Daniela Pirkebner

14-3-3σ Expression Is an Independent Prognostic Parameter for Poor Survival in Colorectal Carcinoma Patients

Purpose: 14-3-3σ is an intracellular, dimeric, phosphoserine binding protein that is expressed in epithelial cells and involved in cancer development. In this study, we examined the expression of 14-3-3σ and evaluated its clinical significance in colorectal carcinoma. Experimental Design: Expression of 14-3-3σ was analyzed by Western blot in nine colorectal carcinoma cell lines, eight paired colorectal carcinoma tissues, and normal mucosas. Immunohistochemistry was used to evaluate expression of 14-3-3σ in tissues of 121 colorectal carcinoma patients and to correlate it with clinical parameters. Results: Western blot analysis of colorectal carcinoma cell lines and tissues revealed strong 14-3-3σ expression in four of eight cell lines and 14-3-3σ overexpression in carcinomas compared with normal mucosa in six of eight colorectal carcinoma tissue pairs. Immunohistochemical analysis revealed 14-3-3σ overexpression in 38.8% of colorectal carcinoma samples. Furthermore, highly positive immunoreactivity was significantly correlated with tumor differentiation (P < 0.001) and pT stage (P < 0.003). In Kaplan-Meier analysis, 14-3-3σ overexpression was associated with a significantly decreased survival time compared with negatively stained or low stained cases (P < 0.0096). In multivariate regression analysis, 14-3-3σ expression emerged as a significant independent parameter (P < 0.037). Conclusions: These results provide evidence that 14-3-3σ expression increases during carcinoma progression in a subset of colorectal carcinoma. The overexpression of this antigen identifies patients at high risk. It is tempting to suggest that 14-3-3σ overexpression either promotes tumor proliferation and/or prevents apoptotic signal transduction in colorectal carcinoma. Thus, targeting 14-3-3σ might be a new therapeutic strategy in colorectal carcinoma.

Dkk-3 expression in the tumor endothelium: a novel prognostic marker of pancreatic adenocarcinomas.

Dkk‐3 is proposed to be a new specific marker for tumor endothelial cells. Here we analyzed the clinical relevance of Dkk‐3 expression in pancreas adenocarcinomas and determined its role on endothelial cell growth in vitro. Microvessel density in tumor samples was immunohistochemically determined using Dkk‐3 and CD31 as endothelial cell markers, respectively. Based on the median microvessel density as a cut‐off point, patients were categorized into high and low microvessel density groups and a correlation with survival and clinical parameters was assessed. Moreover, the role of Dkk‐3 expression on chemosensitivity of endothelial cells was analyzed. In contrast to CD31 staining, Dkk‐3‐positive vessels were found only in tumor tissue and Dkk‐3 microvessel density significantly correlated negative with tumor grading. In survival analysis the median survival time was 7 months for patients with Dkk‐3 low, and 15 months for Dkk‐3 high microvessel density (P = 0.0013). Subset analysis of patients receiving gemcitabine therapy showed that overall survival was significantly decreased in Dkk‐3 low tumors than in high tumors (P = 0.009). In Cox regression Dkk‐3 emerged as a significant independent parameter (P = 0.024). Dkk‐3 overexpression in endothelial cells resulted in significantly enhanced growth inhibition after 5‐fluorouracil or gemcitabine treatment compared to control endothelial cells and cancer cell lines. Dkk‐3 low microvessel density was associated with tumor progression and worse clinical outcome. Overexpression of Dkk‐3 enhanced endothelial cell growth inhibition to chemotherapeutic drugs. Therefore, we suggest that Dkk‐3 high microvessel density may help to select patients who may benefit from chemotherapy. (Cancer Sci 2009)

Peroxisome Proliferator-Activated Receptor-α Activation Inhibits Langerhans Cell Function

Epidermal Langerhans cells (LC) play a pivotal role in initiating and maintaining primary immune responses in the skin. In the present study, we asked whether peroxisome proliferator-activated receptor-α (PPARα) activation modulates LC function. Our results show that PPARα is expressed in immature LC and is down-regulated in mature LC suggesting that an early decrease of PPARα expression in LC may allow them to mature after contact with an Ag. We further show that pharmacologic PPARα activation inhibits LC maturation, migratory capacity, cytokine expression, and the ability to drive T cell proliferation. Moreover, PPARα activation inhibits NF-κB but not stress-activated protein kinase/JNK, p38MAPK, and ERK1/2. In conclusion, PPARα activation by endogenous ligands may provide a molecular signal that allows LC to remain in an immature state within the epidermis for extended periods of time despite minor environmental stimuli.

Reduction of intracellular pH inhibits constitutive expression of cyclooxygenase-2 in human colon cancer cells.

Cyclooxygenase‐2 (COX‐2) over‐expression is critically involved in tumor formation. Intracellular pH (pHi) has been shown to be alkaline in cancer cells, and to be an important trigger for cell proliferation. This study therefore analyzed the relationship between pHi and COX‐2 expression. HRT‐18 and Caco‐2 cells cultured in medium with bicarbonate maintained a pHi of ∼7.6, which is higher than that of non‐neoplastic cells. Cells grown in bicarbonate‐free medium with a pH at 6.8 showed a reduction in pHi to approximately 7.0. Importantly, reduction of pHi resulted in a complete inhibition of COX‐2 mRNA and protein expression. When cells were grown in bicarbonate‐supplemented medium at pH 6.8, pHi maintained at ∼7.6 and COX‐2 expression was not inhibited. Additionally, analysis utilizing protein synthesis inhibitor cycloheximide demonstrated that pHi mediated inhibition of COX‐2 mRNA expression requires de novo protein synthesis of regulatory protein(s). These data strongly suggest that an alkaline pHi is an important trigger for constitutive COX‐2 expression. Defining pHi‐mediated mechanisms that govern the constitutive COX‐2 expression may help in developing new strategies to block COX‐2 over‐expression in cancer cells. J. Cell. Physiol. 198: 295–301, 2004© 2003 Wiley‐Liss, Inc.

Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process.1 Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established. Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused “rotary transport system for islets” termed “ROTi.” Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating “standard” shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated “standard” shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

Rotational Transport of Islets: The Best Way for Islets to Get around?

Islet transplantation is a valid treatment option for patients suffering from type 1 diabetes mellitus. To assure optimal islet cell quality, specialized islet isolation facilities have been developed. Utilization of such facilities necessitates transportation of islet cells to distant institutions for transplantation. Despite its importance, a clinically feasible solution for the transport of islets has still not been established. We here compare the functionality of isolated islets from C57BL/6 mice directly after the isolation procedure as well as after two simulated transport conditions, static versus rotation. Islet cell quality was assessed using real-time live confocal microscopy. In vivo islet function after syngeneic transplantation was determined by weight and blood sugar measurements as well as by intraperitoneal glucose tolerance tests. Vascularization of islets was documented by fluorescence microscopy and immunohistochemistry. All viability parameters documented comparable cell viability in the rotary group and the group transplanted immediately after isolation. Functional parameters assessed in vivo displayed no significant difference between these two groups. Moreover, vascularization of islets was similar in both groups. In conclusion, rotary culture conditions allows the maintenance of highest islet quality for at least 15 h, which is comparable to that of freshly isolated islets.