[Cisteinski katepsini, stefini in razgradnja izvenceličnega matriksa med invazijo človeških transformiranih celičnih linij raka dojke]

Background. Human breast cellular model, comprising four cell lines originating from spontaneously immortalized human breast epithelial MCF10A cell line, its c-Ha-ras transfectant, MCF10AT, and two tumourigenic derivatives, cultured from two sequential mouse xenographs, MCF10AT-Ca1a and MCF10AT-Ca1d, were used to compare the relative protein concentration of cathepsins and stefins in single cells. Methods. The relative protein concentration of cathepsins and stefins in single cells was analysed by confocal microscopy, and compared to their protein expression in cell homogenates. Results. The most invasive, MCF10AT cell line contained several fold higher protein concentration of cathepsin B and increased levels of stefins, but similar levels of cathepsin L, compared with the parental MCF10A cells. This was associated with five fold higher endocytosis of Matrigel-DQ-collagen IV (DQC) and a simultaneous increase in signal overlap between DQC and cathepsin L as well as DQC and stefin B, but a decrease in that of DQC and cathepsin B overlap in the MCF10AT cells. Simultaneously, increased signal overlaps between both cathepsins and between cathepsins-stefins pairs, were observed in this cell line. Conclusions. These results suggest that the increased collagen endocytosis and degradation in theinvasive phenotype significantly affect also the subcellular localization of cysteine cathepsins and stefins. Based on these and the reports of other authors, we hypothesize that the intracellular degradation may also be assoeiated with cathepsin L, whereas cathepsin B in the ras transformed breastcells is involved in both, the intracellular and pericellular degradation of extracellular matrix during cell migration and invasion

Circlulation of tumor cells and their effect on breast cancer – reviewing the results of previous studies

Circulating tumor cells (CTCs) are epithelial tumor cells detected in the peripheral blood of patients with solid malignant tumors. They originate from the primary tumor or metastatic sites. New techniques have been developed to isolate and characterise these cells, including the FDA-approved CellSearch, which are using mainly cytometric/antibody-based and molecular approaches. Recent advances in theories regarding metastasis support the role of early release of tumor cells in the neoplastic process. It has been found that phenotypic variation exists between the primary tumor and CTCs. Of particular interest is the difference found between primary tumor and CTC HER-2 status in both metastatic and early breast cancer. CTC enumeration has been incorporated into different fields of oncology as a prognostic marker, a tool to monitor therapy response, and a method to understand basic tumor characteristics. But currently, there is still no role for CTCs in clinical practice

Cirkulirajoče tumorske celice in njihov pomen pri raku dojke: pregled izsledkov dosedanjih raziskav

Circulating tumor cells (CTCs) are epithelial tumor cells detected in the peripheral blood of patients with solid malignant tumors. They originate from the primary tumor or metastatic sites. New techniques have been developed to isolate and characterise these cells, including the FDA-approved Cell-Search, which are using mainly cytometric/antibody-based and molecular approaches. Recent advances in theories regarding metastasis support the role of early release of tumor cells in the neoplastic process. It has been found that phenotypic variation exists between the primary tumor and CTCs. Of particular interest is the difference found between primary tumor and CTC HER-2 status in both metastatic and early breast cancer. CTC enumeration has been incorporated into different fields of oncology as a prognostic marker, a tool to monitor therapy response, and a method to understand basic tumor characteristics. But currently, there is still no role for CTCs in clinical practice.Ni abstrakta

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

BACKGROUND: Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. METHODS: Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. RESULTS: The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. CONCLUSIONS: The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents