DNA-SYNTHESIS PRECEDES GLIOTOXIN-INDUCED APOPTOSIS

The toxin gliotoxin induces apoptosis or programmed cell death in a va riety of immune cells including thymocytes. Apoptosis induced by gliot oxin in thymocytes is unaffected by protein synthesis inhibitors nor i s it associated with early changes in intracellular calcium levels (Be aver and Waring, 1994). This work shows that the cell lines P815 and W EHI 7 and murine thymocytes when treated with gliotoxin show an early incorporation of tritiated thymidine over the concentration range whic h causes apoptosis. Proliferating cell nuclear antigen (PCNA), a marke r for S phase, is elevated in cells following gliotoxin treatment and S phase DNA content is increased. Thymidine incorporation is inhibited by hydroxyurea, an inhibitor of replicative DNA synthesis not repair. Free radical scavangers have no effect on apoptosis induced by glioto xin in thymocytes. Hydrogen peroxide-treated cells showed no enhanced thymidine incorporation and no apoptosis. Thus oxidative stress does n ot appear to be a factor in gliotoxin-induced apoptosis. Thymocytes tr eated with gliotoxin show increased phosphorylation of a 16.3 kDa prot ein, and apoptosis is inhibited by the tyrosine kinase inhibitor genis tein, which also inhibited the increased thymidine incorporation in P8 15 cells, We conclude that one mechanism by which gliotoxin can cause apoptosis may be the induction of inappropriate entry of cells into th e cell cycle followed by death.