A robust method for estimating gene expression states using Affymetrix microarray probe level data

<p>Abstract</p> <p>Background</p> <p>Microarray technology is a high-throughput method for measuring the expression levels of thousand of genes simultaneously. The observed intensities combine a non-specific binding, which is a major disadvantage with microarray data. The Affymetrix GeneChip assigned a mismatch (MM) probe with the intention of measuring non-specific binding, but various opinions exist regarding usefulness of MM measures. It should be noted that not all observed intensities are associated with expressed genes and many of those are associated with unexpressed genes, of which measured values express mere noise due to non-specific binding, cross-hybridization, or stray signals. The implicit assumption that all genes are expressed leads to poor performance of microarray data analyses. We assume two functional states of a gene - expressed or unexpressed - and propose a robust method to estimate gene expression states using an order relationship between PM and MM measures.</p> <p>Results</p> <p>An indicator 'probability of a gene being expressed' was obtained using the number of probe pairs within a probe set where the PM measure exceeds the MM measure. We examined the validity of the proposed indicator using Human Genome U95 data sets provided by Affymetrix. The usefulness of 'probability of a gene being expressed' is illustrated through an exploration of candidate genes involved in neuroblastoma prognosis. We identified the candidate genes for which expression states differed (un-expressed or expressed) when compared between two outcomes. The validity of this result was subsequently confirmed by quantitative RT-PCR.</p> <p>Conclusion</p> <p>The proposed qualitative evaluation, 'probability of a gene being expressed', is a useful indicator for improving microarray data analysis. It is useful to reduce the number of false discoveries. Expression states - expressed or unexpressed - correspond to the most fundamental gene function 'On' and 'Off', which can lead to biologically meaningful results.</p

TAK1 Is Required for Survival of Mouse Fibroblasts Treated with TRAIL, and Does So by NF-κB Dependent Induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance

Identification of novel vascular targets in lung cancer

Background: Lung cancer remains the leading cause of cancer-related death, largely owing to the lack of effective treatments. A tumour vascular targeting strategy presents an attractive alternative; however, the molecular signature of the vasculature in lung cancer is poorly explored. This work aimed to identify novel tumour vascular targets in lung cancer. Methods: Enzymatic digestion of fresh tissue followed by endothelial capture with Ulex lectin-coated magnetic beads was used to isolate the endothelium from fresh tumour specimens of lung cancer patients. Endothelial isolates from the healthy and tumour lung tissue were subjected to whole human genome expression profiling using microarray technology. Results: Bioinformatics analysis identified tumour endothelial expression of angiogenic factors, matrix metalloproteases and cellsurface transmembrane proteins. Predicted novel tumour vascular targets were verified by RNA-seq, quantitative real-time PCR analysis and immunohistochemistry. Further detailed expression profiling of STEAP1 on 82 lung cancer patients confirmed STEAP1 as a novel target in the tumour vasculature. Functional analysis of STEAP1 using siRNA silencing implicates a role in endothelial cell migration and tube formation. Conclusions: The identification of cell-surface tumour endothelial markers in lung is of interest in therapeutic antibody and vaccine development

Altering Chemosensitivity by Modulating Translation Elongation

BACKGROUND: The process of translation occurs at a nexus point downstream of a number of signal pathways and developmental processes. Modeling activation of the PTEN/AKT/mTOR pathway in the Emu-Myc mouse is a valuable tool to study tumor genotype/chemosensitivity relationships in vivo. In this model, blocking translation initiation with silvestrol, an inhibitor of the ribosome recruitment step has been showed to modulate the sensitivity of the tumors to the effect of standard chemotherapy. However, inhibitors of translation elongation have been tested as potential anti-cancer therapeutic agents in vitro, but have not been extensively tested in genetically well-defined mouse tumor models or for potential synergy with standard of care agents. METHODOLOGY/PRINCIPAL FINDINGS: Here, we chose four structurally different chemical inhibitors of translation elongation: homoharringtonine, bruceantin, didemnin B and cycloheximide, and tested their ability to alter the chemoresistance of Emu-myc lymphomas harbouring lesions in Pten, Tsc2, Bcl-2, or eIF4E. We show that in some genetic settings, translation elongation inhibitors are able to synergize with doxorubicin by reinstating an apoptotic program in tumor cells. We attribute this effect to a reduction in levels of pro-oncogenic or pro-survival proteins having short half-lives, like Mcl-1, cyclin D1 or c-Myc. Using lymphomas cells grown ex vivo we reproduced the synergy observed in mice between chemotherapy and elongation inhibition and show that this is reversed by blocking protein degradation with a proteasome inhibitor. CONCLUSION/SIGNIFICANCE: Our results indicate that depleting short-lived pro-survival factors by inhibiting their synthesis could achieve a therapeutic response in tumors harboring PTEN/AKT/mTOR pathway mutations

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors, The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density