Joanna D. Holbrook

Resistin is expressed in human macrophages and directly regulated by PPARγ activators

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

Comprehensive Predictive Biomarker Analysis for MEK Inhibitor GSK1120212

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. Mol Cancer Ther; 11(3); 720–9. ©2011 AACR.

Deep sequencing of gastric carcinoma reveals somatic mutations relevant to personalized medicine

BackgroundGlobally, gastric cancer is the second most common cause of cancer-related death, with the majority of the health burden borne by economically less-developed countries.MethodsHere, we report a genetic characterization of 50 gastric adenocarcinoma samples, using affymetrix SNP arrays and Illumina mRNA expression arrays as well as Illumina sequencing of the coding regions of 384 genes belonging to various pathways known to be altered in other cancers.ResultsGenetic alterations were observed in the WNT, Hedgehog, cell cycle, DNA damage and epithelial-to-mesenchymal-transition pathways.ConclusionsThe data suggests targeted therapies approved or in clinical development for gastric carcinoma would be of benefit to ~22% of the patients studied. In addition, the novel mutations detected here, are likely to influence clinical response and suggest new targets for drug discovery.

The role of positive selection in determining the molecular cause of species differences in disease

BackgroundRelated species, such as humans and chimpanzees, often experience the same disease with varying degrees of pathology, as seen in the cases of Alzheimers disease, or differing symptomatology as in AIDS. Furthermore, certain diseases such as schizophrenia, epithelial cancers and autoimmune disorders are far more frequent in humans than in other species for reasons not associated with lifestyle. Genes that have undergone positive selection during species evolution are indicative of functional adaptations that drive species differences. Thus we investigate whether biomedical disease differences between species can be attributed to positively selected genes.ResultsWe identified genes that putatively underwent positive selection during the evolution of humans and four mammals which are often used to model human diseases (mouse, rat, chimpanzee and dog). We show that genes predicted to have been subject to positive selection pressure during human evolution are implicated in diseases such as epithelial cancers, schizophrenia, autoimmune diseases and Alzheimers disease, all of which differ in prevalence and symptomatology between humans and their mammalian relatives.In agreement with previous studies, the chimpanzee lineage was found to have more genes under positive selection than any of the other lineages. In addition, we found new evidence to support the hypothesis that genes that have undergone positive selection tend to interact with each other. This is the first such evidence to be detected widely among mammalian genes and may be important in identifying molecular pathways causative of species differences.ConclusionOur dataset of genes predicted to have been subject to positive selection in five species serves as an informative resource that can be consulted prior to selecting appropriate animal models during drug target validation. We conclude that studying the evolution of functional and biomedical disease differences between species is an important way to gain insight into their molecular causes and may provide a method to predict when animal models do not mirror human biology.

Characterisation of 5‐HT3C, 5‐HT3D and 5‐HT3E receptor subunits: evolution, distribution and function

The 5‐HT3 receptor is a member of the ‘Cys‐loop’ family of ligand‐gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5‐HT3 receptors originating from homomeric assemblies of 5‐HT3A or heteromeric assembly of 5‐HT3A and 5‐HT3B. Novel genes encoding 5‐HT3C, 5‐HT3D, and 5‐HT3E have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5‐HT3C, 5‐HT3D, and 5‐HT3E are not human–specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5‐HT3C, 5‐HT3D, and 5‐HT3E were all non‐functional when expressed alone. Co‐transfection studies to determine potential novel heteromeric receptor interactions with 5‐HT3A demonstrated that the expression or function of the receptor was modified by 5‐HT3C and 5‐HT3E, but not 5‐HT3D. The lack of distinct effects on current rectification, kinetics or pharmacology of 5‐HT3A receptors does not however provide unequivocal evidence to support a direct contribution of 5‐HT3C or 5‐HT3E to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5‐HT3 receptor antagonists have major clinical usage, therefore remains to be fully determined.

Immunomodulatory effects of etanercept in a model of brain injury act through attenuation of the acute-phase response.

TNF‐α has proved to be a successful target in the treatment of many peripheral inflammatory diseases, but the same interventions worsen immune‐mediated CNS disease. However, anti‐TNF‐α strategies may offer promise as therapy for non‐immune CNS injury. In this study, we have microinjected IL‐1β or lipopolysaccharide (LPS) into the rat brain as a simple model of brain injury and have systemically administered the TNF‐α antagonist etanercept to discover whether hepatic TNF‐α, produced as part of the acute‐phase response to CNS injury, modulates the inflammatory response in the brain. We report a significant reduction in neutrophil numbers recruited to the IL‐1β‐ or LPS‐challenged brain as a result of TNF‐α inhibition. We also show an attenuation in the levels of hepatic mRNA including TNF‐α mRNA and of TNF‐α‐induced genes, such as the chemokines CCL‐2, CXCL‐5, and CXCL‐10, although other chemokines elevated by the injury were not significantly changed. The reduction in hepatic chemokine synthesis results in reduced numbers of circulating neutrophils, and also a reduction in the numbers recruited to the liver as a consequence of brain injury. These findings suggest that TNF‐α inhibitors may reduce CNS inflammatory responses by targeting the hepatic acute‐phase response, and thus therapies for brain injury need not cross the blood–brain barrier to be effective.

Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium‐derived factor: Two novel biomarkers of Alzheimer's disease in human plasma

Emerging disease modifying therapeutic strategies for Alzheimers disease (AD) have generated a critical need for biomarkers of early stage disease. Here, we describe the identification and assessment of a number of candidate biomarkers in patients with mild to moderate probable AD. Plasma from 47 probable Alzheimers patients and 47 matched controls were analysed by proteomics to define a significant number of proteins whose expression appeared to be associated with AD. These were compared to a similar proteomic comparison of a mouse transgenic model of amyloidosis, which showed encouraging overlap with the human data. From these studies a prioritised list of 31 proteins were then analysed by immunoassay and/or functional assay in the same human cohort to verify the changes observed. Eight proteins continued to show significance by either immunoassay or functional assay in the human plasma and these were tested in a further set of 100 probable AD patients and 100 controls from the original cohort. From our data it appeared that two proteins, serpin F1 (pigment epithelium‐derived factor) and complement C1 inhibitor are down‐regulated in plasma from AD patients.

Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor

Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

A comparative analysis of the evolutionary relationship between diet and enzyme targeting in bats, marsupials and other mammals

The subcellular distribution of the enzyme alanine : glyoxylate aminotransferase (AGT) in the livers of different mammals appears to be related to their natural diets. Thus, AGT tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. To what extent this relationship is an incidental consequence of phylogenetic structure or an evolutionarily meaningful adaptive response to changes in dietary selection pressure is unknown. In order to distinguish between these two possibilities, we have determined the subcellular distribution of AGT in the livers of 22 new mammalian species, including members of three orders not studied before. In addition, we have analysed the statistical relationship between AGT distribution and diet in all 77 mammalian species, from 12 different orders, for which the distribution is currently known. Our analysis shows that there is a highly significant correlation between AGT distribution and diet, independent of phylogeny. This finding is compatible with the suggestion that the variable intracellular targeting of AGT is an adaptive response to episodic changes in dietary selection pressure. To our knowledge, this is the first example of such a response being manifested at the molecular and cellular levels across the breadth of Mammalia.

New methods for researching accessory proteins.

Receptor activity-modifying proteins (RAMPs) control the pharmacology of the receptors for the calcitonin family of peptide hormones. There are five of these peptides: calcitonin, calcitonix/calcitixin gene-related peptide (CGRP), adrenomedullin, amylin, and now adrenomedullin 2. The calcitonin receptor is specific for calcitonin when expressed alone but it can function as an amylin or CGRP receptor when co-expressed with a RAMP. The calcitonin receptor-like receptor (CRLR) will not reach the cell surface without any one of the three RAMP proteins to function as either a CGRP or adrenomedullin receptor. This system was discovered more than 6 yr ago. At the time, it was reasonable to think that nature would employ accessory proteins, such as the RAMPs, to enable flexible signaling systems for other ligand families and that these would be discovered in time. In reality, many more new peptide ligands have been discovered than accessory proteins. Why is this? Developments in bioinformatics facilitate the discovery of both seven transmembrane ligands and accessory proteins. Proteomics and transcriptomics can be used together to define likely accessory proteins that can be experimentally tested. Comparative genomics was used in the discovery of adrenomedullin 2. The existence of multiple RAMPs within several species of fish suggests an alternative endocrinology. Finally, genetics offers a direct view of receptors, ligands, and accessory proteins in human disease-either as causative or contributing factors.