INDUCTION OF NEURONAL APOPTOSIS BY CAMPTOTHECIN, AN INHIBITOR OF DNA TOPOISOMERASE-I - EVIDENCE FOR CELL CYCLE-INDEPENDENT TOXICITY

Camptothecin is an S-phase-specific anticancer agent that inhibits the activity of the enzyme DNA topoisomerase-I (topo-I). Irreversible DNA double-strand breaks are produced during DNA synthesis in the presenc e of camptothecin, suggesting that this agent should not be toxic to n ondividing cells, such as neurons. Unexpectedly, camptothecin induced significant, dose-dependent cell death of postmitotic rat cortical neu rons in vitro; astrocytes were more resistant. Aphidicolin, an inhibit or of DNA polymerase or, did not prevent camptothecin-induced neuronal death, while death was prevented by actinomycin D and 5,6-dichloro-1- beta-D-ribofuranosyl benzimidazole as well as cycloheximide and anisom ycin, inhibitors of RNA and protein synthesis, respectively. Camptothe cin-induced neuronal death was apoptotic, as characterized by chromati n condensation, cytoplasmic shrinking, plasma membrane blebbing, and f ragmentation of neurites. DNA fragmentation was also confirmed by the use of the in situ DNA end labeling assay, In addition, aurintricarbox ylic acid, an inhibitor of the apoptotic endonuclease, partially prote cted against camptothecin-induced neuronal death. The toxicity of ster eoisomers of a camptothecin analogue was stereospecific, demonstrating that toxicity was a result of inhibition of topo-I. The difference in sensitivity to camptothecin between neurons and astrocytes correlated with their transcriptional activity and level of topo-I protein expre ssion. These data indicate important roles for topo-I in postmitotic n eurons and suggest that topo-I inhibitors can induce apoptosis indepen dent of DNA synthesis, We suggest a model based on transcriptionally m ediated DNA damage, a novel mechanism of action of topo-I poisons.