Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress.

The combined deletion of chromosomal arms 1p and 19q has been described as a prognostic marker for oligodendroglial tumors. These tumors show a better response to chemotherapy and radiotherapy. Recently, we found a lower abundance of peroxiredoxin 1 (PRDX1) in oligodendroglial tumors with 1p/19q deletion, suggesting a potential role of this enzyme in the clearance of therapy induced reactive oxygen species (ROS). Here, we confirmed the importance of PRDX1 in tumor cell survival by PRDX1 knockdown and overexpression in A-172 cells treated with the alkylating agent bis-chloroethyl nitrosourea (BCNU). Overexpression of PRDX1 resulted in a higher resistance of cells to BCNU treatment. In addition, BCNU challenged cells showed higher levels of ROS in PRDX1 knockdown cells. We applied a modified version of the redox two dimensional difference gel electrophoresis approach to analyze ROS mediated effects on protein thiols after BCNU treatment by labeling protein thiols with fluorescent dyes. Altogether eleven proteins were identified showing PRDX1 dependent altered labeling, many of them have been previously linked to stress response processes. Furthermore, 30 additional potentially redox active proteins were identified. The majority of them is involved in therapy associated processes like cellular stress response, DNA damage and regulation of cell death and therewith suggests that tumor cells maintain a network of redox sensitive proteins to escape chemotherapy. This article is part of a Special Issue entitled: Medical Proteomics.

Differential proteome analysis of human gliomas stratified for loss of heterozygosity on chromosomal arms 1p and 19q.

Combined deletion of chromosomal arms 1p and 19q is an independent prognostic marker in patients with oligodendroglial brain tumors, including oligodendrogliomas and oligoastrocytomas. However, the relevant genes in these chromosome arms and the molecular mechanisms underlying the prognostic significance of 1p/19q deletion are yet unknown. We used two-dimensional difference gel electrophoresis followed by mass spectrometry to perform a proteome-wide profiling of low-grade oligoastrocytomas stratified for the presence or absence of 1p/19q deletions. Thereby, we identified 22 different proteins showing differential expression in tumors with or without combined deletions of 1p and 19q. Four of the differentially expressed proteins, which are vimentin, villin 2 (ezrin), annexin A1, and glial fibrillary acidic protein, were selected for further analysis. Lower relative expression levels of these proteins in 1p/19q-deleted gliomas were confirmed at the protein level by Western blot analysis and immunohistochemistry. Furthermore, sequencing of sodium bisulfite-treated tumor DNA revealed more frequent methylation of 5'-CpG islands associated with the VIM and VIL2 genes in 1p/19q-deleted gliomas when compared with gliomas without these deletions. In summary, we confirm proteome-wide profiling as a powerful means to identify candidate biomarkers in gliomas. In addition, our data support the hypothesis that 1p/19q-deleted gliomas frequently show epigenetic down-regulation of multiple genes due to aberrant methylation of the 5'-CpG islands.

Identification and functional characterization of microRNAs involved in the malignant progression of gliomas.

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma WHO grade III or secondary glioblastoma WHO grade IV. We explored the role of microRNAs (miRNAs) in glioma progression by investigating the expression profiles of 157 miRNAs in four patients with primary WHO grade II gliomas that spontaneously progressed to WHO grade IV secondary glioblastomas. Thereby, we identified 12 miRNAs (miR-9, miR-15a, miR-16, miR-17, miR-19a, miR-20a, miR-21, miR-25, miR-28, miR-130b, miR-140 and miR-210) showing increased expression, and two miRNAs (miR-184 and miR-328) showing reduced expression upon progression. Validation experiments on independent series of primary low-grade and secondary high-grade astrocytomas confirmed miR-17 and miR-184 as promising candidates, which were selected for functional analyses. These studies revealed miRNA-specific influences on the viability, proliferation, apoptosis and invasive growth properties of A172 and T98G glioma cells in vitro. Using mRNA and protein expression profiling, we identified distinct sets of transcripts and proteins that were differentially expressed after inhibition of miR-17 or overexpression of miR-184 in glioma cells. Taken together, our results support an important role of altered miRNA expression in gliomas, and suggest miR-17 and miR-184 as interesting candidates contributing to glioma progression.

Proteomic analysis of astroglial connexin43 silencing uncovers a cytoskeletal platform involved in process formation and migration.

Connexin43 (Cx43) is the most abundant gap junction protein of the brain, where it is predominantly expressed in astrocytes. Recent studies imply a role of Cx43 in the regulation of important cellular processes, including migration, proliferation, and shape formation. These processes are assumed to be reflected by the proteome of the Cx43 expressing cells. To analyze the influence of Cx43 on the astrocytic proteome, we used RNA interference to downregulate the expression of this connexin in cultures of mouse astrocytes. We applied difference gel electrophoresis (DIGE) to compare silenced astrocytes with control cells. The differential proteome analysis revealed 15 significantly regulated proteins (between 1.2- and 1.6-fold), of which six are known to belong to a group of cytoskeletal proteins involved in cortical platform formation. Astrocytes treated with Cx43 small interfering (si)RNA showed an increased expression of the cytoskeletal proteins: actin, tropomyosin, microtubule-associated protein RP/EB1, transgelin, and GFAP, and a decreased expression of cofilin-1. Quantitative immunocytochemistry and Western blotting revealed similar results showing an upregulation of actin, tubulin, tropomyosin, EB1, transgelin and GFAP, and a downregulation of Ser-3-phosphorylated cofilin. Furthermore, Cx43 silencing led to phenotypical changes in cell morphology, migratory activity, and cell adhesion. Our results provide mechanistic clues for an understanding of Cx43 interaction with cellular motor activities such as migration and process formation in astrocytes.

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis.

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high-molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high-salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2-D DIGE, MS and Western blotting.