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1.  Cancer-related genes in the transcription signature of facioscapulohumeral dystrophy myoblasts and myotubes 
Muscular dystrophy is a condition potentially predisposing for cancer; however, currently, only Myotonic dystrophy patients are known to have a higher risk of cancer. Here, we have searched for a link between facioscapulohumeral dystrophy (FSHD) and cancer by comparing published transcriptome signatures of FSHD and various malignant tumours and have found a significant enrichment of cancer-related genes among the genes differentially expressed in FSHD. The analysis has shown that gene expression profiles of FSHD myoblasts and myotubes resemble that of Ewing's sarcoma more than that of other cancer types tested. This is the first study demonstrating a similarity between FSHD and cancer cell expression profiles, a finding that might indicate the existence of a common step in the pathogenesis of these two diseases.
doi:10.1111/jcmm.12182
PMCID: PMC3930408  PMID: 24341522
cancer; rhabdomyosarcoma; Ewing's sarcoma; FSHD; muscular dystrophy
2.  Integrated molecular portrait of non-small cell lung cancers 
BMC Medical Genomics  2013;6:53.
Background
Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC.
Methods
Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC.
Results
At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944.
Conclusions
Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.
doi:10.1186/1755-8794-6-53
PMCID: PMC4222074  PMID: 24299561
NSCLC; AC; SCC; LCC; Systems biology
3.  BMP7 Expression Correlates with Secondary Drug Resistance in Mantle Cell Lymphoma 
PLoS ONE  2013;8(9):e73993.
Purpose
We designed a gene profiling experiment to identify genes involved in secondary drug resistance in mantle cell lymphomas (MCL).
Experimental Design
We obtained paired tissue samples collected from the same patients before treatment and after relapse or progression. Variations in gene expression between the 2 samples were estimated for 5 patients. For each gene, the mean variation was estimated for patients with a refractory primary tumor and for responders who developed secondary drug resistance. Nine genes of interest were selected on the basis of the magnitude and statistical significance of the variation of expression in responders and non-responders.
Results
BMP7 was the only one with significantly increased expression at relapse in patients who developed secondary resistance. Validation of BMP7 as a key gene involved in secondary resistance was performed using cultures of cell line. Incubation of BMP7 with MCL cell lines increased their resistance to bortezomib and cytarabine, while inhibition of BMP7 expression by siRNA correlated with increased cell death linked to drug application.
Conclusion
Variations in gene expression after treatment point out BMP7 as a key gene involved in secondary resistance in mantle cell lymphoma.
doi:10.1371/journal.pone.0073993
PMCID: PMC3771972  PMID: 24069261
4.  Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes 
BMC Genomics  2013;14:265.
Background
miRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis.
Results
Here, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation.
Conclusions
This simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.
doi:10.1186/1471-2164-14-265
PMCID: PMC3639941  PMID: 23597168
microRNA; Skeletal muscle; Myogenesis; Myoblast
5.  Independent transcriptional reprogramming and apoptosis induction by cisplatin 
Cell Cycle  2012;11(18):3472-3480.
Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part “private,” i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance
doi:10.4161/cc.21789
PMCID: PMC3466557  PMID: 22918244
N-acetyl-cysteine; autophagy; bongkrekic acid; cyclosporine A; glutathione; large-amplitude swelling
6.  Network enrichment analysis: extension of gene-set enrichment analysis to gene networks 
BMC Bioinformatics  2012;13:226.
Background
Gene-set enrichment analyses (GEA or GSEA) are commonly used for biological characterization of an experimental gene-set. This is done by finding known functional categories, such as pathways or Gene Ontology terms, that are over-represented in the experimental set; the assessment is based on an overlap statistic. Rich biological information in terms of gene interaction network is now widely available, but this topological information is not used by GEA, so there is a need for methods that exploit this type of information in high-throughput data analysis.
Results
We developed a method of network enrichment analysis (NEA) that extends the overlap statistic in GEA to network links between genes in the experimental set and those in the functional categories. For the crucial step in statistical inference, we developed a fast network randomization algorithm in order to obtain the distribution of any network statistic under the null hypothesis of no association between an experimental gene-set and a functional category. We illustrate the NEA method using gene and protein expression data from a lung cancer study.
Conclusions
The results indicate that the NEA method is more powerful than the traditional GEA, primarily because the relationships between gene sets were more strongly captured by network connectivity rather than by simple overlaps.
doi:10.1186/1471-2105-13-226
PMCID: PMC3505158  PMID: 22966941
7.  Mesenchymal Transition and PDGFRA Amplification/Mutation Are Key Distinct Oncogenic Events in Pediatric Diffuse Intrinsic Pontine Gliomas 
PLoS ONE  2012;7(2):e30313.
Diffuse intrinsic pontine glioma (DIPG) is one of the most frequent malignant pediatric brain tumor and its prognosis is universaly fatal. No significant improvement has been made in last thirty years over the standard treatment with radiotherapy. To address the paucity of understanding of DIPGs, we have carried out integrated molecular profiling of a large series of samples obtained with stereotactic biopsy at diagnosis. While chromosomal imbalances did not distinguish DIPG and supratentorial tumors on CGHarrays, gene expression profiling revealed clear differences between them, with brainstem gliomas resembling midline/thalamic tumours, indicating a closely-related origin. Two distinct subgroups of DIPG were identified. The first subgroup displayed mesenchymal and pro-angiogenic characteristics, with stem cell markers enrichment consistent with the possibility to grow tumor stem cells from these biopsies. The other subgroup displayed oligodendroglial features, and appeared largely driven by PDGFRA, in particular through amplification and/or novel missense mutations in the extracellular domain. Patients in this later group had a significantly worse outcome with an hazard ratio for early deaths, ie before 10 months, 8 fold greater that the ones in the other subgroup (p = 0.041, Cox regression model). The worse outcome of patients with the oligodendroglial type of tumors was confirmed on a series of 55 paraffin-embedded biopsy samples at diagnosis (median OS of 7.73 versus 12.37 months, p = 0.045, log-rank test). Two distinct transcriptional subclasses of DIPG with specific genomic alterations can be defined at diagnosis by oligodendroglial differentiation or mesenchymal transition, respectively. Classifying these tumors by signal transduction pathway activation and by mutation in pathway member genes may be particularily valuable for the development of targeted therapies.
doi:10.1371/journal.pone.0030313
PMCID: PMC3289615  PMID: 22389665
8.  Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation 
Stem Cells (Dayton, Ohio)  2009;27(10):2373-2382.
Gliomas, the most frequent primitive CNS tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. TGFα, an EGF family member, is frequently over-expressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGFα is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGFα de-differentiating effects are associated with cancerous transforming effects, we grafted intra-cerebrally de-differentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo and did not give birth to tumors. When astrocytes de-differentiated with TGFα were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGFα after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress.
doi:10.1002/stem.155
PMCID: PMC3245240  PMID: 19544474
Animals; Astrocytes; drug effects; metabolism; radiation effects; Brain Neoplasms; chemically induced; physiopathology; Cell Dedifferentiation; drug effects; physiology; radiation effects; Cell Transformation, Neoplastic; chemically induced; metabolism; radiation effects; Cells, Cultured; Culture Media, Serum-Free; pharmacology; Gamma Rays; adverse effects; Glioma; chemically induced; physiopathology; Mice; Mice, Inbred C57BL; Mice, Nude; Stem Cell Transplantation; Stem Cells; drug effects; metabolism; radiation effects; Stress, Physiological; physiology; radiation effects; Transforming Growth Factor alpha; metabolism; pharmacology; EGF; transdifferentiation; metaplasia; radial glia; erbB
9.  Chromosomal Minimal Critical Regions in Therapy-Related Leukemia Appear Different from Those of De Novo Leukemia by High-Resolution aCGH 
PLoS ONE  2011;6(2):e16623.
Therapy-related acute leukemia (t-AML), is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop de novo acute leukemia (p-AML). Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case) in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case). In primary leukemia with a previously “normal” karyotype, 18% exhibited a previously undetected CNA, whereas in the (few) t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs) were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.
doi:10.1371/journal.pone.0016623
PMCID: PMC3038855  PMID: 21339820
10.  Infantile Convulsions with Paroxysmal Dyskinesia (ICCA Syndrome) and Copy Number Variation at Human Chromosome 16p11 
PLoS ONE  2010;5(10):e13750.
Background
Benign infantile convulsions and paroxysmal dyskinesia are episodic cerebral disorders that can share common genetic bases. They can be co-inherited as one single autosomal dominant trait (ICCA syndrome); the disease ICCA gene maps at chromosome 16p12-q12. Despite intensive and conventional mutation screening, the ICCA gene remains unknown to date. The critical area displays highly complicated genomic architecture and is the site of deletions and duplications associated with various diseases. The possibility that the ICCA syndrome is related to the existence of large-scale genomic alterations was addressed in the present study.
Methodology/Principal Findings
A combination of whole genome and dedicated oligonucleotide array comparative genomic hybridization coupled with quantitative polymerase chain reaction was used. Low copy number of a region corresponding to a genomic variant (Variation_7105) located at 16p11 nearby the centromere was detected with statistical significance at much higher frequency in patients from ICCA families than in ethnically matched controls. The genomic variant showed no apparent difference in size and copy number between patients and controls, making it very unlikely that the genomic alteration detected here is ICCA-specific. Furthermore, no other genomic alteration that would directly cause the ICCA syndrome in those nine families was detected in the ICCA critical area.
Conclusions/Significance
Our data excluded that inherited genomic deletion or duplication events directly cause the ICCA syndrome; rather, they help narrowing down the critical ICCA region dramatically and indicate that the disease ICCA genetic defect lies very close to or within Variation_7105 and hence should now be searched in the corresponding genomic area and its surrounding regions.
doi:10.1371/journal.pone.0013750
PMCID: PMC2966418  PMID: 21060786
11.  Portrait of Ependymoma Recurrence in Children: Biomarkers of Tumor Progression Identified by Dual-Color Microarray-Based Gene Expression Analysis 
PLoS ONE  2010;5(9):e12932.
Background
Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown.
Methodology/Principal Findings
To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression.
Conclusions/Significance
The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors.
doi:10.1371/journal.pone.0012932
PMCID: PMC2945762  PMID: 20885975
12.  High Resolution Genome-Wide Analysis of Chromosomal Alterations in Burkitt's Lymphoma 
PLoS ONE  2009;4(9):e7089.
Additional chromosomal abnormalities are currently detected in Burkitt's lymphoma. They play major roles in the progression of BL and in prognosis. The genes involved remain elusive. A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt's lymphoma-derived cell lines and primary tumors. More than half of the 145 CNAs<2 Mb were mapped to Mendelian CNVs, including GSTT1, glutathione s-transferase and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs >2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 13q (4/27), 6p (3/27), 17p (3/27), 6q (2/27),11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2 and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. The 13q31.3q32.1 <89.58–96.81> MCR contained an amplicon and ABCC4 might be the driver of this amplicon. The 40 Kb 2p16.1 <60.96–61> MCR was the smallest gained MCR and specifically encompassed the REL oncogene which is already implicated in B cell lymphomas. The most frequently deleted MCR was 3p14.1 <60.43–60.53> that removed the fifth exon of FHIT. Further investigations which combined gene expression and functional studies are essential to understand the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies.
doi:10.1371/journal.pone.0007089
PMCID: PMC2739276  PMID: 19759907
13.  Inhibition of Chk1 Kills Tetraploid Tumor Cells through a p53-Dependent Pathway 
PLoS ONE  2007;2(12):e1337.
Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.
doi:10.1371/journal.pone.0001337
PMCID: PMC2131784  PMID: 18159231
14.  αEβ7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis 
Various T cell adhesion molecules and their cognate receptors on target cells promote T cell receptor (TCR)–mediated cell killing. In this report, we demonstrate that the interaction of epithelial cell marker E-cadherin with integrin αE(CD103)β7, often expressed by tumor-infiltrating lymphocytes (TILs), plays a major role in effective tumor cell lysis. Indeed, we found that although tumor-specific CD103+ TIL-derived cytotoxic T lymphocyte (CTL) clones are able to kill E-cadherin+/intercellular adhesion molecule 1− autologous tumor cells, CD103− peripheral blood lymphocyte (PBL)-derived counterparts are inefficient. This cell killing is abrogated after treatment of the TIL clones with a blocking anti-CD103 monoclonal antibody or after targeting E-cadherin in the tumor using ribonucleic acid interference. Confocal microscopy analysis also demonstrated that αEβ7 is recruited at the immunological synapse and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis. Moreover, we report that the CD103− profile, frequently observed in PBL-derived CTL clones and associated with poor cytotoxicity against the cognate tumor, is up-regulated upon TCR engagement and transforming growth factor β1 treatment, resulting in strong potentiation of antitumor lytic function. Thus, CD8+/CD103+ tumor-reactive T lymphocytes infiltrating epithelial tumors most likely play a major role in antitumor cytotoxic response through αEβ7–E-cadherin interactions.
doi:10.1084/jem.20061524
PMCID: PMC2137907  PMID: 17325197
15.  Robust interlaboratory reproducibility of a gene expression signature measurement consistent with the needs of a new generation of diagnostic tools 
BMC Genomics  2007;8:148.
Background
The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters.
Results
We report the results of an interlaboratory comparison of gene expression array measurements on the same microarray platform, in which the RNA amplification and labeling, hybridization and wash, and slide scanning were each individually varied. Identical input RNA was used for all experiments. While some sources of variation have measurable influence on individual microarray signals, they showed very low influence on sample-to-reference ratios based on averaged triplicate measurements in the two-color experiments. RNA labeling was the largest contributor to interlaboratory variation.
Conclusion
Despite this variation, measurement of one particular breast cancer gene expression signature in three different laboratories was found to be highly robust, showing a high intralaboratory and interlaboratory reproducibility when using strictly controlled standard operating procedures.
doi:10.1186/1471-2164-8-148
PMCID: PMC1904205  PMID: 17553173
16.  NF-κB and p53 Are the Dominant Apoptosis-inducing Transcription Factors Elicited by the HIV-1 Envelope 
The coculture of cells expressing the HIV-1 envelope glycoprotein complex (Env) with cells expressing CD4 results into cell fusion, deregulated mitosis, and subsequent cell death. Here, we show that NF-κB, p53, and AP1 are activated in Env-elicited apoptosis. The nuclear factor κB (NF-κB) super repressor had an antimitotic and antiapoptotic effect and prevented the Env-elicited phosphorylation of p53 on serine 15 and 46, as well as the activation of AP1. Transfection with dominant-negative p53 abolished apoptosis and AP1 activation. Signs of NF-κB and p53 activation were also detected in lymph node biopsies from HIV-1–infected individuals. Microarrays revealed that most (85%) of the transcriptional effects of HIV-1 Env were blocked by the p53 inhibitor pifithrin-α. Macroarrays led to the identification of several Env-elicited, p53-dependent proapoptotic transcripts, in particular Puma, a proapoptotic “BH3-only” protein from the Bcl-2 family known to activate Bax/Bak. Down modulation of Puma by antisense oligonucleotides, as well as RNA interference of Bax and Bak, prevented Env-induced apoptosis. HIV-1–infected primary lymphoblasts up-regulated Puma in vitro. Moreover, circulating CD4+ lymphocytes from untreated, HIV-1–infected donors contained enhanced amounts of Puma protein, and these elevated Puma levels dropped upon antiretroviral therapy. Altogether, these data indicate that NF-κB and p53 cooperate as the dominant proapoptotic transcription factors participating in HIV-1 infection.
doi:10.1084/jem.20031216
PMCID: PMC2213296  PMID: 14993250
Bax; mitochondria; NF-κB; Puma; Bak
17.  Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects 
Journal of Clinical Investigation  2004;114(3):379-388.
Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell–dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-γ production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.
doi:10.1172/JCI200421102
PMCID: PMC489961  PMID: 15286804
18.  Survey of Human Genes of Retroviral Origin: Identification and Transcriptome of the Genes with Coding Capacity for Complete Envelope Proteins 
Journal of Virology  2003;77(19):10414-10422.
Sequences of retroviral origin occupy approximately 8% of the human genome. Most of these “retroviral” genes have lost their coding capacities since their entry into our ancestral genome millions of years ago, but some reading frames have remained open, suggesting positive selection. The complete sequencing of the human genome allowed a systematic search for retroviral envelope genes containing an open reading frame and resulted in the identification of 16 genes that we have characterized. We further showed, by quantitative reverse transcriptase PCR using specifically devised primers which discriminate between coding and noncoding elements, that all 16 genes are expressed in at least some healthy human tissues, albeit at highly different levels. All envelope genes disclose significant expression in the testis, three of them have a very high level of expression in the placenta, and a fourth is expressed in the thyroid. Besides their primary role as key molecules for viral entry, the envelope genes of retroviruses can induce cell-cell fusion, elicit immunosuppressive effects, and even protect against infection, and as such, endogenous retroviral envelope proteins have been tentatively identified in several reports as being involved in both normal and pathological processes. The present study provides a comprehensive survey of candidate genes and tools for a precise evaluation of their involvement in these processes.
doi:10.1128/JVI.77.19.10414-10422.2003
PMCID: PMC228468  PMID: 12970426
19.  Epigenetic regulation of an IAP retrotransposon in the aging mouse: progressive demethylation and de-silencing of the element by its repetitive induction 
Nucleic Acids Research  2002;30(11):2365-2373.
The recent insertion of a murine intracisternal A-particle (IAP) retrotransposon within one of the introns of a housekeeping gene, the circadian m.nocturnin gene, revealed a singular expression profile, both throughout the daytime and the mouse life span. Measurement of the levels of transcripts from this element by quantitative real-time RT–PCR, in organs of 1–24-month-old mice, disclosed that the inserted element—which is part of a large family of otherwise severely repressed mobile elements—becomes active upon aging, specifically in the liver where the m.nocturnin housekeeping gene is expressed in a circadian manner and induces a circadian expression of the IAP sequence. This age-dependent induction is cell-autonomous, as it persists in hepatocytes in primary culture. We further show, using methylation-sensitive enzymes, a correlation between the life-time kinetics of this process and a liver-specific demethylation of the IAP promoter. These results strongly support a model whereby the progressive demethylation and turning on of the IAP sequence is the sole result of the transient, daily activation—throughout the mouse life span—of its promoter. This phenomenon, which develops on a timescale of months to years in the aging mouse, might reveal a general epigenetic—and stochastic—process, which could account for a large series of events associated with cell and animal aging.
PMCID: PMC117196  PMID: 12034823

Results 1-19 (19)