Topotecan tpt

Dose–response curves for glow and spark PAW, and topotecan (TPT) (Sigma-Aldrich, Arklow, Ireland) were established. TPT was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Arklow, Ireland) at a final concentration of 1 mM and stored at −20 °C. This stock was subsequently used to make the working standard solutions in media. The highest concentration of DMSO used was a 5 µM final concentration. Existing media were removed from each well and cells were treated with either PAW, TPT or solvent control (DMSO) and incubated for 72 h. No deleterious effects were observed from the control solvent (range of concentrations between 0.02 nM and 5 µM). Dose–response curves were established for the final % (v/v) of PAW (spark discharge—1 to 10%; glow discharge—5 to 40%), and the final concentration (nM) of TPT (500.00, 250.00, 125.00, 62.50, 31.25, 15.62, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03, 0.015, 0.008, 0.004 and 0.002 for U-251mg cell line and 250.00, 125.00, 62.50, 31.25, 15.62, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03, 0.015, 0.008 and 0.004 for A172 cell line) in each well.

In order to exacerbate the formation of R-loops and determine whether R-loop could form in post-mitotic tissues upon DNA damage accumulation, ARCA mouse models were treated with Topotecan (TPT) (Sigma-Aldrich), a water-soluble derivative of camptothecin for a period of 9 days. Topotecan was administered daily (approximately at 10:00 am) as an intraperitoneal injection following a brief anaesthesia using Isofluorane gas (2.5% at 3 L/min). Isofluorane is one of the safest methods recognised for rodents anaesthesia. For each genotype group, 3 mice were injected daily either with carrier solution (Sterile Injection Water BP, Pfizer) or TPT (2 mg/Kg/Day) and weight was monitored and recorded daily prior and 24 hours post injection. Animal weights were recorded prior, during, and after the treatment regimen. General health and behaviour of the mice was monitored daily until the end of the treatment when the animals were sacrificed to assess for R-loop formation, apoptosis levels and proliferation status.