Jürgen Hammer

Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer

Abstract: Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve diagnosis and treatment, key mechanisms of deregulated molecular functions have to be identified. Using microarray analysis, the expression patterns of 5600 human genes were assessed in PDAC by comparison with the normal pancreas and chronic pancreatitis (CP). The expression of 467 of 5600 genes was increased in PDAC in comparison to the normal pancreas, and the expression of 120 of these genes was not increased in CP. In addition, 341 of 5600 genes were expressed at decreased levels in PDAC tissues, of which 96 were decreased in comparison to both normal and CP tissues. Thus, a total of 808 of 5600 human genes were differentially expressed in pancreatic cancer. The identification of a large panel of altered genes in PDAC will stimulate additional studies that will lead to improved understanding of the molecular mechanisms underlying pancreatic malignant growth.

Role of Mononuclear Cells and Inflammatory Cytokines in Pancreatic Cancer-Related Cachexia

Background and Purpose: The mechanism behind aggressive development of cachexia in patients suffering from pancreatic cancer is not well understood. In this study, we investigated which factors are associated with the cachectic status of the patients and evaluated cachexia-promoting capacity of cancer and inflammatory cells. Experimental Design: DNA microarray analysis and quantitative reverse transcription-PCR were used to screen for cachexia-associated factors in pancreatic specimens obtained from noncachectic and cachetic patients diagnosed with pancreatic ductal adenocarcinoma. The expression pattern of the most prominently altered cachexia-associated factor, interleukin-6 (IL-6), was further analyzed in patients sera by ELISA, in pancreatic specimens by immunohistochemistry, and in a coculture system by quantitative reverse transcription-PCR using pancreatic cancer cell lines T3M4 (IL-6 positive) and Panc-1 (IL-6 negative) and peripheral blood mononuclear cells (PBMC) obtained from donors and noncachectic and cachectic patients. Results: Among numerous analyzed factors, IL-6 was significantly overexpressed in pancreatic specimens and elevated in serum of cachectic patients. The coculture system revealed that pancreatic cancer T3M4 cells but not Panc-1 cells were able to stimulate IL-6 exclusively in cachectic PBMC (by 14-fold) and this triggering was reduced by half in the presence of IL-6-neutralizing antibodies. Conclusion: IL-6 represents a prominent cachexia-associated factor in pancreatic cancer. IL-6 overexpression in cachectic patients is related to the ability of certain tumors to sensitize PBMC and induce cytokine expression in cachectic PBMC.

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules

We have identified a heptapeptide with high affinity to rheumatoid arthritis–associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Expression and functional significance of CDC25B in human pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths. Deregulation of cell-cycle control is thought to be a crucial event in malignant transformation, and CDC25 phosphatases are a family of cyclin-dependent kinase activators, which act at different points of the cell cycle, including G1–S and G2–M transition. Here, we investigated the expression and functional significance of CDC25s in PDAC. CDC25B mRNA expression levels in human pancreatic tissue samples were analysed by cDNA array, quantitative PCR and Northern blotting. Immunohistochemistry was carried out to localize and quantify CDC25B expression. Two specific CDC25B inhibitors were utilized to determine the functional relevance of CDC25B. By quantitative RT–PCR, CDC25B mRNA was overexpressed in pancreatic cancer (7.5-fold) in comparison to the normal pancreas. Strong nuclear CDC25B immunoreactivity was present in both pancreatic and metastatic cancer samples, and there was a marked increase of the percentage of positive cells in primary cancer (48.6±16.3%) and metastatic tissues (71.7±3.1%) compared to normal samples (8.3±1.8%). Two CDC25B inhibitors reduced the growth of pancreatic cancer cell lines, resulting in the accumulation of phosphorylated CDC2 and G2/M arrest. These findings demonstrate an important role of CDC25B in cell-cycle progression, raising the possibility that inhibition of CDC25B may have therapeutic potential in pancreatic cancer.

Identification of Disease-specific Genes in Chronic Pancreatitis Using DNA Array Technology

ObjectiveTo use DNA arrays to analyze the differential gene expression patterns in the normal pancreas and in pancreatic diseases. Summary Background DataGenome-wide gene expression analysis will provide new insights into gene function and cause of disease. MethodsRNA was extracted from eight normal pancreatic specimens, eight specimens with chronic pancreatitis (CP), and eight pancreatic cancer (PCa) tissues. Poly A(+) RNA was purified, reverse-transcribed, and converted into cRNA using biotinylated nucleotides. The HuGeneFL DNA array containing 5,600 full-length human genes was used for analysis. ResultsFirst, normal pancreatic tissues were analyzed in comparison with a panel of other normal tissues (colon, liver, prostate, lung, lymph node). This analysis revealed 11 signature genes that were selectively expressed in the pancreas (e.g., pancreatic elastase-IIA). Comparison of the expression of 5,600 genes between the normal pancreas, CP, and PCa specimens showed that the expression of 34 genes was decreased in CP tissues compared with normal pancreatic tissues, and that the expression of all of these genes was simultaneously decreased in PCa. In addition, the expression of 157 genes was increased in CP tissues compared with the normal pancreas. Of those, 152 genes were simultaneously increased in PCa. Thus, only 5 of 5,600 genes were significantly overexpressed in CP compared with both normal pancreas and PCa. ConclusionsThe majority of alterations observed in CP are present in PCa, and the number of genes whose expression is selectively deregulated in CP is surprisingly small. These results may provide new insight into the pathobiology of CP and help identify certain molecular alterations that might serve as targets for new diagnostic tools and disease-specific therapy.

In Vitro and In Vivo Induction of a Th Cell Response Toward Peptides of the Melanoma-Associated Glycoprotein 100 Protein Selected by the TEPITOPE Program

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4+ cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-γ expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.

Microarray Analysen beim Pankreaskarzinom: Identifizierung von Schlüsselgenen

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is extremely poor. To improve diagnosis and treatment in this dismal disorder, key mechanisms of deregulated molecular functions have to be identified. Presently, microarray analysis was applied to simultaneously assess the expression pattern of 5600 human genes in PDAC by comparison with the normal pancreas and chronic pancreatitis (CP). RNA was extracted from 8 PDAC tissues, 8 normal pancreatic specimens, and 8 CP tissues. For expression analysis, poly-A(+) RNA was purified, converted into cRNA using biotinylated nucleotides, and hybridized onto oligonucleotide microarrays representing 5600 full-length human genes. The expression of 467 of 5600 genes was increased in PDAC in comparison to the normal pancreas, and the expression of 120 of these genes was not increased in CP. In addition, 341 of 5600 genes were expressed at decreased levels in PDAC tissues, and 96 of these genes were significantly decreased in comparison to normal pancreatic and CP tissues. Thus, a total of 808 of 5600 human genes (14.4%) were differentially expressed in pancreatic cancer samples. Identification of a large panel of altered genes in pancreatic cancer will initiate future studies that will lead to a better understanding of the molecular mechanisms of pancreatic cancer growth. These studies will help to delineate new molecular markers for diagnostic, prognostic, and therapeutic use in this disease.