Detection of disease resistance and susceptibility alleles in pigs using oligonucleotide microarray hybridization.

A multiplex DNA microarray chip aimed at the identification of allelic polymorphisms was developed for simultaneous detection of swine disease resistance genes underlying malignant hyperthermia (RYR), postweaning diarrhea, edema disease (FUT1), neonatal diarrhea (MUC4), and influenza (MX1). The on-chip detection was performed with fragmented polymerase chain reaction (PCR)-amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required. The targets were biotin labeled during the PCR reaction, and the arrays were detected using a colorimetric methodology. Target recognition was provided by specific capture probes designed for each susceptible or resistant allelic variant. Sequencing was chosen as the gold standard to assess chip accuracy. All genotypes retrieved from the microarray (476) fit with sequencing data despite the fact that each pig was heterozygote for at least 1 target gene.

Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPARgamma-independent mechanism, whereas TNFalpha-induced de-differentiation is PPARgamma dependent.

Impairment of mitochondrial activity affects lipid-metabolizing tissues and mild mitochondrial uncoupling has been proposed as a possible strategy to fight obesity and associated diseases. In this report, we characterized the 3T3-L1-adipocyte ;de-differentiation' induced by carbonyl cyanide (p-trifluoromethoxy)-phenylhydrazone (FCCP), a mitochondrial uncoupler. We found a decrease in triglyceride (TG) content in adipocytes incubated with this molecule. We next analyzed the expression of genes encoding adipogenic markers and effectors and compared the differentially expressed genes in adipocytes treated with FCCP or TNFalpha (a cytokine known to induce adipocyte de-differentiation). Furthermore, a significant decrease in the transcriptional activity of PPARgamma and C/EBPalpha transcription factors was found in adipocytes with impaired mitochondrial activity. However, although these modifications were also found in TNFalpha-treated adipocytes, rosiglitazone and 9-cis retinoic acid (PPARgamma and RXR ligands) were unable to prevent triglyceride loss in FCCP-treated cells. Metabolic assays also revealed that TG reduction could be mediated by a downregulation of lipid synthesis rather than an upregulation of fatty acid oxidation. Finally, lipolysis stimulated by the uncoupler also seems to contribute to the TG reduction, a process associated with perilipin A downregulation. These results highlight some new mechanisms that might potentially be involved in adipocyte de-differentiation initiated by a mitochondrial uncoupling.

Gene expression silencing with 'specific' small interfering RNA goes beyond specificity - a study of key parameters to take into account in the onset of small interfering RNA off-target effects.

RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments.

Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity.

BACKGROUND: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. RESULTS: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposide-induced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1alpha by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. CONCLUSION: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents.

Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Expression profiling of ATP-binding cassette transporters in childhood T-cell acute lymphoblastic leukemia.

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance.

ABCA3 as a possible cause of drug resistance in childhood acute myeloid leukemia.

BACKGROUND: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases. EXPERIMENTAL DESIGN: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance. RESULTS: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin. CONCLUSION: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

DualChip microarray as a new tool in cancer research.

Over the last 5 years, the emergence of gene expression profiling using high-density DNA microarrays led to a better understanding of tumor development and identified new prognostic markers. However, high-density microarrays failed to leap from the researcher's bench to the clinical practice due to their cost, data management and lack of standardization. DualChip low-density DNA microarrays were developed as a new flexible tool that is able to reliably quantify the expression of a limited number of genes of clinical relevance. This review will illustrate how DualChip technology can be applied to tumor diagnosis and tumor-acquired drug resistance.

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes.

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein alpha (C/EBPalpha), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Microarray Expression Profiling of ABC Transporters in Human Breast Cancer.

The ATP-binding cassette (ABC) transporters are highly conserved genes involved in the translocation of molecules through biological membranes. Several of them are involved in tumor drug resistance, and it is thought that many others may contribute to the development of the tumor phenotype in a still unknown way. A low-density DNA microarray was recently developed for the analysis of 38 ABC-transporter genes and 3 other transporters. In the present pilot study, clinical samples from 16 breast cancer patients were tested. Of the 41 transporters analyzed, 10 were not or very seldom expressed, while 23 were found to be expressed, sometimes at very high levels, in the majority of the tumors. Comparison of the treated and untreated tumors showed an unexpected similarity of results. The signal obtained on the capture probes for ABCC6/8/9 was, however, found to be higher in the treated samples. The microarray data were validated on 15 ABC-transporter genes by real-time PCR. The present results showed that the expression of the majority of the ABC transporters was a clear feature of breast tumors, whether treated or not.

Molecular characterization of breast cancer cell lines by a low-density microarray.

We designed a low-density microarray carrying 132 DNA capture sequences highly specific for genes known to be differentially expressed among breast tumors and BCC lines or associated with specific tumor properties (cell-cycle alteration, proteolysis, adhesion, hormone sensitivity, etc). We analyzed gene expression in 11 BCC lines among which 6 had already been extensively studied (BT-474, Hs578T, MCF-7, MDA-MB-231, MDA-MB-453, T-47D) and 5 were still poorly characterized (Evsa-T, IBEP-1, IBEP-2, IBEP-3, KPL-1). Some data obtained were verified or extended by real-time polymerase chain reaction (real-time PCR), Northern blotting, Western blotting, immunohistochemistry and cell growth studies. Clustering analysis of the low-density microarray data allowed the sorting of BCC lines into two classes and supported a major discriminatory role for ER alpha, confirming data from previous studies. A few genes that are highly and specifically expressed in one cell line were identified, such as MGB1 (mammaglobin 1) in Evsa-T cells, and PIP (prolactin-inducible protein) in MDA-MB-453 BCC, suggesting an apocrine origin for these latter cells. Two BCC lines (IBEP-1 and IBEP-3) that had been previously characterized as ER alpha-negative, were classified by the low-density microarray among ER alpha-positive lines (MCF-7, T-47D, IBEP-2, BT-474, KPL-1) and were indeed confirmed as receptor-positive (at both mRNA and protein levels) and hormone-responsive cells. In conclusion, our results support the utility of a low-density microarray approach in cases where the cost and exhaustiveness of high-density microarrays may constitute a drawback; for instance, in obtaining a rapid phenotype evaluation in cell populations freshly isolated from breast tumors.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway.

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (non-proapoptotic) exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were markedly expressed: growth arrest, senescence-associated beta-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress- and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/apolipoprotein J (apo J) and transforming growth factor-beta1 (TGF-beta1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-beta1 or its receptor II (TbetaRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-beta1 in UVB-induced premature senescence. Both the latent and active forms of TGF-beta1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.

Microarray-based detection of multidrug resistance in human tumor cells by expression profiling of ATP-binding cassette transporter genes.

Different mechanisms of drug resistance, including ATP-binding cassette (ABC) transporters, are responsible for treatment failure of tumors. We developed a low-density DNA microarray which contains 38 genes of the ABC transporter gene family. This tool has been validated with three different multidrug-resistant sublines (CEM/ADR5000, HL60/AR, and MCF7/CH1000) known to overexpress either the ABCB1 (MDR1), ABCC1 (MRP1), or ABCG2 (MXR and BCRP) genes. When compared with their drug-sensitive parental lines, we observed not only the overexpression of these genes in the multidrug-resistant cell lines but also of other ABC transporter genes pointing to their possible role in multidrug resistance. These results were corroborated by quantitative real-time reverse transcription-PCR. As the microarray allows the determination of the expression profile of many ABC transporters in a single hybridization experiment, it may be useful as a diagnostic tool to detect drug resistance in clinical samples.

Gene expression and regulation in H2O2-induced premature senescence of human foreskin fibroblasts expressing or not telomerase.

We compared the DNA-binding activity of transcription factors and gene expression patterns in BJ human diploid fibroblasts (HDFs) expressing or not telomerase (hTERT) in stress-induced premature senescence (SIPS). Senescent BJ cells were also studied. Hydrogen peroxide (H2O2)-induced SIPS modulated gene expression in both BJ and hTERT-BJ1 cells. Increased p21(WAF-1) mRNA level was amongst the common gene expression changes in BJ and hTERT-BJ1 cells induced by SIPS. Telomerase expression markedly changed gene expression in non-stressful conditions. Expression patterns of senescent BJ cells partially overlapped those of BJ and hTERT-BJ1 cells in SIPS. The basal levels of DNA-binding activity of NF-kappaB and phosphorylated ATF-2 were different in BJ and hTERT-BJ1 cells. Both cell lines displayed a higher DNA-binding activity of p53 and HIF-1 72 h after H2O2 exposure. Our results indicate that similar mechanisms involving p21(WAF-1) and probably p53 are at work in BJ and hTERT-BJ1 HDFs under H2O2-induced SIPS, suggesting that generalized DNA damage rather than telomere length/telomerase plays a crucial role in H2O2induced SIPS. We propose that H2O2-induced SIPS involves a rearrangement of proliferative and apoptotic pathways. The marked changes in gene expression induced by telomerase suggest that apart from immortalization of HDFs, telomerase also alters the normal cellular functions but does not protect against SIPS.

No increase in senescence-associated beta-galactosidase activity in Werner syndrome fibroblasts after exposure to H2O2.

Normal human diploid fibroblasts (HDFs) exposed to a single H(2)O(2) subcytotoxic stress display features of premature senescence, termed stress-induced premature senescence (SIPS). In this work, our aim was to study SIPS in Werner syndrome (WS) fibroblasts, derived from a patient with WS, a disease resembling accelerated aging. The subcytotoxic dose for WS fibroblasts was found to be inferior to that of normal HDFs, indicating WS fibroblasts are more sensitive to hydrogen peroxide than normal HDFs. SA beta-gal activity has been shown to occur both in vitro and in vivo, and we studied the proportion of WS cells positive for SA beta-gal. Intriguingly, the percentage of positive cells did not increase with the dose of H(2)O(2) used. Contrary to other HDFs, the DNA-binding activity of p53 in WS fibroblasts did not increase in SIPS. We found, based on our results, that WS fibroblasts feature an altered stress response and do not reach SIPS from H(2)O(2). We suggest that the proportion of cells that in normal HDFs would enter SIPS instead die in WS fibroblasts. Last, we propose that aging derives from a loss of integrity of the chromatin structure, which occurs faster in WS patients.

An evaluation of a low-density DNA microarray using cytochrome P450 inducers.

The aim of this study was to validate a low-density DNA microarray "Rat HepatoChip", which contains 59 genes from a range of potential toxic markers and drug metabolism-related genes. Liver mRNA was isolated from rats dosed with six different chemicals, dexamethasone, troleandomycin, miconazole, clotrimazole, and methylclofanapate, which are all known to induce different cytochrome P450 genes, and isoniazid, which does not cause histopathological changes. Replicate microarrays were used to measure the variability in the chips and in the process. The average variability in signal between different chips observed in triplicate experiments was 33% ranging from 21 to 39% depending on genes. We also demonstrated a strong correlation between the liver histopathology and the gene expression profiles indicating that the gene expression profile reflects histopathological changes. These results suggest that the Rat HepatoChip microarray may provide a fast and effective tool for assessing the toxicity profile of developmental drug candidates during the drug discovery process.

Use of a low-density microarray for studying gene expression patterns induced by hepatotoxicants on primary cultures of rat hepatocytes.

In the field of gene expression analysis, DNA microarray technology is having a major impact on many different areas including toxicology. For instance, a number of studies have shown that transcription profiling can generate the information needed to assign a compound to a mode-of-action class. In this study, we investigated whether compounds inducing similar toxicological endpoints produce similar changes in gene expression. In vitro primary rat hepatocytes were exposed to 11 different hepatotoxicants: acetaminophen, amiodarone, clofibrate, erythromycin estolate, isoniazid, alpha-naphtylylisothiocyanate, beta-naphtoflavone, 4-pentenoic acid, phenobarbital, tetracycline, and zileuton. These molecules were selected on the basis of their variety of hepatocellular effects observed such as necrosis, cholestasis, steatosis, and induction of CYP P450 enzymes. We used a low-density DNA microarray containing 59 genes chosen as relevant toxic and metabolic markers. The in vitro gene expression data generated in this study were generally in good agreement with the literature, which mainly concerns in vivo data. Furthermore, gene expression profiles observed in this study have been confirmed for several genes by real-time PCR assays. All the tested drugs generated a specific gene expression profile. Our results show that even with a relatively limited gene set, gene expression profiling allows a certain degree of classification of compounds with similar hepatocellular toxicities such as cholestasis, necrosis. The clustering analysis revealed that the compounds known to cause steatosis were linked, suggesting that they functionally regulate similar genes and possibly act through the same mechanisms of action. On the other hand, the drugs inducing necrosis and cholestasis were pooled in the same cluster. The drugs arbitrarily classified as the CYP450 inducers formed individual clusters. In conclusion, this study suggests that low-density microarrays could be useful in toxicological studies.

New chips for molecular biology and diagnostics.

The DNA chips are arrays of DNA probes immobilized on solid support for simultaneous identification of many target DNA sequences. DNA chips applied to diagnosis aims to detect genomic DNA or RNA after PCR amplification. This review provides an overview of DNA chip technology, focusing on diagnostic applications. A comparison between high density and low density microarrays is given showing that low density chips are more suitable for routine applications due to their simplicity, good reproducibility, easy data management and low cost.

Gene expression profiling of drug metabolism and toxicology markers using a low-density DNA microarray.

DNA microarrays are useful tools to study changes of gene expression in response to a treatment with drugs. Here, we describe the optimization of conditions for the cDNA synthesis and hybridization protocols to be used for a low-density DNA microarray called 'Rat HepatoChips.' This DNA microarray with 59 carefully selected genes could be used to study changes in gene expression levels due to a treatment with xenobiotic. These 59 genes (including 8 housekeeping genes) have been selected among potential toxic markers involved in basic cellular processes and drug metabolism related genes. Using the optimized conditions, the results were shown to be reproducible, with 6% variation between the duplicated spots and 10% between arrays. Conditions were optimized to allow quantification with a dynamic range of four log units. In order to demonstrate the major advantage of these tool for studying gene expression, samples of control rat liver were compared with those of animals dosed with phenobarbital (PB) or pregnenolone-16 alpha-carbonitrile (PCN), two compounds well known to induce cytochrome P450 isoforms of 2B and 3A subfamilies, respectively. This microarray has shown that other genes apart from the corresponding CYP P450 genes have been changed due to PB and PCN treatment. Apoptosis-related genes have shown to be changed due to PB and PCN treatment, which confirms results from previous work.