SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione Stransferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G0/G1 and S in HEK293 cells, whereas HEK293/SET showed G2/M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.9-100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.

Effectiveness, against tuberculosis, of pseudo-ternary complexes: Peptide-DNA-cationic liposome

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-alpha-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1 M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery. (C) 2011 Elsevier Inc. All rights reserved.

Fragmentation of extracellular DNA by long-term exposure to radiation from uranium in aquatic environments

Persistent harmful scenarios associated with disposal of radioactive waste, high-background radiation areas and severe nuclear accidents are of great concern regarding consequences to both human health and the environment. Of particular concern is the extracellular DNA in aquatic environments contaminated by radiological substances. Strand breaks induced by radiation promote decrease in the transformation efficiency for extracellular DNA. The focus of this study is the quantification of DNA damage following long-term exposure (over one year) to low doses of natural uranium (an alpha particle emitter) to simulate natural conditions, since nothing is known about alpha radiation induced damage to extracellular DNA. A high-resolution Atomic Force Microscope was used to evaluate DNA fragments. Double-stranded plasmid pBS as a model for extracellular DNA was exposed to different amounts of natural uranium. It was demonstrated that low concentrations of U in water (50 to 150 ppm) produce appreciable numbers of double strand breaks, scaling with the square of the average doses. The importance of these findings for environment monitoring of radiological pollution is addressed.

Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome micro-dissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini.

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Background: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300Vcm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215 000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.