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.

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.

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.

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.

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.