Deciphering transcription factor networks from microarray data remains difficult. This study presents a simple method to infer the regulation of transcription factors from microarray data based on well-characterized target genes. We generated a catalog containing transcription factors associated with 2720 target genes and 6401 experimentally validated regulations. When it was available, a distinction between transcriptional activation and inhibition was included for each regulation. Next, we built a tool (www.tfacts.org) that compares submitted gene lists with target genes in the catalog to detect regulated transcription factors. TFactS was validated with published lists of regulated genes in various models and compared to tools based on in silico promoter analysis. We next analyzed the NCI60 cancer microarray data set and showed the regulation of SOX10, MITF and JUN in melanomas. We then performed microarray experiments comparing gene expression response of human fibroblasts stimulated by different growth factors. TFactS predicted the specific activation of Signal transducer and activator of transcription factors by PDGF-BB, which was confirmed experimentally. Our results show that the expression levels of transcription factor target genes constitute a robust signature for transcription factor regulation, and can be efficiently used for microarray data mining.

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The online version of this article contains a supplementary appendix. BackgroundChimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases. Design and MethodsWe used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation. ResultsIn contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation. ConclusionsWe have shown that chimeric receptor tyrosine kinases escape ubiquitination and downregulation and that their stabilization is critical to efficient stimulation of cell proliferation.Key words: PDGF receptor, oncogenes, protein degradation, ubiquitin.Citation: Toffalini F, Kallin A, Vandenberghe P, Pierre P, Michaux L, Cools J, and Demoulin J-B. The fusion proteins TEL-PDGFRβ and FIP1L1-PDGFRα escape ubiquitination and degradation Haematologica 2009;94:1085-1093.doi:10.3324/haematol.2008 This is an open-access paper. The fusion proteins TEL-PDGFRβ and FIP1L1-PDGFRα escape ubiquitination and degradation

the secondary malignancies and cardiovascular events, may not directly relate to SCT itself.Long-term follow-up should also consider issues of quality of life (QOL). This initial analysis shows that chronic graft-versushost disease rates and the duration of required immune suppression were lower after RIC. Chronic graft-versus-host disease is a dominant factor determining QOL, suggesting better long-term QOL with RIC; however, formal QOL assessment is required to determine this question.The major limitation of this study is the nonrandomized comparison and the selection biases in allocating patients to one of the regimens. However, because patients were selected for RIC based on high risk for non-relapse mortality, and because OS was similar among these patients given RIC and eligible patients given MAC, it is possible that results in standard risk patients may be similar. Randomized studies, for patients in CR (preferentially CR1) with long-term follow-up and assessment of late complication and QOL, will be needed to determine the best approach in this setting. MAC and possibly the new reduced toxicity myeloablative regimens are still the best approach in patients with active disease.

The fusion of TEL with platelet-derived growth factor receptor (PDGFR) ␤ (TP␤) is found in a subset of patients with atypical myeloid neoplasms associated with eosinophilia and is the archetype of a larger group of hybrid receptors that are produced by rearrangements of PDGFR genes. TP␤ is activated by oligomerization mediated by the pointed domain of TEL/ETV6, leading to constitutive activation of the PDGFR␤ kinase domain. The receptor transmembrane (TM) domain is retained in TP␤ and in most of the described PDGFR␤ hybrids. Deletion of the TM domain (⌬TM-TP␤) strongly impaired the ability of TP␤ to sustain growth factor-independent cell proliferation. We confirmed that TP␤ resides in the cytosol, indicating that the PDGFR␤ TM domain does not act as a transmembrane domain in the context of the hybrid receptor but has a completely different function. The ⌬TM-TP␤ protein was expressed at a lower level because of increased degradation. It could form oligomers, was phosphorylated at a slightly higher level, co-immunoprecipitated with the p85 adaptor protein, but showed a much reduced capacity to activate STAT5 and ERK1/2 in Ba/F3 cells, compared with TP␤. In an in vitro kinase assay, ⌬TM-TP␤ was more active than TP␤ and less sensitive to imatinib, a PDGFR inhibitor. In conclusion, we show that the TM domain is required for TP␤-mediated signaling and proliferation, suggesting that the activation of the PDGFR␤ kinase domain is not enough for cell transformation.

Dynamic follow up and analysis of isolated single cells that display non-adhering behaviour is hindered by the fact that they float in the suspension medium and thus requires the implementation of systems that physically entrap the cells for successful analysis. We describe the potential of digital microfluidic (DMF) chip technology for conducting analysis of cells in suspension at the single cell resolution. More specifically, we demonstrate the use of DMF technology for the analysis of a group of single individual protoplasts from Arabidopsis thaliana plants that are labelled with magnetic particles and are immobilized on the DMF chip by magnetic forces. By transporting droplets with different osmotic conditions to the site where the cells are trapped, we challenge the cells and monitor their responses dynamically with a camera. The use of DMF technology for performing water potential measurements has the following advantages: (i) solid particles such as cells and magnetic beads are manipulated on the chip without the risk of clogging channels, (ii) low shear stress during droplet unit operations like mixing and transport that characterize DMF are particularly suited for analysis of delicate cells types that lack cell walls such as protoplasts, (iii) the throughput of the analysis is strongly increased as multiple protoplasts are analysed simultaneously, in contrast with the traditional methods that can handle and challenge only one cell at a time. We show that the DMF analysis platform is effective in creating the steep osmotic gradients required for calculation of water permeability coefficients (P) as the values found are comparable with previously reported ones thus validating the suitability of the technology for such studies. This work illustrates a proof of concept for the applicability of DMF as an unparalleled and promising system for implementing single cells studies on non-adhering cells in an automated way.

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα(D842V) mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα(D842V) inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34(+) hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα(D842V) and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.

Aptamers are short, single stranded DNA/RNA oligonucleotides with the ability to fold into well-defined three-dimensional structures, which are fundamental for their highly selective target recognition, rivaling thus antibodies in terms of affinity and specificity. Thanks to these properties, they have been integrated into different micro-and nanoscale transducers, resulting in a new type of biosensors, known as 'aptasensors', used in a truly new generation of diagnostic tools. Here, an overview will be given of different aptamer selection approaches, with the focus on lab-on-a-chip technology based methods, different concepts reported for integrating aptamers into micro-and nanobiosystems aiming at the development of sensitive diagnostics concluding with an outlook on the future of aptamers in theranostics.