The main purpose of this study was to determine whether enhancement of repair capacity would attenuate mitochondrial DNA oxidative damage and result in greater cell survival under stressful conditions. analysis of Annexin V and DNA degradation measured by the Comet assay. Another notable obtaining was that ectopic expression of either dOgg1 or RpS3 in mitochondria increased cell survival after exposure to the nitric oxide donor SNAP. These results suggest that ectopic expression of one of the constituents of the DNA repair system in mitochondria may cause a perturbation in the base excision repair pathway and lower, rather than enhance, survivability. mutant [14,15]. Heterologous expression of RpS3 has been previously demonstrated to enhance the removal of 8-oxodG in human cells [16,17]. The main purpose of the present study was to determine whether oxidative damage to mitochondrial DNA can be attenuated by the ectopic expression of DNA glycosylase/AP lyase within the mitochondrial matrix. Specifically, stable S2 transfectant cell lines, expressing dOgg1 or RpS3 proteins in mitochondria, were generated and tested for DNA damage and cell viability under normal and nerve-racking conditions. Materials and methods Generation of Drosophila S2 cells expressing dOgg1 and RpS3 in the mitochondria EST clone LD19945 made up of a cDNA corresponding to the dOgg1 gene in a pBluescript vector and EST clone LD 47488 made up of a cDNA corresponding to the RpS3 gene in a pOT2 vector were obtained from Research Genomics (Huntsville, AL, USA). The 22 amino-terminal codons of the ornithine aminotransferase (OAT) gene, including a putative mitochondrial presequence, was attached to the N-termini of 10129-56-3 the coding regions of the dOgg1 and RpS3 genes, replacing the start codons, using a two-step splicing by overlapping extension (SOE) PCR amplification approach. In the first set of reactions, PCR products made up of OAT and dOgg1 or RpS3 fusion sequences were generated. Primers for the 10129-56-3 generation of the OAT-derived PCR product were 5-gatattggtaccatcSchneider cells were maintained in total DES Expression medium (Invitrogen) supplemented with 10% FBS and 50 g/ml penicillin/streptomycin (Cellgro). Cells were transfected with 19 g of plasmid DNA using the Calcium Phosphate Transfection Kit followed by selection of stable transfectant cell lines according to the manufacturer’s manual (Invitrogen). After selection, cells were maintained in a DES medium made up of 30 g/ml blasticidin. All cell lines were transferred to new medium every 3C5 days at 1:3C1:5 dilution retaining one-third of the conditioned medium. Localization of recombinant OAT-dOgg1 and OAT-RpS3 proteins in mitochondria was assessed by immunoblot analysis of isolated cell fractions. Experimentally induced stress and cell viability For viability assays, overnight cell cultures that reached 1 106 cells/ml density were exposed to 20 mM hydrogen peroxide (Sigma), 10 mM paraquat (Sigma), or 1 mM cells expressing dOgg1 and RpS3 in mitochondria On the basis of these results, it can be concluded that the recombinant dOgg1 and RpS3 glycosylases targeted to the mitochondrial matrix are functional and that the 8-oxodG excision in mt-dOgg1- and mt-RpS3-transfected S2 cells is usually more efficient than in control cells. The effect of ectopic expression of dOgg1 and RpS3 in mitochondria on cell viability To determine whether the ectopic expression of dOgg1 and RpS3 translates into enhanced cellular survival after oxidative stress, cells were subjected to 20 mM hydrogen 10129-56-3 peroxide, 10 mM paraquat (superoxide anion donor), and 1 mM SNAP (nitric oxide donor), followed by evaluation of cell viability by trypan blue exclusion (Fig. 3). Fig. 3 Viability of 10129-56-3 cells after H2O2, paraquat, and SNAP treatment. Cells were exposed to (A) 20 mM H2O2, (B) 10 mM paraquat, and (C) 1 mM SNAP and percentage cell survival was decided at various time intervals. The survival percentages are shown as the means … Under unchallenged (normal) conditions, cells ectopically expressing either dOgg1 or RpS3 enzyme were as viable as the controls (95C100%). However, compared to the control cells, the transfectant were more sensitive to H2O2 and paraquat, but experienced higher viability when treated with SNAP. Incidence of apoptosis To investigate further the effect of an increase in sensitivity to H2O2 and paraquat, apoptosis-associated DNA fragmentation was quantified in cells expressing RpS3 or dOgg1 in mitochondria. No DNA degradation was observed in samples isolated from your untreated control cells, but a typical internucleosomal fragmentation pattern was observed in untreated dOgg1 and RpS3 transfectants (Fig. 4). We also observed an increase in DNA fragmentation in mitochondrial preparations isolated from cells overexpressing dOgg1 or RpS3 compared to control; however, we have not seen substantial differences in DNA laddering between untreated cells or cells exposed to H2O2, paraquat, or SNAP. Fig. Rabbit polyclonal to UGCGL2 4 DNA fragmentation analysis of mt-dOgg1 and mt-RpS3 cell lines. DNA.