Réparation, Radiations et Thérapies innovantes anticancer

Publications de l’équipe

Année de publication : 2010

Pauline Rimmelé, Jun Komatsu, Philippe Hupé, Christophe Roulin, Emmanuel Barillot, Marie Dutreix, Emmanuel Conseiller, Aaron Bensimon, Françoise Moreau-Gachelin, Christel Guillouf (2010 Jul 28)

Spi-1/PU.1 oncogene accelerates DNA replication fork elongation and promotes genetic instability in the absence of DNA breakage.

Cancer research : 6757-66 : DOI : 10.1158/0008-5472.CAN-09-4691 En savoir plus

The multistage process of cancer formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Using a well-defined multistep leukemia model driven by Spi-1/PU.1 overexpression in the mouse and Spi-1/PU.1-overexpressing human leukemic cells, we investigated the relationship between DNA replication and cancer progression. Here, using DNA molecular combing and flow cytometry methods, we show that Spi-1 increases the speed of replication by acting specifically on elongation rather than enhancing origin firing. This shortens the S-phase duration. Combining data from Spi-1 knockdown in murine cells with Spi-1 overexpression in human cells, we provide evidence that inappropriate Spi-1 expression is directly responsible for the replication alteration observed. Importantly, the acceleration of replication progression coincides with an increase in the frequency of genomic mutations without inducing DNA breakage. Thus, we propose that the hitherto unsuspected role for spi-1 oncogene in promoting replication elongation and genomic mutation promotes blastic progression during leukemic development.

M Dutreix, J M Cosset, J-S Sun (2010 Jan 14)

Molecular therapy in support to radiotherapy.

Mutation research : 182-9 : DOI : 10.1016/j.mrrev.2010.01.001 En savoir plus

Approximately half of all cancer patients are treated with radiation therapy. However, some tumor cells can escape the lethal effects of irradiation by hypoxia, deregulation of the cell cycle or apoptosis or by increasing their ability to repair the DNA damage induced, resulting in recurrence of disease. In order to overcome these resistance mechanisms, various strategies have been developed. Over the last decade, extensive progress in human genomics and genetic tools has been made. Several methods using DNA or RNA molecules have been developed to target angiogenesis or other cellular functions in order to restore sensitivity to irradiation. In this review, we focus on five classes of nucleic acid-based approaches, (i) gene transfer by recombinant plasmid or virus, (ii) immune-stimulating oligonucleotides, (iii) antisense oligonucleotides, (iv) siRNA and shRNA, and (v) siDNA (signal interfering DNA), which target specific proteins or pathways involved in radioresistance. We review the results of the preclinical studies and clinical trials conducted to date by combining nucleic acid-based molecular therapy and radiotherapy.


Année de publication : 2009

Matteo Campana, Benoit Maury, Marie Dutreix, Nadine Peyriéras, Alessandro Sarti (2009 Sep 29)

Methods toward in vivo measurement of zebrafish epithelial and deep cell proliferation.

Computer methods and programs in biomedicine : 103-17 : DOI : 10.1016/j.cmpb.2009.08.008 En savoir plus

We present a strategy for automatic classification and density estimation of epithelial enveloping layer (EVL) and deep layer (DEL) cells, throughout zebrafish early embryonic stages. Automatic cells classification provides the bases to measure the variability of relevant parameters, such as cells density, in different classes of cells and is finalized to the estimation of effectiveness and selectivity of anticancer drug in vivo. We aim at approaching these measurements through epithelial/deep cells classification, epithelial area and thickness measurement, and density estimation from scattered points. Our procedure is based on Minimal Surfaces, Otsu clustering, Delaunay Triangulation, and Within-R cloud of points density estimation approaches. In this paper, we investigated whether the distance between nuclei and epithelial surface is sufficient to discriminate epithelial cells from deep cells. Comparisons of different density estimators, experimental results, and extensively accuracy measurements are included.

Maria Quanz, Danielle Chassoux, Nathalie Berthault, Céline Agrario, Jian-Sheng Sun, Marie Dutreix (2009 Jul 22)

Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

PloS one : e6298 : DOI : 10.1371/journal.pone.0006298 En savoir plus

Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an « all-or-none » pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a « false » DNA damage signaling that disorganizes the DNA repair system.

Maria Quanz, Nathalie Berthault, Christophe Roulin, Maryline Roy, Aurélie Herbette, Céline Agrario, Christophe Alberti, Véronique Josserand, Jean-Luc Coll, Xavier Sastre-Garau, Jean-Marc Cosset, Lionel Larue, Jian-Sheng Sun, Marie Dutreix (2009 Feb 3)

Small-molecule drugs mimicking DNA damage: a new strategy for sensitizing tumors to radiotherapy.

Clinical cancer research : an official journal of the American Association for Cancer Research : 1308-16 : DOI : 10.1158/1078-0432.CCR-08-2108 En savoir plus

Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage.


Année de publication : 2005

G Mercier, N Berthault, N Touleimat, F Képès, G Fourel, E Gilson, M Dutreix (2005 Dec 3)

A haploid-specific transcriptional response to irradiation in Saccharomyces cerevisiae.

Nucleic acids research : 6635-43 En savoir plus

Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. We used global transcriptome analysis to investigate the role of ploidy and mating-type in inducing the response to damage in various Saccharomyces cerevisiae strains. We observed a response to DNA damage specific to haploid strains that seemed to be controlled by chromatin regulatory proteins. Consistent with these microarray data, we found that mating-type factors controlled the chromatin-dependent silencing of a reporter gene. Both these analyses demonstrate the existence of an irradiation-specific response in strains (haploid or diploid) with only one mating-type factor. This response depends on the activities of Hdf1 and Sir2. Overall, our results suggest the existence of a new regulation pathway dependent on mating-type factors, chromatin structure remodeling, Sir2 and Hdf1 and independent of Mec1 kinase.


Année de publication : 2004

G Mercier, N Berthault, J Mary, J Peyre, A Antoniadis, J-P Comet, A Cornuejols, C Froidevaux, M Dutreix (2004 Jan 15)

Biological detection of low radiation doses by combining results of two microarray analysis methods.

Nucleic acids research : e12 En savoir plus

The accurate determination of the biological effects of low doses of pollutants is a major public health challenge. DNA microarrays are a powerful tool for investigating small intracellular changes. However, the inherent low reliability of this technique, the small number of replicates and the lack of suitable statistical methods for the analysis of such a large number of attributes (genes) impair accurate data interpretation. To overcome this problem, we combined results of two independent analysis methods (ANOVA and RELIEF). We applied this analysis protocol to compare gene expression patterns in Saccharomyces cerevisiae growing in the absence and continuous presence of varying low doses of radiation. Global distribution analysis highlights the importance of mitochondrial membrane functions in the response. We demonstrate that microarrays detect cellular changes induced by irradiation at doses that are 1000-fold lower than the minimal dose associated with mutagenic effects.


Année de publication : 2003

Elodie Biet, Lionel Larue, Marie Dutreix (2003 May 14)

[Homologous recombination and gene targeting].

Comptes rendus biologies : 51-64 En savoir plus

Gene therapy and the production of mutated cell lines or model animals both require the development of efficient, controlled gene-targeting strategies. Classical approaches are based on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. The low frequency of homologous recombination in mammalian cells leads to inefficient targeting. Here, we review the limiting steps of classical approaches and the new strategies developed to improve the efficiency of homologous recombination in gene-targeting experiments.

Elodie Biet, Christophe Alberti, Paolo Faccella, Jian Sheng Sun, Marie Dutreix, Lionel Larue (2003 Mar 8)

Tyrosinase gene correction using fluorescent oligonucleotides.

Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society : 133-8 En savoir plus

Gene therapy and production of mutated cell lines or animal models should be improved significantly once efficient controlled gene targeting strategies are developed. We used short single-stranded oligodeoxynucleotides (ODN), in some cases coupled to the fluorescent dye fluorescein isothiocyanate (FITC), to correct an endogenic natural point mutation in melanocytes in culture. The addition of the FITC molecule to the 5′ extremity of the ODN did not interfere with the efficiency of the reversion of the mutation and did not have any deleterious side-effects. The use of fluorescent ODN could lead to great improvement in the technique. In particular, it may facilitate sorting of the transfected cells in the treated population, and thereby significantly increase the percentage of corrected cells.

Elodie Biet, Jian-Sheng Sun, Marie Dutreix (2003 Feb 1)

Stimulation of D-loop formation by polypurine/polypyrimidine sequences.

Nucleic acids research : 1006-12 En savoir plus

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.


Année de publication : 2002

C G Gendrel, M Dutreix (2002 Jan 10)

(CA/TG) microsatellite sequences escape the inhibition of recombination by mismatch repair in Saccharomyces cerevisiae.

Genetics : 1539-45 En savoir plus

Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, whereas failure to repair mismatches results in postmeiotic segregation (PMS). By examining the conversion and PMS in yeast strains deficient in various MMR genes and heterozygous for large inserts (107 bp) with either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we demonstrate that: (1) the inhibition of conversion by large inserts depends upon a complex containing both Msh2 and Pms1 proteins; (2) conversion is not inhibited if the single-stranded DNA loop in the heteroduplex is the microsatellite sequence; and (3) large heteroduplex loops with random sequence or repetitive sequence might be repaired by two complexes, containing either Msh2 or Pms1. Our results suggest that inhibition of recombination by heterologous inserts and large loop repair are not processed by the same MMR complexes. We propose that the inhibition of conversion by large inserts is due to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand interactions in the heteroduplex loop.


Année de publication : 2001

G Mercier, Y Denis, P Marc, L Picard, M Dutreix (2001 Dec 12)

Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae.

Mutation research : 157-72 En savoir plus

We investigated the inhibition of cell-cycle progression and replication and the induction of the transcriptional response in diploid budding yeast populations exposed to two different doses of gamma-rays resulting in 15 and 85% survival respectively. We studied the kinetics of the cellular response to ionizing treatment during the period required for all of the surviving cells to achieve at least one cell division. The length of these periods increased with the dose. Irradiated populations arrested as large-budded cells containing partially replicated chromosomes. The extent of the S-phase was proportional to the amount of damage and lasted 3 or 7h depending on the irradiation dose. In parallel to the division study, we carried out a kinetic analysis of the expression of 126 selected genes by use of dedicated microarrays. About 26 genes were induced by irradiation and displayed various pattern of expression. Interestingly, 10 repair genes (RAD51, RAD54, CDC8, MSH2, RFA2, RFA3, UBC5, SRS2, SPO12 and TOP1), involved in recombination and DNA synthesis, display similar regulation of expression in the two irradiated populations. Their pattern of expression were confirmed by Northern analysis. At the two doses, the expression of this group of genes closely followed the extended replication period, and their expression resumed when replication restarted. These results suggest that the damage-induced response and DNA synthesis are closely regulated during repair. The analysis of the promoter regions indicates a high occurrence of the three MCB, HAP and UASH regulatory boxes in the promoters of this group of genes. The association of the three boxes could confer an irradiation-replication specific regulation.


Année de publication : 2000

C G Gendrel, A Boulet, M Dutreix (2000 May 19)

(CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis.

Genes & development : 1261-8 En savoir plus

One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes.


Année de publication : 1998

E Biet, J Sun, M Dutreix (1998 Dec 24)

Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure.

Nucleic acids research : 596-600 En savoir plus

Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.

H Debrauwere, C G Gendrel, S Lechat, M Dutreix (1998 Feb 18)

Differences and similarities between various tandem repeat sequences: minisatellites and microsatellites.

Biochimie : 577-86 En savoir plus

Tandemly repetitive DNA sequences are abundantly interspersed in the genome of practically all eukaryotic species studied. The relative occurrence of one type of repetitive sequence and its location in the genome appear to be species specific. A common property of repetitive sequences within the living world is their ability to give rise to variants with increased or reduced number of repeats. This instability depends upon numerous parameters whose exact role is unclear: the number of repeats, their sequence content, their chromosomal location, the mismatch repair capability of the cell, the developmental stage of the cell (mitotic or meiotic) and/or the sex of the transmitting parent. It is now apparent that mutations in repetitive sequences are a common cause of human disease, including cancer and disorders which may exhibit a dominant mode of inheritance. Two mechanisms have been proposed to explain the instability of repetitive sequences: DNA polymerase slippage, which may account for the instability of short repeats and unequal recombination which reshuffles repeat variants and maintains repeat heterogeneity in minisatellites. The purpose of this review is to show that no general rule can explain the instability of repetitive sequence. Each sequence of repeats is under the influence of local and general biological activities that determine its level of instability.