Search

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program. [ABSTRACT FROM AUTHOR]

Blokzijl A, Friedman M, Pontén F, Landegren U (From the Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden). Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine. (Review) J Intern Med 2010; 268: 232–245. The ability to detect very low levels of expressed proteins has enormous potential for early diagnostics and intervention at curable stages of disease. An extended range of targets such as interacting or post-translationally modified proteins can further improve the potential for diagnostics and patient stratification, and for monitoring response to treatment. These are critical building blocks for personalized treatment strategies to manage disease. The past few decades have seen a remarkably improved understanding of the molecular basis of disease in general, and of tumour formation and progression in particular. This accumulated knowledge creates opportunities to develop drugs that specifically target molecules or molecular complexes critical for survival and expansion of tumour cells. However, tumours are highly variable between patients, necessitating the development of diagnostic tools to individualize treatment through parallel analysis of sets of biomarkers. The proximity ligation assay (PLA) can address many of the requirements for advanced molecular analysis. The method builds on the principle that recognition of target proteins by two, three or more antibodies can bring in proximity DNA strands attached to the antibodies. The DNA strands can then participate in ligation reactions, giving rise to molecules that are amplified for highly sensitive detection. PLA is particularly well suited for sensitive, specific and multiplexed analysis of protein expression, post-translational modifications and protein–protein interactions. The analysis of this extended range of biomarkers will prove critical for the development and implementation of personalized medicine. [ABSTRACT FROM AUTHOR]

Background: IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways. Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases. Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, multiple sclerosis (MS). Methods: We genotyped nine single nucleotide polymorphisms and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case-control cohorts from Spain and Sweden, and a set of MS trio families from Finland. Results: Two single nucleotide polymorphism ISNPs) (rs4728142, rs3807306), and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with values of p<0.001 when the data from all cohorts were combined. The predisposing alleles were present on the same common haplotype in all populations. Using electrophoretic mobility shift assays we observed allele specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel. Conclusion: These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population. Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases. [ABSTRACT FROM AUTHOR]

Abstract. Landegren U (Rudbeck Laboratory, Uppsala, Sweden) Gene-based diagnostics (Internal Medicine in the 21st Century). J Intern Med 2000; 248: 271–276. Gene-based diagnostics has been slow to enter medical routine practice in a grand way, but it is now spurred on by three important developments: the total genetic informational content of humans and most of our pathogens is rapidly becoming available; a very large number of genetic factors of diagnostic value in disease are being identified; and such factors include the identity of genes frequently targeted by mutations in specific diseases, common DNA sequence variants associated with disease or responses to therapy, and copy number alterations at the level of DNA or RNA that are characteristic of specific diseases. Finally, improved methodology for genetic analysis now brings all of these genetic factors within reach in clinical practice. The increasing opportunities for genetic diagnostics may gradually influence views on health and normality, and on the genetic plasticity of human beings, provoking discussions about some of the central attributes of genetics. [ABSTRACT FROM AUTHOR]

Cyclosporin A (CyA) interferes with immune responses and prevents growth factor release by stimulated T cells. However, it is not known whether this is due to an effect on the accessory cells, required for T-cell responses, whether antigen recognition cannot occur, or whether later steps, leading to lymphokine production, are blocked. For this reason, the effect of CyA on homogeneous populations of T tumour cells was investigated. The immunosuppressive compound efficiently prevented T-cell growth factor (TCGF) (interleukin 2) release by stimulated tumour cells. Still, the cells retained the surface antigen T.V known to be involved in T-cell stimulation, after treatment with CyA. Furthermore, CyA failed to affect the inhibition of proliferation, observed in a T-cell tumour in response to stimulation, indicating that the cells had received the stimulatory impetus. TCGF release, induced by treatment with a phorbol ester, was only partly sensitive to inhibition by CyA, demonstrating that CyA will interfere with discrete aspects of the stimulation of a T-cell. [ABSTRACT FROM AUTHOR]

Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.Results: For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: Estrogen is an established endometrial carcinogen. One of the most important mediators of estrogenic action is the estrogen receptor alpha. We have investigated whether polymorphic variation in the estrogen receptor alpha gene (ESR1) is associated with endometrial cancer risk.Methods: In 702 cases with invasive endometrial cancer and 1563 controls, we genotyped five markers in ESR1 and used logistic regression models to estimate odds ratios (OR) and 95 percent confidence intervals (CI).Results: We found an association between rs2234670, rs2234693, as well as rs9340799, markers in strong linkage disequilibrium (LD), and endometrial cancer risk. The association with rs9340799 was the strongest, OR 0.75 (CI 0.60-0.93) for heterozygous and OR 0.53 (CI 0.37-0.77) for homozygous rare compared to those homozygous for the most common allele. Haplotype models did not fit better to the data than single marker models.Conclusion: We found that intronic variation in ESR1 was associated with endometrial cancer risk. [ABSTRACT FROM AUTHOR]