Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13-acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.

Identification of genes differentially regulated by the P210 BCR/ABL1 fusion oncogene using cDNA microarrays

Objective: The t(9;22) translocation is associated with more than 95% of cases of chronic myeloid leukemia. The resulting fusion of the BCR and ABL1 loci produces the constitutively active BCR/ABL1 tyrosine kinase. A wide range of signal transduction molecules are activated by BCR/ABL1, including MYC, PI-3 kinase, and different STAT molecules. In contrast, relatively few genes are known to be regulated by BCR/ABL1 at the level of transcription.

Materials and Methods: In an effort to better understand the transcriptional program activated by BCR/ABL1, we used cDNA microarrays to evaluate the relative expression of approximately 6450 human genes in U937 myelomonocytic cells expressing P210 BCR/ABL1 via a tetracycline-inducible promoter.

Results: We confirmed the previously reported up-regulation of the PIM1 and JUN oncogenes by BCR/ABL1. In addition, we identified 59 more genes up-regulated by BCR/ABL1. Interestingly, roughly one third of these were genes previously reported to be interferon (IFN)-responsive, including the OAS1, IFIT1, IFI16, ISGF3G, and STAT1 genes. An additional seven BCR/ABL1-regulated genes were found to be IFN-responsive in U937 cells. The expression profile also included genes encoding transcription factors, kinases, and signal transduction molecules, as well as genes regulating cell growth, differentiation, apoptosis, and cell adhesion, features previously suggested to be affected by BCR/ABL1.

Conclusion: These observations shed novel insight into the mechanism of BCR/ABL1 action and provide a range of targets for further investigation.

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type-1 and type-2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type-1 and type-2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type-2 response and two linked with an extreme type-1 cytokine phenotype. Predictions about liver fibrosis,  apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining  coordinated gene expression profiles but are also highly effective for molecularly “fingerprinting” diseased tissues. Moreover, they illustrate the potential of genome-wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious  disease pathogenesis.

New approaches to gene discovery with animal models of obesity and diabetes

DNA-based approaches to the discovery of genes contributing to the development of type 2 diabetes have not been very successful despite substantial investments of time and money. The multiple gene-gene and gene-environment interactions that influence the development of type 2 diabetes mean that DNA approaches are not the ideal tool for defining the etiology of this complex disease. Gene expression-based technologies may prove to be a more rewarding strategy to identify diabetes candidate genes. There are a number of RNA-based technologies available to identify genes that are differentially expressed in various tissues in type 2 diabetes. These include differential display polymerase chain reaction (ddPCR), suppression subtractive hybridization (SSH), and cDNA microarrays. The power of new technologies to detect differential gene expression is ideally suited to studies utilizing appropriate animal models of human disease. We have shown that the gene expression approach, in combination with an excellent animal model such as the Israeli sand rat (Psammomys obesus), can provide novel genes and pathways that may be important in the disease process and provide novel therapeutic approaches. This paper will describe a new gene discovery, beacon, a novel gene linked with energy intake. As the functional characterization of novel genes discovered in our laboratory using this approach continues, it is anticipated that we will soon be able to compile a definitive list of genes that are important in the development of obesity and type 2 diabetes.

Obesity and diabetes gene discovery approaches

New treatments are currently required for the common metabolic diseases obesity and type 2 diabetes. The identification of physiological and  biochemical factors that underlie the metabolic disturbances observed in obesity and type 2 diabetes is a key step in developing better therapeutic outcomes. The discovery of new genes and pathways involved in the  pathogenesis of these diseases is critical to this process, however  identification of genes that contribute to the risk of developing these diseases represents a significant challenge as obesity and type 2 diabetes are complex diseases with many genetic and environmental causes. A number of diverse approaches have been used to discover and validate potential new targets for obesity and diabetes. To date, DNA-based approaches using candidate gene and genome-wide linkage analysis have had limited success in identifying genomic regions or genes involved in the development of these diseases. Recent advances in the ability to evaluate linkage analysis data from large family pedigrees using variance components based linkage analysis show great promise in robustly identifying genomic regions associated with the development of obesity and diabetes. RNA-based technologies such as cDNA microarrays have identified many genes differentially expressed in tissues of healthy and diseased subjects. Using a combined approach, we are endeavouring to focus attention on differentially expressed genes located in chromosomal regions previously linked with obesity and / or diabetes. Using this strategy, we have identified Beacon as a potential new target for obesity and diabetes.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA.

Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression

Polyunsaturated fatty acids (PUFA) are essential structural components of the central nervous system. Their role in controlling learning and memory has been well documented. A nutrigenomic approach with high-density microarrays was used to reveal brain gene-expression changes in response to different PUFA-enriched diets in rats. In aged rats fed throughout life with PUFA-enriched diets, genes with altered expressions included transthyretin, α-synuclein, and calmodulins, which play important roles in synaptic  plasticity and learning. The effect of perinatal omega-3 PUFA supply on gene expression later in life also was studied. Several genes showed similar changes in expression in rats fed omega-3-deficient diets in the perinatal period, regardless of whether they or their mothers were fed omega-3 PUFA-sufficient diets after giving birth. In this experiment, among the down-regulated genes were a kainate glutamate receptor and a DEAD-box polypeptide. Among the up-regulated genes were a chemokine-like factor, a tumor necrosis factor receptor, and cytochrome c. The possible involvement of the genes with altered expression attributable to different diets in different brain regions in young and aged rats and the possible mode of regulatory action of PUFA also are discussed. We conclude that PUFA-enriched diets lead to significant changes in expression of several genes in the central nervous tissue, and these effects appear to be mainly independent of their effects on membrane composition. The direct effects of PUFA on transcriptional modulators, the downstream developmentally and tissue-specifically activated elements might be one of the clues to understanding the beneficial effects of the omega-3 PUFA on the nervous system.

Global and targeted gene expression and protein content in skeletal muscle of young men following short-term creatine monohydrate supplementation

Creatine monohydrate (CrM) supplementation has been shown to increase fat-free mass and muscle power output possibly via cell swelling. Little is known about the cellular response to CrM. We investigated the effect of short-term CrM supplementation on global and targeted mRNA expression and protein content in human skeletal muscle. In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, nonobese men were supplemented with either a placebo (PL) or CrM (loading phase, 20 g/day x 3 days; maintenance phase, 5 g/day x 7 days) for 10 days. Following a 28-day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and were assessed for mRNA expression (cDNA microarrays + real-time PCR) and protein content (Kinetworks KPKS 1.0 Protein Kinase screen). CrM supplementation significantly increased fat-free mass, total body water, and body weight of the participants (P < 0.05). Also, CrM supplementation significantly upregulated (1.3- to 5.0-fold) the mRNA content of genes and protein content of kinases involved in osmosensing and signal transduction, cytoskeleton remodeling, protein and glycogen synthesis regulation, satellite cell proliferation and differentiation, DNA replication and repair, RNA transcription control, and cell survival. We are the first to report this large-scale gene expression in the skeletal muscle with short-term CrM supplementation, a response that suggests changes in cellular osmolarity.

Microarray classification using statistical learning and decision tree algorithms

This considers the challenging task of cancer prediction based on microarray data for the medical community. The research was conducted on mostly common cancers (breast, colon, long, prostate and leukemia) microarray data analysis, and suggests the use of modern machine learning techniques to predict cancer.

Molecular genetic approaches to the identification of commercial fish species

A DNA database was developed to enable the identification and discrimination of commercially important fish species. Three genetic techniques were compared for efficiency, accuracy and reliability. It is envisaged this study will aid authorities to identify mislabelled fish fillets and provide greater consumer confidence in the Australian Seafood Industry.

An embedded two-layer feature selection approach for microarray data analysis

Feature selection is an important technique in dealing with application problems with large number of variables and limited training samples, such as image processing, combinatorial chemistry, and microarray analysis. Commonly employed feature selection strategies can be divided into filter and wrapper. In this study, we propose an embedded two-layer feature selection approach to combining the advantages of filter and wrapper algorithms while avoiding their drawbacks. The hybrid algorithm, called GAEF (Genetic Algorithm with embedded filter), divides the feature selection process into two stages. In the first stage, Genetic Algorithm (GA) is employed to pre-select features while in the second stage a filter selector is used to further identify a small feature subset for accurate sample classification. Three benchmark microarray datasets are used to evaluate the proposed algorithm. The experimental results suggest that this embedded two-layer feature selection strategy is able to improve the stability of the selection results as well as the sample classification accuracy.

Chicken anemia virus : an understanding of the in-vitro host response over time

Chicken anemia virus (CAV) is an economically important virus affecting the chicken meat and egg industry. CAV is characterized by anemia, lymphoid depletion, and immunosuppression. Microarrays were used to investigate the response of MDCC-MSB1 cells (MSB1) to infection with CAV at 24 and 48 h post-infection (hpi). The major genes responding to CAV infection include genes involved in inflammation, apoptosis, and antiviral activity. Several cytokines were differentially regulated at either 24 or 48 hpi, including interleukin 2 (IL-2), interleukin receptors IL-1R, IL-22R, IL-18R, and IL-7R, and interferon-α (IFN-α). While there were many genes differentially regulated in this experiment, only two genes were common to both time points, suggesting a dramatic change in gene expression over the two time points studied. The present study is the first microarray experiment to investigate CAV, and we identified a number of key pathways involved in viral infection. Overall, there were more genes upregulated at 24 hpi than at 48 hpi, including genes involved in cytokine signaling, apoptosis, and antiviral activity. The two time points were vastly different in their gene expression patterns, in that at 24 hpi there were many genes involved in the response to infection, whereas at 48 hpi there were many genes associated with apoptosis and immunosuppression.

MicroRNA-194 modulates glucose metabolism and its skeletal muscle expression is reduced in diabetes

BACKGROUND: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. METHODS: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. RESULTS: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. CONCLUSIONS: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.