Cornelia Hasel

Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells

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 beta-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 beta1-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 beta-catenin, which leads to an increasing expression of the beta-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.

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.

Sex-specific telomere length profiles and age-dependent erosion dynamics of individual chromosome arms in humans

During aging, telomeres are gradually shortened, eventually leading to cellular senescence. By T/C-FISH (telomere/centromere-FISH), we investigated human telomere length differences on single chromosome arms of 205 individuals in different age groups and sexes. For all chromosome arms, we found a linear correlation between telomere length and donor age. Generally, males had shorter telomeres and higher attrition rates. Every chromosome arm had its individual age-specific telomere length and erosion pattern, resulting in an unexpected heterogeneity in chromosome- specific regression lines. This differential erosion pattern, however, does not seem to be accidental, since we found a correlation between average telomere length of single chromosome arms in newborns and their annual attrition rate. Apart from the above-mentioned sex-specific discrepancies, chromosome arm-specific telomere lengths were strikingly similar in men and women. This implies a mechanism that arm specifically regulates the telomere length independent of gender, thus leading to interchromosomal telomere variations.

Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay

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.

Quantifying Telomere Lengths of Human Individual Chromosome Arms by Centromere-Calibrated Fluorescence in Situ Hybridization and Digital Imaging

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.

Effects of ventilation with 100% oxygen during early hyperdynamic porcine fecal peritonitis.

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 13CO2 enrichments during continuous intravenous 1,2,3,4,5,6-13C6-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.

Old Meets Modern: The Use of Traditional Cryoprobes in the Age of Molecular Biology

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.

Biallelic deletion within 16p13.13 including SOCS-1 in Karpas1106P mediastinal B-cell lymphoma line is associated with delayed degradation of JAK2 protein.

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.

Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease

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.

MedB-1, a human tumor cell line derived from a primary mediastinal large B-cell lymphoma

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 α‐chains and β2microglobulin 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/κ 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.