Julia Poland

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.

We report on a new silver stain especially developed for staining large gels (25 cm× 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 νg per gel.

Study of Therapy Resistance in Cancer Cells with Functional Proteome Analysis

Abstract 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 unkown, 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.

Comparison of protein expression profiles between monolayer and spheroid cell culture of HT-29 cells revealed fragmentation of CK18 in three-dimensional cell culture.

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.

Characterization of the differential protein expression associated with thermoresistance in human gastric carcinoma cell lines

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.

Association of genomic imbalances with drug resistance and thermoresistance in human gastric carcinoma cells

Therapy resistance is the major obstacle to advances in successful cancer treatment. To characterize chromosomal alterations associated with different types of acquired MDR and thermoresistance, we applied CGH to compare a unique panel of human gastric carcinoma cells consisting of the parental, drug‐sensitive and thermosensitive cancer cell line EPG85‐257P, the atypical MDR variant EPG85‐257RNOV, the classical MDR subline EPG85‐257RDB and their thermoresistant counterparts EPG85‐257P‐TR, EPG85‐257RNOV‐TR and EPG85‐257RDB‐TR. CGH with genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included various members of the families of ABC transporters and molecular chaperones. The importance of these cell variant‐specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.

High correlation of the proteome patterns in bone marrow and peripheral blood blast cells in patients with acute myeloid leukemia

BackgroundWhen comparing myelogenous blasts from bone marrow and peripheral blood, immunophenotyping usually show a strong correlation of expression of surface antigens. However, it remains to be determined, whether this correlation also exists on the level of protein expression.MethodTherefore, we investigated both bone marrow and peripheral blood blast cells from six patients with newly diagnosed acute myeloid leukemia (AML) using conventional two-dimensional electrophoresis in the first dimension and linear polyacrylamide gels (12%) in the second dimension. Proteins were visualized using the silver staining method and image analysis was performed using the PDQuest system.ResultsFor each patient over 80 proteins were evaluated in the sample from peripheral blood and bone marrow. We could demonstrate that the protein expression profile of bone marrow did not significantly differ from the expression patterns of peripheral blast cells.ConclusionThe proteome-set of leukemic blast cells from marrow and blood, does not differ substantially when drawn from AML patients with over 80 percent blast cells in both compartments. This indicates that in AML, blasts from peripheral blood samples can be considered suitable for investigations of the proteome using 2D-electrophoresis.

Use of proteomics to study chemosensitivity.

Chemoresistance remains an unresolved problem in clinical oncology. Therefore it is important to identify molecular factors that lead to an understanding of the mechanisms of drug resistance in cancer cells. On the protein-expression level, this can be done using proteomics, which has become the focus of significant interest and research over the past decade. We describe an easy and practicable standardized technique that can be used to study global protein expression in chemosensitive and chemoresistant cancer cells to find candidate proteins that are potentially associated with the drug-resistant phenotype. As an example, fractionation of human neuroblastoma cells using two-dimensional polyacrylamide electrophoresis, spot detection, image analysis, and finally protein identification is illustrated.