Biomedical subjects

Guy Cavet

Publications and source records attributed to Guy Cavet.

13 recordsLinked to original sources

PubMed2005

Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients.

Significant improvements in the outcome of non-small cell lung carcinoma (NSCLC) have been reported in patients treated with the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. To discover biomarkers for the enrichment of patients who might benefit from treatment, a pharmacogenomic approach was used to identify gene signatures that may predict erlotinib activity using in vitro model systems. Erlotinib sensitivity in a panel of 42 NSCLC cell lines was determined by EGFR-mediated proliferative potential, EGFR mutations, and/or EGFR gene amplification, thus supporting an underlying biological mechanism of receptor activation. A strong multigene signature indicative of an epithelial to mesenchymal transition (EMT) was identified as a determinant of insensitivity to erlotinib through both supervised and unsupervised gene expression approaches. This observation was further supported by expression analysis of classic EMT marker proteins, including E-cadherin and vimentin. To investigate the clinical relevance of these findings, we examined expression of the epithelial marker E-cadherin by immunohistochemistry on primary tumor samples from subjects enrolled in a randomized NSCLC clinical trial in which erlotinib in combination with chemotherapy previously failed to show clinical activity. The majority (75%) of the 87 subjects tested showed strong E-cadherin staining and exhibited a significantly longer time to progression (hazard ratio, 0.37; log rank P=0.0028) and a nonsignificant trend toward longer survival with erlotinib plus chemotherapy treatment versus chemotherapy alone. These data support a potential role for EMT as a determinant of EGFR activity in NSCLC tumor cells and E-cadherin expression as a novel biomarker predicting clinical activity of the EGFR inhibitor erlotinib in NSCLC patients.

Antineoplastic Agents↗

PubMed2005

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Animals↗

PubMed2004

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.

Animals↗

PubMed2004

A comprehensive transcript index of the human genome generated using microarrays and computational approaches.

BACKGROUND: Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22. RESULTS: The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale. CONCLUSIONS: These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized.

Chromosomes, Human, Pair 20↗

PubMed2004

A large-scale RNAi screen in human cells identifies new components of the p53 pathway.

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.

Cell Division↗

PubMed2004

A microarray platform comparison for neuroscience applications.

To address the need for high sensitivity in gene expression profiling of small neural tissue samples ( approximately 100 ng total RNA), we compared a novel RT-PCR-IVT protocol using fluor-reverse pairs on inkjet oligonucleotide microarrays and an RT-IVT protocol using 33P labeling on nylon cDNA arrays. The comparison protocol was designed to evaluate these systems for sensitivity, specificity, reproducibility, and linearity. We developed parameters, thresholds, and testing conditions that could be used to differentiate various systems that spanned detection chemistry and instrumentation; probe number and selection criteria; and sample processing protocols. We concluded that the inkjet system had better performance in sensitivity, specificity, and reproducibility than the nylon system, and similar performance in linearity. Between these two platforms, the data indicates that the inkjet system would perform better for the transcriptional profiling of 100 ng total RNA samples for neuroscience studies.

Animals↗

PubMed2004

Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.

Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

Drug Evaluation, Preclinical↗

PubMed2003

Microarray standard data set and figures of merit for comparing data processing methods and experiment designs.

MOTIVATION: There is a very large and growing level of effort toward improving the platforms, experiment designs, and data analysis methods for microarray expression profiling. Along with a growing richness in the approaches there is a growing confusion among most scientists as to how to make objective comparisons and choices between them for different applications. There is a need for a standard framework for the microarray community to compare and improve analytical and statistical methods. RESULTS: We report on a microarray data set comprising 204 in-situ synthesized oligonucleotide arrays, each hybridized with two-color cDNA samples derived from 20 different human tissues and cell lines. Design of the approximately 24 000 60mer oligonucleotides that report approximately 2500 known genes on the arrays, and design of the hybridization experiments, were carried out in a way that supports the performance assessment of alternative data processing approaches and of alternative experiment and array designs. We also propose standard figures of merit for success in detecting individual differential expression changes or expression levels, and for detecting similarities and differences in expression patterns across genes and experiments. We expect this data set and the proposed figures of merit will provide a standard framework for much of the microarray community to compare and improve many analytical and statistical methods relevant to microarray data analysis, including image processing, normalization, error modeling, combining of multiple reporters per gene, use of replicate experiments, and sample referencing schemes in measurements based on expression change. AVAILABILITY/SUPPLEMENTARY INFORMATION: Expression data and supplementary information are available at http://www.rii.com/publications/2003/HE_SDS.htm

Base Sequence↗

PubMed2003

Expression profiling reveals off-target gene regulation by RNAi.

RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.

Base Sequence↗

PubMed2003

Genetics of gene expression surveyed in maize, mouse and man.

Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma. One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression. Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes. Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes. Furthermore, we find that these obesity subtypes are under the control of different loci.

Animals↗

PubMed2003

Development of a DNA microarray for toxicology based on hepatotoxin-regulated sequences.

Toxicogenomics is an emerging field combining genomics and bioinformatics to identify and characterize mechanisms of toxicity of compounds. One of the main tools used in toxicogenomics is DNA microarrays. We have used a novel strategy to create a library highly enriched for genes expressed in the liver under hepatotoxic conditions. Using this library, we have created a new oligonucleotide microarray dedicated to the study of rat liver function. Oligonucleotide probes for these genes were designed and used in experimental hybridization studies to deduce the correct sequence orientation and to determine those sequences exhibiting differential regulation under a variety of toxicity-related treatments and conditions. The final array was benchmarked on treatments with 3-methylcholanthrene, Aroclor 1254, and cyclopropane carboxylic acid. Our results showed that the subtractive hybridization greatly enriched for genes regulated during a hepatotoxic response. Overall, our strategy for design of a new rat toxicology microarray can be applied to other systems as well and should aid greatly in the development of microarrays targeted for specific scientific areas.

Animals↗

PubMed2002

Modulation of TCR-induced transcriptional profiles by ligation of CD28, ICOS, and CTLA-4 receptors.

Signals generated by T cell receptor (TCR) and CD28 engagement are required for optimal T cell activation, but how these signals integrate within the cell is still largely unknown. We have used near genome-scale expression profiling to monitor T cell signal transduction pathways triggered via TCR and/or costimulatory receptors. Ligation of CD28 alone induced a set of short-lived early response transcripts in both Jurkat T cells and primary CD4 T cells, thus providing evidence that CD28 engagement can affect gene regulation independently of TCR engagement. Simultaneous signaling through both the TCR and CD28 resulted in altered expression of several thousand genes following several distinct temporal patterns. Most of these gene regulations were induced by TCR signaling alone and were augmented to varying degrees by CD28 costimulation. CD28 and ICOS costimulation had nearly identical effects on gene regulation, but a few transcripts (e.g., IL2, IL9) were significantly more affected by CD28. Therefore, the distinctive functional outcomes of costimulation via CD28 and ICOS are accompanied by relatively few distinct differences in gene expression. Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement selectively blocked augmentation of gene regulations by CD28-mediated costimulation, but did not ablate gene regulation induced by TCR triggering alone.

Abatacept↗

PubMed2002

Quantitation of the distribution and flux of myosin-II during cytokinesis.

BACKGROUND: During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring. RESULTS: The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex. CONCLUSION: The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.

Animals↗