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Biomedical subjects

Charles Auffray

Publications and source records attributed to Charles Auffray.

At least 19 recordsLinked to original sources
PubMed2007

A transgenic mouse model engineered to investigate human brain-derived neurotrophic factor in vivo.

Brain-derived neurotrophic factor (BDNF) is an attractive component for the treatment of various neurodegenerative diseases such as Alzheimer's or Parkinson's disease. Innovative non-invasive therapeutic approaches involve appropriate pharmacological induction of endogenous BDNF synthesis in brain. A transgenic mouse model has been established to study human BDNF gene expression and permit the screening of compounds capable of stimulating its activity. A 145-kb yeast artificial chromosome carrying the human BDNF gene has been engineered to produce the transgene which contains the extended BDNF promoter and 3' flanking regions and has integrated the enhanced green fluorescent protein (E-GFP) coding sequence in place of the BDNF coding exon. Five transgenic lines have been obtained through microinjection of the YAC into fertilized mouse oocytes. From the three lines expressing the transgene, one displays the specific pattern of BDNF expression. Faithful tissue-restricted transcription of BDNF 5' exons and localization of the fluorescent reporter gene product in the expected brain subregions are reported. This line constitutes an exploitable system for investigating human BDNF gene regulation in vivo.

Animals↗
PubMed2006

Deciphering cellular states of innate tumor drug responses.

BACKGROUND: The molecular mechanisms underlying innate tumor drug resistance, a major obstacle to successful cancer therapy, remain poorly understood. In colorectal cancer (CRC), molecular studies have focused on drug-selected tumor cell lines or individual candidate genes using samples derived from patients already treated with drugs, so that very little data are available prior to drug treatment. RESULTS: Transcriptional profiles of clinical samples collected from CRC patients prior to their exposure to a combined chemotherapy of folinic acid, 5-fluorouracil and irinotecan were established using microarrays. Vigilant experimental design, power simulations and robust statistics were used to restrain the rates of false negative and false positive hybridizations, allowing successful discrimination between drug resistance and sensitivity states with restricted sampling. A list of 679 genes was established that intrinsically differentiates, for the first time prior to drug exposure, subsequently diagnosed chemo-sensitive and resistant patients. Independent biological validation performed through quantitative PCR confirmed the expression pattern on two additional patients. Careful annotation of interconnected functional networks provided a unique representation of the cellular states underlying drug responses. CONCLUSION: Molecular interaction networks are described that provide a solid foundation on which to anchor working hypotheses about mechanisms underlying in vivo innate tumor drug responses. These broad-spectrum cellular signatures represent a starting point from which by-pass chemotherapy schemes, targeting simultaneously several of the molecular mechanisms involved, may be developed for critical therapeutic intervention in CRC patients. The demonstrated power of this research strategy makes it generally applicable to other physiological and pathological situations.

Antineoplastic Agents↗
PubMed2006

Integration of Myeloblastosis Associated Virus proviral sequences occurs in the vicinity of genes encoding signaling proteins and regulators of cell proliferation.

AIMS: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8-10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS: Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. RESULTS: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. CONCLUSION: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors.

Journal Article↗
PubMed2005

Coordination of intrinsic, extrinsic, and endoplasmic reticulum-mediated apoptosis by imatinib mesylate combined with arsenic trioxide in chronic myeloid leukemia.

A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce markedly more cell apoptosis than imatinib mesylate alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic/additive action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing map (CPP-SOM), and methods of protein biochemistry to study cell apoptosis induced by imatinib mesylate, ATO, and the combination of the 2 agents in the CML cell line K562. Numerous features with temporospatial relationships were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. Imatinib mesylate appears to induce mainly the intrinsic pathway of cell apoptosis, whereas ATO induces the endoplasmic reticulum (ER) stress-mediated pathway of cell apoptosis, and the combination of the 2 agents seems to more effectively induce the intrinsic, extrinsic, and ER stress-mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in K562 cells. This finding appears to be supported also by data derived from bone marrow cells of 2 patients with CML, one in chronic phase and the other in blast-crisis phase of the disease.

Antineoplastic Agents↗
PubMed2005

'The 39 steps' in gene expression profiling: critical issues and proposed best practices for microarray experiments.

Gene expression microarrays have been used widely to address increasingly complex biological questions and to produce an unprecedented amount of data, but have yet to realize their full potential. The interpretation of microarray data remains a major challenge because of the complexity of the underlying biological networks. To gather meaningful expression data, it is crucial to develop standardized approaches for vigilant study design, controlled annotation of resources, careful quality control of experiments, robust statistics, and data registration and storage. This article reviews the steps needed in the design and execution of valid microarray experiments so that global gene expression data can play a major role in the pursuit of future biological discoveries that will impact drug development.

Drug Design↗
PubMed2005

Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia.

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML-RARalpha oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.

Antineoplastic Agents↗
PubMed2005

Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.

While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.

Cell Line↗
PubMed2005

Modifications in the myogenic program induced by in vivo and in vitro aging.

In this study, we have used high density cDNA arrays to assess age-related changes in gene expression in the myogenic program of human satellite cells and to elucidate modifications in differentiation capacity that could occur throughout in vitro cellular aging. We have screened a collection of 2016 clones from a human skeletal muscle 3'-end cDNA library in order to investigate variations in the myogenic program of myotubes formed by the differentiation of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and induced to differentiate at different stages of their lifespan (early proliferation, presenescence and senescence). Although our analysis has not been able to underline specific changes in the expression of genes encoding proteins involved in muscle structure and/or function, we have demonstrated an age-related induction of genes involved in stress response and a down-regulation of genes involved both in mitochondrial electron transport/ATP synthase and in glycolysis/TCA cycle. From this global approach of post-mitotic cell aging, we have identified 2 potential new markers of presenescence for human myotubes, both strongly linked to carbohydrate metabolism, which could be useful in developing therapeutic strategies.

Aged↗
PubMed2005

Sex-linked recombination variation and distribution of disease-related genes.

Analysis of the distribution of recombination along human chromosomes and correlation with sequence features and genes have been previously performed on one genetic map for a given chromosome, limiting therefore their validity and precision. In this paper, we circumvent these issues: (1) by testing the correlation between recombination frequency in sex-specific versions of three genetic maps of chromosome 21 and their content in disease-related loci compared to the distribution of genes along the chromosome, and (2) by reanalysing the previously reported chromosome 22 results (Chelala et al., J. Biol. Syst. 10 (2002) 303-317) with updated version of the sequence and mapping tools. Recombination hot zones were detected and analysed on each genetic map. Despite local differences, for chromosome 21, recombination hot zones were found relatively enriched in disease-related genes on the male genetic maps. This contrasts with the previously described enrichment of the chromosome 22 female genetic map hot zones in disease-related loci (Chelala et al., J. Biol. Syst. 10 (2002) 303-317), which was confirmed with the updated data and tools. Our study demonstrates that the use of different data sets and tools have only a local impact on the distribution of genetic recombination hot zones and provides evidence for gender-specific differences in enrichment in disease-related loci in relation with recombination frequency. Automation of such analyses and extension to the entire human genome will be required in order assess the general character of these observations and to advance in the understanding of genome-wide recombination patterns to help the process of identifying disease-causing genes.

Chromosome Mapping↗
PubMed2005

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.

Database Management Systems↗
PubMed2004

Characterization of a new human isoform of the enigma homolog family specifically expressed in skeletal muscle.

We have identified a fourth member of the enigma homolog (ENH) family within a pool of human transcripts specifically expressed in skeletal muscle tissue. This new ENH isoform of 215 amino acids is the shorter of the family, it lacks the C-terminal LIM domains present in ENH1 but contains the N-terminal PDZ domain. Northern blot analysis confirmed the muscle specificity of ENH4. Western blot studies of muscle tissues using a non-isoform-specific anti-ENH antibody revealed that ENH4 is present only in skeletal muscle and that there is a specific distribution of ENH members between skeletal and cardiac muscles, which is different in human and mouse. ENH4 was found to co-localize in the sarcomeric Z-band and to interact with alpha-actinin like the other members of the ENH family. Two additional new ENH4 partners of about 34 and 54kDa were also identified. These results bring new lights on the ENH protein family members.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Alternative usage of 5' exons in the chicken nerve growth factor gene: refined characterization of a weakly expressed gene.

Nerve growth factor (NGF) is the prototype member of the neurotrophin family. Identification of transcript structures and promoter regions is described here in view of clarifying the molecular basis of chicken NGF gene regulation. Chicken NGF complementary DNA (cDNA) was amplified from heart and brain mRNA using the single-strand ligation to cDNA (SLIC) procedure. Several cloning and sequencing rounds were necessary to elucidate the diversity of NGF transcripts. The chicken NGF gene was shown to possess, in addition to its unique 3' coding exon, five 5' exons grouped into two clusters that have been entirely sequenced. The first cluster encompasses three leader exons (1a, 1b and 1c) and is separated from the second cluster by a approximately 15 kilobases (kb) intronic sequence. "Exon walking" based on reverse transcription-polymerase chain reaction (RT-PCR) allowed to ascertain the length of the three leader exons. The second cluster contains exons 2 and 3, separated from each other by a approximately 2.4 kb intron, and lies approximately 0.5 kb upstream from coding exon 4. Combination of several mechanisms, such as differential usage of leader and internal exons, alternative transcription start inside exon 1b, second donor and acceptor sites in exon 1c and 4, respectively, leads to the production of at least 21 different transcripts. This remarkable diversity may represent a common feature largely underestimated for other weakly expressed genes. Preliminary RT-PCR expression study in a panel of chicken tissues shows that transcripts containing exon 1b are present in most tissues tested. Transcripts containing exon 1a are represented mainly in heart and reproductive organs, whereas transcripts containing exon 1c are mostly represented in peripheral organs other than heart. Complementary data are published as a Web supplement available at.

Alternative Splicing↗
PubMed2003

Gene expression profiling of human satellite cells during muscular aging using cDNA arrays.

It is well established that biological aging is associated with functional deficits at the cellular, tissue, organ and system levels, but the molecular mechanisms that control lifespan and age-related phenotypes are still not well understood. In order to investigate the molecular mechanisms underlying myoblast aging, we have used quantitative hybridization of a cDNA array of 2016 clones from a human skeletal muscle 3'-end cDNA library to monitor gene expression patterns of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and at different stages of proliferation (early, presenescent and senescent). We have shown that expression profiles in satellite cells vary with donor age, with an up-regulation of genes involved in muscle structure, muscle differentiation and in metabolism in the newborn, and a down-regulation of genes involved in protein renewal in adults. We have also observed that myoblasts isolated from subjects of different ages have typical expression profiles at the beginning of their proliferative lifespan. However, this phenomenon progressively disappears as the cells approach senescence. In addition, even though some of the modifications are similar to those observed in other cell types, we have observed that many changes in gene expression are characteristic of the myoblasts, confirming the hypothesis that the program of replicative senescence is specific for each cell type. Finally, we have identified four potential new markers of presenescence for human myoblasts, which could be useful in developing therapeutic strategies.

Aged↗
PubMed2003

Self-organized living systems: conjunction of a stable organization with chaotic fluctuations in biological space-time.

Living systems have paradoxical thermodynamic stability, the intrinsic property of self-organization, fluctuation and adaptation to their changing environment. Knowledge accumulated in the analytical reductionist framework has provided useful systematic descriptions of biological systems which appear to be insufficient to gain deep understanding of their behaviour in physiologic conditions and diseases. A state-of-the-art functional genomics study in yeast points to the current inability to appraise 'biological noise', leading to focus on few genes, transcripts and proteins subject to major detectable changes, while currently inaccessible small fluctuations may be major determinants of the behaviour of biological systems. We conjecture that biological systems self-organize because they operate as a conjunction between the relatively variable part of a stable organization and the relatively stable part of a chaotic network of fluctuations, and in a space with a changing number of dimensions: biological space-time. We propose to complement the precepts of the analytical reductionist framework with those of the biosystemic paradigm, in order to explore these conjectures for systems biology, combining in an iterative mode systemic modelling of biological systems, to generate hypotheses, with a high level of standardization of high-throughput experimental platforms, enabling detection of small changes of low-intensity signals, to test them.

Adaptation, Physiological↗
PubMed2003

ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes.

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.

Actinin↗
PubMed2003

Transcriptome analysis of two bovine muscles during ontogenesis.

Macro-arrays, on which 1339 human skeletal muscle cDNA clone inserts had been spotted as PCR products, were used to make large-scale measurement of gene expression in bovine muscles during ontogenesis. Ten complex cDNA targets derived from two mixed muscle samples, Rectus abdominis (rather red oxidative muscle, RA) and Semitendinosus (rather white glycolytic muscle, ST), were taken from foetuses at 4 different stages (110, 180, 210, and 260 days post-conception) and from 15-month-old young bulls to generate differential expression patterns. Each sample analysed was prepared from a pool of RNA extracted from muscle tissues sampled from at least 6 different animals. Approximately 200 expression signals were validated and taken into account to provide a first "bovine" muscle gene repertoire. Despite the relatively small number of probes and the heterologous approach, this made it possible to identify up to 7 genes differentially expressed between RA and ST, depending on age. From 110 days post-conception to 15 months of age, differences in the expression levels of 110 genes were detected in the four comparisons between two consecutive ages. By comparing 260 days post-conception foetal muscles and adult muscles, up to 87 genes were overexpressed, whereas only 7 genes were shown to be down-regulated. Among these genes, 33% have unknown biological functions. Taken together, the results reported here underline the importance of the last three months of gestation in muscle myogenesis, and highlight new genes involved in this process.

Adult↗
PubMed2002

Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma.

To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance.

Antineoplastic Agents↗