Tests for measuring DNA damage following exposure to ionising radiation and genotoxicity testing of chemicals and products such as pharmaceuticals, pesticides, food additives, and cosmetics are important for the assessment of health hazard and are also regulatory requirements. We developed a new genotoxicity test – a cell-based biosensor system - based on a p21 promoter-dependent DsRED reporter gene assay with stably transformed human hepatoma HepG2 cells. The performance and sensitivity of our cell-based biosensor system for the detection of DNA damage has been evaluated with genotoxic agents with known mechanisms of action, such as radiation and chemical agents cisplatin, methyl methanesulphonate (MMS), benzo(a)pyrene (BaP) and vinblastine (VLB). Our preliminary results show that radiation, as well as all tested agents, induces the increase in DsRED fluorescence above a certain dose (concentration) in a dose-dependent manner. Based on the results, we demonstrated that this novel assay can be used as a fast and simple biosensor system for the detection of genetic damage
COBISS.SI-ID: 1366651
The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12. Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter. In this study p21 promoter was proven to be a suitable candidate for radiation induced transcriptional targeting. As a proof of principle the therapeutic value was demonstrated with the radio-inducible interleukin 12 plasmid providing a synergistic antitumor effect to radiotherapy alone, which makes this approach feasible for the combined treatment with radiotherapy.
COBISS.SI-ID: 1643131
One of the new approaches for cancer gene therapy represent development of vectors which will ensure the expression of transgene into the specific cell types. Article represents preparation of plasmid DNA encoding green fluorescent protein (GFP) under the control of human endothelin-1 promoter (pENDO-EGFP). Significantly higher expression of GFP was observed in endothelial cells, which were specific for promoter compared to non-endothelial cells. Improved transfection efficacy of pENDO-EGFP into endothelial cells was reached after optimization of gene electrotransfer parameters. Based on these results we can prepare therapeutic plasmid DNA which could be appropriate for antiangiogenic cancer gene therapy.
COBISS.SI-ID: 512362809
Contrary to extensive in vitro studies demonstrating increased gene electrotransfer by changing the electric field direction during the pulse delivery, little is known about the efficiency of both polarities pulses in vivo. Therefore the aim of our study was to evaluate the effect of pulse polarity and orientation on the efficacy of gene electrotransfer in the murine fibrosarcoma tumor model by using the luciferase and GFP reporter gene expression plasmids. Our results demonstrated no significant difference in luciferase activity, GFP transfected area or fluorescence intensity between different sets of electric pulses. Inversion of the pulse polarity did not result in the increase of gene transfer, but non-significant enhancement up to 7-fold was detected by changing the electric field orientation in perpendicular direction. Also, transfection of surrounding skin tissue was observed, meaning that intratumoral gene electrotransfer could also result in a systemic effect. Our data indicate that tested modifications of electric pulses are not significantly affecting the efficiency of gene electrotransfer to LPB tumors. Biological factors, like tissue composition, might be of greater importance and should therefore also be taken into account when selecting the electric pulse parameters for specific tissues.
COBISS.SI-ID: 1687675
Aplication of electric pulses is an effective method for gene transfer in vitro and in vivo. Currently, the mechanism by whichthe DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. The aim of the study was to elucidate whether inhibitors of endocytosis an impair transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. Tibialis cranialis in mice. We show that methyl-β-cyclodextrin- general inhibitor of endocitosis- can almost prevent GET of EGFP-N1 plasmid in vitro, that Concanavalin A- inhibitor clathrin mediated endocytosis- also abrogates GET but to lesser extent, and when using Dynasore- reversible inhibitor of dynamin- there is no effect on GET efficacy. The result of this study shows that endocytosis is probably the main mechanism od entrnce of DNA in vitro and also in vivo.
COBISS.SI-ID: 1852795