We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

Neuroblastoma, one of the most common pediatric solid tumors, originates from the peripheral sympathetic nervous system and is responsible for approximately 15% of all childhood cancer deaths. Among the several antineoplastic drugs used in neuroblastoma chemotherapeutic protocols, topoisomerase inhibitors (i.e., etoposide) represent the most commonly used. Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs, mainly reducing the ability of neoplastic cells to start programmed cell death when exposed to antineoplastic drugs. The aim of this study was to determine, by means of proteomics, potential markers of etoposide resistance in human neuroblastoma cell lines as well as to investigate protein levels and modifications possibly involved in the onset of resistance. The etoposide resistant clone showed overexpression of the following proteins: peroxiredoxin 1, beta-galactoside soluble lectin binding protein, vimentin (three protein spots), heat shock 27 kDa protein (two protein spots) and heterogeneous nuclear ribonucleoprotein K. In addition, we also found down-regulation of dUTP pyrophosphatase. This investigation might represent a first step towards the development of novel prognostic markers of neuroblastoma chemotherapy.

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

Resistance to chemotherapeutic agents is one of the major problems faced during palliative therapy of tumor cells. Thus, chemotherapy is frequently combined with other modes of therapy such as radiation therapy and/or hyperthermia. Tumor cells respond to heat stress with development of thermotolerance and the interactions between chemo- and thermoresistance phenomena are not clearly understood. In this paper, we analyze the differential protein expression in vitro in human stomach cancer cells, their chemoresistant and thermoresistant counterparts using proteomics. The immediate aim was to identify sets of proteins that may lead to the development of thermoresistance. Based on these results, we aim to develop functional tests and methods for the modulation of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.

Malignant melanomas have poor prognosis since treatment with anti-neoplastic agents is mostly ineffective. The biological mechanisms of this strong intrinsic therapy resistance are unknown. In order to identify new molecular factors potentially associated with the drug-resistant phenotype of malignant melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to four commonly used anticancer drugs in melanoma treatment, i.e., vindesine, etoposide, cisplatin, and fotemustine, was characterized using proteomic tools (two-dimensional electrophoresis for protein fractionation and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry for protein identification). In the neutral and weak acidic milieu (pH 4.0-8.0) a total number of 14 proteins showed alterations in expression whereas 20 proteins were differentially expressed in the basic milieu (pH 8.0-11.0). Besides proteins with unknown physiologic function, several factors were identified that show chaperone activity. Moreover, proteins involved in drug detoxification, metabolism, and regulation of apoptotic pathways could be identified. The possible role of these proteins in the development of chemoresistance is discussed, although detailed functional tests with these proteins have still to be performed. Nevertheless, it is clear that this proteomic approach for studying chemoresistance phenomena is a prerequisite before further investigation can yield insight into the biology and development of drug resistance in malignant melanoma.

Different types of cancer are naturally resistant to many anticancer drugs. Additionally, these tumours develop acquired drug resistance, which includes the classical multidrug resistance (MDR) accompanied by the synthesis of P-glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. Furthermore, atypical MDR is mediated by several different, some unknown, mechanisms. To overcome chemoresistance problems, antineoplastic drugs are often combined with other modes of therapy, e.g. hyperthermia, where good response has been reported in several experimental tumour models and in advanced cancer patients. The success of this combined anticancer treatment may be limited by an increase in chemoresistance and thermoresistance. A model system to study resistance phenomena is the use of chemoresistant and thermoresistant cancer cell lines. We have established chemoresistant cancer cell lines (gastric and pancreatic carcinoma, fibrosarcoma, melanoma) and now thermoresistant cell lines derived from gastric and pancreatic carcinoma cells and their counterparts that were resistant towards daunorubicin (classical MDR) and mitoxantrone (atypical MDR). Using proteomics, in this paper we evaluate the drug resistance of chemoresistant melanoma cells (parental cell line MeWo and sublines exhibiting drug resistance towards etoposide, cisplatin, fotemustine and vindesine) as a paradigm for analysis of drug resistance phenomena. Additionally, we investigate heat resistance and the interaction of chemoresistance and thermoresistance to identify common pathways using the parental and drug resistant stomach cancer cell lines EPG85-257, EPG85-257RNOV, EPG85-257RDB and their thermoresistant counterparts. Possible implications of differential protein expression will be discussed.

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysisThe number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,

The use of three-dimensional cell culture models, so-called multicellular tumor spheroids, is a special approach in experimental cancer research, because spheroids are similar to in vivo tumors in structural as well as functional sense. Cells grown in spheroids exhibit alterations of cell cycle regulation, induction of apoptosis and differentiation and can acquire multidrug resistance. In this study we investigated the protein expression in human colorectal cancer cells grown in monolayer and in spheroid cultures using proteomics. Evaluation by computer-assisted image analysis revealed overexpression of three cytokeratin 18 fragments that were generated in vivo. Cytokeratin 18 has previously been described as a target for caspase-mediated cleavage during apoptosis and our results indicate that apoptosis may take place in spheroids. Other proteins upregulated in spheroids include calreticulin precursor, a rho GDP dissociation inhibitor variant, several cytokeratins and peroxiredoxin 4. Some of these proteins have already been linked to chemoresistance and apoptotic phenomena.