Analysis of gene expression for studying tumor progression: the case of glucocorticoid administration

Background

Glucocorticoids are commonly used as adjuvant treatment for side-effects and have anti-proliferative activity in several tumors but, on the other hand, their proliferative effect has been reported in several studies, some of them involving the spread of cancer. We shall attempt to reconcile these incongruities from the genomic and tissue-physiology perspectives with our findings.

Methods

An accurate phenotype analysis of microarray data can help to solve multiple paradoxes derived from tumor-progression models. We have developed a new strategy to facilitate the study of interdependences among the phenotypes defined by the sample clusters obtained by common clustering methods (HC, SOTA, SOM, PAM). These interdependences are obtained by the detection of non-linear expression-relationships where each fluctuation in the relationship implies a phenotype change and each relationship typology implies a specific phenotype interdependence. As a result, multiple phenotypic changes are identified together with the genes involved in the phenotype transitions. In this way, we study the phenotypic changes from microarray data that describe common phenotypes in cancer from different tissues, and we cross our results with biomedical databases to relate the glucocorticoid activity to the phenotypic changes.

Results

11,244 significant non-linear expression relationships, classified into 11 different typologies, have been detected from the data matrix analyzed. From them, 415 non-linear expression relationships were related to glucocorticoid activity. Studying them, we have found the possible reason for opposite effects of some stressor agents like dexamethasone on tumor progression and it has been confirmed by literature. This hidden reason has resulted in being linked with the type of tumor progression of the tissues. In the first type of tumor progression found, new cells can be stressed during proliferation and stressor agents increase tumor proliferation. In the second type, cell stress and tumor proliferation are antagonists so, therefore, stressor agents stop tumor proliferation in order to stress the cells. The non-linear expression relationships among DUSP6, FERMT2, FKBP5, EGFR, NEDD4L and CITED2 genes are used to synthesize these findings.     

Human adenosine deaminase as an allosteric modulator of human A(1) adenosine receptor: abolishment of negative cooperativity for [H](R)-pia binding to the caudate nucleus

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them A(1) adenosine receptors (A(1)Rs). Bovine ADA interacts with A(1)Rs from many species and regulates agonists binding to receptors in an activity-independent form. However, it was not known whether human ADA exerted any effect on the agonist binding to human A(1)Rs, because of both technical difficulties in obtaining pure human ADA and tissues containing human A(1)Rs. In this study, human ADA was purified to homogeneity. Taking in consideration that A(1)Rs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, which allows to calculate ligand dissociation constants and the degree of cooperativity between the two subunits in the dimer, here it is demonstrated that human ADA markedly enhances the agonist and antagonist affinity and abolishes the negative cooperativity on agonist binding to human striatal A(1)Rs. ADA also increases the ability of the agonist to decrease the forskolin-induced cAMP levels. The results show that human ADA, apart from reducing the adenosine concentration and thus preventing A(1)R desensitization, binds to A(1)R behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. The physiological role of the interaction is to make receptors more sensitive to adenosine. This powerful regulation has important implications for the physiology and pharmacology of neuronal A(1)Rs.     

Regulation of the kinase activity of the MIK GCK-like MAP4K by alternative splicing

Alternative splicing of introns is essential to ensure the complexity of mammalian genome functions. In particular, the generation of a high number of different isoforms by alternative splicing is an important characteristic of genes coding for signalling proteins such as mitogen activated protein kinases (MAPKs). This is thought to allow these proteins to transduce multiple stimuli in a highly regulated manner. Plant genes are also subjected to alternative splicing. Nevertheless, clear examples of the functional consequences of this phenomenon are still scarce in plants. MIK is a maize gene coding for a GCK-like MAP4K that can be activated by interaction with maize atypical receptor kinase (MARK), an atypical receptor kinase. Here we show that MIK is subjected to alternative splicing. Expression of MIK leads to, at least, 4 different mature mRNAs that accumulate with particular expression profiles during maize development. Our results show that the polypeptides encoded by the different MIK mRNAs display different kinase activity and are differentially activated by interaction with the MARK receptor. Two MIK isoforms display constitutive kinase activity, one isoform is inactive but can be activated by MARK, and the fourth MIK isoform is inactive and cannot be activated by MARK. Our results constitute a clear example of the biochemical consequences of alternative splicing in plants. The selective conservation during evolution of the intron–exon structure of the region coding for the regulator domain of MIK, as well as the maintenance in maize, rice and Arabidopsis of the alternative splicing of some of these introns, are strong indications of its functional importance.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Ontogenetic behavior of Siberian sturgeon, Acipenser baerii: A synthesis between laboratory tests and field data

We have reviewed field data and studies on the behavior and development of Siberian sturgeon at early-life intervals and related them to different ecologically relevant environmental factors that may play a role in the distribution, recruitment, and survival of young fish. Four behavioral phases (swimming-up, rheotactism, shoaling, and foraging) are observed from hatching to the juvenile phase. Each behavior is associated with an early-life interval and might allow fish to occupy different river habitats, directly influencing their distribution, survival, and recruitment. River current intensity, substrate typology, food resources, and predation pressure seem to be the most important factors affecting the distribution of Siberian sturgeon free embryos and larvae, while juveniles and adult fish disperse and migrate according to food abundance and reproduction. Mechanisms involved in regulating downstream migration during Siberian sturgeon early life stages are different than those observed in anadromous sturgeon species. In all large Siberian rivers, with the exception of the Lake Baikal, the Siberian sturgeon is represented by population continuums, and in many cases the foraging range also includes the spawning areas. Ontogenetic changes in Siberian sturgeon behavior could be interpreted as a species-specific mechanism to maintain the population continuums described in this species without significant mixture of local populations within the river.     

Effector CD4+ T cell expression signatures and immune-mediated disease associated genes

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in na?ve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to na?ve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.     

Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.