acino-ductal metaplasia and pancreatic intraepithelial neoplasia (PanIN) are frequently seen in CP. Using laser capture microdissection, cDNA microarrays and Ingenuity Pathways Analysis, we found an altered Notch pathway in the ectatic ducts of CP. The microarray data was further validated by real-time PCR. We also found elevated transcripts of Notch receptors, Notch1 and Notch3 in microdissected ectatic ducts of CP. The Notch pathway ligands, Jagged/Delta-like and a Notch target, HES-related repressor protein (HERP), were up-regulated in ectatic compared to normal pancreatic ducts, while another target of Notch, hairy/enhancer of split (HES), was down-regulated. The transcripts of Delta-like1 and Jagged1 were increased 3.7-fold and 1.3-fold, respectively, while those of HERP1 were elevated 2.4-fold in the ectatic ducts of CP, compared to normal ducts. Immunohistochemistry showed that Jagged1 was not expressed in normal pancreatic ducts, while it was highly expressed in ectatic ducts. This pattern of Notch component alteration in ectatic ducts was mimicked to some extent in vitro in a human pancreatic duct epithelial (HPDE) cell line, when subjected to a pressure of 200 mmHg for 24 h. Therefore, we conclude that in the ectatic ducts of CP, PDH activates signalling pathways such as Notch, which have transforming potential.

Objectives Acute pancreatitis is associated with substantial alterations of the immunologic host response which has been claimed to promote remote organ dysfunction, septic complications, and mortality. Treatment with immunomodulating substances has been subject of few experimental studies with still conflicting results. Methods We used the taurocholate-induced model of severe acute pancreatitis (SAP) in rats which were assigned to different treatment regimen: isotonic saline (SAP-S) for nontreated controls, recombinant rat interferon-gamma for immunostimulation (SAP-IFN-gamma), and FK506 for immunosuppression (SAP-FK506). Animals were killed after 3, 6, and 24 hours as well as 3 and 7 days, and parameters of local and systemic severity were assessed. Results Treatment with IFN-gamma and FK506 attenuated the progression of intrapancreatic necrosis within the first 24 hours after pancreatitis induction along with a substantial reduction of subsequent neutrophil tissue infiltration as shown by decreased myeloperoxidase activity. Enhanced cell death by apoptosis during the postacute course was reduced in FK506-treated animals only. Pancreatic interleukin (IL) 1beta messenger RNA up-regulation occurred early and was slightly suppressed in both treatment groups; tumor necrosis factor alpha (TNF-alpha) and IL-2 messenger RNA expression paralleled the onset of apoptosis and was markedly decreased in IFN-gamma- and FK506-treated rats. The 2 therapeutic regimens had similar effects on intrapancreatic and systemic IL-1beta and TNF-alpha protein release; however, the profiles of both cytokines were differently influenced. Whereas IFN-gamma and FK506 treatment lead to an enhanced intrapancreatic and systemic TNF-alpha protein release during the early course, IL-1beta concentrations were significantly reduced within the late intervals. Seven-day mortality was 44% in saline-, 29% in IFN-gamma-, and 25% in FK506-treated rats (P = not significant). Conclusions Severe acute pancreatitis is associated with early alterations of the immune response comprising overt T-cell activation and impaired monocyte/macrophage function alike. Targeting either immunologic derangement improves local pancreatic damage and systemic severity. However, because mortality was not improved, a therapeutic benefit of immunomodulating substances in clinical SAP remains to be defined.

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive gastrointestinal cancers and is thought to arise from noninvasive precursors-pancreatic intraepithelial neoplasia (PanIN). Aberrantly prolonged cell survival due to apoptosis suppression is likely to contribute to carcinogenesis and carcinoma progression where the inhibitor of apoptosis proteins (IAPs) may play an important role. IAPs specifically inhibit caspases 3, 7, and 9 and prevent apoptosis. Survivin is a unique member of the IAPs family that is expressed in most human cancers including PDA but is not expressed in most normal adult tissues. To measure survivin transcript levels in normal pancreatic ducts, PanINs, and PDA, we used laser capture microdissection and real-time polymerase chain reaction. Survivin protein expression in normal pancreatic ducts, PanINs, PDA, and its metastases to lymph nodes were evaluated by immunohistochemistry. In microdissected tissues, we found a steady and close to exponential increase in survivin transcript levels from low-grade lesions (PanINs-1) to high-grade lesions (PanINs-2 and 3) and further to PDA. This observation was strictly mirrored by survivin protein expression. In addition, survivin was localized to the nucleus in high-grade lesions (starting at PanIN-2 stage), PDA, and nodal metastases, suggesting that nuclear translocation of survivin may be an early event in transformation to malignancy.

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling-1 (SOCS-1). We have previously shown that SOCS-1 is biallelically mutated in the primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1, resulting in impaired JAK2 degradation and sustained phospho-JAK2 action. SOCS-1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS-1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB-1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB-1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS-1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL.

We describe cyclic hydrostatic pressure of 200/100 mmHg with a frequency of 85/min as a hemodynamically relevant pathological condition enforcing apoptosis in endothelial cells (EC) after 24 h of treatment. This went along with an increase of CD95 and CD95L surface expression, shedding of CD95L into the supernatant, cleavage of caspase-3 and caspase-8, and elevated JNK-2, c-Jun, and CD95L mRNA expression. Furthermore, increased DNA-binding activity of the AP-1 transcription factor family members FRA-1 and c-Jun was observed. This activation was reduced by inhibition of JNK, which subsequently prevented elevated CD95L mRNA expression. Caspase inhibitors and a CD95L-neutralizing antibody also reduced EC apoptosis. Most of the pressure-induced events were most prominent at 24 and 48 h. However, after 48 h, the CD95/CD95L expression pattern switched back to CD95−/CD95L+ and the specific death rate decreased. Cyclic pathological hydrostatic pressure is a novel type of stress to EC that renders them susceptible to CD95/CD95L-mediated autoapoptosis and/or paracrine apoptosis accompanied by upregulation of intracellular molecules known to trigger both apoptosis and survival.

There are several indications that classical Hodgkin lymphoma (cHL) and at least a proportion of cases of Primary Mediastinal B cell Lymphoma (PMBL) are derived from B cells at similar stages of differentiation and share common pathogenic mechanisms. The first indication was the existence of mediastinal grey zone lymphomas as identified in the 4th International Symposium on HL, with clinical, histological and immunohistochemical features intermediate between cHL and PMBL. Second, both tumor types resemble a cell that is developmentally situated in-between the germinal center reaction and a plasma cell. Third, cHL and PMBL were found to have similar gene expression profiles, including the lack of immunoglobulin expression and low levels of B cell receptor signalling molecules, and the secretion of molecules like the chemokine TARC and the prominent expression of IL-13 receptors. Fourth, both entities were found to have common genomic aberrancies, notably in 2p15 and 9p24, the sites of the REL oncogene and the tyrosine kinase gene JAK2, respectively. Further comparison of both lymphoma types may provide further insight in the pathogenic mechanisms and allow the design of diagnostic algorithms to sort out the small number of so-called mediastinal grey zone lymphomas, that appear to be intermediate between PMBL and cHL.

Primary mediastinal B-cell lymphoma (PMBL) is a highly aggressive tumour with a unique pattern of clinical, morphological, immunological and genetic features distinct from other diffuse large B-cell lymphomas. PMBLs are characterized by a mature B-cell phenotype, but they typically lack immunoglobulin (Ig) gene expression. The PMBL cell line MedB-1 shares many characteristic properties of the primary tumour, including low-level Ig production despite a functionally rearranged IgVH gene and absence of 'crippling' mutations. In this study, a search was undertaken for reasons for downregulated Ig expression. Similar levels of the B-cell-specific transcription factors BOB.1/OBF.1 and PU.1 were found in MedB-1 cells to those in the Ig-producing UM-1 lymphoblastoid cell line. However, MedB-1 lacked the Oct2 transcription factor. Reporter assays showed that Ig-type promoters were active in MedB-1 cells. In contrast, activity of the intronic heavy chain enhancer was dramatically reduced. Ectopic expression of Oct2 was able partially to restore enhancer activity but transcription from the endogenous IgVH gene could not be rescued. Therefore, the role of epigenetic factors in the downregulation of Ig was investigated. Methylated histone 3 lysine 9, a reliable marker of chromatin silencing, was not detected in MedB-1 promoter and enhancer regions. Inhibition of DNA methyltransferase and of histone deacetylases also did not reactivate Ig production. These data suggest the existence of alternative mechanisms of Ig inhibition in MedB-1 cells, different from chromatin silencing and the lack of Oct2.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptosis. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL-R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL-R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptosis were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-gamma. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner.

Acinar regression in chronic pancreatitis may be due to immune attack in parenchymal areas neoexpressing HLA-DR molecules. CD4+Th1 cytotoxic T cells induce apoptosis of their targets via oligomerizing CD95 (APO-1/Fas) death receptors on target cells by their CD95 ligand (CD95L). We determined the expression of CD95 and CD95L in epithelia of normal and chronically inflamed pancreatic tissues. We applied RT-PCR and Western blotting for CD95L expression profiles, serial frozen section immunohistochemistry to detect CD95, CD95L, and HLA-DR molecules, CD3, CD4, CD11c, and S-100 protein (S100p). Normal pancreases and chronic pancreatitis contain CD95L message and protein. Immunohistochemistry revealed a mutually exclusive expression of CD95 and CD95L. Physiologically, acini were CD95-/CD95L+, ducts were CD95-/CD95L-, and islets were CD95-/CD95L+. In areas of lymphohistiocytic infiltration, mainly consisting of CD3+CD4+ T cells and CD11c+, CD4+/-, S100p+ interstitial dendritic cells, and in areas of initial fibrosis, acini and ducts were HLA-DR+, acini CD95+/CD95L-, and ducts CD95+/CD95L-. Islet cells were CD95-/CD95L+ in both conditions. IFNgamma levels in protein lysates, as measured by an immunoassay, were significantly higher in chronic pancreatitis than in normal pancreas (p < 0.0003). In vitro, IFNgamma down-modulated CD95L message and protein in ASPC1 and BxPc3 pancreatic carcinoma cells. In conclusion, pancreatic epithelia differentially express CD95 and CD95L in a mutually exclusive manner. In chronic pancreatitis the CD95-/CD95L+ status is conserved in islet cells even in the vicinity of lymphohistiocytic infiltrates, whereas it is lost in acini coexpressing HLA-DR. As a potential consequence, and possibly triggered by local release of IFNgamma, CD4-Th1 cells may cognately interact with and successfully attack exocrine cells by triggering CD95 on their target without being killed by epithelial, CD95L-mediated, counterattack.

In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L+ cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L+ cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry. Cell Death and Differentiation (2001) 8, 273–278

Primary mediastinal B-cell lymphoma is a locally highly aggressive but poorly disseminating tumor composed of medium sized or large cells most probably of thymic medullary origin. It has a mature B-cell phenotype, typically lacks immunoglobulin expression and has variable defects in expression of HLA-molecules. We present here a cell line, MedB-1, derived from such a tumor. As is frequently found in mediastinal B-cell lymphomas in situ, MedB-1 is CD10(-), CD19(+), CD21(-), CD22(+), CD23(+), CD25(-), CD37(+), CD38(-), CD39(+), CD40(+), CD54(+), CD95(+). Like the parental tumor, MedB-1 lacks HLA-A,B,C alpha-chains and beta(2)microglobulin and expresses HLA-D molecules at decreased levels. Both parental tumor and MedB-1 cells are clonally related as shown by immunoglobulin heavy chain gene rearrangement analysis. Unlike the parental tumor tissue, the MedB-1 cell line cytoplasmically expresses IgG/kappa in a very small subset of cells under standard culture conditions. MedB-1 does not contain any Epstein-Barr virus DNA. In a tissue adhesion assay MedB-1 cells showed an extensive binding to the medullary region of normal thymus. Altogether, MedB-1 is a suitable tool for functional and molecular analysis of this distinct lymphoma entity.

Objective: Early goal-directed therapy aims at balancing tissue oxygen delivery and demand. Hyperoxia (i.e., pure oxygen breathing) has not been studied in this context, since sepsis increases oxygen radical production, which is believed to be directly related to the oxygen tension. On the other hand, oxygen breathing improved survival in various shock models. Therefore, we hypothesized that hyperoxia may be beneficial during early septic shock. Design: Laboratory animal experiments. Setting: Animal research laboratory at university medical school. Subjects: Twenty domestic pigs of either gender. Interventions: After induction of fecal peritonitis, anesthetized and instrumented pigs were ventilated with either 100% oxygen or supplemental oxygen as needed to maintain arterial hemoglobin oxygen saturation ≥90%. Normotensive and hyperdynamic hemodynamics were achieved using hydroxyethyl starch and norepinephrine infusion. Measurements and Main Results: Before and at 12,18, and 24 hrs of peritonitis, we measured lung compliance; systemic, pulmonary, and hepatosplanchnic hemodynamics; gas exchange; acid-base status; blood isoprostanes; nitrates; DNA strand breaks; and organ function. Gluconeogenesis and glucose oxidation were calculated from blood isotope and expiratory 13 CO 2 enrichments during continuous intravenous 1,2,3,4,5,6- 13 C 6 -glucose. Apoptosis in lung and liver was assessed postmortem (TUNEL staining). Hyperoxia did not affect lung mechanics or gas exchange but redistributed cardiac output to the hepatosplanchnic region, attenuated regional venous metabolic acidosis, increased glucose oxidation, improved renal function, and markedly reduced the apoptotic death rate in liver and lung. Conclusions: During early hyperdynamic porcine septic shock, 100% oxygen improved organ function and attenuated tissue apoptosis without affecting lung function and oxidative or nitrosative stress. Therefore, it might be considered as an additional measure in the first phase of early goal-directed therapy.

In chronic pancreatitis (CP), fibrous replacement of exocrine tissue spares islets. There is local production of IFNgamma and death ligands by inflammatory cells as well as TGFbeta and TRAIL by pancreatic stellate cells (PSCs), along with functional death receptor neo-expression and apoptosis in exocrine but not in endocrine cells. Moreover, islets are strongly induced for TRAIL-receptor(R)-4 lacking a functional death domain. TRAIL-R4 signalling in T-cells induces NFkappaB transcription factors which activate anti-apoptotic programs. Whether TRAIL elicits this response in endocrine cells, we tested human insulinoma cell line CM and determined NFkappaB subunits transcripts and NFkappaB dependent inhibitor of apoptosis proteins (IAPs) in normal pancreas (NP) and CP. We treated CM with cytokines, determined TRAIL-R expression by flow cytometry, graded degree of fibrosis in CP specimens, microdissected epithelial compartments, performed real time PCRs for NFkappaB subunits transcripts, and immunohistochemistry for IKK-gamma, IkappaB-alpha, RelA, survivin, and cIAP1. In CM, TGFbeta/IFNgamma/TRAIL induced TRAIL-R4 surface expression. TRAIL/ IFNgamma, upregulated NFkappaB subunits and survivin while down-modulating 1kappaBalpha. NP epithelia had low RNA levels of NFkappaB subunits. These were increased in parenchymal areas of CP with severe fibrosis and most intensely in islets. The NFkappaB regulated proteins IkappaBalpha, survivin, and cIAP1 were found in corresponding sites, again, at highest levels in islets surrounded by fibrosis. In CP, islets not only evade immune attack by non-exposure of functional death receptors in presence of TRAIL-R4. They also neo-express NFkappaB subunits, survivin, and cIAP1. This apoptosis-inhibitory security program might be enforced by PSC-derived TRAIL.

The effect of the characteristic desmoplastic reaction of pancreatic cancer on tumor progression is largely unknown. We investigated whether pancreatic stellate cells, which are responsible for the desmoplastic reaction, support tumor progression. Immunohistology revealed that matrix metalloproteinase-2 (MMP-2), which is suggested to promote pancreatic cancer progression, is present in stellate cells adjacent to cancer cells. In vitro, stellate cells exhibited a much higher basal expression of MMP-2 compared with cancer cells. Panc1-, MiaPaCa2- and SW850-conditioned media stimulated MMP-2 release of stellate cells as detected by zymography. Cancer cells expressed and released basigin [BSG, extracellular matrix metalloproteinase inducer (EMMPRIN), CD147], a glycoprotein that is known to stimulate MMP-2 in mesenchymal cells, as detected by immunostaining, western blot and reverse transcription-polymerase chain reaction. Tumor cell-conditioned medium and BSG purified by affinity chromatography from supernatants of cancer cells, but not supernatants depleted from BSG, stimulated expression of MMP-1 and MMP-2 of stellate cells as demonstrated by western blot and zymography. Moreover, the interaction of stellate cells and cancer cells promoted the invasiveness of Panc-1 cells in the chorioallantoic membrane assay and increased the weight of tumors induced by all carcinoma cell lines in nude mice by 2.1-3.7-fold. Our findings support the assumption that the interaction of stellate cells and cancer cells promotes progression of pancreatic cancer.

Chronic pancreatitis is now thought to have a multifactorial etiology. New concepts integrating cellular, molecular and genetic knowledge of the disease have been proposed to explain its pathogenesis. However, the mechanisms responsible for early exocrine parenchymal destruction and preservation of endocrine islets were unexplored until recently. In the course of chronic inflammation, pancreatic acini lose their "immunoprotective" status by neo-expressing death receptors. Therefore, they become susceptible to apoptosis that is triggered by their respective ligands expressed on lymphocytes and released by pancreatic stellate cells. By contrast, islets retain their immunoprotective status and activate nuclear factor-kappaB (NF-kappaB)-induced anti-apoptotic factors, thus enabling survival. This knowledge might be exploited for devising therapeutic approaches to retard acinar loss and to prolong islet survival.

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein β-catenin, which correlates with collagen type I–dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with β1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of β-catenin, which leads to an increasing expression of the β-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin–mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells. (Cancer Res 2006; 66(9): 4662-71)

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling-1 (SOCS-1). We have previously shown that SOCS-1 is biallelically mutated in the primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1, resulting in impaired JAK2 degradation and sustained phospho-JAK2 action. SOCS-1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS-1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB-1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB-1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS-1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL.

In advanced chronic pancreatitis (CP), islets are preserved even in the midst of scarring. We recently showed in CP local production of interferon (IFN)gamma, transforming growth factor (TGF)beta and death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), along with functional death receptor neoexpression and apoptosis in exocrine but not in endocrine cells. However, islets are strongly induced for TRAIL-receptor (R)-4 lacking the functional death domain. TRAIL-R4 signaling in T cells induces NFkappaB, which activates antiapoptotic programs. Here, we demonstrate that in insulinoma cells CM, TGFbeta/IFNgamma/TRAIL in combination induced TRAIL-R4 surface expression. TRAIL/IFNgamma upregulated NFkappaB subunits and its target gene survivin while downmodulating IkappaB alpha mRNA. RelA transcriptional activity increased upon stimulation with IFNgamma and IFNgamma/TRAIL. In situ, normal pancreatic epithelia had low mRNA levels of NFkappaB subunits. These were higher in parenchymal areas of CP with severe fibrosis and highest in islets. NFkappaB-regulated proteins IkappaB alpha, survivin and another apoptosis inhibitor, cIAP1, were found in corresponding sites, again at highest levels in islets surrounded by fibrosis. In conclusion, islets in CP not only evade immune attack by nonexposure of functional death receptors in the presence of TRAIL-R4 but also additionally neoexpress NFkappaB and its target genes, survivin and cIAP1, to protect themselves from apoptosis.

Background: Loss of CD95 expression in tumour cells occurs frequently in colon carcinoma and may be associated with disease progression. On the other hand, neo-expression of CD95L in tumour cells may contribute to immune evasion. Aims: We aimed at further exploring the functional role and prognostic significance of the CD95/CD95L death inducing system in colon carcinomas. Patients and methods: CD95 and CD95L expression was examined by immunohistochemistry in 128 R0 resected UICC (International Union against Cancer) stage II/III colon carcinomas and correlated with disease free survival. Results: CD95 expression in tumour cells was observed in only 30 carcinomas (23.4%) whereas the others had at least a minor subpopulation of CD95 negative cells. Loss of CD95 in tumour cells was related to adverse prognosis in uni- and multivariate analysis (p = 0.046 and p = 0.036, respectively). Tumour infiltrating lymphocytes (TIL) were the major source of CD95L in colon carcinomas. CD95L+TIL were present in 83% of cases whereas CD95L was found in tumour cells in only 12% of cases. Moreover, a high rate of CD95L+TIL correlated with prolonged disease free survival in patients with UICC stage II (p = 0.05) but not in those with stage III. Conclusions: Loss of CD95 in tumour cells may be an independent prognostic factor in colon carcinomas. The CD95L counterattack is not a relevant feature in colon carcinoma but CD95L+TIL may contribute to tumour control in the early stages of the disease, exerting a concurrent selection pressure in the direction of CD95 abrogation/resistance.

Primary mediastinal B-cell lymphoma (PMBL) is a highly aggressive tumour with a unique pattern of clinical, morphological, immunological and genetic features distinct from other diffuse large B-cell lymphomas. PMBLs are characterized by a mature B-cell phenotype, but they typically lack immunoglobulin (Ig) gene expression. The PMBL cell line MedB-1 shares many characteristic properties of the primary tumour, including low-level Ig production despite a functionally rearranged IgVH gene and absence of 'crippling' mutations. In this study, a search was undertaken for reasons for downregulated Ig expression. Similar levels of the B-cell-specific transcription factors BOB.1/OBF.1 and PU.1 were found in MedB-1 cells to those in the Ig-producing UM-1 lymphoblastoid cell line. However, MedB-1 lacked the Oct2 transcription factor. Reporter assays showed that Ig-type promoters were active in MedB-1 cells. In contrast, activity of the intronic heavy chain enhancer was dramatically reduced. Ectopic expression of Oct2 was able partially to restore enhancer activity but transcription from the endogenous IgVH gene could not be rescued. Therefore, the role of epigenetic factors in the downregulation of Ig was investigated. Methylated histone 3 lysine 9, a reliable marker of chromatin silencing, was not detected in MedB-1 promoter and enhancer regions. Inhibition of DNA methyltransferase and of histone deacetylases also did not reactivate Ig production. These data suggest the existence of alternative mechanisms of Ig inhibition in MedB-1 cells, different from chromatin silencing and the lack of Oct2.

Primary mediastinal B-cell lymphoma (PMBL) is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene because expression profiling showed high Janus kinase-2 (JAK2) transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harboring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not overexpressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the suppressor of cytokine signaling-1 (SOCS-1) gene in this cell, which abrogates SOCS box function of the protein. Ectopic expression of wild-type (wt) SOCS-1 in MedB-1 leads to growth arrest and dramatic reduction of phospho-JAK2 and its downstream partner phospho–signal transducer and activator of transcription-5 (phospho-STAT5). Ultimately, the target gene cyclin D1 is repressed in transfectants while RB1, which is silenced in MedB-1, is induced. We conclude that, in MedB-1, action of phospho-JAK2 is sustained due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, SOCS-1 mutations are also present in the parental tumor of MedB-1 and were detected in 9 of 20 PMBLs.

Structural aberrations of the short arm of chromosome 2, mostly resulting in gains of 2p13 approximately 16, have recently been described as being highly recurrent in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). As these gains consistently lead to increased copy numbers of the REL oncogene locus, we investigated the expression of the c-Rel protein in a series of 30 cHL cases with known genomic REL status as determined by comparative genomic hybridization and interphase cytogenetics. Expression of the c-Rel protein was investigated in 26 biopsies by immunohistochemistry. Distinct patterns were observed in HRS cells with no staining, cytoplasmic, and/or nuclear staining for c-Rel. All 13 samples with additional copies of the REL locus displayed nuclear staining for c-Rel, while 13 cHL samples lacking chromosome 2 (2p) gains displayed a significantly lower proportion or complete absence of HRS cells with nuclear c-Rel expression. Detailed analysis using combined immunophenotyping and interphase cytogenetics of individual HRS cells demonstrated that REL gains correlated with the presence of nuclear c-Rel staining. Additionally, in 2 cHL samples with translocation breakpoints in 2p13 approximately 16, nuclear staining of c-Rel was observed; in one of them the staining pattern was indicative of a truncated c-Rel protein. The correlation between structural aberrations involving the REL locus and nuclear c-Rel accumulation in HRS cells qualifies REL as a target gene of the frequent gains in 2p in cHL. The data suggest that REL aberrations are a genetic mechanism contributing to constitutive nuclear factor (NF)-kappa B/Rel activation in cHL.

The peptide hormone leptin plays a major role in the regulation of energy intake and expenditure and is predominantly expressed in mature adipocytes but not in preadipocytes. Using bisulfite genomic sequencing, we found that 32 CpGs, distributed within a 317-bp sequence of the proximal leptin promoter, were highly methylated in human preadipocytes (73.4% +/- 9.0%). During maturation toward terminally differentiated adipocytes, this promoter region was extremely demethylated (9.4% +/- 4.4%). CpG methylation-dependent transcriptional activity of the promoter fragment was determined in transfection experiments using a set of 5'-truncated mock-, HhaI-, and SssI-methylated promoter-reporter constructs. Whereas the methylated CpG within the CCAAT/enhancer-binding protein alpha recognition site down-regulated reporter expression, methylated CpGs proximal to the TATA motif and/or in a further upstream region abrogated promoter activity completely. These distinct promoter CpG sequences were found unmethylated in leptin-expressing mature adipocytes. As evidenced by electrophoretic mobility shift assays, nuclear protein complexes were specifically formed on methylated oligonucleotide probes corresponding to the dedicated promoter sequences, indicating that methyl-CpG binding proteins participate in transcriptional repression and regulation of the human leptin gene.

In cell stress research, there is still a need to apply long-term hydrostatic pressure without changing any other environmental condition. We present here a new, open, pressurized chamber system allowing long-term sustained and dynamic application of hydrostatic pressure with the option of additional tension. Based on the computer-controlled Flexcell Strain Unit, we designed a pressurized chamber with a dynamic airflow and a defined membrane extension, which can be regulated by spacers. During operation up to 26.6 kPa, O2 partial pressures and pH in the cell-culture medium do not change compared to control cultures kept at normal atmosphere.

Background & Aims: Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and induces apoptosis by cross-linking either of the 2 TRAIL receptors containing a death domain (TRAIL-R1 or TRAIL-R2). TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. The aim of this study was to investigate the expression and function of TRAIL and its receptors in normal colonic epithelium. Methods: TRAIL and TRAIL receptor expression was studied by reverse-transcriptase polymerase chain reaction and immunohistochemistry. TRAIL sensitivity of epithelial cells was determined in vitro. Results: Normal colonic epithelial cells express TRAIL, TRAIL-R1, TRAIL-R2, and TRAIL-R4. Interestingly, TRAIL and TRAIL-R2 are coexpressed mostly in the luminal surface epithelium. Despite the expression of apoptosis-mediating TRAIL receptors, the normal colonic crypt epithelium is completely resistant to TRAIL-induced apoptosis in vitro. Using an infection model with restricted human cytomegalovirus gene expression or productive adenovirus infection in the colon carcinoma cell line Caco-2, we show that TRAIL sensitivity of colonic epithelial cells is induced on virus infection along with an up-regulation of TRAIL-R1 and TRAIL-R2 on the cell surface. Conclusions: We conclude that the TRAIL system may play a role in the early elimination of virus-infected epithelial cells in the normal gut. GASTROENTEROLOGY 2002;122:659-666

We describe sustained hyposmotic stress as a novel type of environmental condition enforcing apoptosis. In a dose- and time-dependent fashion, hyposmotic stress leads to a delayed type of apoptosis with considerable variations in constitutive sensitivity among different cell types. For example, after 48 h at 84 mosmol/l, the death rate ranged from 10.8 ± 0.7% in AsPc1 human pancreatic carcinoma cells to 72.0 ± 1.6% in HK-2 human kidney tubule cells. Caspase inhibitors rendered cells more resistant to hyposmolar stress; the caspase 3 inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde was the most efficient. After 24 h of stress, HT-29 colon carcinoma and HK-2 cells had increased their mitochondrial mass. This went along with an increase in mitochondrial membrane potential in HT-29 cells but with a decrease in HK-2 cells. Starting at 2 h of stress, we detected transient CD95L transcription followed by surface expression of CD95L in HT-29 but not in HK-2 cells. Inhibitory CD95L antibody partially inhibited specific death in HT-29 but not in HK-2 cells. Thus, as in other types of stress-induced apoptosis, the CD95/CD95L system is one of the different routes to suicide optionally used by hyposmotically stressed cells. Our findings may have clinical implications for the prevention and treatment of tissue damage caused by severe hyposmolar states.

Purpose In previous experiments we have shown a proliferative effect of platelet derived growth factor (PDGF) on smooth muscle cells (SMC) of the bladder of the rat. Thereby the signalling pathway is lipid raft (cholesterol-enriched microdomains) dependent and can be manipulated reversibly by altering membrane cholesterol levels. This study we performed to prove the role of PDGF as a potential regulative mitogen of SMC of the human bladder. Material and Methods Specimens from detrusor and trigone were taken from patients in the course of an ureteral reimplant procedure. Primary SMC-cultures were created. The proliferative effect of growth factors was determined by BrdU incorporation. In select experiments SMC were treated with predefined pressure and stretch for 1 hour. Gene expression was measured by real-time PCR. Results Under full serum conditions both SMC cultures showed different proliferation with a higher growth rate of the trigone. SMC of the trigone showed also a higher proliferation rate in response to the growth factor PDGF. After mechanical load both SMC showed a significant increase in gene expression of PDGF receptor (PDGFR) alpha and beta. PDGF itself was not expressed in this SMC. Conclusions Gene expression of PDGFR after short-term mechanical load of SMC of the bladder supports a regulatory role for PDGF in terms of pressure-induced hypertrophy of the human bladder. Since PDGF is not expressed in SMC, it may act on a paracrinic way with expression in the urothelium. Otherwise in vascular SMC PDGFR is known to be activated in response to stretch, in the absence of PDGF.

The coincidence of a gastrointestinal stromal tumor (GIST) and a neuroendocrine tumor (NET) in neurofibromatosis type 1 (NF1) is described only five times within the literature. We report on a 63 year old Caucasian female with the rare condition of neurofibromatosis type 1 coinciding with recurrent gastrointestinal stromal tumor plus bilateral pheochromocytoma (PCC). After a history of palpitations and dizziness that lasted for years, a left adrenal mass was detected by CT. Laparotomy revealed a pheochromocytoma of the left adrenal gland while an ileoterminal GIST was found incidentally intraoperatively. After six months contralateral PCC and multiple recurrent GIST were resected again. After four years the patient is doing well without any signs of further recurrent tumors. Discussion includes review of the literature.

Telomere length analysis has aroused considerable interest in biology and oncology. However, most published data are pan-genomic Southern-blot-based estimates. We developed T/C-FISH (telomere/centromere-FISH), allowing precise measurement of individual telomeres at every single chromosome arm. Metaphase preparations are co-hybridized with peptide nucleic acid probes for telomeric sequences and the chromosome 2 centromere serving as internal reference. Metaphase images are captured and karyotyped using dedicated software. A software module determines the absolute integrated fluorescence intensities of the p- and q-telomeres of each chromosome and the reference signal. Normalized data are derived by calculating the ratio of absolute telomere and reference signal intensities, and descriptive statistics are calculated. T/C-FISH detects even small differences in telomere length. Using T/C-FISH we have discovered an epigenetic process occurring in the human male postzygote or early embryo: in umbilical cord blood lymphocytes, telomeres on male Xqs are around 1100 bp shorter than female Xqs.