The study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1-0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides ofmolecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS-containing gels and a simple spot test on thin gel layers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05-0.1 ng protein per band. Total staining time with the new procedure is 75 min.

Purpose:The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients. Experimental Design: To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using twodimensional gel electrophoresis followed by MALDI-ToF analysis.The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays. Results: In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/ progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival. Conclusions: Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo.Breast cancer, the most common malignancy in females, kills f300,000 women worldwide every year and is the main reason for cancer-related death in the postoperative development of distant metastasis in secondary organs (1). Many of the patients who are primarily diagnosed as having apparently localized or regional breast cancer eventually develop distant metastases. Clinical experience showed that in early clinical stages, cells could already dissociate from the tumor's parenchyma via the hematogenous route and colonize the bone marrow, the clinically most relevant site of metastatic disease in patients with solid epithelial tumors (2). Often, many years after resection of the primary tumor and treatment of cancer patients, micrometastatic cancer cells can grow out to form overt metastases. Indeed, evidence is accumulating that the presence of occult carcinoma cells of epithelial origin in the bone marrow is an independent risk factor in breast cancer (3 -5). Understanding the biology of these dormant tumor cells in the bone marrow is therefore pivotal for the development of novel therapeutic approaches for the t...