Kerstin David

Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision

The aim of this study was to determine the impact of ischemia on gene and protein expression profiles of healthy and malignant colon tissue and, thus, on screening studies for identification of molecular targets and diagnostic molecular patterns. Healthy and malignant colon tissue were snap-frozen at various time points (3-30 min) after colon resection. Gene and protein expression were determined by microarray (HG-U133A chips) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) technology (CM10 chips, SAX2 chips, and IMAC3Ni chips), respectively. Real-time reverse transcription PCR (RT-PCR) was used for comparative measurement of expression of particular genes. Initial changes of gene and protein expression profiles were already observed 5-8 min after colon resection. Fifteen minutes after surgery, 10%-15% of molecules, and after 30 min, 20% of all detectable genes and proteins, respectively, differed significantly from the baseline values. Significant changes of expression were found in most functional groups. As confirmed by real-time RT-PCR, this included not only known hypoxia-related molecules (HIF-1 alpha, c-fos, HO-1) but also cytoskeletal genes (e.g., CK20) and tumor-associated antigens (e.g., CEA). In conclusion, preanalytical factors, such as tissue ischemia time, dramatically affect molecular data. Control of these variables is mandatory to obtain reliable data in screening programs for molecular targets and diagnostic molecular patterns.

What establishes a protein as an allergen

There is little known about the factors that determine the allergenicity of food proteins. Apparently, the ability of a food protein to induce an allergic response requires its presence in substantial amounts in the food supply, its durability during food processing, and its resistance to digestion in the gastrointestinal tract. In addition to the mode and degree of exposure, structural characteristics appear to play an important role for the capacity of a protein to modulate the immune response towards allergic reactions. Until now, however, there has been no indication for common structural characteristics of linear T cell or linear IgE (B cell) epitopes and the knowledge of structural characteristics of conformational IgE binding sites is very limited. Experimental data point only to certain surface areas of allergenic proteins which are important for IgE binding. Therefore, it is not possible to suggest any structural motif or conformational sequence pattern common to all allergenic proteins. Furthermore, glycosylation appears not to be a common critical determinant of allergenicity since food allergens comprise both glycoproteins and nonglycosylated proteins. Based on the few published three-dimensional structures of allergenic proteins including food proteins, one unifying feature of allergens appears to be their spherical shape. The three-dimensional structures of many more allergens have to be determined, however, to allow for a better understanding of the molecular basis of allergenicity. Most recently, new ideas have been introduced as to why certain biochemical or biologic functions such as enzymatic activities may predispose a protein to become an allergen. Proteolytically active allergens have been demonstrated to irritate the human mucosal surface, to enhance their own transmucosal uptake, and to augment IgE production. Therefore, the functional activity of some allergens may play a role among other factors in the process of sensitization and allergic responses.

Key role of mitochondria in cerulenin-mediated apoptosis.

Cerulenin, a fungal metabolite, is known to be a specific inhibitor of fatty acid synthase. Here we report that cerulenin is an effective inducer of apoptosis in different wild-type p53 and mutant p53 tumor cell lines, whereas normal human keratinocytes and fibroblasts are resistant to the apoptotic effect. To get more insight into the mechanisms of how cerulenin induces apoptosis we investigated several signal transduction molecules, including p53, p73, p21/WAF1, Bax, cytochrome c, and caspases 3 and 9. Our data strongly indicate that mitochondria play a key role in the cerulenin-mediated pathway. Bax overexpression correlated with the extent of apoptosis and appears to be regulated in a p53-independent manner. The significance of the mitochondrial pathway for the cerulenin-mediated apoptosis was confirmed by the rapid mitochondrial release of cytochrome c both in wild-type p53 and mutant cell lines. Interestingly, the rapid release of cytochrome c was not accompanied by a breakdown of the mitochondrial potential. Instead, the complete disruption of the mitochondrial function coincided with the appearance of a p18 kDa cleavage product of Bax.

Combined targeting of AKT and mTOR using MK-2206 and RAD001 is synergistic in the treatment of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3‐kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration‐dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001‐mediated anti‐tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK‐2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan‐AKT inhibitor MK2206 in the CCA cell line TFK‐1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK‐2206 might be a new, effective strategy for the treatment of CCA.

Prediction of individual response to anticancer therapy: historical and future perspectives

Since the introduction of chemotherapy for cancer treatment in the early 20th century considerable efforts have been made to maximize drug efficiency and at the same time minimize side effects. As there is a great interpatient variability in response to chemotherapy, the development of predictive biomarkers is an ambitious aim for the rapidly growing research area of personalized molecular medicine. The individual prediction of response will improve treatment and thus increase survival and life quality of patients. In the past, cell cultures were used as in vitro models to predict in vivo response to chemotherapy. Several in vitro chemosensitivity assays served as tools to measure miscellaneous endpoints such as DNA damage, apoptosis and cytotoxicity or growth inhibition. Twenty years ago, the development of high-throughput technologies, e.g. cDNA microarrays enabled a more detailed analysis of drug responses. Thousands of genes were screened and expression levels were correlated to drug responses. In addition, mutation analysis became more and more important for the prediction of therapeutic success. Today, as research enters the area of -omics technologies, identification of signaling pathways is a tool to understand molecular mechanism underlying drug resistance. Combining new tissue models, e.g. 3D organoid cultures with modern technologies for biomarker discovery will offer new opportunities to identify new drug targets and in parallel predict individual responses to anticancer therapy. In this review, we present different currently used chemosensitivity assays including 2D and 3D cell culture models and several –omics approaches for the discovery of predictive biomarkers. Furthermore, we discuss the potential of these assays and biomarkers to predict the clinical outcome of individual patients and future perspectives.

Surgical procedures and postsurgical tissue processing significantly affect expression of genes and EGFR-pathway proteins in colorectal cancer tissue.

An understanding of tissue data variability in relation to processing techniques during and postsurgery would be desirable when testing surgical specimens for clinical diagnostics, drug development, or identification of predictive biomarkers. Specimens of normal and colorectal cancer (CRC) tissues removed during colon and liver resection surgery were obtained at the beginning of surgery and postsurgically, tissue was fixed at 10, 20, and 45 minutes. Specimens were analyzed from 50 patients with primary CRC and 43 with intrahepatic metastasis of CRC using a whole genome gene expression array. Additionally, we focused on the epidermal growth factor receptor pathway and quantified proteins and their phosphorylation status in relation to tissue processing timepoints. Gene and protein expression data obtained from colorectal and liver specimens were influenced by tissue handling during surgery and by postsurgical processing time. To obtain reliable expression data, tissue processing for research and diagnostic purposes needs to be highly standardized.

Mechanisms of in vivo antineuroblastoma activity of human natural IgM

Normal human sera of healthy adults contain natural IgM antibodies which are cytotoxic for human neuroblastoma cells. In this study, we evaluated the anti-neuroblastoma activity of these natural IgM antibodies in nude rats bearing solid human neuroblastoma tumours. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM in the tumours with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 h. Five consecutive i.v. injections of purified cytotoxic IgM into neuroblastoma-bearing animals resulted in complete growth arrest of even large established solid tumours which lasted for several weeks after discontinuation of the injections, whereas tumours of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-neuroblastoma IgM may have a potential as a novel therapeutic modality in the treatment of human neuroblastoma.

Detection of colorectal adenoma and cancer based on transthyretin and C3a‐desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.

Expression of a 260 kDa Neuroblastoma Surface Antigen, the Target of Cytotoxic Natural Human IgM: Correlation to MYCN Amplification and Effects of Retinoic Acid

Human neuroblastoma cells contain a 260 kDa surface-associated antigen (NB-p260) that is recognised by natural cytotoxic IgM antibodies. In this study we demonstrate that NB-p260 is expressed in vivo in a neuroblastoma tumour specimen but not in normal human tissues of neuronal origin. Since MYCN amplification is a clinical marker of neuroblastoma disease progression, we analysed the expression of NB-p260 in human neuroblastoma cell lines with different MYCN amplification status. However, both amplified and non-amplified neuroblastoma cell lines exhibited comparable NB-p260 expression. Treatment of neuroblastoma cells with the differentiation-inducing agent retinoic acid (RA) also had no effect on the expression of NB-p260. Collectively, the data suggest that expression of NB-p260 on human neuroblastoma cells is independent of malignancy and differentiation status of neuroblastoma.

Determination of phosphorylated proteins in tissue specimens requires high-quality samples collected under stringent conditions.

For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in‐vivo phosphorylation states.