CAMPTOTHECIN INDUCTION OF A TIME-DEPENDENT AND CONCENTRATION-DEPENDENT DECREASE OF TOPOISOMERASE-I AND ITS IMPLICATION IN CAMPTOTHECIN ACTIVITY

Camptothecin (CPT) has been shown to induce protein-linked DNA breaks (PLDB), which are stabilized intermediates of topoisomerase I (TOP1) a ctivity. Due to the reversible nature of PLDB and the need for replica tion fork movement for CPT toxicity, both the time of CPT exposure and TOP1 levels are determinants of CPT toxicity. Therefore, the effects of CPT exposure on TOP1 over time were examined in an established huma n cell line, KB. Using an in vivo KCI-SDS co-precipitation assay, it w as determined that 1 hr of CPT exposure resulted in a concentration-de pendent increase in PLDB that reached a maximum at 5 mu M CPT. However , prolonged incubations with CPT revealed a concentration- and time-de pendent decrease in CPT-induced PLDB formation. The most rapid loss of PLDB was within 6 hr. Neither aphidicolin nor cycloheximide cotreatme nts altered the PLDB decrease induced by CPT. Immunoblot analysis reve aled a reduction in TOP1 protein upon CPT exposure, whereas RNA analys is revealed no changes, which suggested a post-transciptional mechanis m of TOP1 down-regulation. The CPT-induced reduction was specific for TOP1, because actin and tubulin levels were unaltered by CPT exposure. Finally, clonogenic assays revealed a small but statistically signifi cant decrease in CPT toxicity throughout the CPT exposure period. Beca use PLDB formation based on TOP1 levels is an important step in the to xicity of CPT, the CPT-induced TOP1 reduction could be a transient mec hanism of resistance for cells to avoid toxic levels of PLDB.