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 G, 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 G(2) cells. Fluorescence-activated cell sorter analysis demonstrated that the S-phase cells did not incorporate BrdU and likely represented cells that arrested in G, 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 G(2) arrest at 24 h, but at 48 and 72 h there were also decreases in the level of G(2) cells and concomitant increases in the levels of S, G(0)/G(1), and sub-G(0) 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 G, 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 G, 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.