Françoise de Longueville

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.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 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 β-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-β1 (TGF-β1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-β1 or its receptor II (TβRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-β1 in UVB-induced premature senescence. Both the latent and active forms of TGF-β1 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.

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.

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 α (C/EBPα), 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.

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 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.

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

Backgroundit 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.Resultsin 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-1α by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression.Conclusionthese 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. [Mol Cancer Ther 2006;5(8):1986–94]

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.

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.