Insulin is stored in vivo inside the pancreatic β-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic β-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic β-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone.

• MeSH
• beta-buňky * MeSH
• elektronová mikroskopie MeSH
• inzulin MeSH
• Langerhansovy ostrůvky * MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. METHODS: We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. RESULTS: Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. CONCLUSIONS: Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

• MeSH
• 2D gelová elektroforéza MeSH
• alkoholoxidoreduktasy metabolismus   MeSH
• chromatografie afinitní MeSH
• cisplatina aplikace a dávkování   MeSH
• deoxycytidin aplikace a dávkování   analogy a deriváty   MeSH
• dospělí MeSH
• fenotyp MeSH
• fluorouracil aplikace a dávkování   analogy a deriváty   MeSH
• glutathion-S-transferasa fí metabolismus   MeSH
• karboplatina aplikace a dávkování   MeSH
• kolorektální nádory farmakoterapie   metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• nádory hlavy a krku farmakoterapie   metabolismus   patologie   MeSH
• nádory plic farmakoterapie   metabolismus   patologie   MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• neurofyziologie MeSH
• organoplatinové sloučeniny aplikace a dávkování   MeSH
• paclitaxel aplikace a dávkování   MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• výsledek terapie MeSH
• western blotting MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

BACKGROUND: Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences. METHODS: We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4-7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. RESULTS: Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased. CONCLUSION: We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

• MeSH
• 2D gelová elektroforéza MeSH
• dospělí MeSH
• epitelové buňky metabolismus   MeSH
• financování organizované MeSH
• lidé středního věku MeSH
• lidé MeSH
• metastázy nádorů patofyziologie   patologie   MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny metabolismus   MeSH
• nádory prsu metabolismus   MeSH
• peptidové mapování MeSH
• proteomika MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• srovnávací studie MeSH

Human MDA-MB-231 derived breast cancer cell lines 1833 and 4175 have different metastatic potentials in terms of their tissue tropisms and aggressiveness. Cell line 1833 is specifically metastatic to the bone. The highly aggressive cell line 4175 is specific to the lung. We performed 2-DE analysis of the cell lines. We found 16 significantly changed protein spots, 14 protein spots were identified. Expression of cathepsin D, triosephosphate isomerase, phosphoglycerate kinase 1, heme binding protein 1 and annexin 2 could be correlated with the in vitro aggressiveness of the respective cell lines. Interstitial collagenase and dimethylargininase 2 were exclusive to the cell line 1833 and might contribute to its bone specificity. Serpin B9, cathepsin B chain b, galectin 3 and HSP 27 were changed in the lung specific cell line 4175. The possible contribution of identified proteins to differences in metastatic behavior of the cell lines is discussed.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• financování organizované MeSH
• hmotnostní spektrometrie metody   MeSH
• invazivní růst nádoru MeSH
• kathepsin D biosyntéza   MeSH
• kolagenasy biosyntéza   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové buněčné linie MeSH
• nádory prsu metabolismus   patologie   MeSH
• peptidy chemie   MeSH
• počítačové zpracování obrazu MeSH
• regulace genové exprese u nádorů MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.

• MeSH
• 2D gelová elektroforéza MeSH
• duktální karcinom prsu chemie   MeSH
• fenotyp MeSH
• financování organizované MeSH
• kmenové buňky chemie   MeSH
• lidé MeSH
• mléčné žlázy lidské cytologie   chemie   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny chemie   MeSH
• nádory prsu chemie   MeSH
• proteom chemie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

• Publikační typ
• abstrakty MeSH

• MeSH
• 2D gelová elektroforéza * metody   přístrojové vybavení   využití   MeSH
• biomedicínské technologie metody   trendy   MeSH
• hmotnostní spektrometrie * metody   využití   MeSH
• karcinoembryonální antigen izolace a purifikace   MeSH
• kokultivační techniky metody   využití   MeSH
• lékařská onkologie trendy   MeSH
• lidé MeSH
• mamografie metody   využití   MeSH
• muciny diagnostické užití   izolace a purifikace   MeSH
• nádorové biomarkery izolace a purifikace   MeSH
• nádory prsu * diagnóza   MeSH
• plošný screening metody   využití   MeSH
• statistika jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH