Construction and validation of the APOCHIP, a spotted oligo-microarray for the study of beta-cell apoptosis

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a autoimmune disease caused by a long-term negative balance between immune-mediated beta-cell damage and beta-cell repair/regeneration. Following immune-mediated damage the beta-cell fate depends on several genes up- or down-regulated in parallel and/or sequentially. Based on the information obtained by the analysis of several microarray experiments of beta-cells exposed to pro-apoptotic conditions (e.g. double stranded RNA (dsRNA) and cytokines), we have developed a spotted rat oligonucleotide microarray, the APOCHIP, containing 60-mer probes for 574 genes selected for the study of beta-cell apoptosis. RESULTS: The APOCHIP was validated by a combination of approaches. First we performed an internal validation of the spotted probes based on a weighted linear regression model using dilution series experiments. Second we profiled expression measurements in ten dissimilar rat RNA samples for 515 genes that were represented on both the spotted oligonucleotide collection and on the in situ-synthesized 25-mer arrays (Affymetrix GeneChips). Internal validation showed that most of the spotted probes displayed a pattern of reaction close to that predicted by the model. By using simple rules for comparison of data between platforms we found strong correlations (r(median)= 0.84) between relative gene expression measurements made with spotted probes and in situ-synthesized 25-mer probe sets. CONCLUSION: In conclusion our data suggest that there is a high reproducibility of the APOCHIP in terms of technical replication and that relative gene expression measurements obtained with the APOCHIP compare well to the Affymetrix GeneChip. The APOCHIP is available to the scientific community and is a useful tool to study the molecular mechanisms regulating beta-cell apoptosis

Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

<p>Abstract</p> <p>Background</p> <p>Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated.</p> <p>Methods</p> <p>MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy.</p> <p>Results</p> <p>MSI was found in 43 (13.8%) tumors. Absence of repair protein expression was assessed in 52 (17.0%) tumors, which had primarily lost hMLH1 in 39 (12.7%), hMSH2 in 5 (1.6%), and hMSH6 in 8 (2.6%) tumors. In multivariate analysis MSI (instable) compared to MSS (stable) tumors were significantly associated with lower risk of recurrence (hazard ratio (HR) = 0.3; 95% CI: 0.2–0.7; P = 0.0007) and death (HR = 0.4; 95% CI: 0.2–0.9; P = 0.02) independently of the TS and DPD expressions. A direct relationship between MSI and TS intensity (P = 0.001) was found, while there was no significant association with DPD intensity (P = 0.1).</p> <p>Conclusion</p> <p>The favourable outcome of MSI colorectal carcinomas is ascribed mainly to the tumor biology and to a lesser extent to antitumor response to 5-fluorouracil therapy. There is no evidence that differential TS or DPD expression may account for these outcome characteristics.</p

Pressure Load: The Main Factor for Altered Gene Expression in Right Ventricular Hypertrophy in Chronic Hypoxic Rats

BACKGROUND: The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading. METHODOLOGY/PRINCIPAL FINDINGS: To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment. CONCLUSION/SIGNIFICANCE: Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure

Inbreeding by Environmental Interactions Affect Gene Expression in Drosophila melanogaster

Genomewide gene expression patterns were investigated in inbred and noninbred Drosophila melanogaster lines under benign and stressful (high temperature) environmental conditions in a highly replicated experiment using Affymetrix gene chips. We found that both heat-shock protein and metabolism genes are strongly affected by temperature stress and that genes involved in metabolism are differentially expressed in inbred compared with noninbred lines, and that this effect is accentuated after heat stress exposure. Furthermore we show that inbreeding and temperature stress cause increased between-line variance in gene expression patterns. We conclude that inbreeding and environmental stress both independently and synergistically affect gene expression patterns. Interactions between inbreeding and the environment are often observed at the phenotypic level and our results reveal some of the genes that are involved at the individual gene level. Our observation of several metabolism genes being differentially expressed in inbred lines and more so after exposure to temperature stress, together with lower fitness in the investigated inbred lines, supports the hypothesis that superiority of heterozygous individuals partly derives from increased metabolic efficiency

Genome-Wide Analysis on Inbreeding Effects on Gene Expression in Drosophila melanogaster

The deleterious consequences of inbreeding, especially in the form of inbreeding depression, are well known. However, little is known about how inbreeding affects genome-wide gene expression. Here, we show that inbreeding changes transcription levels for a number of genes. Gene expression profiles of Drosophila melanogaster lines inbred to F ≈ 0.67 at different rates changed relative to those of noninbred lines, but the rate of inbreeding did not significantly affect gene expression patterns. Genes being differentially expressed with inbreeding are disproportionately involved in metabolism and stress responses, suggesting that inbreeding acts like an environmental stress factor

Identification of novel cytokine-induced genes in pancreatic beta-cells by high-density oligonucleotide arrays.

Type 1 diabetes is an autoimmune disease resulting from the selective destruction of insulin-producing beta-cells. Cytokines may contribute to pancreatic beta-cell death in type 1 diabetes. beta-cell exposure to interleukin (IL)-1beta induces functional impairment, whereas beta-cell culture for 6-9 days in the presence of IL-1beta and interferon (INF)-gamma leads to apoptosis. To clarify the mechanisms involved in these effects of cytokines, we studied the general pattern of cytokine-induced gene expression in beta-cells. Primary rat beta-cells were fluorescence-activated cell sorter-purified and exposed for 6 or 24 h to control condition, IL-1beta + INF-gamma, or IL-1beta alone (24 h only). Gene expression profile was analyzed in duplicate by oligonucleotide arrays. Nearly 3,000 transcripts were detected in controls and cytokine-treated beta-cells. Of these, 96 and 147 displayed changes in expression after 6 and 24 h, respectively, of exposure to IL-1beta + INF-gamma, whereas 105 transcripts were modified after a 24-h exposure to IL-1beta. The cytokine-responsive genes were clustered according to their biological functions. The major clusters observed were metabolism, signal transduction, transcription factors, protein synthesis/ processing, hormones, and related receptors. These modifications in gene expression may explain some of the cytokine effects in beta-cells, such as decreased protein biosynthesis and insulin release. In addition, there was induction of diverse cytokines and chemokines; this suggests that beta-cells may contribute to mononuclear cell homing during insulitis. Several of the cytokine-induced genes are potentially regulated by the transcription factor NF-kappaB. Clarification of the function of the identified cytokine-induced gene patterns may unveil some of the mechanisms involved in beta-cell damage and repair in type 1 diabetes.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe