PubMed · 14054607
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A POREBSKA, K ZEMBUROWA. [SPECIFIC GEL PRECIPITATION].. https://pubmed.ncbi.nlm.nih.gov/14054607/
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In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.
Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.
Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.