Laurent Bracco

The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression

Wild‐type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline‐rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline‐rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence‐specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53‐responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a proline‐rich domain‐mediated cellular activation of p53 DNA binding.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53−/− human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53−/− human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

The relevance of alternative RNA splicing to pharmacogenomics

The importance of alternative RNA splicing in the generation of genetic diversity is now widely accepted. This article highlights how alternative RNA splicing can have an impact on drug efficacy and safety, and demonstrates its potential pharmacogenomic value. The analysis of the repertoire of alternative RNA splicing events could potentially identify markers of pharmacogenomic relevance with high sensitivity and specificity and also provides a route through which genes can be selected for single nucleotide polymorphism (SNP) genotyping. Recent methodological advances, including microarray and splice-dedicated expression profiling, have made it possible to perform high-throughput alternative splicing analyses.

Toward an Alzheimer's disease diagnosis via high-resolution blood gene expression.

There is a significant need for reliable molecular biomarkers to aid in Alzheimers disease (AD) clinical diagnosis.

Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity.

RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.

Studies on Nucleotide Chemistry 15. Synthesis of Oligodeoxynucleotides Using Amidine Protected Nucleosides

Abstract An in situ method is described for synthesizing DNA which incorporates a new series of amidine protected deoxy-nucleosides and bis-dialkylaminophosphines as phosphitylating agents. These procedures were used to synthesize d(GGGAATTCCC) which was digested by EcoRI.

High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

BackgroundCommercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity.ResultsThe human Genome-Wide SpliceArray™ (GWSA), a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC) Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform.ConclusionSignificant changes were detected independent of transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

Methods and platforms for the quantification of splice variants' expression.

The relatively limited number of human protein encoding genes highlights the importance of the diversity generated at the level of the mRNA transcripts. As alternative RNA splicing plays a key role in mediating this diversity, it becomes critical to develop the tools and platforms that will deliver quantitative information on the specific expression levels associated with splice isoforms. This chapter describes the constraints generated by this global transcriptome analysis and the state-of-the-art techniques and products available to the scientific community.

Alternative isoform discrimination by the next generation of expression profiling microarrays

Microarray expression profiling has revolutionised the way that many therapeutic targets have been identified over the past 10 years. High-density microarrays have allowed scientists to simultaneously scrutinise the expression of all genes encoded on a given genome. Although the data collected from classically designed microarrays greatly enriched the pool of information available to help guide the selection and design of new therapeutic strategies, they were unable to tell the complete story. The major limitation with most array designs is that they can only produce a global expression value for all transcripts produced from a specific locus and cannot monitor each individual alternative isoform produced from the interrogated locus. Recently, new array designs have been described, and become commercially available, that can efficiently monitor individual alternatively spliced isoforms produced from a single locus, allowing the research community to get a more accurate picture of the biological landscape of the expressed transcripts.

Pharmacogenomics and Personalised Medicine

Deciphering the human genome will undoubtedly constitute the next evolution, if not revolution, in human healthcare. Genetics and genomics will play a critical role in increasing productivity of the pharmaceutical R&D process from the identification of molecular targets causative of human diseases to the segmentation of the population with respect to succesful therapies and less frequent adverse drug reactions (ADRs). SMI’s Pharmacogenomics and Personalised Medicine was held in London to gather some of the stakeholders in the field, from pharmaceutical and biotechnology companies to intellectual property offices. Several of the hurdles (technical, ethical, legal and economic) that need to be overcome to make pharmacogenomics a success were discussed. Discussion