ABSTRACT
Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower 'efficacy dose/toxicity dose' ratio as compared with Dbait/PEI, coDbait was selected for clinical trials.
Subject(s)
Nanoparticles/chemistry , Oligodeoxyribonucleotides , Animals , Animals, Genetically Modified , Cell Line, Transformed , Female , Genetic Vectors , Kaplan-Meier Estimate , Mice , Mice, Nude , Nanoparticles/administration & dosage , Nanoparticles/toxicity , Neoplasms/genetics , Neoplasms/mortality , Neoplasms/therapy , Oligodeoxyribonucleotides/analysis , Oligodeoxyribonucleotides/chemical synthesis , Transfection , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
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.
Subject(s)
DNA Damage , DNA Repair , Gene Expression Regulation, Bacterial , Haploidy , Saccharomyces cerevisiae/genetics , Antigens, Nuclear/physiology , Chromatin/metabolism , Chromosomes, Bacterial , DNA-Binding Proteins/physiology , Diploidy , Gene Silencing , Genes, Bacterial , Histone Deacetylases/physiology , Ku Autoantigen , Promoter Regions, Genetic , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/radiation effects , Silent Information Regulator Proteins, Saccharomyces cerevisiae/physiology , Sirtuin 2 , Sirtuins/physiology , Transcription, Genetic/radiation effectsABSTRACT
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.
