In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

BACKGROUND: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. METHOD AND RESULTS: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6-HER2 protein-protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). CONCLUSION: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2-PTK6 complexes are of prognostic relevance

Comparing the Epidermal Growth Factor Interaction with Four Different Cell Lines: Intriguing Effects Imply Strong Dependency of Cellular Context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of 125I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®

Application guide for omics approaches to cell signaling

Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations

Novel and highly recurrent chromosomal alterations in Sezary syndrome.

Contains fulltext : 71220.pdf (publisher's version ) (Closed access)This study was designed to identify highly recurrent genetic alterations typical of Sezary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sezary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.Ola Söderberg and Ulf Landegren jointly supervised this work</p

TREM-1 multimerization is essential for its activation on monocytes and neutrophils

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells. By promoting the amplification of inflammatory signals that are initially triggered by Toll-like receptors (TLRs), TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases, such as septic shock, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. However, the molecular events leading to the activation of TREM-1 in innate immune cells remain unknown. Here, we show that TREM-1 is activated by multimerization and that the levels of intracellular Ca2+release, reactive oxygen species, and cytokine production correlate with the degree of TREM-1 aggregation. TREM-1 activation on primary human monocytes by LPS required a two-step process consisting of upregulation followed by clustering of TREM-1 at the cell surface, in contrast to primary human neutrophils, where LPS induced a rapid cell membrane reorganization of TREM-1, which confirmed that TREM-1 is regulated differently in primary human neutrophils and monocytes. In addition, we show that the ectodomain of TREM-1 is able to homooligomerize in a concentration-dependent manner, which suggests that the clustering of TREM-1 on the membrane promotes its oligomerization. We further show that the adapter protein DAP12 stabilizes TREM-1 surface expression and multimerization. TREM-1 multimerization at the cell surface is also mediated by its endogenous ligand, a conclusion supported by the ability of the TREM-1 inhibitor LR12 to limit TREM-1 multimerization. These results provide evidence for ligand-induced, receptor-mediated dimerization of TREM-1. Collectively, our findings uncover the mechanisms necessary for TREM-1 activation in monocytes and neutrophils