RESUMO
Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.
Assuntos
Biomarcadores Tumorais/análise , Processamento de Imagem Assistida por Computador , Proteínas de Neoplasias/análise , Neoplasias/química , Análise Serial de Proteínas/métodos , Animais , Biópsia , Western Blotting/métodos , Neoplasias da Mama/química , Carcinoma/química , Feminino , Fixadores , Formaldeído , Congelamento , Humanos , Camundongos , Camundongos Nus , Nanomedicina , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Proteômica , Receptor ErbB-2/análise , Fixação de TecidosRESUMO
Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones. To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins. Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain. Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth. Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex. In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches. Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain. The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions. By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp.