Exposure of Lymphocytes to High Doses of Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Induces Rapid Onset of Apoptosis-Mediated DNA Fragmentation

We have shown previously that treatment of human lymphocytes with the Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) results in dose-dependent G₂ arrest, followed 24 h later by apoptotic cell death. Here we demonstrated that for Jurkat cells exposed to high concentrations of Cdt (>0.2 ng/ml) there was a dose-dependent increase in the level of S-phase cells and a concomitant decrease in the level of G₂ cells. Fluorescence-activated cell sorter analysis demonstrated that the S-phase cells did not incorporate BrdU and likely represented cells that arrested in G₂ and underwent significant DNA fragmentation. Analysis of the kinetics of the appearance of both S-phase cells and apoptotic cells supported this interpretation. Cells exposed to low doses of toxin exhibited G₂ arrest at 24 h, but at 48 and 72 h there were also decreases in the level of G₂ cells and concomitant increases in the levels of S, G₀/G₁, and sub-G₀ cells; these changes were paralleled by increased numbers of apoptotic cells. Cells exposed to high doses of toxin exhibited these changes 24 to 48 h earlier. We also examined the relationship between G₂ arrest, DNA fragmentation, and activation of the apoptotic cascade. We employed two inhibitors of apoptosis, overexpression of Bcl-2 and the caspase-3 inhibitor zvad. Both inhibitors blocked Cdt-induced apoptosis, Cdt-induced DNA fragmentation, and phosphorylation of the histone H2AX. However, the cells retained the ability to undergo G₂ arrest in the presence of the toxin. Thus, it appears that high doses of Cdt induce rapid onset of DNA degradation resulting from activation of the apoptotic cascade.