Effects of bleomycin on PHA-stimulated lymphocytes and isolated hepatic nuclei.

Abstract

Experiments were carried out in which lymphocytes stimulated by phytohemagglutinin were exposed during 3-day culture to concentrations of bleomycin varying from 0.0 to 300 μg/ml. Inhibition of one hour 3H-thymidine incorporation in these 72-hour cultures increased as the concentration of bleomycin increased from 1.5μg/ml (14.1 % inhibition) to 300μg/ml (96.4% inhibition), and was a linear function when dose and percent control 3H-thymidine uptake were plotted logarithmically. In 3 of 9 experiments, 3H-thymidine uptake at the 1.5μg/ml dose was increased. This increase was not statistically significant, but the possibility of sublethal DNA damage followed by repair was raised. Exposure of resting lymphocytes to bleomycin in the absence of PHA showed no significant increase above baseline thymidine uptake. Addition of bleomycin to cultures at 24 and 48 hours after the addition of PHA produced the same quantitative reduction in uptake of thymidine at 70 hours as the same concentration added at zero time, suggesting that the effect of bleomycin is independent of the initial stimulatory events resulting from the addition of PHA. Evidence of a repair-like process following damage by bleomycin was observed in a different system utilizing isolated hepatic nuclei. A two to five fold increase of thymidine tri-phosphate incorporation was obtained in response to addition of bleomycin in this isolated nuclear system (rat liver nuclei). This process was time and dose related and continued for at least one hour in the presence of bleomycin. These results demonstrate that bleomycin causes dose-dependent reduction of DNA synthesis in PHA-stimulated lymphocytes. Furthermore, they suggest that DNA damage caused by low concentrations of bleomycin may be repaired, and that when damage exceeds the cell's ability to repair, cell death results.