[Tumour-stroma interactions in urothelial cancer]. / Tumor-Stroma-Interaktionen im Harnblasenkarzinom.

BACKGROUND: The histopathological structure of malignant tumours involves two essential compartments - the tumour parenchyma with the actual transformed cells, and the supportive tumour stroma. The latter consists of specialized mesenchymal cells, such as fibroblasts, macrophages, lymphocytes and vascular cells, as well as of their secreted products, including components of the extracellular matrix, matrix modifying enzymes and numerous regulatory growth factors and cytokines. In consequence, the tumour stroma has the ability to influence virtually all aspects of tumour development and progression, including therapeutic response. AIM: In this article we review the current knowledge of tumor stroma interactions in urothelial carcinoma and present various experimental systems that are currently in use to unravel the biological basis of these heterotypic cell interactions. RESULTS: For urothelial carcinoma, an extensive tumour stroma is quite typical and markers of activated fibroblasts correlate significantly with clinical parameters of advanced disease. Another clinically important variable is provided by the stromal expression of syndecan-1. CONCLUSION: Integration of markers of activated stroma into clinical risk evaluation could aid to better stratification of urothelial bladder carcinoma patients. Elucidation of biological mechanisms underlying tumour-stroma interactions could provide new therapeutical targets.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

A myosin I isoform in the nucleus.

A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.

Statistical evaluation of colocalization patterns in immunogold labeling experiments.

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.

Evidence for the presence of myosin I in the nucleus.

We produced and affinity-purified polyclonal antibodies to adrenal myosin I. These antibodies recognize adrenal myosin I by Western blot analysis (116 kDa) and inhibit the actin-activated ATPase activity of purified adrenal myosin I. They also recognize a 120-kDa protein in extracts prepared from many different cell lines. Fluorescence microscopy demonstrated the presence of immunoreactive material in the perinuclear region, the leading edges, and the nuclei of 3T3 cells. Fluorescence microscopy also demonstrated nuclear staining in mouse oocytes at the germinal vesicle stage and in the pronuclei during fertilization. Confocal and immunoelectron microscopy confirmed the intranuclear localization. Electron microscopy also demonstrated staining of structures in nucleoli that are thought to be associated with rDNA transcription. Western blot analyses revealed the presence of the 120-kDa protein in extracts prepared from nuclei that are apparently free of cytosolic contamination. The same nuclear protein binds 125I-calmodulin and is photoaffinity labeled with [alpha-32P]ATP. The 120-kDa protein was partially purified from twice washed nuclei using ammonium sulfate fractionation and gel filtration chromatography. Column fractions containing 120-kDa protein as revealed by Western blot analysis also contain K+-EDTA ATPase activity. The 120-kDa protein was also shown to bind actin in the absence, but not the presence, of ATP. Since K+-EDTA ATPase activity, actin, and ATP binding are defining features of the members of the myosin superfamily of proteins, we propose that the 120-kDa protein is a previously undescribed myosin I isoform that is an intranuclear actin-based molecular motor.